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Journal Cover Journal of Cellular Physiology
  [SJR: 1.842]   [H-I: 139]   [5 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
   Published by John Wiley and Sons Homepage  [1611 journals]
  • Dermal-Epidermal Cross-Talk: Differential Interactions With Microvascular
           Endothelial Cells
    • Authors: Eleonora Bassino; Edoardo Vallariello, Franco Gasparri, Luca Munaron
      Abstract: The biological importance of circulatory blood supply and angiogenesis for hair growth is now well recognized, but the their regulatory mechanisms require more mechanistic investigation. In vitro cocultures and tricultures can be successfully employed to greatly improve our knowledge on paracrine crosstalk between cell types that populate the dermal-epidermal interface and cutaneous vasculature. Here we report that human dermal fibroblasts (NHDF) promote viability and proliferation of microvascular endothelial cells (HMVEC), while HMVEC are not mitogenic for NHDF. In triculture setup, conditioned media obtained by cocultures (HMVEC/NHDF or HMVEC/follicle fibroblasts) differently modulate growth and proliferation of keratinocytes and alter the expression of metabolic and pro-inflammatory markers. In conclusion, tricultures were successfully employed to characterize in vitro dermal-epithelial and endothelial interactions and could integrate ex vivo and in vivo approaches by the use of high-throughput and standardized protocols in controlled conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-20T18:36:10.658839-05:
      DOI: 10.1002/jcp.25657
  • ADP-Induced Ca2+ Signalling and Proliferation of Rat Ventricular
           Myofibroblasts Depend on Phospholipase C-Linked TRP Channels Activation
           Within Lipid Rafts
    • Authors: Mariana Certal; Adriana Vinhas, Aurora Barros-Barbosa, Fátima Ferreirinha, Maria Adelina Costa, Paulo Correia-de-Sá
      Abstract: Nucleotides released during heart injury affect myocardium electrophysiology and remodelling through P2 purinoceptors activation in cardiac myofibroblasts. ATP and UTP endorse [Ca2+]i accumulation and growth of DDR-2/a-SMA-expressing myofibroblasts from adult rat ventricles via P2Y4 and P2Y2 receptors activation, respectively. Ventricular myofibroblasts also express ADP-sensitive P2Yi, P2Yi2 and P2Yi3 receptors as demonstrated by immunofluorescence confocal microscopy and western blot analysis, but little information exists on ADP effects in these cells. ADP (0.003-3mM) and its stable analogue, ADPBS (100uM), caused fast [Ca2+]i transients originated from thapsigargin-sensitive internal stores, which partially declined to a plateau sustained by capacitative Ca2+ entry through transient receptor potential (TRP) channels inhibited by 2-APB (50uM) and flufenamic acid (100uM). Hydrophobic interactions between Gq/11-coupled P2Y purinoceptors and TRP channels were suggested by prevention of the ADP-induced [Ca2+]i plateau following PIP2 depletion with LiCl (10mM) and cholesterol removal from lipid rafts with methyl-B-cyclodextrin (2mM). ADP [Ca2+]i transients were insensitive to P2Y1, P2Y12 and P2Y13 receptor antagonists, MRS2179 (10uM), AR-C66096 (0.1uM) and MRS2211 (10uM), respectively, but were attenuated by suramin and reactive blue-2 (100uM) which also blocked P2Y4 receptors activation by UTP. Cardiac myofibroblasts growth and type I collagen production were favoured upon activation of MRS2179-sensitive P2Y1 receptors with ADP or ADPBS (30uM). In conclusion, ADP exerts a dual role on ventricular myofibroblasts: [Ca2+]i transients are mediated by fast-desensitizing P2Y4 receptors, whereas the pro-fibrotic effect of ADP involves the P2Y1 receptor activation. Data also show that ADP-induced capacitative Ca2+ influx depends on phospholipase C-linked TRP channels opening in lipid raft microdomains. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-18T11:55:22.363946-05:
      DOI: 10.1002/jcp.25656
  • Neuroprotection and Blood-Brain Barrier Restoration by Salubrinal After a
           Cortical Stab Injury
    • Authors: M. Asunción Barreda-Manso; Natalia Yanguas-Casás, Manuel Nieto-Sampedro, Lorenzo Romero-Ramírez
      Abstract: Following a central nervous system (CNS) injury, restoration of the blood-brain barrier (BBB) integrity is essential for recovering homeostasis. When this process is delayed or impeded, blood substances and cells enter the CNS parenchyma, initiating an additional inflammatory process that extends the initial injury and causes so-called secondary neuronal loss. Astrocytes and profibrotic mesenchymal cells react to the injury and migrate to the lesion site, creating a new glia limitans that restores the BBB. This process is beneficial for the resolution of the inflammation, neuronal survival and the initiation of the healing process. Salubrinal is a small molecule with neuroprotective properties in different animal models of stroke and trauma to the CNS. Here, we show that salubrinal increased neuronal survival in the neighbourhood of a cerebral cortex stab injury. Moreover, salubrinal reduced cortical blood leakage into the parenchyma of injured animals compared with injured controls. Adjacent to the site of injury, salubrinal induced immunoreactivity for platelet-derived growth factor subunit B (PDGF-B), a specific mitogenic factor for mesenchymal cells. This effect might be responsible for the increased immunoreactivity for fibronectin and the decreased activation of microglia and macrophages in injured mice treated with salubrinal, compared with injured controls. The immunoreactivity for PDGF-B colocalized with neuronal nuclei (NeuN), suggesting that cortical neurons in the proximity of the injury were the main source of PDGF-B. Our results suggest that after an injury, neurons play an important role in both, the healing process and the restoration of the BBB integrity. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-18T06:40:27.39664-05:0
      DOI: 10.1002/jcp.25655
  • Src Family Kinase Links Insulin Signaling to Short Term Regulation of
           Na,K-ATPase in Nonpigmented Ciliary Epithelium
    • Authors: Mohammad Shahidullah; Amritlal Mandala, Nicholas A. Delamere
      Abstract: Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2 fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2 and p38 activation, all of which were prevented by a PI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates PI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-17T06:44:20.795008-05:
      DOI: 10.1002/jcp.25654
  • p62 Promotes Amino Acid Sensitivity of mTOR Pathway and Hepatic
           Differentiation in Adult Liver Stem/Progenitor Cells
    • Authors: Masakazu Sugiyama; Tomoharu Yoshizumi, Yoshihiro Yoshida, Yuki Bekki, Yoshihiro Matsumoto, Shohei Yoshiya, Takeo Toshima, Toru Ikegami, Shinji Itoh, Norifumi Harimoto, Shinji Okano, Yuji Soejima, Ken Shirabe, Yoshihiko Maehara
      Abstract: Autophagy is a homeostatic process regulating turnover of impaired proteins and organelles, and p62 (sequestosome-1, SQSTM1) functions as the autophagic receptor in this process. p62 also functions as a hub for intracellular signaling such as that in the mammalian target of rapamycin (mTOR) pathway. Liver stem/progenitor cells have the potential to differentiate to form hepatocytes or cholangiocytes. In this study, we examined effects of autophagy, p62 and associated signaling on hepatic differentiation. Adult stem/progenitor cells were isolated from the liver of mice with chemically-induced liver injury. Effects of autophagy, p62 and related signaling pathways on hepatic differentiation were investigated by silencing the genes for autophagy protein 5 (ATG5) and/or SQSTM1/p62 using small interfering RNAs. Hepatic differentiation was assessed based on increased albumin and hepatocyte nuclear factor 4α, as hepatocyte markers, and decreased cytokeratin 19 and SOX9, as stem/progenitor cell markers. These markers were measured using quantitative RT-PCR, immunofluorescence and western blotting. ATG5 silencing decreased active LC3 and increased p62, indicating inhibition of autophagy. Inhibition of autophagy promoted hepatic differentiation in the stem/progenitor cells. Conversely, SQSTM1/p62 silencing impaired hepatic differentiation. A suggested mechanism for p62-dependent hepatic differentiation in our study was activation of the mTOR pathway by amino acids. Amino acid activation of mTOR signaling was enhanced by ATG5 silencing and suppressed by SQSTM1/p62 silencing. Our findings indicated that promoting amino acid sensitivity of the mTOR pathway is dependent on p62 accumulated by inhibition of autophagy and that this process plays an important role in the hepatic differentiation of stem/progenitor cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-17T06:44:12.967902-05:
      DOI: 10.1002/jcp.25653
  • The Natural cAMP Elevating Compound Forskolin in Cancer Therapy: Is it
    • Authors: Luigi Sapio; Monica Gallo, Michela Illiano, Emilio Chiosi, Daniele Naviglio, Annamaria Spina, Silvio Naviglio
      Abstract: Cancer is a major public health problem and the second leading cause of mortality around the world. Although continuous advances in the science of oncology and cancer research are now leading to improved outcomes for many cancer patients, novel cancer treatment options are strongly demanded. Naturally occurring compounds from a variety of vegetables, fruits and medicinal plants have been shown to exhibit various anticancer properties in a number of in vitro and in vivo studies and represent an attractive research area for the development of new therapeutic strategies to fight cancer.Forskolin is a diterpene produced by the roots of the Indian plant Coleus forskohlii. The natural compound forskolin has been used for centuries in traditional medicine and its safety has also been documented in conventional modern medicine. Forskolin directly activates the adenylate cyclase enzyme, that generates cAMP from ATP, thus raising intracellular cAMP levels.Notably, cAMP signaling, through the PKA-dependent and/or independent pathways, is very relevant to cancer and its targeting has shown a number of antitumor effects, including the induction of mesenchymal-to-epithelial transition, inhibition of cell growth and migration and enhancement of sensitivity to conventional antitumor drugs in cancer cells.Here, we describe some features of cAMP signaling that are relevant to cancer biology and address the state of the art concerning the natural cAMP elevating compound forskolin and its perspectives as an effective anticancer agent. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-14T03:21:46.56839-05:0
      DOI: 10.1002/jcp.25650
  • Increased Chondrogenic Potential of Mesenchymal Cells from Adipose Tissue
           Versus Bone Marrow Derived Cells in Osteoarthritic In Vitro Models
    • Authors: Stefania Pagani; Veronica Borsari, Francesca Veronesi, Andrea Ferrari, Simona Cepollaro, Paola Torricelli, Giuseppe Filardo, Milena Fini
      Abstract: ObjectivesPrimarily, to compare the behavior of human mesenchymal stem cells (MSCs) derived from bone marrow (hBMSCs) and adipose tissue (hADSCs) in an osteoarthritic (OA) microenvironment; secondly, to investigate the reaction of these cell types in two alternative in vitro culture systems, obtained by using TNFα and/or IL1β as inflammation mediators, or by using synovial fluid harvested by OA patients (OSF) to simulate the complex inflamed knee microenvironment.Methods3D micromass cultures of hBMSCs or hADSCs were grown in chondrogenic medium (CTR), in the presence of TNFα and/or IL1β, or synovial fluid from OA patients. After 1 month of culture, the chondrogenic differentiation of micromasses was evaluated by gene expression, matrix composition and organization.ResultsBoth hMSCs types formed mature micromasses in CTR, but a better response of hADSCs to the inflammatory environment was documented by micromass area and Bern score evaluations. The addition of OSF elicited a milder reaction than with TNFα and/or IL1β by both cell types, probably due to the presence of both catabolic and protective factors. In particular, SOX9 and ACAN gene expression and GAG synthesis were more abundant in hADSCs than hBMSCs when cultured in OSF. The expression of MMP1 was increased for both hMSCs in inflammatory conditions, but in particular by hBMSCs.ConclusionhADSCs showed an increased chondrogenic potential in inflammatory culture systems, suggesting a better response of hADSCs in the OA environment, thus underlining the importance of appropriate in vitro models to study MSCs and potential advantages of using these cells for future clinical applications. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-14T03:21:41.163181-05:
      DOI: 10.1002/jcp.25651
  • Maintenance of Bone Homeostasis by DLL1-Mediated Notch Signaling
    • Authors: Yukari Muguruma; Katsuto Hozumi, Hiroyuki Warita, Takashi Yahata, Tomoko Uno, Mamoru Ito, Kiyoshi Ando
      Abstract: Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta-like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast-specific manner, we investigated the ligand-specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1-induced Notch signaling was responsible for the expansion of the bone-forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1-Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast-specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast-osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1-mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand-specific activation of Notch signaling in the maintenance of bone homeostasis. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:55:59.336038-05:
      DOI: 10.1002/jcp.25647
  • Role of microRNAs on the Regulation of Mitochondrial Biogenesis and
           Insulin Signaling in Skeletal Muscles
    • Authors: Tanes I. Lima; Hygor N. Araujo, Eveline S. Menezes, Carlos H. Sponton, Michel B. Araújo, Lucas H. M. Bomfim, André L. Queiroz, Madla A. Passos, Thais Amaral e Sousa, Sandro M. Hirabara, Amanda R. Martins, Helena C.L.B. Sampaio, Alice Rodrigues, Rui Curi, Everardo M. Carneiro, Antônio C. Boschero, Leonardo R. Silveira
      Abstract: Mitochondria play a critical role in several cellular processes and cellular homeostasis. Mitochondrion dysfunction has been correlated with numerous metabolic diseases such as obesity and type 2 diabetes. MicroRNAs are non-coding RNAs that have emerged as key regulators of cell metabolism. The microRNAs act as central regulators of metabolic gene networks by leading to the degradation of their target messenger RNA or repression of protein translation. In addition, vesicular and non-vesicular circulating miRNAs exhibit a potential role as mediators of the cross-talk between the skeletal muscle and other tissues/organs. In this review, we will focus on the emerging knowledge of miRNAs controlling mitochondrial function and insulin signaling in skeletal muscle cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:47:09.296418-05:
      DOI: 10.1002/jcp.25645
  • PPARα Antagonist AA452 Triggers Metabolic Reprogramming and Increases
           Sensitivity to Radiation Therapy in Human Glioblastoma Primary Cells
    • Authors: E Benedetti; M d'Angelo, A Ammazzalorso, G Gravina, C Laezza, A Antonosante, G Panella, B Cinque, L Cristiano, AC Dhez, C Astarita, R Galzio, MG Cifone, R Ippoliti, R Amoroso, E Di Cesare, A Giordano, A Cimini
      Abstract: Glioblastoma is the most-common cancer in the brain and with an increasing incidence. Despite major advances in the field, there is no curative therapy for GB to date. Many solid tumors, including GB, experienced metabolic reprogramming in order to sustain uncontrolled proliferation, hypoxic conditions and angiogenesis. PPARs, member of the steroid hormone receptor superfamily, are particularly involved in the control of energetic metabolism, particularly lipid metabolism, which has been reported deregulated in gliomas. PPARα was previously indicated by us as a potential therapeutic target for this neoplasm, due to the malignancy grade dependency of its expression, being particularly abundant in GB. In this work we used a new PPARα antagonist on patient-derived GB primary cells with particular focus on the effects on lipid metabolism and response to radiotherapy. The results obtained demonstrated that blocking PPARα results in cell death induction, increase of radiosensitivity and decrease of migration. Therefore, AA452 arises as a potent drug adjuvanting the gold standard therapies for GB, opening the possibility for pre-clinical and clinical trials for this class of compounds. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:47:07.048024-05:
      DOI: 10.1002/jcp.25648
  • Primary Cilium-Regulated EG-VEGF Signaling Facilitates Trophoblast
    • Authors: Chia-Yih Wang; Hui-Ling Tsai, Jhih-Siang Syu, Ting-Yu Chen, Mei-Tsz Su
      Abstract: Trophoblast invasion is an important event in embryo implantation and placental development. During these processes, endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is the key regulator mediating the crosstalk at the feto-maternal interface. The primary cilium is a cellular antenna receiving environmental signals and is crucial for proper development. However, little is known regarding the role of the primary cilium in early human pregnancy. Here, we demonstrate that EG-VEGF regulates trophoblast cell invasion via primary cilia. We found that EG-VEGF activated ERK1/2 signaling and subsequent upregulation of MMP2 and MMP9, thereby facilitating cell invasion in human trophoblast HTR-8/SVneo cells. Inhibition of ERK1/2 alleviated the expression of MMPs and trophoblast cell invasion after EG-VEGF treatment. In addition, primary cilia were observed in all the trophoblast cell lines tested and, more importantly, in human first-trimester placental tissue. The receptor of EG-VEGF, PROKR1, was detected in primary cilia. Depletion of IFT88, the intraflagellar transporter required for ciliogenesis, inhibited primary cilium growth, thereby ameliorating ERK1/2 activation, MMP upregulation, and trophoblast cell invasion promoted by EG-VEGF. These findings demonstrate a novel function of primary cilia in controlling EG-VEGF-regulated trophoblast invasion and reveal the underlying molecular mechanism. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:46:59.819024-05:
      DOI: 10.1002/jcp.25649
  • Arabinogalactan Proteins from Baobab and Acacia Seeds Influence Innate
           Immunity of Human Keratinocytes in vitro
    • Authors: Abderrakib Zahid; Julie Despres, Magalie Benard, Eric Nguema-Ona, Jerome Leprince, David Vaudry, Christophe Rihouey, Maité Vicré-Gibouin, Azeddine Driouich, Marie-Laure Follet-Gueye
      Abstract: Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However little is currently known regarding their potential activity towards skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:41:38.516217-05:
      DOI: 10.1002/jcp.25646
  • Osteogenic Differentiation of Periosteal Cells during Fracture Healing
    • Authors: Tao Wang; Xinping Zhang, Daniel D. Bikle
      Abstract: 5-10% of fractures fail to heal normally leading to additional surgery, morbidity, and altered quality of life. Fracture healing involves the coordinated action of stem cells primarily coming from the periosteum which differentiate into the chondrocytes and osteoblasts, forming first the soft (cartilage) callus followed by the hard (bone) callus. These stem cells are accompanied by a vascular invasion that appears critical for the differentiation process and which may enable the entry of osteoclasts necessary for the remodeling of the callus into mature bone. However, more research is needed to clarify the signaling events that activate the osteochondroprogenitor cells of periosteum and stimulate their differentiation into chondrocytes and osteoblasts. Ultimately a thorough understanding of the mechanisms for differential regulation of these osteochondroprogenitors will aid in the treatment of bone healing and the prevention of delayed union and nonunion of fractures. In this review, evidence supporting the concept that the periosteal cells are the major cell sources of skeletal progenitors for the fracture callus will be discussed. The osteogenic differentiation of periosteal cells manipulated by Wnt/β-catenin, TGF/BMP, Ihh/PTHrP, and IGF-1/PI3K–Akt signaling in fracture repair will be examined. The effect of physical (hypoxia and hyperoxia) and chemical factors (reactive oxygen species) as well as the potential coordinated regulatory mechanisms in the periosteal progenitor cells promoting osteogenic differentiation will also be discussed. Understanding the regulation of periosteal osteochondroprogenitors during fracture healing could provide insight into possible therapeutic targets and thereby help to enhance future fracture healing and bone tissue engineering approaches. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:40:55.646886-05:
      DOI: 10.1002/jcp.25641
  • New Roles of Lkb1 in Regulating Adipose Tissue Development and
    • Authors: Ziye Xu; Jiaqi Liu, Tizhong Shan
      Abstract: Adipose tissues regulate energy metabolism and reproduction. There are three types of adipocytes (brown, white and beige adipocytes) in mammals. White adipocytes store energy and are closely associated with obesity and other metabolic diseases. The beige and brown adipocytes have numerous mitochondria and high levels of UCP1 that dissipates lipid to generate heat and defend against obesity. The global epidemic of obesity and its associated metabolic diseases urge an imperative need for understating the regulation of adipogenesis. Liver kinase B1 (Lkb1), also called STK11, is a master kinase of the AMPK subfamily and plays crucial roles in regulating glucose and energy homeostasis in various metabolic tissues. In this review we focus on the regulatory roles of Lkb1 in regulating preadipocyte differentiation, adipose tissue development and thermogenesis. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:54.490641-05:
      DOI: 10.1002/jcp.25643
  • Low Shear Stress Attenuates COX-2 Expression Induced by Resistin in Human
           Osteoarthritic Chondrocytes
    • Authors: Yu-Ping Su; Cheng-Nan Chen, Hsin-I Chang, Kuo-Chin Huang, Chin-Chang Cheng, Fang-Yao Chiu, Ko-Chao Lee, Chun-Min Lo, Shun-Fu Chang
      Abstract: Low shear stress has been proposed to play a reparative role in modulating cartilage homeostasis. Recently, epidemiological studies have found a positive correlation between the resistin level in serum and synovial fluid and osteoarthritis (OA) severity in patients. However, the effect of moderate shear stress on the catabolic stimulation of resistin in OA chondrocytes remains unclear. Hence, this study was to investigate whether low shear stress could regulate resistin-induced catabolic cyclooxygenase (COX)-2 expression in human OA chondrocytes and the underlying mechanism. Human OA chondrocytes and SW1353 chondrosarcoma cells were used in this study. Two modes of low shear stress (2 dyn/cm2), pre-shear and post-shear, were applied to the chondrocytes. A specific activator and siRNAs were used to investigate the mechanism of low shear stress-regulated COX-2 expression of resistin induction. We found that human OA chondrocytes exposed to different modes of low shear stress elicit an opposite effect on resistin-induced COX-2 expression: pre-shear for a short duration attenuates the resistin effect by inhibiting the transcription factor nuclear factor (NF)-κB-p65 subunit and the cAMP response element binding protein; however, post-shear over a longer duration enhances the resistin effect by activating only the NF-κB-p65 subunit. Moreover, our results demonstrated that the regulation of both shear modes in resistin-stimulated COX-2 expression occurs through increasing AMP-activated protein kinase activation and then sirtuin 1 expression. This study elucidates the detailed mechanism of low shear stress regulating the resistin-induced catabolic COX-2 expression and indicates a possible reparative role of moderate shear force in resistin-stimulated OA development. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:50.402155-05:
      DOI: 10.1002/jcp.25644
  • Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is
           Involved in Cell Cycle Progression
    • Authors: Alessandro Poli; Roberta Fiume, Gianluca Baldanzi, Daniela Capello, Stefano Ratti, Marco Gesi, Lucia Manzoli, Andrea Graziani, Pann-Ghill Suh, Lucio Cocco, Matilde Y. Follo
      Abstract: Phosphatidylinositol (PI) signalling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signalling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:43.293952-05:
      DOI: 10.1002/jcp.25642
  • Peripheral Blood Mononuclear Cells Infiltration Downregulates Decidual
           FAAH Activity in an LPS-Induced Embryo Resorption Model
    • Authors: Manuel Luis Wolfson; Julieta Aisemberg, Fernando Correa, Ana María Franchi
      Abstract: Background and PurposeMaternal infections with Gram-negative bacteria are associated with miscarriage and are one of the most common complications during pregnancy. Previous studies from our group have shown that lipopolysaccharide (LPS)-activated infiltrating peripheral blood mononuclear cells (PBMC) into decidual tissue play an important role on the establishment of a local inflammatory process that result in embryo cytotoxicity and early embryo resorption. Moreover, we have also shown that an increased endocannabinoid tone mediates LPS-induced deleterious effects during early pregnancy loss. Here we sought to investigate whether the infiltrating PBMC modulates the decidual endocannabinoid tone and the molecular mechanisms involved.Experimental approachPBMC isolated from 7-days pregnant mice subjected to different treatments were co-cultured in a transwell system with decidual tissue from control 7-days pregnant mice. Decidual fatty acid amide hydrolase (FAAH) activity was measure by radioconvertion, total decidual protein nitration by western blot (WB) and decidual FAAH nitration by immuneprecipitation followed by WB.Key ResultsWe found that co-culture of PBMC obtained from LPS-treated mice increased the level of nitration of decidual FAAH, which resulted in a negative modulation of decidual FAAH activity. Interestingly, co-treatment with progesterone or aminoguanidine prevented this effect.Conclusions & ImplicationsWe found that LPS-treated PBMC release high amounts of nitric oxide (NO) which causes tyrosine nitration of decidual FAAH, diminishing its enzymatic activity. Inactivation of FAAH, the main degrading enzyme of anandamide and similar endocannabinoids, could lead to an increased decidual endocannabinoid tone with embryotoxic effects. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:34.357677-05:
      DOI: 10.1002/jcp.25640
  • The Role of Cardiac Myosin Binding Protein C3 in Hypertrophic
           Cardiomyopathy- Progress and Novel Therapeutic Opportunities
    • Authors: Iman A. Mohamed; Navaneethakrishnan T. Krishnamoorthy, Gheyath K. Nasrallah, Sahar Da'as
      Abstract: Hypertrophic cardiomyopathy (HCM) is a common autosomal dominant genetic cardiovascular disorder marked by genetic and phenotypic heterogeneity. Mutations in the gene encodes the cardiac myosin-binding protein C, cMYBPC3 is amongst the various sarcomeric genes that are associated with HCM. These mutations produce mutated mRNAs and truncated cMyBP-C proteins. In this review, we will discuss the implications and molecular mechanisms involved in MYBPC3 different mutations. Further, we will highlight the novel targets that can be developed into potential therapeutics for the treatment of HMC. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:31:00.403813-05:
      DOI: 10.1002/jcp.25639
  • Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Regulates
           Osteoclast Differentiation via the Ca2+ /NFATc1 Axis
    • Authors: Ya-Hui Chiu; Edward Schwarz, Dongge Li, Yuexin Xu, Tzong-Ren Sheu, Jinbo Li, Karen L. de Mesy Bentley, Changyong Feng, Baoli Wang, Jhih-Cheng Wang, Liz Albertorio-Saez, Ronald Wood, Minsoo Kim, Wensheng Wang, Christopher T. Ritchlin
      Abstract: DC-STAMP is a multi-pass transmembrane protein essential for cell-cell fusion of osteoclast precursors during osteoclast (OC) development. DC-STAMP-/- mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP suggested a potential signaling function but the absence of known DC-STAMP ligand has hindered examination of downstream signaling pathways. To address this problem, we engineered a light-activatable DC-STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca2+ signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca2+ flux. We also found that decreased NFATc1 expression in DC-STAMP-/- cells was restored by DC-STAMP over-expression. Multiple biological phenotypes including cell-cell fusion, bone erosion, cell mobility, DC-STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of tyrosine residues in the ITIM showed no effect on DC-STAMP function. Collectively, our results suggest that ITIM on DC-STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1 / Ca2+ axis. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-10T06:31:04.676478-05:
      DOI: 10.1002/jcp.25638
  • Dact1, a Wnt-Pathway Inhibitor, Mediates Human Mesangial Cell
           TGF-β1-Induced Apoptosis
    • Authors: Daniele Pereira Jardim; Paula Cristina Eiras Poço, Alexandre Holthausen Campos
      Abstract: Chronic kidney disease (CKD) is a worldwide public health problem that affects millions of men and women of all ages and racial groups. Loss of mesangial cells (MC) represents an early common feature in the pathogenesis of CKD. Transforming growth factor-β1 (TGF-β1) is a key inducer of kidney damage and triggers several pathological changes in renal cells, notably MC apoptosis. However, the mechanism of MC apoptosis induced by TGF-β1 remains elusive. Here we demonstrate for the first time a novel regulatory pathway in which the disheveled-binding antagonist of beta-catenin 1 (Dact1) gene is upregulated by TGF-β1, inducing MC apoptosis. We also show that the inhibitory effect of Dact1 and TGF-β1 on the transcriptional activation of the pro-survival Wnt pathway is the mechanism of death induction. In addition, Dact1 mRNA/protein levels are increased in kidney remnants from 5/6 nephrectomized rats and strongly correlate with TGF-β1 expression. Together, our results point to Dact1 as a novel element controlling MC survival that is causally related to CKD progression. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:49:27.48411-05:0
      DOI: 10.1002/jcp.25636
  • Inhibitory Effects of Quercetin on Progression of Human Choriocarcinoma
    • Authors: Whasun Lim; Changwon Yang, Sunwoo Park, Fuller W. Bazer, Gwonhwa Song
      Abstract: As a major dietary flavonol, quercetin mitigates proliferation and progression of cancer due to its anti-angiogenic, anti-inflammatory, anti-oxidant and apoptotic biological effects on cells. Although its apoptotic effects have been reported for various cancers, little is known of the functional role of quercetin in gestational choriocarcinoma. Results of the present study indicated that quercetin reduced proliferation and induced cell death in two choriocarcinoma cell lines, JAR and JEG3 cells, with an increase in the sub-G1 phase of the cell cycle. In addition, quercetin induced mitochondrial dysfunction significantly reduced mitochondrial membrane potential (MMP) and increased production of reactive oxygen species (ROS) in both JAR and JEG3 cells. Further, quercetin inhibited phosphorylation of AKT, P70S6K and S6 proteins whereas it increased phosphorylation of ERK1/2, P38, JNK and P90RSK proteins in JAR and JEG3 cells. The decrease in viability of choriocarcinoma cells treated with quercetin was confirmed by using combinations of quercetin and pharmacological inhibitors of the PI3K and MAPK signaling pathways. Classical chemotherapeutic agents, cisplatin (a platinum-based drug) and paclitaxel (a taxene-based drug), inhibited proliferation of JAR and JEG3 cells and when combined with quercetin, the antiproliferative effects of cisplatin and paclitaxel were enhanced for both choriocarcinoma cell lines. Collectively, these results suggest that quercetin prevents development of choriocarcinoma and may be a valuable therapeutic agent for treatment of choriocarcinoma through its regulation of PI3K and MAPK signal transduction pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:49:16.88812-05:0
      DOI: 10.1002/jcp.25637
  • Osteoclast Fusion: Time-Lapse Reveals Involvement of CD47 and Syncytin-1
           at Different Stages of Nuclearity
    • Authors: Anaïs Marie Julie Møller; Jean-Marie Delaissé, Kent Søe
      Abstract: Investigations addressing the molecular keys of osteoclast fusion are primarily based on end-point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multinucleated cells, the number of nuclei per multinucleated cell or TRAcP activity. But end-point analyses do not show how the fusion came about. This would not be a problem if fusion of osteoclasts is a random process and occurs by the same molecular mechanism from beginning to end. However, we and others have in the recent period published data suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed individual fusion events using time-lapse and antagonists of CD47 and syncytin-1. All time-lapse recordings have been studied by two independent observers. A total of 1808 fusion events were analyzed. The present study shows that CD47 and syncytin-1 have different roles in osteoclast fusion depending on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono-nucleated pre-osteoclasts, syncytin-1 promotes fusion of two multi-nucleated osteoclasts, but also reduces the number of fusions between mono-nucleated pre-osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through broad contact surfaces between the partners' cell membrane while syncytin-1 mediate fusion through phagocytic-cup like structure. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:36:16.164329-05:
      DOI: 10.1002/jcp.25633
  • Essential Role of CFTR in PKA-Dependent Phosphorylation, Alkalinization
           and Hyperpolarization During Human Sperm Capacitation
    • Authors: Lis C. Puga Molina; Nicolás A. Pinto, Paulina Torres Rodríguez, Ana Romarowski, Alberto Vicens Sanchez, Pablo E. Visconti, Alberto Darszon, Claudia L. Treviño, Mariano G. Buffone
      Abstract: Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3- is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm and membrane hyperpolarization. However, how HCO3- is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3--entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:36:13.303942-05:
      DOI: 10.1002/jcp.25634
  • Short- and Long-Term Hindlimb Immobilization and Reloading: Profile of
           Epigenetic Events in Gastrocnemius
    • Authors: Alba Chacon-Cabrera; Joaquim Gea, Esther Barreiro
      Abstract: Skeletal muscle dysfunction and atrophy are characteristic features accompanying chronic conditions. Epigenetic events regulate muscle mass and function maintenance. We hypothesized that the pattern of epigenetic events (muscle-enriched microRNAs and histone acetylation) and acetylation of transcription factors known to signal muscle wasting may differ between early- and late-time points in skeletal muscles of mice exposed to hindlimb immobilization (I) and recovery following I. Body and muscle weights, grip strength, muscle-enriched microRNAs, histone deacetylases (HDACs), acetylation of proteins, histones, and transcription factors (TF), myogenic TF factors, and muscle phenotype were assessed in gastrocnemius of mice exposed to periods (1, 2, 3, 7, 15 and 30 days, I groups) of hindlimb immobilization, and in those exposed to reloading for different periods of time (1, 3, 7, 15, and 30 days, R groups) following 7-day immobilization. Compared to non-immobilized controls, muscle weight, limb strength, microRNAs, especially miR-486, SIRT1 levels, and slow- and fast-twitch cross-sectional areas were decreased in mice of I groups, whereas Pax7, and acetylated FoxO1 and FoxO3 levels were increased. Muscle reloading following splint removal improved muscle mass loss, strength, and fiber atrophy, by increasing microRNAs, particularly miR-486, and SIRT1 content, while decreasing acetylated FoxO1 and FoxO3 levels. In this mouse model of disuse muscle atrophy, muscle-enriched microRNAs, especially miR-486, through Pax7 regulation delayed muscle cell differentiation following unloading of gastrocnemius muscle. Acetylation of FoxO1 and 3 seemed to drive muscle mass loss and atrophy, while deacetylation of these factors through SIRT1 would enable the muscle fibers to regenerate. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:36:06.967146-05:
      DOI: 10.1002/jcp.25635
  • Challenging of AS160/TBC1D4 Alters Intracellular Lipid Milieu in L6
           Myotubes Incubated with Palmitate
    • Authors: Agnieszka Mikłosz; Bartłomiej Łukaszuk, Małgorzata Żendzian-Piotrowska, Justyna Brańska-Januszewska, Halina Ostrowska, Adrian Chabowski
      Abstract: The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre-PA-silencing, AS160-/PA); 2) palmitate incubation with subsequent AS160 knockdown (post-PA-silencing, PA/AS160-). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP-1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post-PA-silencing of AS160 resulted in a marked decrement in DAG, TAG and PL contents, but increased FFA content (PA/AS160- vs. PA). The opposite effect was observed in the group with pre-PA-silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160-/PA vs. PA). Our results indicate that post-PA-silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:35:57.765285-05:
      DOI: 10.1002/jcp.25632
  • Bone Shaft Revascularization After Marrow Ablation is Dramatically
           Accelerated in BSP-/- Mice, Along With Faster Haematopoietic
    • Authors: Wafa Bouleftour; Renata Neves Granito, Arnaud Vanden-Bossche, Odile Sabido, Bernard Roche, Mireille Thomas, Marie Thérèse Linossier, Jane E Aubin, Marie-Hélène Lafage-Proust, Laurence Vico, Luc Malaval
      Abstract: The bone organ integrates the activity of bone tissue, bone marrow and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the Small Integrin Binding Ligand N-Linked Glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP-/- mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP + /+ and BSP-/- mice. At d3, the ablated area in BSP-/- femurs showed higher vessel density (x4) and vascular volume (x7) than BSP + / +. Vessel numbers in the shaft of ablated BSP + /+ mice reached BSP-/- values only by d8, but with a vascular volume which was twice the value in BSP-/-, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin– (x3) as well as LSK (Lin- IL-7Rα- Sca-1hi c-Kithi, x2) and hematopoietic stem cells (HSC: Flt3- LSK, x2) were counted in BSP-/- marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin- was lower in BSP-/- and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP-/- femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants.In conclusion, bone marrow ablation in BSP-/- mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-05T05:11:10.605707-05:
      DOI: 10.1002/jcp.25630
  • Indispensable Role of Ion Channels and Transporters in the Auditory System
    • Authors: Rahul Mittal; Mayank Aranke, Luca H. Debs, Desiree Nguyen, Amit P. Patel, M'hamed Grati, Jeenu Mittal, Denise Yan, Prem Chapagain, Adrien A. Eshraghi, Xue Zhong Liu
      Abstract: Ear is a complex system where appropriate ionic composition is essential for maintaining the tissue homeostasis and hearing function. Ion transporters and channels present in the auditory system plays a crucial role in maintaining proper ionic composition in the ear. The extracellular fluid, called endolymph, found in the cochlea of the mammalian inner ear is particularly unique due to its electrochemical properties. At an endocochlear potential of about +80 mV, signaling initiated by acoustic stimuli at the level of the hair cells is dependent on the unusually high potassium (K+) concentration of endolymph. There are ion channels and transporters that exists in the ear to ensure that K+ is continually being cycled into the stria media endolymph. This review is focused on the discussion of the molecular and genetic basis of previously and newly recognized ion channels and transporters that support sensory hair cell excitation based on recent knock-in and knock-out studies of these channels. This article also addresses the molecular and genetic defects and the pathophysiology behind Meniere's disease as well as how the dysregulation of these ion transporters can result in severe defects in hearing or even deafness. Understanding the role of ion channels and transporters in the auditory system will facilitate in designing effective treatment modalities against ear disorders including Meniere's disease and hearing loss. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-05T05:11:04.350582-05:
      DOI: 10.1002/jcp.25631
  • Increased Amyloid Precursor Protein and Tau Expression Manifests as Key
           Secondary Cell Death in Chronic Traumatic Brain Injury
    • Authors: Sandra A. Acosta; Naoki Tajiri, Paul R. Sanberg, Yuji Kaneko, Cesar V. Borlongan
      Abstract: In testing the hypothesis of Alzheimer's disease (AD)-like pathology in late stage traumatic brain injury (TBI), we evaluated AD pathological markers in late stage TBI model. Sprague-Dawley male rats were subjected to moderate controlled cortical impact (CCI) injury, and 6 months later euthanized and brain tissues harvested. Results from H&E staining revealed significant 33% and 10% reduction in the ipsilateral and contralateral hippocampal CA3 interneurons, increased MHCII-activated inflammatory cells in many gray matter (8-20 fold increase) and white matter (6-30 fold increased) regions of both the ipsilateral and contralateral hemispheres, decreased cell cycle regulating protein marker by 1.6- and 1-fold in the SVZ and a 2.3 and 1.5 fold reductions in the ipsilateral and contralateral dentate gyrus, diminution of immature neuronal marker by 2- and 1- fold in both the ipsilateral and contralateral SVZ and dentate gyrus, and amplified amyloid precursor protein (APP) distribution volumes in white matter including corpus callosum, fornix, and internal capsule (4-38 fold increase), as well as in the cortical gray matter, such as the striatum hilus, SVZ and dentate gyrus (6-40 fold increase) in TBI animals compared to controls (p's 
      PubDate: 2016-10-04T06:03:23.777999-05:
      DOI: 10.1002/jcp.25629
  • MicroRNA-494 Activation Suppresses Bone Marrow Stromal Cell-Mediated Drug
           Resistance in Acute Myeloid Leukemia Cells
    • Authors: Chen Tian; Guoguang Zheng, Hongqing Zhuang, Xubin Li, Dongzhi Hu, Lei Zhu, Wang Tengteng, M. James You, Yizhuo Zhang
      Abstract: Acute myeloid leukemia (AML) is not sensitive to chemotherapy partially because of the protection of AML cells by mesenchymal stromal cells (MSCs). Our previous studies found that MSCs protected AML cells from apoptosis through the c-Myc-dependent pathway. However, the mechanism by which MSCs regulate c-Myc in AML cells is still unknown. To elucidate the mechanism, we performed microRNA array analysis of AML cell lines and validated by TaqMan realtime PCR. The results showed that the expression of microRNA-494 (miR-494) in AML cells after coculture with MSCs was down-regulated. Reporter gene analysis confirmed miR-494 as one of the regulators of c-Myc. In the coculture system, activation of miR-494 in AML cells suppressed proliferation and induced apoptosis of AML cells in vitro. After addition of mitoxantrone to the coculture system, the proliferation of AML cells with miR-494 activation was suppressed more than that of control cells. After subcutaneous injection of AML cell lines in combination with MSC, tumor growth was suppressed in mice injected with miR-494-overexpressing AML cells. The rate of tumor formation was even lower after mitoxantrone treatment in the miR-494 overexpressing group. Moreover, miR-494 activation resulted in a decrease of leukemic cell counts in peripheral blood and bone marrow and prolonged survival in mice injected with miR-494-overexpressing AML cellls and MSCs compared to the control mice. Our results indicate that miR-494 suppresses drug resistance in AML cells by down-regulating c-Myc through interaction with MSCs and that miR-494 therefore is a potential therapeutic target. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-03T06:35:39.113816-05:
      DOI: 10.1002/jcp.25628
  • Epigenetic Signatures at the RUNX2-P1 and Sp7 Gene Promoters Control
           Osteogenic Lineage Commitment of Umbilical Cord-Derived Mesenchymal Stem
    • Authors: Hugo Sepulveda; Rodrigo Aguilar, Catalina P. Prieto, Francisco Bustos, Sócrates Aedo, José Lattus, Brigitte van Zundert, Veronica Palma, Martin Montecino
      Abstract: Wharton's Jelly mesenchymal stem cells (WJ-MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ-MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies here we analyze the ability of WJ-MSCs to engage osteoblast lineage. In undifferentiated WJ-MSCs, RUNX2/P57 P1 and SP7 promoters are found deprived of significant levels of the histone post-translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ-MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together these results point to critical regulatory steps during epigenetic control of WJ-MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-30T16:25:24.511202-05:
      DOI: 10.1002/jcp.25627
  • Differences in Activation of HIV-1 Replication by Superinfection With
           HIV-1 and HIV-2 in U1 Cells
    • Authors: Xue Wang; Bing Sun, Christelle Mbondji, Santanu Biswas, Jiangqin Zhao, Indira Hewlett
      Abstract: Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir that serve as a viral source for the infection of CD4 T cells. The relationship between HIV-1 latent infection and superinfection in macrophages has not been well studied. Using susceptible U1 cells chronically infected with HIV-1, we studied the effects of HIV superinfection on latency and differences in superinfection with HIV-1 and HIV-2 in macrophages. We found that HIV-1 (MN) superinfection displayed increased HIV-1 replication in a time-dependent manner; while cells infected with HIV-2 (Rod) initially showed increased HIV-1 replication, followed by a decrease in HIV-1 RNA production. HIV-1 superinfection upregulated/activated NF-ĸB, NFAT, AP-1, SP-1, and MAPK Erk through expression/activation of molecules, CD4, CD3, TCRβ, Zap-70, PLCγ1, and PKCΘ in T cell receptor-related signaling pathways; while HIV-2 superinfection initially increased expression/activation of these molecules followed by decreased protein expression/activation. HIV superinfection initially downregulated HDAC1 and upregulated acetyl-histone H3 and histone H3 (K4), while HIV-2 superinfection demonstrated an increase in HDAC1 and a decrease in acetyl-histone H3 and histone H3 (K4) relative to HIV-1 superinfection. U1 cells superinfected with HIV-1 or HIV-2 showed differential expression of proteins, IL-2, PARP-1, YB-1, and LysRS. These findings indicate that superinfection with HIV-1 or HIV-2 has different effects on reactivation of HIV-1 replication. HIV-1 superinfection with high load of viral replication may result in high levels of cytotoxicity relative to HIV-2 superinfection. Cells infected with HIV-2 showed lower level of HIV-1 replication, suggesting that co-infection with HIV-2 may result in slower progression toward AIDS. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-09-30T09:20:42.86365-05:0
      DOI: 10.1002/jcp.25614
  • Hypoglycemia Enhances Epithelial-Mesenchymal Transition and Invasiveness,
           and Restrains the Warburg Phenotype, in Hypoxic HeLa Cell Cultures and
    • Authors: Álvaro Marín-Hernández; Juan Carlos Gallardo-Pérez, Ileana Hernández-Reséndiz, Isis Del Mazo-Monsalvo, Diana Xochiquetzal Robledo-Cadena, Rafael Moreno-Sánchez, Sara Rodríguez-Enríquez
      Abstract: The accelerated growth of solid tumors leads to episodes of both hypoxia and hypoglycemia (HH) affecting their intermediary metabolism, signal transduction, and transcriptional activity. A previous study showed that normoxia (20% O2) plus 24 h hypoglycemia (2.5 mM glucose) increased glycolytic flux whereas oxidative phosphorylation (OxPhos) was unchanged versus normoglycemia in HeLa cells. However, the simultaneous effect of HH on energy metabolism has not been yet examined. Therefore, the effect of hypoxia (0.1–1% O2) plus hypoglycemia on the energy metabolism of HeLa cells was analyzed by evaluating protein content and activity, along with fluxes of both glycolysis and OxPhos. Under hypoxia, in which cell growth ceased and OxPhos enzyme activities, ΔΨm and flux were depressed, hypoglycemia did not stimulate glycolytic flux despite increasing H-RAS, p-AMPK, GLUT1, GLUT3, and HKI levels, and further decreasing mitochondrial enzyme content. The impaired mitochondrial function in HH cells correlated with mitophagy activation. The depressed OxPhos and unchanged glycolysis pattern was also observed in quiescent cells from mature multicellular tumor spheroids, suggesting that these inner cell layers are similarly subjected to HH. The principal ATP supplier was glycolysis for HH 2D monolayer and 3D quiescent spheroid cells. Accordingly, the glycolytic inhibitors iodoacetate and gossypol were more effective than mitochondrial inhibitors in decreasing HH-cancer cell viability. Under HH, stem cell-, angiogenic-, and EMT-biomarkers, as well as glycoprotein-P content and invasiveness, were also enhanced. These observations indicate that HH cancer cells develop an attenuated Warburg and pronounced EMT- and invasive-phenotype. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.Hypoglycemia enhances (i) epithelial-mesenchymal transition, (ii) invasiveness, and (iii) restrains the Warburg phenotype in hypoxic HeLa cells.
      PubDate: 2016-09-30T09:16:31.539215-05:
      DOI: 10.1002/jcp.25617
  • PACAP and VIP Inhibit HIF-1α-Mediated VEGF Expression in a Model of
           Diabetic Macular Edema
    • Authors: Grazia Maugeri; Agata Grazia D'Amico, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata
      Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) exert a protective role against retinal injuries, including diabetic macular edema (DME). The macular damage is induced by hyperglycemia, which damages vessels supplying blood to the retina and induces hypoxia. The microenvironmental changes stimulate the expression of hypoxia-inducible factors (HIFs), which promote the choroidal endothelial cell transmigration across the retinal pigmented epithelium (RPE) into neurosensory retina, where they proliferate into new vessels under stimulation of the vascular endothelial growth factor (VEGF). In the present study, we have investigated whether PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors. In accord to our hypothesis, we have shown that both peptides are able to significantly reduce HIF-1α and increase HIF-3α expression in ARPE-19 cells exposed to hyperglycemic/hypoxic insult. This effect is also related to a reduction of VEGF and its receptors expression. Moreover, both peptides also reduce the activation of p38 mitogen-activated protein kinase (MAPK), a pro-apoptotic signaling pathway, which is activated by VEGFR-1 and 2 receptors. In conclusion, our study has further elucidated the protective role performed by PACAP and VIP, against the harmful combined effect of hyperglycemia/hypoxia characterizing the DME microenvironment. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors; PACAP and VIP act against the harmful combined effect of hyperglycemia/hypoxia.
      PubDate: 2016-09-30T09:16:20.837141-05:
      DOI: 10.1002/jcp.25616
  • MED28 Regulates Epithelial–Mesenchymal Transition Through NFκB in Human
           Breast Cancer Cells
    • Authors: Chun-Yin Huang; Nien-Tsu Hsieh, Chun-I Li, Yu-Ting Weng, Hsiao-Sheng Liu, Ming-Fen Lee
      Abstract: MED28, a mammalian Mediator subunit, was found highly expressed in several types of malignancy, including breast cancer. Recently, we have identified a role of MED28 in regulating both cell growth and migration in human breast cancer cells. In epithelium-derived solid tumor, migration and invasion are preceded by the progression of epithelial–mesenchymal transition (EMT) which calls for downregulation of epithelial markers as well as upregulation of mesenchymal markers, among other features. The objective of this study was to investigate a putative role of MED28 in the progression of EMT in human breast cancer cells. In fibroblast-like MDA-MB-231 cells, suppression of MED28 attenuated the mesenchymal morphology, concomitantly with a reduction of several mesenchymal biomarkers and Snail, a transcriptional repressor of E-cadherin. The suppression effect was also accompanied by downregulation of p-NFκB/p65. However, overexpression of MED28 exhibited in an opposite manner. In epithelial MCF7 cells, administration of Adriamycin®, an experimental EMT induction system, led to a mesenchyme-like appearance correlated with increased expression of MED28, p-p65, and Snail, and a reciprocal change of epithelial and mesenchymal markers. Furthermore, suppression of MED28 attenuated the experimental EMT effect and restored the original expression status of E-cadherin and MMP9 in MCF7 cells. Our data indicate that MED28 modulates the development of EMT through NFκB in human breast cancer cells, further reinforcing the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.MED28 regulates the expression of epithelial markers and mesenchymal markers through NFκB, thereby modulating the development of epithelial–mesenchymal transition. These findings further reinforce the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration.
      PubDate: 2016-09-30T09:09:02.969848-05:
      DOI: 10.1002/jcp.25610
  • Hyperexcitability of Mesencephalic Trigeminal Neurons and Reorganization
           of Ion Channel Expression in a Rett Syndrome Model
    • Authors: Max F. Oginsky; Ningren Cui, Weiwei Zhong, Christopher M. Johnson, Chun Jiang
      Abstract: People with Rett syndrome (RTT) have defects in motor function also seen in Mecp2-null mice. Motor function depends on not only central motor commands but also sensory feedback that is vulnerable to changes in excitability of propriosensory neurons. Here we report evidence for hyperexcitability of mesencephalic trigeminal (Me5) neurons in Mecp2-null mice and a novel cellular mechanism for lowering its impact. In in vitro brain slices, the Me5 neurons in both Mecp2−/Y male and symptomatic Mecp2+/− female mice were overly excitable showing increased firing activity in comparison to their wild-type (WT) male and asymptomatic counterparts. In Mecp2−/Y males, Me5 neurons showed a reduced firing threshold. Consistently, the steady-state activation of voltage-gated Na+ currents (INa) displayed a hyperpolarizing shift in the Mecp2-null neurons with no change in the INa density. This seems to be due to NaV1.1, SCN1B and SCN4B overexpression and NaV1.2 and SCN3B under-expression. In contrast to the hyperexcitability, the sag potential and postinhibitory rebound (PIR) were reduced in Mecp2-null mice. In voltage-clamp, the IH density was deficient by ∼33%, and the steady-state half-activation had a depolarizing shift of ∼10 mV in the Mecp2-null mice. Quantitative PCR analysis indicated that HCN2 was decreased, HCN1 was upregulated with no change in HCN4 in Mecp2−/Y mice compared to WT. Lastly, blocking IH reduced the firing rate much more in WT than in Mecp2-null neurons. These data suggest that the Mecp2 defect causes an increase in Me5 neuronal excitability likely attributable to alterations in INa, meanwhile IH is reduced likely altering neuronal excitability as well. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.Mesencephalic trigeminal V neurons are hyperexcitable in Mecp2-null mice. Our data suggest that the Mecp2 defect that causes an increase in Me5 neuronal excitability is likely attributable to alterations in INa, meanwhile IH is reduced likely altering neuronal excitability as well. Further, the changes in these currents seem to be due to alterations in channel subunit composition.
      PubDate: 2016-09-30T09:09:00.806058-05:
      DOI: 10.1002/jcp.25589
  • Inflammatory Events and Oxidant Production in the Diaphragm,
           Gastrocnemius, and Blood of Rats Exposed to Chronic Intermittent Hypoxia:
           Therapeutic Strategies
    • Authors: Marisol Domínguez-Álvarez; Joaquim Gea, Esther Barreiro
      Abstract: We hypothesized that inflammatory events and reactive oxygen species (ROS) production may be differentially expressed in respiratory and limb muscles, and blood of a chronic intermittent hypoxia (CIH) experimental model and that antioxidants and TNF-alpha blockade may influence those events. In blood, diaphragm, and gastrocnemius of rats non-invasively exposed to CIH (10% hypoxia, 2 h/day, 14 consecutive days) with/without concomitant treatment with either anti-TNF-alpha antibody (infliximab) or N-acetyl cysteine (NAC), inflammatory cytokines, superoxide anion production, muscle structural abnormalities, and fiber-type composition were assessed. Compared to non-exposed controls, in CIH-exposed rats, body weight gain was reduced, TNF-alpha, IL-1beta, IL-6, and interferon-gamma levels were increased in diaphragm, TNF-alpha, and IL-1 beta plasma levels were greater, systemic and muscle superoxide anion production was higher, diaphragm and gastrocnemius inflammatory cells and internal nuclei were higher, and muscle fiber-type and morphometry remained unmodified. CIH rats treated with infliximab further increased TNF-alpha, IL-1beta, IL-6, and interferon-gamma diaphragm levels, whereas NAC induced a reduction only in TNF-alpha and IL-1beta levels in diaphragm and plasma. Infliximab and NAC elicited a significant decline in superoxide anion production in diaphragm, gastrocnemius, and plasma, while inducing a further increase in inflammatory cells and internal nuclei in both muscles. Proinflammatory cytokines are differentially expressed in respiratory and limb muscles and plasma of CIH-exposed rats, while superoxide anion production increased in both muscle types and blood. Infliximab and NAC exerted different effects. These findings may help understand the biology underlying CIH in skeletal muscles and blood of patients with chronic respiratory diseases. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Proinflammatory cytokines are differentially expressed in respiratory and limb muscles and plasma of CIH-exposed rats, while superoxide anion production increased in both muscle types and blood. Infliximab and NAC exerted different effects. These findings may help understand the biology underlying CIH in skeletal muscles and blood of patients with chronic respiratory diseases.
      PubDate: 2016-09-30T09:08:52.834967-05:
      DOI: 10.1002/jcp.25600
  • Role of Protein Kinase G and Reactive Oxygen Species in the Regulation of
           Podocyte Function in Health and Disease
    • Authors: Agnieszka Piwkowska
      Abstract: Podocytes and their foot processes form an important cellular layer of the glomerular barrier involved in the regulation of glomerular permeability. Disturbing the function of podocytes plays a central role in the development of proteinuria in diabetic nephropathy. Retraction of the podocyte foot processes that form slit diaphragms is a common feature of proteinuria; although, the correlation between these events in not well understood. Notably, it is unclear whether podocyte foot processes are able to regulate slit diaphragm permeability and glomerular ultrafiltration. The occurrence of reactive oxygen species generation, insulin resistance, and hyperglycemia characterizes early stages of type 2 diabetes. Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. It is activated in both cGMP-dependent and cGMP-independent manners. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI-dependent signaling pathways in podocytes. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI-dependent signaling pathways in podocytes.
      PubDate: 2016-09-30T08:58:57.845176-05:
      DOI: 10.1002/jcp.25613
  • Risk Differences Between Prediabetes And Diabetes According To Breast
           Cancer Molecular Subtypes
    • Authors: A. Crispo; L.S.A. Augustin, M. Grimaldi, F. Nocerino, A. Giudice, E. Cavalcanti, M. Di Bonito, G. Botti, M. De Laurentiis, M. Rinaldo, E. Esposito, G. Riccardi, A. Amore, M. Libra, G. Ciliberto, D.J.A. Jenkins, M. Montella
      Abstract: Hyperglycemia and hyperinsulinemia may play a role in breast carcinogenesis and prediabetes and diabetes have been associated with increased breast cancer (BC) risk. However, whether BC molecular subtypes may modify these associations is less clear. We therefore investigated these associations in all cases and by BC molecular subtypes among women living in Southern Italy. Cases were 557 patients with non-metastatic incident BC and controls were 592 outpatients enrolled during the same period as cases and in the same hospital for skin-related non-malignant conditions. Adjusted multivariate logistic regression models were built to assess the risks of developing BC in the presence of prediabetes or diabetes. The analyses were repeated by strata of BC molecular subtypes: Luminal A, Luminal B, HER2+, and Triple Negative (TN). Prediabetes and diabetes were significantly associated with higher BC incidence after controlling for known risk factors (OR = 1.94, 95% CI 1.32–2.87 and OR = 2.46, 95% CI 1.38–4.37, respectively). Similar results were seen in Luminal A and B while in the TN subtype only prediabetes was associated with BC (OR = 2.43, 95% CI 1.11–5.32). Among HER2+ patients, only diabetes was significantly associated with BC risk (OR = 3.04, 95% CI 1.24–7.47). Furthermore, when postmenopausal HER2+ was split into hormone receptor positive versus negative, the association with diabetes remained significant only in the former (OR = 5.13, 95% CI 1.53–17.22). These results suggest that prediabetes and diabetes are strongly associated with BC incidence and that these metabolic conditions may be more relevant in the presence of breast cancer molecular subtypes with positive hormone receptors. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-09-30T08:12:46.992363-05:
      DOI: 10.1002/jcp.25579
  • Mitochondria-Targeted Antioxidants and Uncouplers of Oxidative
           Phosphorylation in Treatment of the Systemic Inflammatory Response
           Syndrome (SIRS)
    • Authors: Vlada V. Zakharova; Olga Yu. Pletjushkina, Roman A. Zinovkin, Ekaterina N. Popova, Boris V. Chernyak
      Abstract: Systemic inflammatory response syndrome (SIRS) development is accompanied by mitochondrial dysfunction and excessive ROS production. Mitochondrial dysfunctions also occur in many SIRS-related diseases and may be critical for their pathogenesis; therefore a use of mitochondria-targeted drugs is a promising trend in SIRS research and therapy. Here we review recent studies concerning the application of the mitochondria-targeted antioxidants and uncouplers of oxidative phosphorylation in animal models of SIRS and related diseases. We propose that a new class of uncouplers of oxidative phosphorylation, lipophilic cations could be a base for a new generation of drugs for SIRS treatment. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-29T14:05:12.571562-05:
      DOI: 10.1002/jcp.25626
  • Basic Signaling in Cardiac Fibroblasts
    • Authors: Stéphanie Chacar; Nassim Farès, Patrick Bois, Jean-François Faivre
      Abstract: Cardiac fibroblasts are commonly known as supporting cells of the cardiac network and exert many essential functions that are fundamental for normal cardiac growth as well as for cardiac remodeling process during pathological conditions. This review focuses on the roles of cardiac fibroblasts in the formation and regulation of the extracellular matrix components, and in maintaining structural, biochemical and mechanical properties of the heart. Additionally, though considered as non-excitable cells, we review the functional expression in cardiac fibroblasts of a wide variety of transmembrane ion channels which activity may contribute to key regulation of cardiac physiological processes. All together, cardiac fibroblasts which actively participate to fundamental regulation of cardiac physiology and physiopathology processes may represent pertinent targets for pharmacological approaches of cardiac diseases and lead to new tracks of therapeutic strategies. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-29T14:05:11.464802-05:
      DOI: 10.1002/jcp.25624
  • Exhaustive Training Leads to Hepatic Fat Accumulation
    • Authors: Alisson L. da Rocha; Ana P. Pinto, Giovana R. Teixeira, Bruno C. Pereira, Luciana C. Oliveira, Adriana C. Silva, Gustavo P. Morais, Dennys E. Cintra, José R. Pauli, Adelino S. R. da Silva
      Abstract: Recently, we demonstrated that an overtraining (OT) protocol for mice based on downhill running sessions increased the hepatic phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (S6K1; Thr389), a downstream target of the mammalian target of rapamycin complex 1 (mTORC1). In liver, the overactivation of the Akt/mTORC1 pathway induces lipogenesis via regulation of the action of sterol regulatory element binding protein-1 (SREBP-1) at multiple steps. Herein, we verified the effects of three running OT models with same external load (i.e., the product between intensity and volume of training), but performed in downhill, uphill and without inclination, on the proteins related to the mTORC1 signaling pathway, the protein content of the SREBP-1, ACC and FAS, and the morphological characteristics of C57BL/6 mouse livers. In summary, the downhill running-induced OT model up-regulated the levels of major proteins of the mTORC1 signaling pathway, the protein levels of SREBP-1 (p125 precursor) and induced signs of cell swelling accompanied by acute inflammation. The other two OT protocols performed uphill and without inclination did not modulate the most analyzed molecular proteins, but induced hepatic morphological alterations, suggesting an acute pathological adaptation. The three OT models induced hepatic fat accumulation. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-29T14:05:10.503024-05:
      DOI: 10.1002/jcp.25625
  • New Insights on the Regulation of Ca2+-Activated Chloride Channel TMEM16A
    • Authors: Ke Ma; Hui Wang, Jiankun Yu, Minjie Wei, Qinghuan Xiao
      Abstract: TMEM16A, also known as anoctamin 1, is a recently identified Ca2+-activated chloride channel and the first member of a 10-member TMEM16 family. TMEM16A dysfunction is implicated in many diseases such as cancer, hypertension, and cystic fibrosis. TMEM16A channels are well known to be dually regulated by voltage and Ca2+. In addition, recent studies have revealed that TMEM16A channels are regulated by many molecules such as calmodulin, protons, cholesterol, and phosphoinositides, and a diverse range of stimuli such as thermal and mechanical stimuli. A better understanding of the regulatory mechanisms of TMEM16A is important to understand its physiological and pathological role. Recently, the crystal structure of a TMEM16 family member from the fungus Nectria haematococcaten (nhTMEM16) is discovered, and provides valuable information for studying the structure and function of TMEM16A. In this review, we discuss the structure and function of TMEM16A channels based on the crystal structure of nhTMEM16A and focus on the regulatory mechanisms of TMEM16A channels. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:13.785451-05:
      DOI: 10.1002/jcp.25621
  • Understanding the Pathogenesis of Neurotrophic Keratitis: The Role of
           Corneal Nerves
    • Authors: Leonardo Mastropasqua; Giacomina Massaro-Giordano, Mario Nubile, Marta Sacchetti
      Abstract: Neurotrophic keratitis (NK) is a rare degenerative disease of the cornea caused by trigeminal nerve damage, which leads to loss of corneal sensitivity, corneal epithelium breakdown, and poor healing. Though extremely uncommon, NK is increasingly recognized for its characteristics as a distinct and well-defined clinical entity rather than a rare complication of various diseases that can disrupt trigeminal innervation. Indeed, the defining feature of NK is loss of corneal sensitivity, and its clinical findings do not correlate with the wide range of systemic or ocular conditions that underlie trigeminal nerve damage. Despite increasing awareness of NK as a distinct condition, its management continues to be challenged by the lack of treatments that target nerve regeneration. This review focuses on the role of corneal nerves in maintaining ocular surface homeostasis, the consequences (such as alterations in neuromediators and corneal cell morphology/function) of impaired innervation, and advances in NK diagnosis and management. Novel therapeutic strategies should aim to improve corneal innervation in order support corneal renewal and healing. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:09.942628-05:
      DOI: 10.1002/jcp.25623
  • Identification of a Novel Human E-Cadherin Splice Variant and Assessment
           of its Effects upon EMT-Related Events
    • Authors: María Laura Matos; Lara Lapyckyj, Arina Rosso, María José Besso, María Victoria Mencucci, Clara Isabel Marín Briggiler, Silvina Giustina, Laura Inés Furlong, Mónica Hebe Vazquez-Levin
      Abstract: Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) of human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05%-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them cell acquisition of fibroblastic-like phenotype, increased expression of Twist, Snail, Zeb1 and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding molecular basis of tumor progression and improve cancer diagnosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:07.213371-05:
      DOI: 10.1002/jcp.25622
  • p120-Catenin Is Required for Dietary Calcium Suppression of Oral
           Carcinogenesis in Mice
    • Authors: Zhongjian Xie; Yuan Yuan, Yi Jiang, Chandrama Shrestha, Ying Chen, Liyan Liao, Shangli Ji, Xiaoge Deng, Eryuan Liao, Daniel D. Bikle
      Abstract: Previous studies have shown that dietary calcium suppresses oral carcinogenesis, but the mechanism is unclear. p120-catenin (p120) is a cytoplasmic protein closely associated with E-cadherin to form the E-cadherin-β-catenin complex and may function as a tumor suppressor in the oral epithelium. To determine whether p120 is involved in the mechanism by which dietary calcium suppresses oral carcinogenesis, The normal, low or high calcium diet was fed control mice (designated as floxed p120 mice) or mice in which p120 was specifically deleted in the oral squamous epithelium during the adult stage (designated as p120cKO mice). All mice were exposed to a low dose of oral cancer carcinogen 4-nitroquinoline 1-oxide and rates of oral squamous cell carcinoma (OSCC) and proliferation and differentiation in the cancerous and non-cancerous oral epithelium of these mice were examined. The results showed that the low calcium diet increased rates of OSCC and proliferation of the non-cancerous oral epithelium and decreased differentiation of the non-cancerous oral epithelium, but had no effect on cancerous oral epithelium. In contrast, the high calcium diet had opposite effects. However, the effect of the dietary calcium on the rates of OSCC, proliferation and differentiation of the non-cancerous epithelium were not seen in p120cKO mice. Based on these results, we conclude that p120 is required for dietary calcium suppression of oral carcinogenesis and oral epithelial proliferation and dietary calcium induction of oral epithelial differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:02.467485-05:
      DOI: 10.1002/jcp.25620
  • Diverse Functions and Signal Transduction of the Exocyst Complex in Tumor
    • Authors: Toshiaki Tanaka; Kaoru Goto, Mitsuyoshi Iino
      Abstract: The exocyst complex is a large conserved hetero-oligomeric complex that consists of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 subunits. It has been implicated in the targeting of vesicles for regulated exocytosis in various cell types, and is also important for targeted exocytosis of post-Golgi transport vesicles to the plasma membrane. The exocyst complex is essential for membrane growth, secretion and function during exocytosis and endocytosis. Moreover, the individual components of the complex are thought to act on specific biological processes, such as cytokinesis, ciliogenesis, apoptosis, autophagy and epithelial-mesenchymal transition (EMT). As a result, recent studies suggest that the exocyst complex may be involved in several diseases such as kidney disease, neuropathogenesis, diabetes, and cancer. In this review, we focus on the diverse functions and cellular signaling pathways of the exocyst complex in various tumors. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-26T12:15:22.951594-05:
      DOI: 10.1002/jcp.25619
  • Nephroprotective Effects of Metformin in Diabetic Nephropathy
    • Authors: Sreenithya Ravindran; Vinitha Kuruvilla, Kerry Wilbur, Shankar Munusamy
      Abstract: Metformin, a well-known anti-diabetic agent, is very effective in lowering blood glucose in patients with type 2 diabetes with minimal side-effects. Metformin is also being recommended in the treatment of obesity and polycystic ovary syndrome. Metformin elicits its therapeutic effects mainly via activation of AMP-activated kinase (AMPK) pathway. Renal cells under hyperglycemic or proteinuric conditions exhibit inactivation of cell defense mechanisms such as AMPK and autophagy, and activation of pathologic pathways such as mammalian target of rapamycin (mTOR), endoplasmic reticulum (ER) stress, epithelial-to-mesenchymal transition (EMT), oxidative stress, and hypoxia. As these pathologic pathways are intertwined with AMPK signaling, the potential benefits of metformin therapy in patients with type 2 diabetes would extend beyond its anti-hyperglycemic effects. However, since metformin is eliminated unchanged through the kidneys and some studies have shown the incidence of lactic acidosis with its use during severe renal dysfunction, the use of metformin was contraindicated in patients with renal disease until recently. With more studies indicating the relatively low incidence of lactic acidosis and revealing the additional benefits with metformin therapy, the US FDA has now approved metformin to be administered in patients with established renal disease based on their renal function. The purpose of this review is to highlight the various mechanisms by which metformin protects renal cells that have lost its functionality in a diabetic or non-diabetic setting and to enlighten the advantages and therapeutic potential of metformin as a nephroprotectant for patients with diabetic nephropathy and other non-diabetic forms of chronic kidney disease. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.Metformin, a popular anti-diabetic drug and an AMPK activator, modulates various signaling pathways altered in diabetic nephropathy and exhibits nephroprotective properties in various animal models of diabetes. Additional studies in humans are needed to ascertain its therapeutic potential as a nephroprotectant to treat nephropathy in patients with diabetes.
      PubDate: 2016-09-26T11:05:22.881493-05:
      DOI: 10.1002/jcp.25598
  • MnTBAP Inhibits Bleomycin-Induced Pulmonary Fibrosis by Regulating VEGF
           and Wnt Signaling
    • Authors: Rajkumar Venkatadri; Anand Krishnan V. Iyer, Vani Ramesh, Clayton Wright, Carlos A. Castro, Juan S. Yakisich, Neelam Azad
      Abstract: Cellular oxidative stress is implicated not only in lung injury but also in contributing to the development of pulmonary fibrosis. We demonstrate that a cell-permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger, manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly inhibited bleomycin-induced fibrogenic effects both in vitro and in vivo. Further investigation into the underlying mechanisms revealed that MnTBAP targets canonical Wnt and non-canonical Wnt/Ca2+ signaling pathways, both of which were upregulated by bleomycin treatment. The effect of MnTBAP on canonical Wnt signaling was significant in vivo but inconclusive in vitro and the non-canonical Wnt/Ca2+ signaling pathway was observed to be the predominant pathway regulated by MnTBAP in bleomycin-induced pulmonary fibrosis. Furthermore, we show that the inhibitory effects of MnTBAP involve regulation of VEGF which is upstream of the Wnt signaling pathway. Overall, the data show that the superoxide scavenger MnTBAP attenuates bleomycin-induced pulmonary fibrosis by targeting VEGF and Wnt signaling pathways. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-09-26T10:55:22.029958-05:
      DOI: 10.1002/jcp.25608
  • G Protein-Coupled Receptor Endosomal Signaling and Regulation of Neuronal
           Excitability and Stress Responses: Signaling Options and Lessons from the
           PAC1 Receptor
    • Authors: Victor May; Rodney L. Parsons
      Abstract: Our understanding of G protein coupled receptor (GPCR) mechanisms and functions have evolved considerably. Among the many conceptual realignments, GPCRs can exist in an ensemble of active microstates that have the potential to differentially engage specific downstream signaling events. Furthermore, among GPCR dynamics, GPCR internalization and vesicular trafficking are no longer solely mechanisms for desensitization, but now appreciated to form intricate endosomal signaling complexes that can potentially target second messengers to intracellular compartments with high temporal and spatial resolution. The PACAPergic system is important in the maintenance of physiological homeostasis in the central and peripheral nervous systems and activation of the PACAP-selective PAC1 receptor can generate differential but coordinate plasma membrane and endosomal signals for cellular responses. The integration of these signals can modulate PACAP-induced changes in ionic conductances that gate neuronal excitability. PACAP/PAC1 receptor generation of endosomal ERK signals participate in chronic pain and anxiety-like responses which can be attenuated with endocytosis inhibitors. From the abilities of ligands to stabilize the different GPCR microstates for biased downstream signaling, the development of biased PAC1 receptor agonists and antagonists may provide opportunities to dissociate the homeostatic regulatory signals of PACAP from the maladaptive effects. In particular, the development of biased antagonists to PAC1 receptor-mediated endosomal signaling may offer therapeutic options for chronic pain and stress-related disorders. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-23T17:41:35.805915-05:
      DOI: 10.1002/jcp.25615
  • The Role of Calcium-Sensing Receptors in Endothelin-1-Dependent Effects on
           Adult Rat Ventricular Cardiomyocytes: Possible Contribution to Adaptive
           Myocardial Hypertrophy
    • Authors: Elena Dyukova; Rolf Schreckenberg, Christoph Arens, Guzel Sitdikova, Klaus-Dieter Schlüter
      Abstract: Nitric oxide (NO)-deficiency as it occurs during endothelial dysfunction activates the endothelin-1 system and increases the expression of receptor activity modifying protein (RAMP)-1 that acts as a chaperon for calcium-sensing receptors (CaR) that have recently been identified to improve cardiac function. Here we hypothesized that endothelin-1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP-1, endothelin receptors, and CaR were analyzed by RT-PCR in left ventricular tissues of L-NAME-treated rats. Effects of ET-1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP-1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1, ETB2, and ETA receptors. Non-specific NO synthase inhibition with L-Nitro arginine methyl ester (L-NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of endothelin-1 on cardiomyocytes. Indeed, endothelin-1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Down-regulation of RAMP-1 or selective inhibition of ETA and ETB1 receptors led endothelin-1 reducing cell shortening and attenuates up-regulation of CaR. In conclusion, endothelin-1 causes an adaptive type of hypertrophy by up-regulation of CaR in cardiomyocytes via ETA receptors. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-23T17:41:28.634426-05:
      DOI: 10.1002/jcp.25612
  • Anti-Tumor Effects of Cardiac Glycosides on Human Lung Cancer Cells and
           Lung Tumorspheres
    • Authors: Vivek Kaushik; Juan Sebastian Yakisich, Neelam Azad, Yogesh Kulkarni, Rajkumar Venkatadri, Clayton Wright, Yon Rojanasakul, Anand Krishnan V. Iyer
      Abstract: Lung cancer is a leading cause of cancer-related death in the United States. While several drugs have been developed that target individual biomarkers, their success has been limited due to intrinsic or acquired resistance for the specific targets of such drugs. A more effective approach is to target multiple pathways that dictate cancer progression. Cardiac glycosides demonstrate such multimodal effects on cancer cell survival, and our aim was to evaluate the effect of two naturally occurring monosaccaridic cardiac glycosides – Convallatoxin and Peruvoside on lung cancer cells. While both drugs had significant anti-proliferative effects on H460 and Calu-3 lung cancer cells, Convallatoxin demonstrated two-fold higher activity as compared to Peruvoside using both viability and colony forming assays, suggesting a role for the aglycone region in dictating drug potency. The tumor suppressor p53 was found to be important for action of both drugs – p53-underexpressing cells were less sensitive as compared to p53-positive H460 cells. Further assessment of p53-underexpressing H460 cells showed that drugs were able to arrest cells in the G0/G1 phase of the cell cycle in a dose-dependent manner. Both drugs significantly inhibited migration and invasion of cancer cells and decreased the viability of floating tumorspheres. An assessment of intracellular pathways indicated that both drugs were able to modulate proteins that are involved in apoptosis, autophagy, cell cycle, proliferation and EMT. Our data suggests a promising role for cardiac glycosides in lung cancer treatment, and provides impetus for further investigation of the anti-cancer potential of this class of drugs. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-23T17:35:52.30901-05:0
      DOI: 10.1002/jcp.25611
  • Interfering of the Reelin/ApoER2/PSD95 Signaling Axis Reactivates
           Dendritogenesis of Mature Hippocampal Neurons
    • Authors: Estibaliz Ampuero; Nur Jury, Steffen Härtel, María-Paz Marzolo, Brigitte van Zundert
      Abstract: Reelin, an extracellular glycoprotein secreted in embryonic and adult brains, participates in neuronal migration and neuronal plasticity. Extensive evidence shows that reelin via activation of the ApoER2 and VLDLR receptors promotes dendrite and spine formation during early development. Further evidence suggests that reelin signaling is needed to maintain a stable architecture in mature neurons, but, direct evidence is lacking. During activity-dependent maturation of the neuronal circuitry, the synaptic protein PSD95 is inserted into the postsynaptic membrane to induce structural refinement and stability of spines and dendrites. Given that ApoER2 interacts with PSD95, we tested if reelin signaling interference in adult neurons reactivates the dendritic architecture. Unlike findings in developing cultures, the presently obtained in vitro and in vivo data show, for the first time, that reelin signaling interference robustly increase dendritogenesis and reduce spine density in mature hippocampal neurons. In particular, the expression of a mutant ApoER2 form (ApoER2-tailless), which is unable to interact with PSD95 and hence cannot transduce reelin signaling, resulted in robust dendritogenesis in mature hippococampal neurons in vitro. These results indicate that reelin/ApoER2/PSD95 signaling is important for neuronal structure maintenance in mature neurons. Mechanistically, obtained immunofluorescent data indicate that reelin signaling impairment reduced synaptic PSD95 levels, consequently leading to synaptic re-insertion of NR2B-NMDARs. Our findings underscore the importance of reelin in maintaining adult network stability and reveal a new mode for reactivating dendritogenesis in neurological disorders where dendritic arbor complexity is limited, such as in depression, Alzheimer's disease, and stroke. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-21T12:25:21.899058-05:
      DOI: 10.1002/jcp.25605
  • HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1
    • Authors: Eleonora Foglio; Giovanni Puddighinu, Antonia Germani, Matteo A. Russo, Federica Limana
      Abstract: Exogenous High Mobility Group Box-1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex. In particular, its effect on ischemic cardiomyocytes has been poorly investigated. Aim of the present study was to verify whether and how autophagy and apoptosis were involved in HMGB1-induced heart repair following myocardial infarction (MI). HMGB1 (200 ng) or denatured HMGB1 were injected in the peri-infarcted region of mouse hearts following acute MI. Three days after treatment, an upregulation of autophagy was detected in infarcted HMGB1 treated hearts compared to controls. Specifically, HMGB1 induced autophagy by significantly upregulating the protein expression of LC3, Beclin-1, and Atg7 in the border zone. To gain further insights into the molecular mechanism of HMGB1-mediated autophagy, WB analysis were performed in cardiomyocytes isolated from 3 days infarcted hearts in the presence and in the absence of HMGB1 treatment. Results showed that upregulation of autophagy by HMGB1 treatment was potentially related to activation of AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Accordingly, in these hearts, phospho-Akt signaling pathway was inhibited. The induction of autophagy was accompanied by reduced cardiomyocyte apoptotic rate and decreased expression levels of Bax/Bcl-2 and active caspase-3 in the border zone of 3 days infarcted mice following HMGB1 treatment. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP-activated protein kinase-dependent autophagy. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.Exogenous High Mobility Group Box-1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex; in particular, its effect on ischemic cardiomyocytes has been poorly investigated. HMGB1 (200 ng) or denatured HMGB1 were injected in the peri-infarcted region of mouse hearts following acute MI. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP-activated protein kinase-dependent autophagy.
      PubDate: 2016-09-21T10:55:22.031754-05:
      DOI: 10.1002/jcp.25576
  • VIP Family Members Prevent Outer Blood Retinal Barrier Damage in a Model
           of Diabetic Macular Edema
    • Authors: Grazia Maugeri; Agata Grazia D'Amico, Caterina Gagliano, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata
      Abstract: Diabetic macular edema (DME), characterized by an increase of thickness in the eye macular area, is due to breakdown of the blood-retinal barrier (BRB). Hypoxia plays a key role in the progression of this pathology by activating the hypoxia-inducible factors. In the last years, various studies have put their attention on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in retinal dysfunction. However, until now, no study has investigated their protective role against the harmful combined effect of both hyperglycemia and hypoxia on outer BRB. Therefore, in the present study, we have analyzed the role of these peptides on permeability, restoration of tight junctions expression and inhibition of hyperglycemia/hypoxia-induced apoptosis, in an experimental in vitro model of outer BRB. Our results have demonstrated that the peptides' treatment have restored the integrity of outer BRB induced by cell exposure to hyperglycemia/hypoxia. Their effect is mediated through the activation of phosphoinositide 3 kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling pathways. In conclusion, our study further clarifies the mechanism through which PACAP and VIP perform the beneficial effect on retinal damage induced by hyperglycemic/hypoxic insult, responsible of DME progression. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.PACAP and VIP restore the integrity of outer BRB exposed to hyperglycemia/hypoxia. PACAP and VIP perform their beneficial effect on retinal damage through activation of PI3K/Akt and MAPK/ERK signaling cascades.
      PubDate: 2016-09-21T10:45:25.121149-05:
      DOI: 10.1002/jcp.25510
  • Emerging Roles of Transforming Growth Factor β Signaling in Diabetic
    • Authors: Sarah E. Wheeler; Nam Y. Lee
      Abstract: Diabetic retinopathy (DR) is a serious complication of diabetes mellitus affecting about one third of diabetic adults. Despite its prevalence, treatment options are limited and often implemented only in the later stages of the disease. To date, the pathogenesis of DR has been extensively characterized in the context of elevated glucose, insulin, and VEGF signaling, although a growing number of other growth factors and molecules, including transforming growth factor β (TGF-β) are being recognized as important contributors and/or therapeutic targets. Here, we review the complex roles of TGF-β signaling in DR pathogenesis and progression. J. Cell. Physiol. 9999: 1–4, 2016. © 2016 Wiley Periodicals, Inc.Transforming growth factor β (TGF-β) has a clear role in the pathogenesis of diabetic retinopathy, although the underlying mechanisms are poorly established. Here, we discuss our current but limited understanding of how signaling by TGF-β superfamily ligands contributes to the pathogenesis and progression of diabetic retinopathy.
      PubDate: 2016-09-21T10:40:24.736228-05:
      DOI: 10.1002/jcp.25506
  • Functional Effects of Cigarette Smoke-Induced Changes in Airway Smooth
           Muscle Mitochondrial Morphology
    • Authors: Bharathi Aravamudan; Michael Thompson, Gary C. Sieck, Robert Vassallo, Christina M. Pabelick, Y. S. Prakash
      Abstract: Long-term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). We previously showed that CS destabilizes the networked morphology of mitochondria in human ASM by regulating the expression of mitochondrial fission and fusion proteins via multiple signaling mechanisms. Emerging data link regulation of mitochondrial morphology to cellular structure and function. We hypothesized that CS-induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling. Here, ASM cells were exposed to 1% cigarette smoke extract (CSE) for 48 h to alter mitochondrial fission/fusion, or by inhibiting the fission protein Drp1 or the fusion protein Mfn2. Mitochondrial function was assessed via changes in bioenergetics or altered rates of proliferation and apoptosis. Our results indicate that both exposure to CS and inhibition of mitochondrial fission/fusion proteins affect mitochondrial function (i.e., energy metabolism, proliferation, and apoptosis) in ASM cells. In vivo, the airways in mice chronically exposed to CS are thickened and fibrotic, and the expression of proteins involved in mitochondrial function is dramatically altered in the ASM of these mice. We conclude that CS-induced changes in mitochondrial morphology (fission/fusion balance) correlate with mitochondrial function, and thus may control ASM proliferation, which plays a central role in airway health. J. Cell. Physiol. 9999: 1–16, 2016. © 2016 Wiley Periodicals, Inc.Long-term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). Previous studies show that CS destabilizes the networked morphology of mitochondria in human ASM. We hypothesized that CS-induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling.
      PubDate: 2016-09-21T10:15:28.976808-05:
      DOI: 10.1002/jcp.25508
  • Effects of Conjugated Linoleic Acid Associated With Endurance Exercise on
           Muscle Fibres and Peroxisome Proliferator-Activated Receptor γ
           Coactivator 1 α Isoforms
    • Authors: Rosario Barone; Claudia Sangiorgi, Antonella Marino Gammazza, Daniela D'Amico, Monica Salerno, Francesco Cappello, Cristoforo Pomara, Giovanni Zummo, Felicia Farina, Valentina Di Felice, Filippo Macaluso
      Abstract: Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre-type-specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 μl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6-week experimental period, whereas the placebo groups were gavaged with 35 μl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota-Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2, and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre-type-specific hypertrophy induced by endurance exercise (of types I and IIb muscle fibres). J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres.
      PubDate: 2016-09-21T10:15:23.022164-05:
      DOI: 10.1002/jcp.25511
  • Endothelial STAT3 Activation Increases Vascular Leakage Through
           Downregulating Tight Junction Proteins: Implications for Diabetic
    • Authors: Jang-Hyuk Yun; Sung Wook Park, Kyung-Jin Kim, Jong-Sup Bae, Eun Hui Lee, Sun Ha Paek, Seung U. Kim, Sangkyu Ye, Jeong-Hun Kim, Chung-Hyun Cho
      Abstract: Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro-inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL-6-induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO-1 and occludin. In a co-culture model with microglia and endothelial cells under a high glucose condition, the microglia-derived IL-6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL-6-induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO-1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL-6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.Vascular inflammation is characteristic feature of diabetic retinopathy. We demonstrated that STAT3 in retinal endothelial cells plays a causative role in downregulating ZO-1 and occludin levels and increasing the endothelial permeability through VEGF production in retinal endothelial cells. These results suggest the potential importance of IL-6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy.
      PubDate: 2016-09-21T10:05:28.489171-05:
      DOI: 10.1002/jcp.25575
  • Heat Stress Modulates both Anabolic and Catabolic Signaling Pathways
           Preventing Dexamethasone-Induced Muscle Atrophy In Vitro
    • Authors: Wakako Tsuchida; Masahiro Iwata, Takayuki Akimoto, Shingo Matsuo, Yuji Asai, Shigeyuki Suzuki
      Abstract: It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid-induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid-induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel-like factor 15 (KLF15). Heat stress recovered the dexamethasone-induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress-induced protection against glucocorticoid-induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-20T14:20:23.775655-05:
      DOI: 10.1002/jcp.25609
  • Human Telomerase Reverse Transcriptase (hTERT) Positively Regulates 26S
           Proteasome Activity
    • Authors: Eunju Im; Jong Bok Yoon, Han-Woong Lee, Kwang Chul Chung
      Abstract: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, an RNA-dependent DNA polymerase that elongates telomeric DNA. hTERT displays several extra-telomeric functions that are independent of its telomere-regulatory function, including tumor progression and neuronal cell death regulation. In this study, we evaluated these additional hTERT non-telomeric functions. We determined that hTERT interacts with several 19S and 20S proteasome subunits. The 19S regulatory particle and 20S core particle are part of 26S proteasome complex, which plays a central role in ubiquitin-dependent proteolysis. In addition, hTERT positively regulated 26S proteasome activity independent of its enzymatic activity. Moreover, hTERT enhanced subunit interactions, which may underlie hTERT's ability of hTERT to stimulate the 26S proteasome. Furthermore, hTERT displayed cytoprotective effect against ER stress via the activation of 26S proteasome in acute myeloid leukemia cells. Our data suggest that hTERT acts as a novel chaperone to promote 26S proteasome assembly and maintenance. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-20T14:15:22.299963-05:
      DOI: 10.1002/jcp.25607
  • Cover Image, Volume 232, Number 1, January 2017
    • Authors: Areg Zingiryan; Nicholas H. Farina, Kristiaan H. Finstad, Janet L. Stein, Jane B. Lian, Gary S. Stein
      Abstract: Cover: The cover image, by Nicholas H Farina et al., is based on the From the Bench Paper Dissection of Individual Prostate Lobes in Mouse Models of Prostate Cancer to Obtain High Quality RNA,
      DOI : 10.1002/jcp.25384.
      PubDate: 2016-09-19T14:38:07.232144-05:
  • Pulsed Electromagnetic Field Exposure Reduces Hypoxia and Inflammation
           Damage in Neuron-Like and Microglial Cells
    • Authors: Fabrizio Vincenzi; Annalisa Ravani, Silvia Pasquini, Stefania Merighi, Stefania Gessi, Stefania Setti, Ruggero Cadossi, Pier Andrea Borea, Katia Varani
      Abstract: In the present study the effect of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) has been investigated by using different cell lines derived from neuron-like cells and microglial cells. In particular, the primary aim was to evaluate the effect of PEMF exposure in inflammation- and hypoxia-induced injury in two different neuronal cell models, the human neuroblastoma-derived SH-SY5Y cells and rat pheochromocytoma PC12 cells and in N9 microglial cells. In neuron-like cells, live/dead and apoptosis assays were performed in hypoxia conditions from 2 to 48 h. Interestingly, PEMF exposure counteracted hypoxia damage significantly reducing cell death and apoptosis. In the same cell lines, PEMFs inhibited the activation of the hypoxia-inducible factor 1α (HIF-1α), the master transcriptional regulator of cellular response to hypoxia. The effect of PEMF exposure on reactive oxygen species (ROS) production in both neuron-like and microglial cells was investigated considering their key role in ischemic injury. PEMFs significantly decreased hypoxia-induced ROS generation in PC12, SH-SY5Y and N9 cells after 24 or 48 h of incubation. Moreover, PEMFs were able to reduce some of the most well-known pro-inflammatory cytokines such as tumor necrosis factor -α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 release in N9 microglial cells stimulated with different concentrations of LPS for 24 or 48 h of incubation time. These results show a protective effect of PEMFs on hypoxia damage in neuron-like cells and an anti-inflammatory effect in microglial cells suggesting that PEMFs could represent a potential therapeutic approach in cerebral ischemic conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:30:51.474977-05:
      DOI: 10.1002/jcp.25606
  • Valproic Acid and Lithium Meditate Anti-Inflammatory Effects by
           Differentially Modulating Dendritic Cell Differentiation and Function
    • Authors: Sy-Jye Leu; Yi-Yuan Yang, Hsing-Cheng Liu, Chieh-Yu Cheng, Yu-Chen Wu, Ming-Chyi Huang, Yuen-Lun Lee, Chi-Ching Chen, Winston W. Shen, Ko-Jiunn Liu
      Abstract: Valproic acid (VPA), with inhibition activity mainly towards histone deacetylase (HDAC) and Glycogen Synthase Kinase (GSK)-3, and lithium, with inhibition activity mainly toward GSK-3, are both prescribed in clinical as mood-stabilizers and anticonvulsants for the control of bipolar disorder. This study aims to compare the immuno-modulation activities of VPA and lithium, especially on the differentiation and functions of dendritic cells (DC). Our data show that treatment with VPA or lithium effectively alleviated the severity of collagen-induced arthritis triggered by LPS in mice. Both agents reduced the serum level of IL-6 and IL-10 after LPS challenge in mice. VPA and lithium both induce significant down-regulation of group I CD1 expression and secretion of IL-6 during differentiation of human monocyte-derived immature DC, while they differ in the induction of CD83 and CD86 expression, secretion of IL-8, IL-10, and TNF-α. Upon stimulation of immature DC with LPS, VPA and lithium both reduced the secretion of IL-6 and TNF-α. However, only lithium significantly increased the production of IL-10, while VPA increased the production of IL-8 but substantially reduce the secretion of IL-10 and IL-23. Treatment with VPA resulted in a reduced capacity of LPS-stimulated DC to promote the differentiation of T helper 17 cells that are critical in the promotion of inflammatory responses. Taken together, our results suggest that VPA and lithium may differentially modulate inflammation through regulating the capacity of DC to mediate distinct T cell responses, and they may provide a complementary immunomodulatory effects for the treatment of inflammation-related diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:30:50.294937-05:
      DOI: 10.1002/jcp.25604
  • Human Brain Malignant Glioma (BMG-1) 3D Aggregate Morphology and Screening
           for Cytotoxicity and Anti-Proliferative Effects
    • Authors: Maddaly Ravi; Aarthi Ramesh, Aishwarya Pattabhi
      Abstract: Two interesting aspects of cell lines grown in 3D conditions are their distinct morphology and production of extracellular matrix (ECM). Also, it is known that 3D aggregates have different susceptibilities to damage-inducing agents compared to their 2D monolayer counterparts. We describe the effect of ECM on 3D aggregate morphology, the effect of cisplatin, bleomycin and UV on the 3D aggregates and 2D monolayers of the BMG-1 cell line. We also present a rapid method for analyzing cytotoxicity and anti-proliferative effects of 3D aggregates in 96-well plates. We utilized a single-step protocol using the dye resazurin.BMG-1 cells formed floating aggregates on 1% agarose hydrogels. The extent of ECM formed by them was dependent on number of cells seeded irrespective of the seeding density, which in turn directed the 3D aggregates compactness. The 3D aggregates were less susceptible to cisplatin and UV-induced cytotoxicity compared to 2D counterparts. The IC 50 value of cisplatin was elevated at 210 µg/ml for the aggregates compared to 170µg/ml for the monolayers. Exposure to UV for 10, 20 and 30 minutes gave inhibition values of 2.98%, 8%, 22.99% and 31.8% for the aggregates as compared to 3.06%, 7.5%, 39.4% and 46.7% for the monolayers. While bleomycin-induced effects were unapparent when analyzed by vital staining for the doses used, the rapid, single-step method in 96-well plates was able to provide a dose-response for cytotoxicity and anti-proliferative effects. Also, comparative analysis of results obtained from vital staining and the single-step method demonstrates the reliability of the assay described. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:26:20.762476-05:
      DOI: 10.1002/jcp.25603
  • Early Subchondral Bone Loss at Arthritis Onset Predicted Late Arthritis
           Severity in a Rat Arthritis Model
    • Authors: Guillaume Courbon; Damien Cleret, Marie-Thérèse Linossier, Laurence Vico, Hubert Marotte
      Abstract: Synovitis is usually observed before loss of articular function in rheumatoid arthritis (RA). In addition to the synovium and according to the “Inside-Outside” theory, bone compartment is also involved in RA pathogenesis. Then, we investigated time dependent articular bone loss and prediction of early bone loss to late arthritis severity on the rat adjuvant-induced arthritis (AIA) model. Lewis female rats were longitudinally monitored from arthritis induction (day 0), with early (day 10) and late (day 17) steps. Trabecular and cortical microarchitecture parameters of 4 ankle bones were assessed by microcomputed tomography. Gene expression was determined at sacrifice. Arthritis occurred at day 10 in AIA rats. At this time, bone erosions were detected on 4 ankle bones, with cortical porosity increase (+67%) and trabecular alterations including bone volume fraction (BV/TV: -13%) and trabecular thickness decrease. Navicular bone assessment was the most reproducible and sensitive. Furthermore, strong correlations were observed between bone alterations at day 10 and arthritis severity or bone loss at day 17, including predictability of day 10 BV/TV to day 17 articular index (R2 = 0.76). Finally, gene expression at day 17 confirmed massive osteoclast activation and interestingly provided insights on strong activation of bone formation inhibitor markers at the joint level. In rat AIA, bone loss was already observed at synovitis onset and was predicted late arthritis severity. Our results reinforced the key role of subchondral bone in arthritis pathogenesis, in favour to the “Inside-Outside” theory. Mechanisms of bone loss in rat AIA involved resorption activation and formation inhibition changes. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-16T17:05:52.041277-05:
      DOI: 10.1002/jcp.25601
  • Differential Expression of miR-4520a Associated with Pyrin Mutations
           Suggesting a Role of Autophagy in Familial Mediterranean Fever (FMF)
    • Authors: H. Latsoudis; M.-F. Mashreghi, J.R. Grün, H.-D. Chang, B. Stuhlmueller, A. Repa, I. Gergiannaki, E. Kabouraki, T. Haeupl, A. Radbruch, P. Sidiropoulos, D. Kardassis, D. T. Boumpas, G.N. Goulielmos
      Abstract: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (p = 0.032), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (p = 0.00548). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results support a role of deregulated autophagy in the pathogenesis of FMF. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-16T17:05:47.865817-05:
      DOI: 10.1002/jcp.25602
  • A MicroRNA Cluster miR-23∼24∼27 Is Upregulated by Aldosterone in the
           Distal Kidney Nephron where It Alters Sodium Transport
    • Authors: X. Liu; R.S. Edinger, C.A. Klemens, Y.L. Phua, A.J. Bodnar, W.A. LaFramboise, J. Ho, M.B. Butterworth
      Abstract: The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23∼24∼27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3'-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR-27 in aldosterone's action via a novel target. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-16T17:01:00.182963-05:
      DOI: 10.1002/jcp.25599
  • RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease
           Modeling and Therapy
    • Authors: James Kehler; Marianna Greco, Valentina Martino, Manickam Pachiappan, Hiroko Yokoe, Alice Chen, Miranda Yang, Joel Jessee, Martin Gotte, Luciano Milanesi, Alberto Albertini, Gianfranco Bellipanni, Ileana Zucchi, Rolland A. Reinbold, Antonio Giordano
      Abstract: Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogrammed iPSCs lines may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic and translational research. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-15T11:05:27.599142-05:
      DOI: 10.1002/jcp.25597
  • A Novel Skin Splint for Accurately Mapping Dermal Remodeling and
           Epithelialization During Wound Healing
    • Authors: Shiro Jimi; Francesco De Francesco, Giuseppe A. Ferraro, Michele Riccio, Shuuji Hara
      Abstract: The mouse excisional dorsal full-thickness wound model with a silicon splint fixed on the skin has been widely used to mimic human wound healing. However, the method cannot accurately quantify dermal remodeling, since the initial point of epithelialization on the wound surface is unclear. To overcome this limitation, we have developed a novel mouse excisional wound model to assess the degree of epithelial extension and regeneration, using a plastic ring-shaped splint fixed beneath the surrounding epidermal tissue. At the end of the experiment, tissue samples were fixed in formalin, the splint was excised, and paraffin sections were prepared. Splint holes, corresponding to the prior location of the splint, were evident on the tissue cross-sections, and the epidermis above the holes was considered the initial excision site. The epidermal contraction and epithelial regeneration, as independent essential tissue alterations in wound healing, could be distinguishable and quantified. Compared with previous splint models, this method provides an accurate evaluation of epidermal processes in wound healing, and can be a platform to assess the effects of various wound healing factors. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-14T17:55:47.612886-05:
      DOI: 10.1002/jcp.25595
  • Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal
           Epithelial Cells
    • Authors: Fiorella Di Claudio; Cecilia Muglia, Paola Smaldini, María Lucía Orsini Delgado, Fernando Trejo, Raúl Grigera, Guillermo Docena
      Abstract: Intestinal epithelial cell culture is important for biological, functional and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered-cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio-membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL-25 and TSLP), inhibitory (IL-10 and TGF-β) or pro-inflammatory mediators (IL-1β and TNF) were induced. In conclusion, this novel protocol using cow's collagen-ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell-microorganism interaction studies and stem cell expansion. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-14T17:55:38.907459-05:
      DOI: 10.1002/jcp.25594
  • Identifying Nuclear Matrix–Attached DNA Across the Genome
    • Authors: Jason R. Dobson; Deli Hong, A. Rasim Barutcu, Hai Wu, Anthony N. Imbalzano, Jane B. Lian, Janet L. Stein, Andre J. van Wijnen, Jeffrey A. Nickerson, Gary S. Stein
      Abstract: Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix–attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix–associated genome is highly cell-context dependent. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-14T17:55:32.395329-05:
      DOI: 10.1002/jcp.25596
  • Cyclic Tensile Strain Reduces TNF-α Induced Expression of MMP-13 by
           Condylar Temporomandibular Joint Cells
    • Authors: Hessam Tabeian; Astrid D. Bakker, Beatriz F. Betti, Frank Lobbezoo, Vincent Everts, Teun J de Vries
      Abstract: ObjectiveTo investigate whether the disproportionate degradation of mandibular condyle cartilage in arthritic juvenile temporomandibular joint (TMJ) is related to distinctive responses of TMJ-derived cells to tumor necrosis factor-α (TNF-α), and whether mechanical loading affects this response.MethodsThe effect of TNF-α (0.1-10ng/ml) was tested on juvenile porcine TMJ cells isolated from the condyle, fossa, and disc, grown in 3D agarose gels. Expression of anabolic and catabolic factors was quantified by RT-qPCR and/or immunohistochemistry. Condylar cells were stimulated for 12h with TNF-α (10ng/ml), followed by 8h of 6% cyclic tensile strain, and gene expression of MMPs was quantified.ResultsTNF-α (10ng/ml) reduced the expression of the matrix proteins collagen type I and type II after 6h of incubation. Aggrecan gene expression was increased in the presence of 0.1ng/ml TNF-α. The fossa and disc cells responded to TNF-α with an increased expression of the aggrecanase ADAMTS4. TNF-α enhanced MMP-13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect.ConclusionCells isolated from the different cartilaginous structures reacted differently to TNF-α. Since the disc and fossa contain a very low level of proteoglycans in comparison to the condyle, the role played by ADAMTS4 in degradation of the fossa and disc might be limited. TNF-α induced MMP-13 expression by condylar cells might be involved in the degradation of the juvenile condyle. Since this expression was reduced by mechanical loading, functional loading with oral physiotherapy or orthodontic activators may help to reduce the catabolic effect of TNF-α. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-12T17:50:41.79315-05:0
      DOI: 10.1002/jcp.25593
  • Tight Junction Protein Abundance and Apoptosis During Involution of Rat
           Mammary Glands
    • Authors: Claire V. C. Phyn; Kerst Stelwagen, Stephen R. Davis, Christopher D. Mcmahon, Joanne M. Dobson, Kuljeet Singh
      Abstract: To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement was induced by teat-sealing with an adhesive for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. In non-sealed control glands, histological analysis confirmed a lactating phenotype, indicating suckling by pups throughout the experiment. In contrast, alveoli of teat-sealed glands were distended within 6 h, with maximal luminal size observed by 12 h of non-suckling. By 18 h following teat-sealing, an involuting phenotype was observed, indicated by alveolar lumina engorged with milk vesicles and increased leukocytes. Relative to non-sealed glands, mammary apoptosis was increased in engorged glands 18 h following teat-sealing. The abundance of ZO-1 and occludin proteins was decreased in engorged glands by 12 and 18 h, respectively, following teat-sealing. In contrast, the claudin-1 22 kDa band was increased by 6 h and peaked at 12-18 h, whereas the 28 kDa band declined by 36 h, relative to controls. There were no temporal changes in ZO-1, occludin, and claudin-1 22 kDa proteins within control glands, although there were minor differences in claudin-1 28 kDa. These data indicate that intramammary milk accumulation due to cessation of milk removal is associated with mammary apoptosis. The apoptotic event is preceded by a rapid loss of abundance of ZO-1, occludin and an initial increase in claudin-1. The loss of cell-cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non-suckled glands. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T16:35:38.400884-05:
      DOI: 10.1002/jcp.25591
  • Overexpression of Large-Conductance Calcium-Activated Potassium Channels
           in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration
    • Authors: Paolo Rosa; Luigi Sforna, Silvia Carlomagno, Giorgio Mangino, Massimo Miscusi, Mauro Pessia, Fabio Franciolini, Antonella Calogero, Luigi Catacuzzeno
      Abstract: Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large-conductance, calcium-activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem-like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem-like cells (U87-NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co-expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87-NS cells migration was much more sensitive to BK channel block than U87-MG cells. Our data show that BK channels are highly expressed in GBM stem-like cells, and participate to their high migratory activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T16:35:32.146807-05:
      DOI: 10.1002/jcp.25592
  • Cardiotrophin-1 Regulates Adipokine Production in 3T3-L1 Adipocytes and
           Adipose Tissue from Obese Mice
    • Authors: Miguel López-Yoldi; Beatriz Marcos-Gomez, M. Asunción Romero-Lozano, Neira Sáinz, Jesús Prieto, J. Alfredo Martínez, Matilde Bustos, Maria J. Moreno-Aliaga
      Abstract: Cardiotrophin-1 (CT-1) belongs to the IL-6 family of cytokines. Previous studies of our group revealed that CT-1 is a key regulator of glucose and lipid metabolism. The aim of the present study was to analyze the in vitro and in vivo effects of CT-1 on the production of several adipokines involved in body weight regulation, nutrient metabolism and inflammation. For this purpose, 3T3-L1 adipocytes were incubated with recombinant protein CT-1 (rCT-1) (1- 40 ng/mL) for 1 and 18 h. Moreover, the acute effects of rCT-1 administration (0.2 mg/kg, i.v.) for 30 min and 3 h on adipokines levels were also evaluated in high-fat fed obese mice. In 3T3-L1 adipocytes, rCT-1 treatment downregulated the expression and secretion of leptin, resistin and visfatin. However, rCT-1 significantly stimulated apelin mRNA and secretion. rCT- 1 (18 h) also promoted the activation by phosphorylation of AKT, ERK 1/2 and STAT3. Interestingly, pre-treatment with the PI3K inhibitor LY294002 reversed the stimulatory effects of rCT-1 on apelin expression, suggesting that this pathway could be mediating the effects of rCT-1 on apelin production. In contrast, acute administration of rCT-1 (30 min and 3 h) to diet- induced obese mice downregulated leptin and resistin, without significantly modifying apelin or visfatin mRNA in adipose tissue. Furthermore, CT-1 null mice exhibited altered expression of adipokines in adipose tissue. The present study demonstrates that rCT-1 modulates the production of adipokines in vitro and in vivo, suggesting that the regulation of the secretory function of adipocytes could be involved in the metabolic actions of this cytokine. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T16:30:37.064707-05:
      DOI: 10.1002/jcp.25590
  • Live cell imaging: Assessing the phototoxicity of 488 nm and 546 nm light
           and methods to alleviate it
    • Authors: Stephen Douthwright; Greenfield Sluder
      Abstract: Inlive cellimaging of fluorescent proteins, phototoxicity of the excitation light can be problematical. Cell death is obvious, but reduced cell viability can make the interpretation of observations error prone. We characterized the phototoxic consequences of 488 and 546nm light on untransformed human cells and tested methodsthat have or could be used to alleviate photodamage.Unlabeled RPE1 cells were given single 0.5 to 2.5 minute irradiations in early G1 from a mercury arc lamp on a fluorescence microscope. 488nm lightproduced a dose dependent decrease in the percentage of cells that progressed to mitosis, slowing of the cell cycle for some of those entering mitosis, and a ∼12% incidence of cell deathfor the highest dose. For 546nm light we found a 10-15% reduction in the percentage of cells entering mitosis, no strong dose dependency, and a ∼2% incidence of cell deathfor the longest irradiations. For cells expressing GFP-centrin1 or mCherry-centrin1, fewer entered mitosis for each dose than unlabeled cells. For constant total dose 488nm light irradiations of unlabeled cells, reducing the intensity tenfold or spreading the exposures out as a series of 10 second pulses at 1 minute intervals produced a minor and not consistent improvement in the percentage of cells entering mitosis. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Troloxnoticeably increased the fraction of cells entering mitosis. Thus, for long term imaging there can be value to using RFP constructs and for GFP tagged proteins reducing oxidative processes. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T07:45:36.075005-05:
      DOI: 10.1002/jcp.25588
  • Hyaluronic acid nanohydrogel loaded with Quercetin alone or in combination
           to a macrolide derivative of rapamycin RAD001 (Everolimus) as a new
           treatment for hormone-responsive human breast cancer
    • Authors: V Quagliariello; RV Iaffaioli, E Armenia, O Clemente, M Barbarisi, G Nasti, M Berretta, A Ottaiano, A Barbarisi
      Abstract: The aim of this study is based on the evaluation of anticancer, anti-inflammatory activities and cellular uptake of hyaluronic acid nanohydrogel of quercetin tested alone and in combination to a macrolide derivative of rapamycin RAD001 (everolimus) on hormone-responsive breast cancer cell line MCF-7. Biological investigations were focused on the receptor mediated cellular internalization of the nanohydrogel and its abilities to reduce secretion of several cytokines (IL-8, IL-6, IL-19), VEGF and metalloproteases (MMP-2, MMP-9) under pro-inflammatory conditions. Nanohydrogel show a CD44 dependent endocytosis with evident time dependent cytoplasmatic accumulation with abilities to reduce secretion of all cytokines of ∼60% compared to untreated cells. Combination of formulated quercetin and everolimus leads to a synergistic cytotoxic effects with a Combination Index of 0.38. These results highlights the importance of synergistic effect of the hyaluronic acid nanohydrogel of quercetin with everolimus in the regulation of human breast cancer cell proliferation and emphasize the antitumor and anti-inflammatory properties of the nanocarrier. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T07:35:42.566155-05:
      DOI: 10.1002/jcp.25587
  • Acute ethanol increases IGF-I-induced phosphorylation of ERKs by enhancing
           recruitment of p52-Shc to the Grb2/Shc complex
    • Authors: Matthew Dean; Adam Lassak, Anna Wilk, Adriana Zapata, Luis Marrero, Patricia Molina, Krzysztof Reiss
      Abstract: Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin-like growth factor I receptor (IGF-IR) function. Because the IGF-IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane-associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST-pulldowns following acute (1 - 24 hours) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 hours) ethanol (50mM) enhanced IGF-I-induced phosphorylation of ERKs, without affecting IGF-IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF-IR signaling following 3-week ethanol exposure. We then expressed a GFP-tagged IGF-IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently-labeled cells, we determined that 50mM ethanol decreased the half-time of the IGF-IR-associated FRAP, which implied that cell membrane-associated signaling events could be affected. Indeed, co-immunoprecipitation and GST-pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52-Shc to the Grb2-Shc complex, which is known to engage the Ras-Raf-ERKs pathway following IGF-1 stimulation. These experiments indicate that even a short and low-dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T07:31:17.661918-05:
      DOI: 10.1002/jcp.25586
  • Protective role of GPER agonist g-1 on cardiotoxicity induced by
    • Authors: Ernestina M. De Francesco; Carmine Rocca, Francesco Scavello, Daniela Amelio, Teresa Pasqua, Damiano C. Rigiracciolo, Andrea Scarpelli, Silvia Avino, Francesca Cirillo, Nicola Amodio, Maria C. Cerra, Marcello Maggiolini, Tommaso Angelone
      Abstract: The use of Doxorubicin (Dox), a frontline drug for many cancers, is often complicated by dose-limiting cardiotoxicity in approximately 20% of patients. The G-protein estrogen receptor GPER/GPR30 mediates estrogen action as the cardioprotection under certain stressful conditions. For instance, GPER activation by the selective agonist G-1 reduced myocardial inflammation, improved immunosuppression, triggered pro-survival signaling cascades, improved myocardial mechanical performance and reduced infarct size after ischemia/reperfusion (I/R) injury. Hence, we evaluated whether ligand-activated GPER may exert cardioprotection in male rats chronically treated with Dox. 1 week of G-1 (50 µg/kg/day) intraperitoneal administration mitigated Dox (3 mg/kg/day) adverse effects, as revealed by reduced TNF-α, IL-1β, LDH and ROS levels. Western blotting analysis of cardiac homogenates indicated that G-1 prevents the increase in p-c-jun, BAX, CTGF, iNOS and COX2 expression induced by Dox. Moreover, the activation of GPER rescued the inhibitory action elicited by Dox on the expression of BCL2, pERK and pAKT. TUNEL assay indicated that GPER activation may also attenuate the cardiomyocyte apoptosis upon Dox exposure. Using ex vivo Langendorff perfused heart technique, we also found an increased systolic recovery and a reduction of both infarct size and LDH levels in rats treated with G-1 in combination with Dox respect to animals treated with Dox alone. Accordingly, the beneficial effects induced by G-1 were abrogated in the presence of the GPER selective antagonist G15. These data suggest that GPER activation mitigates Dox-induced cardiotoxicity, thus proposing GPER as a novel pharmacological target to limit the detrimental cardiac effects of Dox treatment. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T07:31:15.491629-05:
      DOI: 10.1002/jcp.25585
  • Erratum: All‐Trans Retinoic Acid Attenuates Ultraviolet
           Radiation‐Induced Down‐Regulation of Aquaporin‐3 and Water
           Permeability in Human Keratinocytes by Cong Cao, Shu Wan, Qin Jiang,
           Ashley Amaral, Shan Lu, Gang Hu, Zhigang Bi, Nicola Kouttab, Wenming Chu
           and Yinsheng Wan
    • PubDate: 2016-09-07T14:30:25.15386-05:0
      DOI: 10.1002/jcp.25479
  • Store-Operated Ca2+ Entry in Oocytes Modulate the Dynamics of
           IP3-Dependent Ca2+ Release From Oscillatory to Tonic
    • Authors: Raphaël Courjaret; Maya Dib, Khaled Machaca
      Abstract: Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store-operated Ca2+ entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release leads to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low-amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3R) results in a pump-leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER-plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3Rs at distant sites, a process we refer to as “Ca2+ teleporting.” Therefore, sustained SOCE activation in the presence of an IP3-dependent “leak” pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.Calcium influx through SOCE inhibits calcium oscillations and favors a tonic mode of signaling. This is achieved through focal calcium entry at SOCE clusters, followed by calcium uptake into the ER through SERCA and re-release through open IP3 Receptors. A process referred to as “calcium teleporting.”
      PubDate: 2016-09-07T14:25:44.356793-05:
      DOI: 10.1002/jcp.25513
  • MiR-145 Regulates Lipogenesis in Goat Mammary Cells Via Targeting INSIG1
           and Epigenetic Regulation of Lipid-Related Genes
    • Authors: Hui Wang; Huaiping Shi, Jun Luo, Yongqing Yi, Dawei Yao, Xueying Zhang, Gongzhen Ma, Juan J. Loor
      Abstract: MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post-transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid-lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs, and 821 pre-miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR-145) had significant differences in expression over the lactation cycle. Further studies revealed that miR-145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR-145. Overexpression of miR-145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR-145 impaired fatty acid synthesis. Inhibition of miR-145 increased methylation levels of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), peroxisome proliferator-activated receptor gamma (PPARG), and sterol regulatory element binding transcription factor 1 (SREBF1). Luciferase reporter assays confirmed that insulin induced gene 1 (INSIG1) is a direct target of miR-145. These findings underscore the need for further studies to evaluate the potential for targeting miR-145 for improving beneficial milk components in ruminant milk. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Mammary lipogenesis is partly regulated by epigenetic mechanisms through miR-145 and INSIG1.
      PubDate: 2016-09-07T14:25:37.979785-05:
      DOI: 10.1002/jcp.25499
  • Vertebrate Lonesome Kinase Regulated Extracellular Matrix Protein
           Phosphorylation, Cell Shape and Adhesion in Trabecular Meshwork Cells
    • Authors: Rupalatha Maddala; Nikolai P. Skiba, Ponugoti Vasantha Rao
      Abstract: Glaucoma, a leading cause of irreversible blindness is commonly associated with elevated intraocular pressure (IOP) due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although dysregulated production and organization of extracellular matrix (ECM) is presumed to increase resistance to AH outflow and elevate IOP by altering TM cell contractile and adhesive properties, it is not known whether regulation of ECM protein phosphorylation via the secretory vertebrate lonesome kinase (VLK) influences TM cellular characteristics. Here we tested this possibility. Experiments carried out in this study reveal that the 32 kDa protein is a prominent VLK isoform detectable in lysates and conditioned media (CM) of human TM cells. Increased levels of VLK were observed in CM of TM cells subjected to cyclic mechanical stretch, or treated with dexamethasone, TGF-β2 and TM cells expressing constitutively active RhoA GTPase. Downregulation of VLK expression in TM cells using siRNA decreased tyrosine phosphorylation (TyrP) of ECM proteins and focal adhesions, and induced changes in cell shape in association with reduced levels of actin stress fibers and phospho-paxillin. VLK was also demonstrated to regulate TGF-β2-induced TyrP of ECM proteins. Taken together, these results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-03T16:00:28.92223-05:0
      DOI: 10.1002/jcp.25582
  • NOD Mice Having a Lyn Tyrosine Kinase Mutation Exhibit Abnormal Neutrophil
    • Authors: Yue Wu; Michael Hannigan, Lijun Zhan, Joseph A. Madri, Chi-Kuang Huang
      Abstract: Neutrophils from NOD (Non-Obese Diabetic) mice exhibited reduced migration speed, decreased frequency of directional changes and loss of directionality during chemotaxis (compared to wild type (WT) C57BL/6 mice). Additionally, F-actin of chemotaxing NOD neutrophils failed to orient toward the chemoattractant gradient and NOD neutrophil adhesion was impaired. A point mutation near the autophosphorylation site of Lyn in NOD mice was identified. Point mutations of G to A (G1412 in LynA and G1199 in LynB) cause a change of amino acid E393 (glutamic acid) to K (lysine) in LynA (E393K) (E372 of LynB), affecting fMLP-induced tyrosine phosphorylation. These data indicate that the Lyn mutation in NOD neutrophils is likely responsible for dysregulation of neutrophil adhesion and directed migration, implying the role of Lyn in modulating diabetic patient's susceptibility to bacterial and fungal infections. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-03T16:00:25.524895-05:
      DOI: 10.1002/jcp.25583
  • TNT-Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of
           Pro-Phagocytic Signals from Apoptotic to Viable Cells
    • Authors: Margarethe Bittins; Xiang Wang
      Abstract: The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequently results in uptake by phagocytes. Here we describe how apoptotic cells can use intercellular membrane nanotubes to transfer exposed PS to neighboring viable cells, and thus deposit an “eat-me” tag on the viable cells. Tunneling nanotubes (TNTs) connected UV-treated apoptotic rat pheochromocytoma PC12 cells with neighboring untreated cells. These TNTs were composed of PS-exposed plasma membrane and facilitated the transfer of the membrane from apoptotic to viable cells. Other pro-phagocytic signals, such as oxidized phospholipids and calreticulin, were also transferred to viable cells. In addition, anti-phagocytic signal CD47 presenting on the plasma membrane of viable cells was masked by the transferred PS-membrane. Confocal imaging revealed an increase of phagocytosis of viable PC12 cells by murine RAW264.7 macrophages when the viable PC12 cells were cocultured with UV-treated PC12 cells. Treatment with 50 nM cytochalasin D would abolish TNTs and correspondingly inhibit this phagocytosis of the viable cells. Our study indicates that exposed-PS membrane is delivered from apoptotic to viable cells through TNTs. This transferred membrane may act as a pro-phagocytic signal for macrophages to induce phagocytosis of viable cells in a situation where they are in the vicinity of apoptotic cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-03T16:00:23.18093-05:0
      DOI: 10.1002/jcp.25584
  • Mouse Oocytes Acquire Mechanisms that Permit Independent Cell Volume
           Regulation at the End of Oogenesis
    • Authors: Samantha Richard; Alina P. Tartia, Detlev Boison, Jay M. Baltz
      Abstract: Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [3H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-02T14:05:28.234774-05:
      DOI: 10.1002/jcp.25581
  • Involvement of CX3CL1 in the Migration of Osteoclast Precursors Across
           Osteoblast Layer Stimulated by Interleukin-1ß
    • Authors: Tsuyoshi Matsuura; Shizuko Ichinose, Masako Akiyama, Yuki Kasahara, Noriko Tachikawa, Ken-ichi Nakahama
      Abstract: The trigger for bone remodeling is bone resorption by osteoclasts. Osteoclast differentiation only occurs on the old bone, which needs to be repaired under physiological conditions. However, uncontrolled bone resorption is often observed in pro-inflammatory bone diseases, such as rheumatoid arthritis. Mature osteoclasts are multinuclear cells that differentiate from monocyte/macrophage lineage cells by cell fusion. Although Osteoclast precursors should migrate across osteoblast layer to reach bone matrix before maturation, the underlying mechanisms have not yet been elucidated in detail. We herein found that osteoclast precursors utilize two routes to migrate across osteoblast layer by confocal- and electro-microscopic observations. The osteoclast supporting activity of osteoblasts inversely correlated with osteoblast density and was positively related to the number of osteoclast precursors under the osteoblast layer. Osteoclast differentiation was induced by IL-1ß, but not by PGE2 in high-density osteoblasts. Osteoblasts and osteoclast precursors expressed CX3CL1 and CX3CR1, respectively, and the expression of CX3CL1 increased in response to interleukin-1ß. An anti-CX3CL1-neutralizing antibody inhibited the migration of osteoclast precursors and osteoclast differentiation. These results strongly suggest the involvement of CX3CL1 in the migration of osteoclast precursors and osteoclastogenesis, and will contribute to the development of new therapies for bone diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-31T16:55:26.96921-05:0
      DOI: 10.1002/jcp.25577
  • Meta-Analysis of Transcriptome Regulation During Induction to Cardiac
           Myocyte Fate from Mouse and Human Fibroblasts
    • Authors: Shima Rastegar-Pouyani; Niusha Khazaei, Ping Wee, Moein Yaqubi, Abdulshakour Mohammadnia
      Abstract: Ectopic expression of a defined set of transcription factors (TFs) can directly convert fibroblasts into a cardiac myocyte cell fate. Beside inefficiency in generating induced cardiomyocytes (iCMs), the molecular mechanisms that regulate this process remained to be well defined. The main purpose of this study was to provide better insight on the transcriptome regulation and to introduce a new strategy for candidating TFs for the transdifferentiation process. Eight mouse and three human high quality microarray data sets were analyzed to find differentially expressed genes (DEGs), which we integrated with TF-binding sites and protein-protein interactions to construct gene regulatory and protein-protein interaction networks. Topological and biological analyses of constructed gene networks revealed the main regulators and most affected biological processes. The DEGs could be categorized into two distinct groups, first, up-regulated genes that are mainly involved in cardiac-specific processes and second, down-regulated genes that are mainly involved in fibroblast-specific functions. Gata4, Mef2a, Tbx5, Tead4 TFs were identified as main regulators of cardiac-specific gene expression program; and Trp53, E2f1, Myc, Sfpi1, Lmo2, and Meis1 were identified as TFs which mainly regulate the expression of fibroblast-specific genes. Furthermore, we compared gene expression profiles and identified TFs between mouse and human to find the similarities and differences. In summary, our strategy of meta-analyzing the data of high-throughput techniques by computational approaches, besides revealing the mechanisms involved in the regulation of the gene expression program, also suggests a new approach for increasing the efficiency of the direct reprogramming of fibroblasts into iCMs. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-31T16:50:24.851247-05:
      DOI: 10.1002/jcp.25580
  • Overexpression of Gremlin1 in Mesenchymal Stem Cells Improves Hindlimb
           Ischemia in Mice by Enhancing Cell Survival
    • Authors: Qiuling Xiang; Dongxi Hong, Yan Liao, Yong Cao, Muyun Liu, Jun Pang, Junjie Zhou, Guang Wang, Renhao Yang, Maosheng Wang, Andy Peng Xiang
      Abstract: Mesenchymal stem cells (MSCs) are a promising cell resource for the treatment of ischemic diseases, partially through paracrine effects. One of the major obstacles of MSC treatment is the poor survival rate and low efficiency of transplanted stem cells due to ischemic or inflammatory environments. Gremlin1 (GREM1), a regulator of growth, differentiation and development, has been identified as a novel proangiogenic factor. However, the role and mechanism of GREM1 in MSCs remains unclear. Therefore, we assessed the putative beneficial effects of GREM1 on MSC-based therapy for hindlimb ischemia. The lentiviral vector, EF1a-GREM1, was constructed using the Multisite Gateway System and used to transduce MSCs. In vitro studies demonstrated increased survival of GREM1-MSCs exposed to H2O2, which is consistent with the activation of caspase-3. Conditional medium from GREM1-MSCs (GREM1-MSC-CM) increased the anti-apoptotic effects of human umbilical vein endothelial cells (HUVECs), and this effect was attenuated by treatment with the PI3K/Akt pathway inhibitor LY294002. MSCs modified with GREM1 could significantly increase blood perfusion of the ischemic hindlimb in vivo in a mouse model, which was correlated to improved MSC survival. This study demonstrates that overexpression of GREM1 in MSCs have greater therapeutic effects against ischemia compared with wild-type MSCs by enhancing the survival of MSCs and ECs, which may provide new tools for studies investigating the treatment of ischemic diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-31T13:45:25.493412-05:
      DOI: 10.1002/jcp.25578
  • Mild Aerobic Exercise Training Hardly Affects the Diaphragm of mdx Mice
    • Authors: Giuseppe Morici; Monica Frinchi, Alessandro Pitruzzella, Valentina Di Liberto, Rosario Barone, Andrea Pace, Valentina Di Felice, Natale Belluardo, Francesco Cappello, Giuseppa Mudò, Maria R Bonsignore
      Abstract: In the mdx mice model of Duchenne Muscular Dystrophy (DMD), mild endurance exercise training positively affected limb skeletal muscles, whereas few and controversial data exist on the effects of training on the diaphragm. The diaphragm was examined in mdx (C57BL/10ScSn-Dmdmdx) and wild type (WT, C57BL/10ScSc) mice under sedentary conditions (mdx-SD, WT-SD) and during mild exercise training (mdx-EX, WT-EX). At baseline, and after 30 and 45 days (training: 5 d/wk for 6 weeks), diaphragm muscle morphology and Cx39 protein were assessed. In addition, tissue levels of the chaperonins Hsp60 and Hsp70 and the p65 subunit of nuclear factor-kB (NF-kB) were measured in diaphragm, gastrocnemius, and quadriceps in each experimental group at all time points. Although morphological analysis showed unchanged total area of necrosis/regeneration in the diaphragm after training, there was a trend for larger areas of regeneration than necrosis in the diaphragm of mdx-EX compared to mdx-SD mice. However, the levels of Cx39, a protein associated with active regeneration in damaged muscle, were similar in the diaphragm of mdx-EX and mdx-SD mice. Hsp60 significantly decreased at 45 days in the diaphragm, but not in limb muscles, in both trained and sedentary mdx compared to WT mice. In limb muscles, but not in the diaphragm, Hsp70 and NF-kB p65 levels were increased in mdx mice irrespective of training at 30 and 45 days. Therefore, the diaphragm of mdx mice showed little inflammatory and stress responses over time, and appeared hardly affected by mild endurance training. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-30T17:45:40.562165-05:
      DOI: 10.1002/jcp.25573
  • Early Growth Response 3 (Egr3) Contributes a Maintenance of C2C12 Myoblast
    • Authors: Mitsutoshi Kurosaka; Yuji Ogura, Toshiya Funabashi, Tatsuo Akema
      Abstract: Satellite cell proliferation is a crucially important process for adult myogenesis. However, its regulatory mechanisms remain unknown. Early growth response 3 (Egr3) is a zinc-finger transcription factor that regulates different cellular functions. Reportedly, Egr3 interacts with multiple signaling molecules that are also known to regulate satellite cell proliferation. Therefore, it is possible that Egr3 is involved in satellite cell proliferation. Results of this study have demonstrated that Egr3 transcript levels are upregulated in regenerating mouse skeletal muscle after cardiotoxin injury. Using C2C12 myoblast culture (a model of activated satellite cells), results show that inhibition of Egr3 by shRNA impairs the myoblast proliferation rate. Results also show reduction of NF-кB transcriptional activity in Egr3-inhibited cells. Inhibition of Egr3 is associated with an increase in annexin V+ cell fraction and apoptotic protein activity including caspase-3 and caspase-7, and Poly-ADP ribose polymerase. By contrast, the reduction of cellular proliferation by inhibition of Egr3 was partially recovered by treatment of pan-caspase inhibitor Z-VAD-FMK. Collectively, these results suggest that Egr3 is involved in myoblast proliferation by interaction with survival signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-30T17:45:37.199268-05:
      DOI: 10.1002/jcp.25574
  • Wnt3A Induces GSK-3β Phosphorylation and β-Catenin Accumulation
           through RhoA/ROCK
    • Authors: Jae-Gyu Kim; Myoung-Ju Kim, Won-Ji Choi, Mi-Young Moon, Hee-Jun Kim, Jae-Yong Lee, Jaebong Kim, Sung-Chan Kim, Seung Goo Kang, Goo-Young Seo, Pyeung-Hyeun Kim, Jae-Bong Park
      Abstract: In canonical pathway, Wnt3A has been known to stabilize β-catenin through the dissociation between β-catenin and glycogen synthase kinase-3β (GSK-3β) that suppresses the phosphorylation and degradation of β-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling, however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3β and accumulation of β-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3β and accumulation of β-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3β phosphorylation and β-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3β in vitro. In addition, Wnt3A induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3β phosphorylation and β-catenin accumulation through RhoA and ROCK activation. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-30T17:45:33.804796-05:
      DOI: 10.1002/jcp.25572
  • Role of thrombin in the pathogenesis of central nervous system
           inflammatory diseases
    • Authors: Safieh Ebrahimi; Najme Jaberi, Amir Avan, Mikhail Ryzhikov, Mohammad Reza Keramati, Mohammad Reza Parizadeh, Seyed Mahdi Hassanian
      Abstract: Thrombin initiates proinflammatory signaling responses through activation of protease-activated receptors (PARs) in in vitro and in vivo systems. Proinflammatory signaling function of thrombin increases secretion of proinflammatory cytokines and chemokines, triggers vascular permeability, promotes leukocyte migration, and induces adhesion molecule expression. Thrombin as a potent signaling molecule is strongly implicated in a number of proinflammatory disorders including severe sepsis, cancer, cardiovascular disease, and of special interest in this review neurodegenerative disorders. This review summarizes the role of thrombin in the pathogenesis of central nervous system (CNS) inflammatory diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), promoting greater understanding and clinical management of these diseases. J. Cell. Physiol. 9999: 1–4, 2016. © 2016 Wiley Periodicals, Inc.Proinflammatory signaling function of thrombin increases secretion of proinflammatory cytokines and chemokines, triggers vascular permeability, promotes leukocyte migration, and induces adhesion molecule expression. This review summarizes the role of thrombin in the pathogenesis of central nervous system (CNS) inflammatory diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), promoting greater understanding and clinical management of these diseases.
      PubDate: 2016-08-29T03:00:22.583247-05:
      DOI: 10.1002/jcp.25501
  • Quantitative Analysis of Endocytic Recycling of Membrane Proteins by
           Monoclonal Antibody-Based Recycling Assays
    • Authors: Gordana Blagojević Zagorac; Hana Mahmutefendić, Senka Maćešić, Ljerka Karleuša, Pero Lučin
      Abstract: In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules.
      PubDate: 2016-08-22T02:21:41.323098-05:
      DOI: 10.1002/jcp.25503
  • Unique Regulatory Mechanisms for the Human Embryonic Stem Cell Cycle
    • Authors: Jennifer J. VanOudenhove; Rodrigo A. Grandy, Prachi N. Ghule, Jane B. Lian, Janet L. Stein, Sayyed K. Zaidi, Gary S. Stein
      Abstract: The cell cycle in pluripotent human embryonic stem cells is governed by unique mechanisms that support unrestricted proliferation and competency for endodermal, mesodermal and ectodermal differentiation. The abbreviated G1 period with retention of uncompromised fidelity for genetic and epigenetic mechanisms operative in control of proliferation support competency for expansion of the pluripotent cell population that is fundamental for initial stages of development. Regulatory events during the G1 period of the pluripotent cell cycle are decisive for the transition from pluripotency to lineage commitment. Recent findings indicate that a G2 cell cycle pause is present in both endodermal and mesodermal lineage cells, and is obligatory for differentiation to endoderm. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-17T17:35:41.441477-05:
      DOI: 10.1002/jcp.25567
  • Uric Acid Amplifies Aβ Amyloid Effects Involved in the Cognitive
           Dysfunction/Dementia: Evidences From an Experimental Model In Vitro
    • Authors: Giovambattista Desideri; Roberta Gentile, Andrea Antonosante, Elisabetta Benedetti, Davide Grassi, Loredana Cristiano, Antonello Manocchio, Sara Selli, Rodolfo Ippoliti, Claudio Ferri, Claudio Borghi, Antonio Giordano, Annamaria Cimini
      Abstract: There is still a considerable debate concerning whether uric acid is neuroprotective or neurotoxic agent. To clarify this topic, we tested the effects of uric acid on neuronal cells biology by using differentiated SHSY5Y neuroblastoma cells incubated with amyloid β to reproduce an in vitro model of Alzheimer's disease. The incubation of cells with uric acid at the dose of 40 µM or higher significantly reduced cell viability and potentiated the proapoptotic effect of amyloid β. Finally, uric acid enhanced the generation of 4-hydroxynonenal and the expression of PPARβ/δ promoted by amyloid β, indicating a prooxidant effects. In conclusion, uric acid could exert a detrimental influence on neuronal biology being this influence further potentiated by the concomitant exposure to neurotoxic stimuli. This effect is evident for uric acid concentrations close to those achievable in cerebrospinal fluid in presence of mild hyperuricemia thus suggesting a potential role of uric acid in pathophysiology of cognitive dysfunction. These effects are influenced by the concentrations of uric acid and by the presence of favoring conditions that commonly occur in neurodegenerative disorders and well as in the aging brain, including increased oxidative stress and exposure to amyloid β. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.The study suggests that uric acid could exert detrimental effects on brain structure and function by directly influencing the viability of neuronal cells and their ability to establish synaptic connections.
      PubDate: 2016-08-16T02:10:50.861306-05:
      DOI: 10.1002/jcp.25509
  • Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of
           Fetal Calf Serum
    • Authors: Asima Karim; Andrew C. Hall
      Abstract: Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood, however, pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology, and clustering of cultured bovine articular chondrocytes within stiff (2% w/v) and soft (0.2% w/v) three-dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; fetal calf serum (FCS) 1–10%;380 mOsm) for up to 7 days. Chondrocytes were fluorescently labeled after 1, 3, and 7 days with 5-chloromethylfluorescein-diacetate (CMFDA) and propidium iodide (PI) or 1,5-bis{[2-(di-methylamino)ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology, and clustering were quantified using Volocity™ software. In stiff gels after 7 d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However, in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P 
      PubDate: 2016-08-16T02:10:42.324137-05:
      DOI: 10.1002/jcp.25507
  • Ascorbic Acid Induces Necrosis in Human Laryngeal Squamous Cell Carcinoma
           via ROS, PKC, and Calcium Signaling
    • Authors: Min-Woo Baek; Heui-Seung Cho, Sun-Hun Kim, Won-Jae Kim, Ji-Yeon Jung
      Abstract: Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N-acetylcysteine blocked ascorbic acid-induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid-induced necrotic cell death was inhibited by Ro-31-8425 (PKC inhibitor) and BAPTA-AM (cytosolic calcium-selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro-31-8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid-treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.Ascorbic acid induced necrotic cell death in human laryngeal squamous cell carcinoma through ROS, PKC, and calcium signaling pathway.
      PubDate: 2016-08-16T02:06:19.909855-05:
      DOI: 10.1002/jcp.25438
  • Extracellular Proteasomes Are Deficient in 19S Subunits as Revealed by
           iTRAQ Quantitative Proteomics
    • Authors: Anna S. Tsimokha; Julia J. Zaykova, Andrew Bottrill, Nikolai A. Barlev
      Abstract: Proteasome-mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well-documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ-based quantitative proteomics (iTRAQ LC/MS-MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. Sixty-two of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP-PIPs were associated with protein biosynthesis and, unlike CP-PIPs, were under represented by chaperon/ATP-binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ-based quantitative proteomics (iTRAQ LC/MS-MS). Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes, which implies that the two species of proteasomes may have different biological functions.
      PubDate: 2016-08-16T02:05:30.033441-05:
      DOI: 10.1002/jcp.25492
  • 3-Methylcholanthrene/Aryl-Hydrocarbon Receptor-Mediated Hypertension
           Through eNOS Inactivation
    • Authors: Chih-Cheng Chang; Yung-Ho Hsu, Hsiu-Chu Chou, Yuan-Chii G. Lee, Shu-Hui Juan
      Abstract: Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)-dependent pathway. We previously reported that 3-methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3-MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt-mediated mechanism. The mechanism underlying the effects of 3-MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3-MC modified heat shock protein 90 (HSP90), caveolin-1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA-dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3-MC reduced eNOS phosphorylation through the AhR/RhoA-mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3-MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin-1. Immunofluorescence and Western blot analysis revealed that 3-MC reduced the amount of membrane-bound activated eNOS, and a modified Griess assay revealed that 3-MC concomitantly reduced NO production. However, simvastatin reduced 3-MC-mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3-MC. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.Our study is the first to report that the AhR ligand 3-MC inactivated eNOS associated with downregulated HSP90 and upregulated caveolin-1 and dynein expression through AhR/RhoA-mediated PI3K/Akt inactivation, leading to hypertension in mice. The blockade of AhR and RhoA by using siAhR, DNRhoA, or statins reversed the inhibitory effects of 3-MC on eNOS by correcting the alterations of eNOS regulatory proteins.
      PubDate: 2016-08-10T00:29:24.094759-05:
      DOI: 10.1002/jcp.25497
  • An Overview on Predictive Biomarkers of Testicular Germ Cell Tumors
    • Authors: Paolo Chieffi
      Abstract: Testicular germ cell tumors (TGCTs) are frequent solid malignant tumors and cause of death in men between 20–40 years of age. Genetic and environmental factors play an important role in the origin and development of TGCTs. Although the majority of TGCTs are responsive to chemotherapy, about 20% of patient presents incomplete response or tumors relapse. In addition, the current treatments cause acute toxicity and several chronic collateral effects, including sterility. The present mini-review collectively summarize the most recent findings on the new discovered molecular biomarkers such as tyrosine kinases, HMGAs, Aurora B kinase, and GPR30 receptor predictive of TGCTs and as emerging new possible molecular targets for therapeutic strategies. J. Cell. Physiol. 9999: 1–5, 2016. © 2016 Wiley Periodicals, Inc.Although the majority of TGCTs are responsive to chemotherapy, about 20% of patients present incomplete response or tumor relapse. In addition, the current treatments cause acute toxicity and several chronic collateral effects, including sterility. The present mini-review collectively summarizes the most recent findings on the newly discovered molecular biomarkers such as tyrosine kinases, HMGAs, Aurora B kinase, and GPR30 receptor predictive of TGCTs and as new possible molecular targets for therapeutic strategies.
      PubDate: 2016-07-29T10:10:23.798428-05:
      DOI: 10.1002/jcp.25482
  • Coumestrol Inhibits Proliferation and Migration of Prostate Cancer Cells
           by Regulating AKT, ERK1/2, and JNK MAPK Cell Signaling Cascades
    • Authors: Whasun Lim; Muhah Jeong, Fuller W. Bazer, Gwonhwa Song
      Abstract: Coumestrol is the one of the major phytoestrogens which is abundant in soybeans, legumes, brussel sprouts, and spinach. The beneficial effects of coumestrol are well known in various biological processes including; neuroprotective effects on the nervous system, function of the female reproductive system, anti-bacterial properties, and anti-cancer effects. Although the anti-tumor activity of coumestrol has been demonstrated for ovarian, breast, lung, and cervical cancers, little is known of its effects on prostate cancer. Therefore, in the present study, we investigated the chemotherapeutic effects of coumestrol on two prostate cancer cell lines, PC3 and LNCaP. Our results showed that coumestrol decreased proliferation and migration and induced apoptosis in both PC3 and LNCaP cells. Moreover, effects of coumestrol on cell signaling pathways were investigated and it increased phosphorylation of ERK1/2, JNK, P90RSK, and P53 proteins in a dose- and time-dependent manner whereas phosphorylation of AKT was reduced by coumestrol under the same conditions for culture of PC3 and LNCaP cells. In addition, mitochondrial dysfunction was induced by coumestrol as evidenced by a significant loss of mitochondrial membrane potential. Furthermore, cleavage of caspase-3 and caspase-9, the apoptotic proteins associated with mitochondria, also changed in response to coumestrol. Coumestrol also caused mitochondrial dysfunction resulting in an increase in ROS production in PC3 and LNCaP cells. These results suggest that coumestrol can inhibit progression of prostate cancer and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signaling pathways. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.Our results indicated that coumestrol can inhibit progression of prostate cancer cells by affecting their viability and mitochondrial functions, and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signal transduction cascades.
      PubDate: 2016-07-28T05:51:13.440339-05:
      DOI: 10.1002/jcp.25494
  • Tissue Inhibitor of Metalloproteinase 1 Influences Vascular Adaptations to
           Chronic Alterations in Blood Flow
    • Authors: Erin R. Mandel; Cassandra Uchida, Emmanuel Nwadozi, Armin Makki, Tara L. Haas
      Abstract: Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). We hypothesized that the loss of TIMP1 would enhance both ischemia and flow-induced vascular remodeling by increasing MMP activity. TIMP1 deficient (Timp1−/−) and wild-type (WT) C57BL/6 mice underwent unilateral femoral artery (FA) ligation or were treated with prazosin, an alpha-1 adrenergic receptor antagonist, in order to investigate vascular remodeling to altered flow. Under basal conditions, Timp1−/− mice had reduced microvascular content as compared to WT mice. Furthermore, vascular remodeling was impaired in Timp1−/− mice. Timp1−/− mice displayed reduced blood flow recovery in response to FA ligation and no arteriogenic response to prazosin treatment. Timp1−/− mice failed to undergo angiogenesis in response to ischemia or prazosin, despite maintaining the capacity to increase VEGF-A and eNOS mRNA. Vascular permeability was increased in muscles of Timp1−/− mice in response to both prazosin treatment and FA ligation, but this was not accompanied by greater MMP activity. This study highlights a previously undescribed integral role for TIMP1 in both vascular network maturation and adaptations to ischemia or alterations in flow. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). This study highlights a previously undescribed integral role for TIMP1 in vascular network maturation and in adaptations to altered blood flow.
      PubDate: 2016-07-28T05:45:36.851462-05:
      DOI: 10.1002/jcp.25491
  • Late Endosomal Recycling of Open MHC-I Conformers
    • Authors: Hana Mahmutefendić; Gordana Blagojević Zagorac, Kristina Grabušić, Ljerka Karleuša, Senka Maćešić, Frank Momburg, Pero Lučin
      Abstract: With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty Ld (eLd) molecules, an open form of murine MHC-I allele, in fibroblast-like cells. Pulse labeling of cell surface eLd with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC-I conformers. Kinetic modeling, using in-house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eLd demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eLd distributed into pre-degradative multivesicular bodies (MVBs), these LE subsets were not a source for eLd recycling. The LE recycling of eLd did not require Rab7 membrane domains, as demonstrated by Rab7-silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eLd in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. J. Cell. Physiol. 9999: 1–16, 2016. © 2016 Wiley Periodicals, Inc.Study of endosomal trafficking of open MHC-I conformers reveals that endosomal recycling may occur from late endosomes, suggesting that the late endosomal system should not be simply considered as a feeder for loading of the degradative tract of the cell.
      PubDate: 2016-07-27T10:50:44.218757-05:
      DOI: 10.1002/jcp.25495
  • Nestin Expressed by Pre-Existing Cardiomyocytes Recapitulated in Part an
           Embryonic Phenotype; Suppressive Role of p38 MAPK
    • Authors: Marc-Andre Meus; Vanessa Hertig, Louis Villeneuve, Jean-Francois Jasmin, Angelino Calderone
      Abstract: Nestin(+)-cardiomyocytes were identified in the ischemically damaged human/rodent heart, albeit the cellular source, and signaling events implicated in the appearance of the intermediate filament protein remained undefined. Expression of the enhanced green fluorescent protein (EGFP) driven by the second intron of the nestin gene identified a subpopulation of EGFP/nestin(+) cells that differentiated to a vascular phenotype in the peri-infarct/infarct region of post-MI mice albeit the transgene was not detected in nestin(+)-cardiomyocytes. α-MHC-driven expression of the reporter mCherry was detected in troponin-T(+)- and nestin(+)-cardiomyocytes in the peri-infarct/infarct region of post-MI mice. However, the cell cycle re-entry of nestin/mCherry(+)-cardiomyocytes was not observed. Nestin staining was identified in a paucity of neonatal rat ventricular cardiomyocytes (NNVM). Exposure to phorbol 12,13-dibutyrate (PDBu) induced NNVM hypertrophy but did not promote nestin expression or Brdu incorporation. PDBu treatment of NNVMs phosphorylated p38 MAPK and HSP27 and HSP27 phosphorylation was abrogated by the p38 MAPK inhibitor SB203580. PDBu/SB203580 co-treatment significantly increased the percentage of NNVMs that expressed nestin and incorporated Brdu. In the heart of embryonic 10.5 day mice, nestin immunoreactivity was observed in cycling troponin-T(+)-cardiomyocytes. Nestin was also detected in embryonic rat ventricular cardiomyocytes and depletion of the intermediate filament protein attenuated cell cycle re-entry. Thus, nestin expressed by pre-existing cardiomyocytes following ischemic damage recapitulated in part an embryonic trait and may provide the requisite phenotype to initiate cell cycle re-entry. However, the overt activation of the p38 MAPK pathway post-MI may in part limit the appearance and inhibit the cell cycle re-entry of nestin(+)-cardiomyocytes. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Nestin was expressed by pre-existing cardiomyocytes following ischemic damage to the adult heart and recapitulated in part an embryonic phenotype. However, nestin-expressing cardiomyocytes were unable to re-enter the cell cycle following ischemic damage because of the suppressive effect of the p38 MAPK pathway.
      PubDate: 2016-07-27T10:50:36.765274-05:
      DOI: 10.1002/jcp.25496
  • Neuronatin Protein in Health and Disease
    • Authors: Priyamvada M. Pitale; Wayne Howse, Marina Gorbatyuk
      Abstract: Neuronatin (NNAT) was first identified as a brain-specific gene crucial for brain development. Over the years, NNAT has been studied in different developing and post-developed tissues and organs. While NNAT manifests functional and structural similarities to the phospholamban gene, its physiological and pathological roles in healthy and diseased tissues have not been precisely identified. Ca2+ signaling, glucose transport, insulin secretion, and inflammation modulated at different pathological conditions have been proposed to be governed by NNAT. This review describes the current findings of cellular molecular pathways known to be modified concomitantly with an alteration in NNAT expression, and it highlights the need to conduct extensive investigation regarding the role of NNAT in health and disease. J. Cell. Physiol. 9999: 1–5, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-07-27T10:50:23.162398-05:
      DOI: 10.1002/jcp.25498
  • Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb
    • Authors: Ahn R. Lee; Wayne Hung, Ning Xie, Liangliang Liu, Leye He, Xuesen Dong
      Abstract: The non-POU-domain-containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site-directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co-immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein–protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non-specific binding affinity to anti-phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co-repression and RNA splicing in gene context-dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co-regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.P54nrb (aka NONO) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although Tyr phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. Our studies demonstrate that the Tyr residues of p54nrb are not phosphorylated, but nevertheless significantly regulate p54nrb function by means of potential temporal and spatial regulation of p54nrb, resulting in altered interaction of p54nrb with various co-regulatory splicing or transcriptional proteins.
      PubDate: 2016-07-27T10:45:26.08248-05:0
      DOI: 10.1002/jcp.25493
  • BAG3 Protein Is Over-Expressed in Endometrioid Endometrial Adenocarcinomas
    • Authors: Veronica Esposito; Carlo Baldi, Pio Zeppa, Michelina Festa, Luana Guerriero, Morena d'Avenia, Massimiliano Chetta, Fulvio Zullo, Vincenzo De Laurenzi, Maria Caterina Turco, Alessandra Rosati, Maurizio Guida
      Abstract: Endometrioid endometrial cancer is the most common gynaecological tumor in developed countries, and its incidence is increasing. The definition of subtypes, based on clinical and endocrine features or on histopathological characteristics, correlate to some extent with patient's prognosis, but there is substantial heterogeneity within tumor types. The search for molecules and mechanisms implied in determining the progression and the response to therapy for this cancer is still ongoing. BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumor types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby, modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. BAG3 expression in human endometrial cancer tissues was not investigated so far. Here, we show that BAG3 protein levels are elevated in tumoral and hyperplastic cells in respect to normal glands. Furthermore, BAG3 subcellular localization appears to be changed in tumoral compared to normal cells. Our results indicate a possible role for BAG3 protein in the maintenance of cell survival in endometrioid endometrial cancer and suggest that this field of studies is worthy of further investigations. J. Cell. Physiol. 9999: 1–3, 2016. © 2016 Wiley Periodicals, Inc.BAG3 protein levels has been analyzed for the first time in human endometrial cancer tissues. BAG3 anti-apoptotic protein is over-expressed in endometrial cancer cells. BAG3 could be a target of value for endometrioid endometrial cancer therapy.
      PubDate: 2016-07-22T05:50:31.02862-05:0
      DOI: 10.1002/jcp.25489
  • Atrogin-1 Increases Smooth Muscle Contractility Through Myocardin
    • Authors: Pavneet Singh; Dong Li, Yu Gui, Xi-Long Zheng
      Abstract: Atrogin-1, an E3 ligase present in skeletal, cardiac and smooth muscle, down-regulates myocardin protein during skeletal muscle differentiation. Myocardin, the master regulator of smooth muscle cell (SMC) differentiation, induces expression of smooth muscle marker genes through its association with serum response factor (SRF), which binds to the CArG box in the promoter. Myocardin undergoes ubiquitylation and proteasomal degradation. Evidence suggests that proteasomal degradation of myocardin is critical for myocardin to exert its transcriptional activity, but there is no report about the E3 ligase responsible for myocardin ubiquitylation and subsequent transactivation. Here, we showed that overexpression of atrogin-1 increased contractility of cultured SMCs and mouse aortic tissues in organ culture. Overexpression of dominant-negative myocardin attenuated the increase in SMC contractility induced by atrogin-1. Atrogin-1 overexpression increased expression of the SM contractile markers while downregulated expression of myocardin protein but not mRNA. Atrogin-1 also ubiquitylated myocardin for proteasomal degradation in vascular SMCs. Deletion studies showed that atrogin-1 directly interacted with myocardin through its amino acids 284–345. Immunostaining studies showed nuclear localization of atrogin-1, myocardin, and the Rpt6 subunit of the 26S proteasome. Atrogin-1 overexpression not only resulted in degradation of myocardin but also increased recruitment of RNA Polymerase II onto the promoters of myocardin target genes. In summary, our results have revealed the roles for atrogin-1 in the regulation of smooth muscle contractility through enhancement of myocardin ubiquitylation/degradation and its transcriptional activity. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.Atrogin-1 is an E3 ligase present in skeletal, cardiac and smooth muscle. Our study has shown that atrogin-1 down-regulates myocardin protein in human vascular smooth muscle cells, but increases transcriptional activity of myocardin on its target genes.
      PubDate: 2016-07-21T11:20:36.573109-05:
      DOI: 10.1002/jcp.25485
  • Integrated Transcriptome Map Highlights Structural and Functional Aspects
           of the Normal Human Heart
    • Authors: Maria Caracausi; Allison Piovesan, Lorenza Vitale, Maria Chiara Pelleri
      Abstract: A systematic meta-analysis of the available gene expression profiling datasets for the whole normal human heart generated a quantitative transcriptome reference map of this organ. Transcriptome Mapper (TRAM) software integrated 32 gene expression profile datasets from different sources returning a reference value of expression for each of the 43,360 known, mapped transcripts assayed by any of the experimental platforms used in this regard. Main findings include the visualization at the gene and chromosomal levels of the classical description of the basic histology and physiology of the heart, the identification of suitable housekeeping reference genes, the analysis of stoichiometry of gene products, and the focusing on chromosome 21 genes, which are present in one excess copy in Down syndrome subjects, presenting cardiovascular defects in 30–40% of cases. Independent in vitro validation showed an excellent correlation coefficient (r = 0.98) with the in silico data. Remarkably, heart/non-cardiac tissue expression ratio may also be used to anticipate that effects of mutations will most probably affect or not the heart. The quantitative reference global portrait of gene expression in the whole normal human heart illustrates the structural and functional aspects of the whole organ and is a general model to understand the mechanisms underlying heart pathophysiology. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.A systematic meta-analysis of the available gene expression profiling datasets for the whole normal human heart retrieved 32 microarray experiments performed on the whole organ. These data were integrated through the Transcriptome Mapper (TRAM) software to obtain a reference typical value of expression for each of the 43,360 known, mapped transcripts. In vitro validation through real-time RT-PCR showed an excellent correlation coefficient (r = 0.98) with the in silico data.
      PubDate: 2016-07-21T11:10:38.808796-05:
      DOI: 10.1002/jcp.25471
  • Can Cholesterol Metabolism Modulation Affect Brain Function and
    • Authors: Veronica Cartocci; Michela Servadio, Viviana Trezza, Valentina Pallottini
      Abstract: Cholesterol is an important component for cell physiology. It regulates the fluidity of cell membranes and determines the physical and biochemical properties of proteins. In the central nervous system, cholesterol controls synapse formation and function and supports the saltatory conduction of action potential. In recent years, the role of cholesterol in the brain has caught the attention of several research groups since a breakdown of cholesterol metabolism has been associated with different neurodevelopmental and neurodegenerative diseases, and interestingly also with psychiatric conditions. The aim of this review is to summarize the current knowledge about the connection between cholesterol dysregulation and various neurologic and psychiatric disorders based on clinical and preclinical studies. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-07-20T12:06:03.33063-05:0
      DOI: 10.1002/jcp.25488
  • Vimentin as a Marker of Early Differentiating, Highly Motile Corneal
           Epithelial Cells
    • Authors: Federico Castro-Muñozledo; Diana G. Meza-Aguilar, Rocío Domínguez-Castillo, Veremundo Hernández-Zequinely, Erika Sánchez-Guzmán
      Abstract: Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+/Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4–5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt+/Vim− cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+/Krt+/ΔNp63α+/Pax-6low/α6 integrin+ phenotype. J. Cell. Physiol. 9999: 1–13, 2016. © 2016 Wiley Periodicals, Inc.During growth and wound healing, highly motile epithelial cells require the expression of a specific cytoskeletal network to sustain organelle translocation during cell movement. Such requirements might be fulfilled by the copolymerization of Vim and Krt at the migratory edges of cells, and its interaction with Extracellular Matrix, a task achieved by relocation of alpha6beta4 integrin into focal adhesion plaques, and its association with plectin as an integrator molecule.
      PubDate: 2016-07-20T12:01:04.868486-05:
      DOI: 10.1002/jcp.25487
  • Expression of P2 Purinergic Receptors in Mesenchymal Stem Cells and Their
           Roles in Extracellular Nucleotide Regulation of Cell Functions
    • Authors: Lin-Hua Jiang; Yunjie Hao, Fatema Mousawi, Hongsen Peng, Xuebin Yang
      Abstract: Extracellular ATP and other nucleotides induce autocrine and/or paracrine purinergic signalling via activation of the P2 receptors on the cell surface, which represents one of the most common signalling mechanisms. Mesenchymal stem cells (MSC) are a type of multipotent adult stem cells that have many promising applications in regenerative medicine. There is increasing evidence to show that extracellular nucleotides regulate MSC functions and P2 receptor-mediated purinergic signalling plays an important role in such functional regulation. P2 receptors comprise ligand-gated ion channel P2X receptors and G-protein-coupled P2Y receptors. In this review, we provide an overview of the current understanding with respect to expression of the P2X and P2Y receptors in MSC and their roles in mediating extracellular nucleotide regulation of MSC proliferation, migration and differentiation. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-07-20T12:00:41.476616-05:
      DOI: 10.1002/jcp.25484
  • The β2-Adrenoceptor Agonist Terbutaline Stimulates Angiogenesis via
           Akt and ERK Signaling
    • Authors: Stefanie Lemmens; Lauren Kusters, Annelies Bronckaers, Nathalie Geurts, Sven Hendrix
      Abstract: Angiogenesis is associated with changes in endothelial cell (EC) proliferation and tube formation, controlled by extracellular receptor-activated kinase (ERK)/mitogen activated protein kinase (MAPK) and Akt signaling. Important regulators of these systems include hormones acting on G-protein-coupled receptors, such as beta 2-adrenoceptors (β2-ARs). In central nervous system (CNS) trauma, the importance of β2-AR modulation has been highlighted, although the effects on revascularization remain unclear. Vascular protection and revascularization are, however, key to support regeneration. We have investigated the angiogenic capacity of the specific β2-AR agonist terbutaline on ECs derived from the CNS, namely bEnd.3-cells. As angiogenesis is a multistep process involving increased proliferation and tube formation of ECs, we investigated the effects of terbutaline on these processes. We show that terbutaline significantly induced bEnd.3 tube formation in a matrigel in vitro assay. Moreover, administration of specific inhibitors of ERK and Akt signaling both inhibited terbutaline-induced tube formation. The proliferation rate of the ECs was not affected. In order to investigate the general effects of terbutaline in an organotypic system, we have used the chick chorioallantoic membrane (CAM)-assay. Most importantly, terbutaline increased the number of blood vessels in this in ovo setting. Although we observed a positive trend, the systemic administration of terbutaline did not significantly improve the functional outcome, nor did it affect revascularization in our spinal cord injury model. In conclusion, these data indicate that terbutaline is promising to stimulate blood vessel formation, underscoring the importance of further research into the angiotherapeutic relevance of terbutaline and β2-AR signaling after CNS-trauma. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Terbutaline is a promising beta 2-adrenoceptor agonist to stimulate blood vessel formation. It significantly promotes tube formation by mouse brain endothelial cells. Most importantly, it increases the number of blood vessels in the organotypic setting of the chorio-allantoic membrane assay.
      PubDate: 2016-07-20T11:50:35.977902-05:
      DOI: 10.1002/jcp.25483
  • The Effects of Acute and Chronic Exercise on Skeletal Muscle Proteome
    • Authors: Bernardo A. Petriz; Clarissa P.C. Gomes, Jeeser A. Almeida, Getulio P. de Oliveira, Filipe M. Ribeiro, Rinaldo W. Pereira, Octavio L. Franco
      Abstract: Skeletal muscle plasticity and its adaptation to exercise is a topic that is widely discussed and investigated due to its primary role in the field of exercise performance and health promotion. Repetitive muscle contraction through exercise stimuli leads to improved cardiovascular output and the regulation of endothelial dysfunction and metabolic disorders such as insulin resistance and obesity. Considerable improvements in proteomic tools and data analysis have broth some new perspectives in the study of the molecular mechanisms underlying skeletal muscle adaptation in response to physical activity. In this sense, this review updates the main relevant studies concerning muscle proteome adaptation to acute and chronic exercise, from aerobic to resistance training, as well as the proteomic profile of natural inbred high running capacity animal models. Also, some promising prospects in the muscle secretome field are presented, in order to better understand the role of physical activity in the release of extracellular microvesicles and myokines activity. Thus, the present review aims to update the fast-growing exercise-proteomic scenario, leading to some new perspectives about the molecular events under skeletal muscle plasticity in response to physical activity. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.Skeletal muscle plasticity and its adaptation to exercise is a topic that is widely discussed and investigated due to its primary role in the field of exercise performance and health promotion. Considerable improvements in proteomic tools and data analysis have broth some new perspectives in the study of the molecular mechanisms underlying skeletal muscle adaptation in response to physical activity. In this sense, the present review aims to update the fast-growing exercise-proteomic scenario, leading to some new perspectives about the molecular events under skeletal muscle plasticity in response to physical activity.
      PubDate: 2016-07-20T11:50:30.408853-05:
      DOI: 10.1002/jcp.25477
  • Evidence for the Role of BAG3 in Mitochondrial Quality Control in
    • Authors: Farzaneh G. Tahrir; Tijana Knezevic, Manish K. Gupta, Jennifer Gordon, Joseph Y. Cheung, Arthur M. Feldman, Kamel Khalili
      Abstract: Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co-chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates clearance of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.BAG3 is critical for homeostasis of skeletal and cardiac tissues. Here we demonstrate that knockdown of BAG3 expression increased the level of Parkin, a key regulator of autophagy in neonatal rat ventricular cardiomyocytes. Furthermore, a decrease in the level of BAG3 promotes translocation of Parkin to the depolarized mitochondria and its degradation. BAG3 suppression significantly decreased the clearance of Tom20, an essential import receptor for mitochondria protein, after treatment with electron uncoupler, suggesting that mitophagy is impaired in the absence of BAG3. These observations ascribe a critical role for BAG3 in the maintenance of mitochondrial function under stress conditions.
      PubDate: 2016-07-19T09:00:53.700909-05:
      DOI: 10.1002/jcp.25476
  • SCD1 Alters Long-Chain Fatty Acid (LCFA) Composition and Its Expression Is
           Directly Regulated by SREBP-1 and PPARγ 1 in Dairy Goat Mammary Cells
    • Authors: Dawei Yao; Jun Luo, Qiuya He, Hengbo Shi, Jun Li, Hui Wang, Huifen Xu, Zhi Chen, Yongqing Yi, Juan J. Loor
      Abstract: Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme for the synthesis of the monounsaturated fatty acids (MUFA) palmitoleic acid and oleic acid. In non-ruminant species, SCD1 expression is known to be tightly regulated by a variety of transcription factors. Although the role of SCD1 and the transcriptional regulatory mechanism by SREBP-1 and PPARs in other species is clear, changes in lipid metabolism related to SCD1 and via the regulation of SREBP-1 or PPARG1 in ruminant mammary tissue remain largely unknown. Here, we demonstrated that SCD1 expression in goat mammary tissue is higher during lactation than the dry period. Overexpression of SCD1 increased the intracellular MUFA content and lipid accumulation, whereas SCD1 silencing resulted in a significant decrease in oleic acid concentration and triacylglycerol (TAG) accumulation. The overexpression of SREBF1 in goat mammary epithelial cells (GMEC) enhanced SCD1 expression and its promoter activity, but that effect was abolished when SREBF1 was silenced. Furthermore, deletion of sterol regulatory element (SRE) and the nuclear factor (NF-Y)-binding sites within a −1713 to +65-base pair region of the SCD1 promoter completely abolished SREBP-1-induced SCD1 transcription. Otherwise, PPARG1 overexpression also stimulated the expression of SCD1 and its transcriptional activity directly via a PPAR response element (PPRE) in the SCD1 promoter. Together, these results indicate that SCD1 could markedly affect the fatty acid composition and rate of TAG synthesis through direct regulation via SREBP-1 and PPARG1, hence, underscoring an important role of the enzyme and this transcription regulator in controlling mammary gland lipid synthesis in the goat. J. Cell. Physiol. 9999: 1–15, 2016. © 2016 Wiley Periodicals, Inc.The function of stearoyl-CoA desaturase 1 gene in goat mammary cells. The regulatory mechanism of SCD1 by SREBP1 and PPARG in dairy goat.
      PubDate: 2016-07-18T11:30:35.5889-05:00
      DOI: 10.1002/jcp.25469
  • Biochemical and Pathophysiological Premises to Positron Emission
           Tomography With Choline Radiotracers
    • Authors: Vincenzo Cuccurullo; Giuseppe Danilo Di Stasio, Laura Evangelista, Gabriella Castoria, Luigi Mansi
      Abstract: Choline is a quaternary ammonium base that represents an essential component of phospholipids and cell membranes. Malignant transformation is associated with an abnormal choline metabolism at a higher levels with respect to those exclusively due to cell multiplication. The use of Positron Emission Tomography/Computed Tomography (PET/CT) with radiocholine (RCH), labeled with 11C or 18F, is widely diffuse in oncology, with main reference to restaging of patients with prostate cancer. The enhanced concentration in neoplasm is based not only on the increasing growing rate, but also on more specific issues, such as the augmented uptake in malignant cells due to the up-regulation of choline kinase. Furthermore the role of hypoxia in decreasing choline's uptake determine an in vivo concentration only in well oxygenated tumors, with a lower uptake when malignancy increases, that is, in tumors positive at 18F-Fluoro-deoxyglucose. In this paper we have analyzed the most important issues related to the possible utilization of RCH in diagnostic imaging of human cancer. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 Wiley Periodicals, Inc.This is a review article on PET choline tracer metabolism and practicalities. This paper create a connection between basic and clinical issues.
      PubDate: 2016-07-18T11:30:31.030582-05:
      DOI: 10.1002/jcp.25478
  • Transcriptomic Analyses of Adipocyte Differentiation From Human
           Mesenchymal Stromal-Cells (MSC)
    • Authors: Antonio Casado-Díaz; Jaouad Anter, Sören Müller, Peter Winter, José Manuel Quesada-Gómez, Gabriel Dorado
      Abstract: Adipogenesis is a physiological process required for fat-tissue development, mainly involved in regulating the organism energetic-state. Abnormal distribution-changes and dysfunctions in such tissue are associated to different pathologies. Adipocytes are generated from progenitor cells, via a complex differentiating process not yet well understood. Therefore, we investigated differential mRNA and miRNA expression patterns of human mesenchymal stromal-cells (MSC) induced and not induced to differentiate into adipocytes by next (second)-generation sequencing. A total of 2,866 differentially expressed genes (101 encoding miRNA) were identified, with 705 (46 encoding miRNA) being upregulated in adipogenesis. They were related to different pathways, including PPARG, lipid, carbohydrate and energy metabolism, redox, membrane-organelle biosynthesis, and endocrine system. Downregulated genes were related to extracellular matrix and cell migration, proliferation, and differentiation. Analyses of mRNA-miRNA interaction showed that repressed miRNA-encoding genes can act downregulating PPARG-related genes; mostly the PPARG activator (PPARGC1A). Induced miRNA-encoding genes regulate downregulated genes related to TGFB1. These results shed new light to understand adipose-tissue differentiation and physiology, increasing our knowledge about pathologies like obesity, type-2 diabetes and osteoporosis. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.Adipogenesis transcriptomics was studied in mesenchymal stem-cells (MSC) and 659 mRNA and 46 miRNA were upregulated in adipocyte differentiation. Metabolic, redox, and endocrinology process were upregulated in adipogenesis and Migration, proliferation, and differentiation to others cell types were downregulated. miRNA that can interact with PPARG and TGFB1 pathways were both downregulated and upregulated.
      PubDate: 2016-07-12T10:43:02.696974-05:
      DOI: 10.1002/jcp.25472
  • Parathyroid Hormone-Related Protein Protects Osteoblastic Cells From
           Oxidative Stress by Activation of MKP1 Phosphatase
    • Authors: Juan A. Ardura; Sergio Portal-Núñez, Irantzu Castelbón-Calvo, Irene Martínez de Toda, Mónica De la Fuente, Pedro Esbrit
      Abstract: Oxidative damage is an important contributor to the morphological and functional changes in osteoporotic bone. Aging increases the levels of reactive oxygen species (ROS) that cause oxidative stress and induce osteoblast apoptosis. ROS modify several signaling responses, including mitogen-activated protein kinase (MAPK) activation, related to cell survival. Both parathyroid hormone (PTH) and its bone counterpart, PTH-related protein (PTHrP), can regulate MAPK activation by modulating MAPK phosphatase-1 (MKP1). Thus, we hypothesized that PTHrP might protect osteoblasts from ROS-induced apoptosis by targeting MKP1. In osteoblastic MC3T3-E1 and MG-63 cells, H2O2 triggered p38, JNK, ERK and p66Shc phosphorylation, and cell apoptosis. Meanwhile, PTHrP (1-37) rapidly but transiently increased ERK and Akt phosphorylation without affecting p38, JNK, or p66Shc activation. H2O2-induced p38 and ERK phosphorylation and apoptosis were both decreased by pre-treatment with specific kinase inhibitors or PTHrP (1-37) in both osteoblastic cell types. These dephosphorylating and prosurvival actions of PTHrP (1-37) were prevented by a phosphatase inhibitor cocktail, the phosphatase MKP1 inhibitor sanguinarine or a MKP1 siRNA. PTHrP (1-37) promptly enhanced MKP1 protein and gene expression and MKP1-dependent catalase activity in osteoblastic cells. Furthermore, exposure to PTHrP (1-37) adsorbed in an implanted hydroxyapatite-based ceramic into a tibial defect in aging rats increased MKP1 and catalase gene expression in the healing bone area. Our findings demonstrate that PTHrP counteracts the pro-apoptotic actions of ROS by a mechanism dependent on MKP1-induced dephosphorylation of MAPKs in osteoblasts. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.PTHrP counteracts the pro-apoptotic actions of ROS by a mechanism dependent on MKP1 dephosphorylation of MAP kinases and decreasing reactive oxygen species accumulation.
      PubDate: 2016-07-12T10:32:04.676822-05:
      DOI: 10.1002/jcp.25473
  • Combined Multidimensional Microscopy as a Histopathology Imaging Tool
    • Authors: Gerald J. Shami; Delfine Cheng, Filip Braet
      Abstract: Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy, thus, facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterize various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high-resolution backscattered scanning electron microscopy, we were able to further characterize the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales, utilising X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy on the same sample. By harnessing such an approach, we characterise various features of the hepatic microvascular network ranging from the tissue level down to the nanometre scale. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations, and experimental models.
      PubDate: 2016-07-09T09:15:35.773428-05:
      DOI: 10.1002/jcp.25470
  • Further Support for ECM Control of Receptor Trafficking and Signaling
    • Authors: Lindsay Clegg; Feilim Mac Gabhann
      Abstract: Recently, Sack et al. presented an interesting, novel data set in JCP examining the effect of substrate stiffness on VEGF processing and signaling. The data represents a clear contribution to the field. However, the authors' conclusion that “extracellular matrix binding is essential for VEGF internalization” conflicts with other knowledge in the field, and is not supported by their data. Instead, their data demonstrates the effect of heparin addition and changing ECM stiffness on both VEGF binding to fibronectin and VEGF binding to endothelial receptors. This is consistent with other work showing that matrix‐ binding reduces VEGF‐VEGFR internalization, shifting downstream signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-20T09:45:37.567799-05:
      DOI: 10.1002/jcp.25464
  • Gene and microRNA Expression Are Predictive of Tumor Response in Rectal
           Adenocarcinoma Patients Treated with Preoperative Chemoradiotherapy
    • Authors: Caterina Millino; Isacco Maretto, Beniamina Pacchioni, Maura Digito, Antonino De Paoli, Vincenzo Canzonieri, Edoardo D'Angelo, Marco Agostini, Flavio Rizzolio, Antonio Giordano, Andrea Barina, Senthilkumar Rajendran, Giovanni Esposito, Gerolamo Lanfranchi, Donato Nitti, Salvatore Pucciarelli
      Abstract: Preoperative chemoradiotherapy (pCRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). However, tumor response to pCRT is not uniform, and there are no effective predictive methods. This study investigated whether specific gene and miRNA expression are associated with tumor response to pCRT.Tissue biopsies were obtained from patients before pCRT and resection. Gene and miRNA expression were analyzed using a one-color microarray technique that compares signatures between responders (R) and non-responders (NR), as measured based on tumor regression grade.Two groups composed of 38 “exploration cohort” and 21 “validation cohort” LARC patients were considered for a total of 32 NR and 27 R patients. In the first cohort, using SAM Two Class analysis, 256 genes and 29 miRNAs that were differentially expressed between the NR and R patients were identified. The anti-correlation analysis showed that the same 8 miRNA interacted with different networks of transcripts. The miR-630 appeared only with the NR patients and was anti-correlated with a single transcript: RAB5B. After PAM, the following 8 transcripts were strong predictors of tumor response: TMEM188, ITGA2, NRG, TRAM1, BCL2L13, MYO1B, KLF7 and GTSE1. Using this gene set, an unsupervised cluster analysis was applied to the validation cohort and correctly assigned the patients to the NR or R group with 85.7% accuracy, 90% sensitivity and 82% specificity. All three parameters reached 100% when both cohorts were considered together.In conclusion, gene and miRNA expression profiles may be helpful for predicting response to pCRT in LARC patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-25T17:40:42.055244-05:
      DOI: 10.1002/jcp.25441
  • Hypoxia Modulates the Swelling‐Activated Cl Current in Human
           Glioblastoma Cells: Role in Volume Regulation and Cell Survival
    • Authors: Luigi Sforna; Marta Cenciarini, Silvia Belia, Antonio Michelucci, Mauro Pessia, Fabio Franciolini, Luigi Catacuzzeno
      Abstract: The malignancy of glioblastoma multiform (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non‐tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling‐activated Cl current (ICl,swell). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia‐activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably the hypoxia‐induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell. Since a hypoxia‐induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia‐activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia‐induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-29T03:36:23.324328-05:
      DOI: 10.1002/jcp.25393
  • Table of Contents, Editor's Choice, Highlights
    • Pages: 1 - 6
      PubDate: 2016-09-19T14:38:00.707127-05:
      DOI: 10.1002/jcp.25571
  • Stiffness of Intact Endothelial Cells from Fresh Aortic Bifurcations of
           Atherosclerotic Rabbits ‐ Atomic Force Microscopic Study
    • Authors: Kozaburo Hayashi; Michitaka Higaki
      First page: 7
      Abstract: Stiffness of intact endothelial cells (ECs) in the abdominal aorta (AA) and in the medial and lateral wall of the common iliac artery (CIA(Medial) and CIA(Lateral), respectively), which were freshly obtained from cholesterol‐fed rabbits, were measured with an atomic force microscopic indentation method. In the areas away from atherosclerotic plaques (Off‐plaque), ECs were significantly stiffer in CIA(Medial) than in the other two locations; this result was similar to that from normal diet‐fed animals. On the other hand, there were no significant differences in the stiffness of ECs located on atherosclerotic plaques (On‐plaque) among the three sites; the stiffness was equal to those in “Off‐plaque” wall of CIA(Lateral) and AA. Moreover, the stiffness of ECs covering plaques decreased with the progression of atherosclerosis. The precise quantification of the stiffness of vascular ECs would provide a better understanding of cellular remodeling and adaptation in atherosclerosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:43:15.558698-05:
      DOI: 10.1002/jcp.25379
  • Dissection of Individual Prostate Lobes in Mouse Models of Prostate Cancer
           to Obtain High Quality RNA
    • Authors: Areg Zingiryan; Nicholas H. Farina, Kristiaan H. Finstad, Janet L. Stein, Jane B. Lian, Gary S. Stein
      Pages: 14 - 18
      Abstract: Genetically engineered mouse models of prostate cancer allow for study of disease progression from localized tumor formation through distal metastasis. The anatomy of the mouse prostate differs dramatically from the human prostate, being composed of four lobe pairs (anterior, dorsal, lateral, and ventral), making the identification and dissection technically challenging. Although the entire murine prostate and surrounding tissue, including urethra, bladder, seminal vesicles, and associated adipose tissue, can be quickly dissected for en bloc analysis, it is necessary to isolate individual prostate lobes for gene expression studies elucidating the molecular mechanisms of prostate cancer. The procedure as described here includes full color images, allowing the researcher to appreciate the unique prostate morphology and tissue manipulation required to harvest individual prostate lobes. Along with removing all extraneous tissue, the procedure allows for direct comparison of the different prostate lobes by established downstream techniques. Importantly, high quality RNA required for next‐generation gene expression analysis can only consistently be obtained from ventral and lateral lobes. Finally, preclinical studies using prostate targeted therapies can be monitored specifically in individual prostate lobes for histological and gene expression studies. J. Cell. Physiol. 232: 14–18, 2017. © 2016 Wiley Periodicals, Inc.Here, we present a procedure with full color images to isolate individual murine prostate lobes, free of adjacent tissue, for use in downstream gene expression studies. All eight murine prostate lobes can be harvested within an hour and processed for histologic analysis and molecular component isolation (RNA, DNA, protein, etc.). Only the lateral and ventral prostate lobes yield non‐degraded RNA.
      PubDate: 2016-04-01T03:22:20.741891-05:
      DOI: 10.1002/jcp.25384
  • Probing Micromechanical Properties of the Extracellular Matrix of Soft
           Tissues by Atomic Force Microscopy
    • Authors: Ignasi Jorba; Juan J. Uriarte, Noelia Campillo, Ramon Farré, Daniel Navajas
      Pages: 19 - 26
      Abstract: The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy (AFM) has unique capabilities to measure ECM mechanics at the scale at which cells probe the mechanical features of their microenvironment. Moreover, AFM measurements can be readily combined with bright field and fluorescence microscopy. Performing reliable mechanical measurements with AFM requires accurate calibration of the device and correct computation of the mechanical parameters. A suitable approach to isolate ECM mechanics from cell contribution is removing the cells by means of an effective decellularization process that preserves the composition, structure and mechanical properties of the ECM. AFM measurement of ECM micromechanics provides important insights into organ biofabrication, cell‐matrix mechanical crosstalk and disease‐induced tissue stiffness alterations. J. Cell. Physiol. 232: 19–26, 2017. © 2016 Wiley Periodicals, Inc.The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy allows the measurement of ECM mechanics at the microscale at which cells probe the mechanical features of their microenvironment.
      PubDate: 2016-06-02T06:06:07.36028-05:0
      DOI: 10.1002/jcp.25420
  • Light Sheet Microscopy to Measure Protein Dynamics
    • Authors: Matthias Rieckher
      First page: 27
      Abstract: Visualizing protein dynamics is the key to a quantitative understanding of molecular mechanisms in biological systems. Recent developments in fluorescent microscopy techniques allow for novel experimental approaches to study stochastic localization, distribution and movement of fluorescently labeled molecules in high resolution as they occur over time in the context of living cells and whole organisms. Particularly suitable for such studies is the application of light sheet microscopy, as it enables rapid in vivo imaging of a wide range and size of specimen, from whole organisms and organs, down to the dynamics of subcellular structures and single molecules. This article summarizes the principles of light sheet microscopy and its advantages over other optical imaging techniques, such as light microscopy and confocal microscopy, and highlights recent innovations that significantly enhance spatio‐temporal resolution. Also, this manuscript contains basic guidelines for the implementation of light sheet microscopy in the laboratory and presents thus far unpublished light sheet microscopy applications demonstrating the ability of this technology to measure protein dynamics in a whole‐organism context. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T01:31:54.123735-05:
      DOI: 10.1002/jcp.25451
  • Animal Models of Congenital Cardiomyopathies Associated With Mutations in
           Z‐Line Proteins
    • Authors: Marie‐Louise Bang
      Pages: 38 - 52
      Abstract: The cardiac Z‐line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. The Z‐line is important for efficient force generation and transmission as well as the maintenance of structural stability and integrity. Furthermore, it is a nodal point for intracellular signaling, in particular mechanosensing and mechanotransduction. Mutations in various genes encoding Z‐line proteins have been associated with different cardiomyopathies, including dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction, and mutations even within the same gene can cause widely different pathologies. Animal models have contributed to a great advancement in the understanding of the physiological function of Z‐line proteins and the pathways leading from mutations in Z‐line proteins to cardiomyopathy, although genotype–phenotype prediction remains a great challenge. This review presents an overview of the currently available animal models for Z‐line and Z‐line associated proteins involved in human cardiomyopathies with special emphasis on knock‐in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z‐line proteins. Pros and cons of mouse models will be discussed and a future outlook will be given. J. Cell. Physiol. 232: 38–52, 2017. © 2016 Wiley Periodicals, Inc.The cardiac Z‐line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. This review presents an overview of the currently available animal models for Z‐line and Z‐line associated proteins involved in human cardiomyopathies with special emphasis on knock‐in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z‐line proteins.
      PubDate: 2016-06-02T10:06:33.346872-05:
      DOI: 10.1002/jcp.25424
  • Targeting the Breast Cancer Kinome
    • Authors: Samantha M. Miller; Daniel R. Goulet, Gary L. Johnson
      Pages: 53 - 60
      Abstract: Protein kinases are highly tractable targets for the treatment of many cancers including breast cancer, due to their essential role in tumor cell proliferation and survival. Sequencing of the breast cancer genome and transcriptome has defined breast cancer as a heterogeneous disease that is classified into five molecular subtypes: luminal A, luminal B, HER2‐enriched, basal‐like, and claudin‐low. Each subtype displays a unique expression profile of protein kinases that can be targeted by small molecule kinase inhibitors or biologics. An understanding of genomic changes, including mutations or copy number variations, for specific protein kinases and dependencies on kinases across breast cancer subtypes is allowing for a more rational design of targeted breast cancer therapies. While specific kinase inhibitors have had success in the clinic, including the CDK4/6 inhibitor palbociclib in combination with aromatase inhibitors in luminal breast cancer, patients often become resistant to treatment. An understanding of the mechanisms allowing cells to bypass targeted kinase inhibition has led to the development of combination therapies that are more durable in pre‐clinical studies. However, the heterogeneity of resistance mechanisms and rapid adaptability of the kinome through feedback regulation greatly inhibit the long‐term efficacy of combination kinase inhibitor therapies. It is becoming apparent that epigenetic inhibitors, such as HDAC and BET bromodomain inhibitors can block the transcriptional adaptability of tumor cells to kinase inhibitors and prevent the onset of resistance. Such novel combination therapies are currently showing promise in preclinical studies to markedly increase the durability of kinase inhibitors in breast cancer. J. Cell. Physiol. 232: 53–60, 2017. © 2016 Wiley Periodicals, Inc.Sequencing of the breast cancer genome and transcriptome has identified specific protein kinases as promising drug targets across the five molecular subtypes. While some kinase inhibitors have had success in the clinic, resistance to treatment often occurs. An understanding of the molecular mechanisms of resistance has allowed for the design of combination therapies which have the potential to block resistance in the clinic.
      PubDate: 2016-05-31T07:45:26.494401-05:
      DOI: 10.1002/jcp.25427
  • Browning of White Fat: Novel Insight into Factors, Mechanisms and
    • Authors: Nevena Jeremic; Pankaj Chatuverdi, Suresh C. Tyagi
      First page: 61
      Abstract: What is more interesting about brown adipose tissue (BAT) is its ability to provide thermogenesis, protection against obesity by clearing triglycerides and mitigating insulin resistance. White adipose tissue (WAT) on the other hand stores excess energy and secretes some endocrine factors like leptin for regulating satiety. For the last decade there has been an increasing interest in the browning of fat keeping in view its beneficial effects on metabolic disorders and protection in the form of perivascular fat. Obesity is one such metabolic disorder that leads to significant morbidity and mortality from obesity‐related disorders such as type 2 diabetes mellitus (T2D) and cardiovascular disease risk. Browning of white fat paves the way to restrict obesity and obesity related disorders. Although exercise has been the most common factor for fat browning, however there are other factors that involve: 1) beta aminoisobutyric acid (BAIBA); 2) gamma amino butyric acid (GABA); 3) PPARɣ agonists; 4) JAK inhibition; 5) IRISIN. In this review we propose two novel factors musclin and TFAM for fat browning. Musclin a myokine released from muscles during exercise activates PPARɣ which induces browning of WAT that has beneficial metabolic and cardiac effects. TFAM is a transcription factor that induces mitochondrial biogenesis. Since BAT is rich in mitochondria, higher expression of TFAM in WAT or TFAM treatment in WAT cells can induce browning of WAT. We propose that fat browning can be used as a therapeutic tool for metabolic disorders and cardiovascular diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:23:39.343408-05:
      DOI: 10.1002/jcp.25450
  • Multifaceted Breast Cancer: The Molecular Connection with Obesity
    • Authors: Feola Antonia; Ricci Serena, Kouidhi Soumaya, Rizzo Antonietta, Penon Antonella, Formisano Pietro, Giordano Antonio, Di Carlo Angelina, Di Domenico Marina
      First page: 69
      Abstract: Obesity is characterized by a disruption in energy balance regulation that results in an excess accumulation of body fat. Its increasing prevalence poses a major public health concern because it is a risk factor for a host of additional chronic conditions, including type 2 diabetes, hypertension, and cardiovascular disease. Obesity is increasingly recognized as a growing cause of cancer risk. In particular excessive adipose expansion during obesity causes adipose dysfunction and inflammation that can regulate tumor growth. In obesity, dysregulated systemic metabolism and inflammation induce hyperinsulinemia, hyperglycemia, dyslipidemia and enhance sex hormone production with increased secretion of proinflammatory adipokine that impact breast cancer development and progression. This review describes how adipose inflammation that characterizes obesity is responsible of microenvironment to promote cancer, and discuss how steroid hormones, that are essential for the maintenance of the normal development, growth and differentiation of the cells, influence the induction and progression of breast cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-01T05:36:16.572172-05:
      DOI: 10.1002/jcp.25475
  • Cartilage‐specific and Cre‐dependent Nkx3.2 overexpression in vivo
           causes skeletal dwarfism by delaying cartilage hypertrophy
    • Authors: Da‐Un Jeong; Je‐Yong Choi, Dae‐Won Kim
      First page: 78
      Abstract: Nkx3.2, the vertebrate homologue of Drosophila bagpipe, has been implicated as playing a role in chondrogenic differentiation. In brief, Nkx3.2 is initially expressed in chondrocyte precursor cells and, later during cartilage maturation, its expression is diminished in hypertrophic chondrocytes. In addition to Nkx3.2 expression analyses, previous studies using ex vivo chick embryo cultures and in vitro cell cultures have suggested that Nkx3.2 can suppress chondrocyte hypertrophy. However, it has never been demonstrated that Nkx3.2 functions in regulating chondrocyte hypertrophy during cartilage development in vivo. Here, we show that cartilage‐ specific and Cre‐dependent Nkx3.2 overexpression in mice results in significant postnatal dwarfism in endochondral skeletons, while intramembranous bones remain unaltered. Further, we observed significant delays in cartilage hypertrophy in conditional transgenic ciTg‐Nkx3.2 mice. Together, these findings confirm that Nkx3.2 is capable of controlling hypertrophic maturation of cartilage in vivo, and this regulation plays a significant role in endochondral ossification and longitudinal bone growth. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T12:28:17.938636-05:
      DOI: 10.1002/jcp.25446
  • Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in
           Murine Primary Splenocytes
    • Authors: Aurore Collin; Audrey Noacco, Jérémie Talvas, Florence Caldefie‐Chézet, Marie‐Paule Vasson, Marie‐Chantal Farges
      Pages: 101 - 109
      Abstract: Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation, and survival. Leptin receptor is integrated inside cholesterol‐rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl‐β‐cyclodextrin (β‐MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/ml). Unlike β‐MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. J. Cell. Physiol. 232: 101–109, 2017. © 2016 Wiley Periodicals, Inc.This article suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling.
      PubDate: 2016-04-07T03:41:38.328614-05:
      DOI: 10.1002/jcp.25394
  • Cannabinoid Receptor 1 Mediates Homing of Bone Marrow‐Derived
           Mesenchymal Stem Cells Triggered by Chronic Liver Injury
    • Authors: Lin Wang; Le Yang, Lei Tian, Ping Mai, Shuangshuang Jia, Lin Yang, Liying Li
      Pages: 110 - 121
      Abstract: Cannabinoid receptors (CBs) have been implicated in the pathogenesis of various liver diseases, including liver fibrosis. Our previous studies have demonstrated that after liver injury, mouse bone marrow‐derived mesenchymal stem cells (BMSCs) can migrate to the injured liver and differentiate to myofibroblasts, contributing to hepatic fibrogenesis. However, the role of CBs in the homing of BMSCs in liver injury is yet unclear. In this study, we found that both CB1 and CB2 were expressed in BMSCs. Migration assays were performed by transwell chambers. CB1 agonist ACEA promoted the migration of BMSCs, but CB2 agonist JWH133 had no effect. Pharmacological or genetic ablation of CB1 reduced ACEA‐induced migration, whereas CB2 did not. Moreover, activation of CB1 increased active GTP‐bound Rac1, RhoA, and Cdc42 protein levels. The elevated GTP‐bound Rac1 and RhoA protein levels were decreased by CB1 antagonist AM281 treatment, but not Cdc42. In addition, ACEA‐induced migration was suppressed by NSC23766 (Rac1 inhibitor) or C3 transferase (RhoA inhibitor), whereas MLS‐573151 (Cdc42 inhibitor) had no effect. Consistent with these data, Rac1 or RhoA knock‐down significantly blocked CB1‐mediated migration. Meanwhile, CB1‐mediated migration was associated with cytoskeletal remodeling. In vivo, administration of CB1 antagonist AM281 markedly inhibited the recruitment of BMSCs to the injured liver using fluorescence‐activated cell sorting. Furthermore, blockade of CB1 significantly attenuated liver fibrosis. In conclusion, our results suggest that CB1 plays a crucial role in liver fibrosis through mediating the homing of BMSCs to damaged liver, which may provide new insight into the pathogenesis and treatment of liver fibrosis. J. Cell. Physiol. 232: 110–121, 2017. © 2016 Wiley Periodicals, Inc.The present study aims to evaluate the impact of Cannabinoid receptors (CBs) on the migration of BMSCs in liver injury and investigate the mechanisms involved. We found that BMSCs expressed CB1 and CB2. CB1 rather than CB2 mediated the migration of BMSCs via the Rac1 and RhoA pathways. And CB1‐mediated migration was associated with cytoskeletal remodeling. Furthermore, pharmacological blockade of CB1 markedly reduced the recruitment of BMSCs to injured liver and attenuated liver fibrosis. These results may contribute to the better understanding of the involvement of CB1 in liver injury and open new perspectives for development of potential therapeutics for liver disease.
      PubDate: 2016-04-08T00:12:05.373199-05:
      DOI: 10.1002/jcp.25395
  • MG132 Induces Expression of Monocyte Chemotactic Protein‐Induced Protein
           1 in Vascular Smooth Muscle Cells
    • Authors: Xi Tan; Jie Gao, Zhan Shi, Shi Tai, Leona Loretta Chan, Yang Yang, Dao‐Quan Peng, Duan‐Fang Liao, Zhi‐Sheng Jiang, Ying‐Zi Chang, Yu Gui, Xi‐Long Zheng
      Pages: 122 - 128
      Abstract: Monocyte chemoattractant protein‐1 (MCP‐1) has been reported to induce the expression of monocyte chemotactic protein‐induced protein 1 (MCPIP1), which undergoes ubiquitination degradation. Therefore, we predict that in vascular smooth muscle (VSMCs), MCPIP1 may be induced by MCP‐1 and undergo degradation, which can be inhibited by the proteasome inhibitor, MG132. Our results showed that treatment of human VSMCs with MCP‐1 did not increase the expression of MCPIP1. Treatment with MG132, however, elevated MCPIP1 protein levels through stimulation of the gene transcription, but not through increasing protein stability. MCPIP1 expression induced by MG132 was inhibited by α‐amanitin inhibition of gene transcription or cycloheximide inhibition of protein synthesis. Our further studies showed that MCPIP1 expression induced by MG132 was inhibited by the inhibitors of AKT and p38 kinase, suggesting a role of the AKT‐p38 pathway in MG132 effects. We also found that treatment with MG132 induces apoptosis, but overexpression of MCPIP1 inhibited bromodeoxyuridine (BrdU) incorporation of human VSMCs without induction of significant apoptosis. In summary, MCPIP1 expression is induced by MG132 likely through activation of the AKT‐p38 pathway. MCPIP1 inhibits SMC proliferation without induction of apoptosis. J. Cell. Physiol. 232: 122–128, 2017. © 2016 Wiley Periodicals, Inc.Monocyte chemoattractant protein‐1 (MCP‐1) has been reported to induce the expression of monocyte chemotactic protein‐induced protein 1 (MCPIP1), which undergoes ubiquitination degradation. In cultured human vascular smooth muscle, MCPIP1 expression is induced by MG132, a proteasome inhibitor, through signal transduction and gene transcription. Overexpression of MCPIP1 results in inhibition of smooth muscle cell proliferation.
      PubDate: 2016-04-14T02:00:50.27102-05:0
      DOI: 10.1002/jcp.25396
  • SRC Family Kinase Inhibition in Ewing Sarcoma Cells Induces p38 MAP
           Kinase‐Mediated Cytotoxicity and Reduces Cell Migration
    • Authors: Paola Indovina; Nadia Casini, Iris Maria Forte, Tiziana Garofano, Daniele Cesari, Carmelina Antonella Iannuzzi, Leonardo Del Porro, Francesca Pentimalli, Luca Napoliello, Silvia Boffo, Silvia Schenone, Maurizio Botta, Antonio Giordano
      Pages: 129 - 135
      Abstract: Ewing sarcoma (ES) is a highly aggressive bone and soft tissue cancer, representing the second most common primary malignant bone tumor in children and adolescents. Although the development of a multimodal therapy, including both local control (surgery and/or radiation) and systemic multidrug chemotherapy, has determined a significant improvement in survival, patients with metastatic and recurrent disease still face a poor prognosis. Moreover, considering that ES primarily affects young patients, there are concerns about long‐term adverse effects of the therapy. Therefore, more rational strategies, targeting specific molecular alterations underlying ES, are required. Recent studies suggest that SRC family kinases (SFKs), which are aberrantly activated in most cancer types, could represent key therapeutic targets also for ES. Here, we challenged ES cell lines with a recently developed selective SFK inhibitor (a pyrazolo[3,4‐d]pyrimidine derivative, called SI221), which was previously shown to be a valuable proapoptotic agent in other tumor types while not affecting normal cells. We observed that SI221 significantly reduced ES cell viability and proved to be more effective than the well‐known SFK inhibitor PP2. SI221 was able to induce apoptosis in ES cells and also reduced ES cell clonogenic potential. Furthermore, SI221 was also able to reduce ES cell migration. At the molecular level, our data suggest that SFK inhibition through SI221 could reduce ES cell viability at least in part by hindering an SFK‐NOTCH1 receptor‐p38 mitogen‐activated protein kinase (MAPK) axis. Overall, our study suggests a potential application of specific SFK inhibition in ES therapy. J. Cell. Physiol. 232: 129–135, 2017. © 2016 Wiley Periodicals, Inc.In this study, we challenged Ewing sarcoma (ES) cell lines with SI221, a recently developed selective SRC family kinase (SFK) inhibitor. We observed that this compound significantly reduced ES cell viability, clonogenic potential and migration. At the molecular level, our data suggest that SI221 could reduce ES cell viability at least in part by hindering an SFK‐NOTCH1 receptor‐p38 mitogen‐activated protein kinase (MAPK) axis.
      PubDate: 2016-04-19T03:41:36.652493-05:
      DOI: 10.1002/jcp.25397
  • Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and
           Epithelial Factors
    • Authors: Nicolas Tobar; Omar Porras, Patricio C. Smith, L. Felipe Barros, Jorge Martínez
      Pages: 136 - 144
      Abstract: Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long‐term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre‐culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co‐culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. J. Cell. Physiol. 232: 136–144, 2017. © 2016 Wiley Periodicals, Inc.In a human stromal mammary cell line, glucose pre‐culture and soluble epithelial factors constitute a stimulus for lactate production. In a co‐culture setting, a vectorial flux of lactate from stroma to epithelial cells were stablished. The uptake of lactate by epithelial cells correlates with the degree of malignancy.
      PubDate: 2016-04-28T02:20:45.897269-05:
      DOI: 10.1002/jcp.25398
  • Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of
           Osteoblast Growth and Differentiation
    • Authors: Eri Morimoto; Meng Li, Aysha B. Khalid, Susan A. Krum, Nyam‐Osor Chimge, Baruch Frenkel
      Pages: 145 - 153
      Abstract: Inhibition of Runx2 is one of many mechanisms that suppress bone formation in glucocorticoid (GC)‐induced osteoporosis (GIO). We profiled mRNA expression in ST2/Rx2dox cells after treatment with doxycycline (dox; to induce Runx2) and/or the synthetic GC dexamethasone (dex). As expected, dex typically antagonized Runx2‐driven transcription. Select genes, however, were synergistic stimulated and this was confirmed by RT‐qPCR. Among the genes synergistically stimulated by GCs and Runx2 was Wnt inhibitory Factor 1 (Wif1), and Wif1 protein was readily detectable in medium conditioned by cultures co‐treated with dox and dex, but neither alone. Cooperation between Runx2 and GCs in stimulating Wif1 was also observed in primary preosteoblast cultures. GCs strongly inhibited dox‐driven alkaline phosphatase (ALP) activity in control ST2/Rx2dox cells, but not in cells in which Wif1 was silenced. Unlike its anti‐mitogenic activity in committed osteoblasts, induction of Runx2 transiently increased the percentage of cells in S‐phase and accelerated proliferation in the ST2 mesenchymal pluripotent cell culture model. Furthermore, like the inhibition of Runx2‐driven ALP activity, dex antagonized the transient mitogenic effect of Runx2 in ST2/Rx2dox cultures, and this inhibition eased upon Wif1 silencing. Plausibly, homeostatic feedback loops that rely on Runx2 activation to compensate for bone loss in GIO are thwarted, exacerbating disease progression through stimulation of Wif1. J. Cell. Physiol. 232: 145–153, 2017. © 2016 Wiley Periodicals, Inc.The work by Morimoto et al. demonstrates target gene‐dependent outcomes of the interaction between Runx2 and glucocorticoids (GCs) in preosteoblasts. While Runx2‐driven gene expression is generally antagonized by GCs, a few genes are synergistically stimulated by Runx2 and GCs. One of them, Wif1, potentially thwarts a homeostatic feedback loop and exacerbates GC‐induced osteoporosis.
      PubDate: 2016-04-26T03:35:45.489811-05:
      DOI: 10.1002/jcp.25399
  • Differentiation of Keratinocytes Modulates Expression of Epidermal
           Elements of the Cutaneous Analog of Hypothalamus‐Pituitary‐Adrenal
    • Authors: Justyna M. Wierzbicka; Michał A. Żmijewski, Jakub Antoniewicz, Michal Sobjanek, Andrzej T. Slominski
      First page: 154
      Abstract: It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus‐pituitary‐adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA).Expression of CRF, UCN1‐3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies.Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from Day 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium.Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-06T17:02:14.046921-05:
      DOI: 10.1002/jcp.25400
  • Metabolic Adaptation of the Small Intestine to Short‐ and Medium‐Term
           High‐Fat Diet Exposure
    • Authors: Rosmarie Clara; Manuel Schumacher, Deepti Ramachandran, Shahana Fedele, Jean‐Philippe Krieger, Wolfgang Langhans, Abdelhak Mansouri
      Pages: 167 - 175
      Abstract: The small intestine is the main organ involved in the digestion and absorption of nutrients. It is in an ideal position to sense the availability of energy in the lumen in addition to its absorptive function. Consumption of a high‐fat diet (HFD) influences the metabolic characteristics of the small intestine. Therefore, to better understand the metabolic features of the small intestine and their changes in response to dietary fat, we characterized the metabolism of duodenal, jejunal, and hepatic cell lines and assessed the metabolic changes in the enterocytes and the liver after short‐term (3 days) or medium‐term (14 days) HFD feeding in mice. Experiments with immortalized enterocytes indicated a higher glycolytic capacity in the duodenal cell line compared to the other two cell lines, whereas the jejunal cell line exhibited a high oxidative metabolism. Short‐term HFD feeding induced changes in the expression of glucose and lipid metabolism‐related genes in the duodenum and the jejunum of mice, but not in the liver. When focusing on fatty acid oxidation both, short‐ and medium‐term HFD feeding induced an upregulation of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A, the key enzyme of ketogenesis, at the protein level in the intestinal epithelial cells, but not in the liver. These results suggest that HFD feeding induces an early adaptation of the small intestine rather than the liver in response to a substantial fat load. This highlights the importance of the small intestine in the adaptation of the body to the metabolic changes induced by HFD exposure. J. Cell. Physiol. 232: 167–175, 2017. © 2016 Wiley Periodicals, Inc.Characterization of intestinal cell lines from the proximal and distal part of the small intestine, showed a differential metabolism in response to metabolic modulators. Short‐ and medium‐term access to high‐fat‐diet induced the expression of HMG‐CoAS2, the ketogenesis key‐enzyme, specifically in the jejunum but not in the duodenum or in the liver of mice.
      PubDate: 2016-04-28T02:21:10.59553-05:0
      DOI: 10.1002/jcp.25402
  • Antibodies Against Mimotopes of Simian Virus 40 Large T Antigen, the
           Oncoprotein, in Serum Samples From Elderly Healthy Subjects
    • Authors: Elisa Mazzoni; Giovanni Guerra, Maria Vittoria Casali, Silvia Pietrobon, Ilaria Bononi, Andrea Puozzo, Andrea Tagliapietra, Pier Francesco Nocini, Mauro Tognon, Fernanda Martini
      Pages: 176 - 181
      Abstract: Simian Virus 40 (SV40), a monkey polyomavirus, was administered to human populations by early anti‐poliomylitis vaccines contaminated by this small DNA tumor virus. Data on SV40 infection in humans remain controversial. Elderly subjects represent an interesting cohort to investigate, because they were not immunized with SV40‐contaminated vaccines. Taking advantage of the Italian population, the second oldest worldwide, elderly subjects (n = 237) up to 100 years old were enrolled in this study. Their sera were analyzed, by ELISA tests with synthetic peptides mimicking the viral epitopes, for IgG antibodies reacting with SV40 large Tumor antigen (Tag), the viral oncoprotein. An overall seroprevalence of 22% was revealed in subjects aged 66–100 years, ranging from 19% in individuals 66–74 years old, to 24% in subjects 82–100 years old, with a lower SV40 titer detected in the oldest group. Our data show that: (i) SV40 infection is not frequent in old individuals; (ii) the infection rate increases in elderly with the age; (iii) the antibody titer of SV40 Tag decreases with the age. In conclusion, SV40 infection seems to spread in old subjects independently from SV40‐contaminated vaccines. This study seems to confirm that SV40 is also a human virus. J. Cell. Physiol. 232: 176–181, 2017. © 2016 Wiley Periodicals, Inc.Specific antibodies reacting against Simian Virus 40 large T antigen mimotopes were detected in serum samples from elderly healthy subjects, up to 100 years old, with a prevalence of 22%. Immunological data were obtained with a novel indirect ELISA with synthetic peptides, which mimic the immunogenic epitopes of the viral oncoprotein of SV40. Our data suggests that the exposure to Simian Virus 40 infection is not frequent in old individuals. The immunological results showed that (i) the infection rate increases in elderly with the age; (ii) the proportion of elderly subjects with high Simian Virus 40 large T antigen antibody levels decreases with age; (iii) Simian Virus 40 infection spreads in old subjects independently from SV40‐contaminated vaccines; (iv) this monkey polyomavirus is also a viral agent in humans.
      PubDate: 2016-04-29T05:20:52.781399-05:
      DOI: 10.1002/jcp.25405
  • Runx2/DICER/miRNA Pathway in Regulating Osteogenesis
    • Authors: Leilei Zheng; Qisheng Tu, Shu Meng, Lan Zhang, Liming Yu, Jinlin Song, Yun Hu, Lei Sui, Jin Zhang, Michel Dard, Jessica Cheng, Dana Murray, Yin Tang, Jane B. Lian, Gary S. Stein, Jake Chen
      Pages: 182 - 191
      Abstract: DICER is the central enzyme that cleaves precursor microRNAs (miRNAs) into 21–25 nucleotide duplex in cell lineage differentiation, identity, and survival. In the current study, we characterized the specific bone metabolism genes and corresponding miRNAs and found that DICER and Runt‐related transcription factor 2 (Runx2) expressions increased simultaneously during osteogenic differentiation. Luciferase assay showed that Runx2 significantly increased the expression levels of DICER luciferase promoter reporter. Our analysis also revealed weaker DICER expression in embryos of Runx2 knock out mice (Runx2 −/−) compared with that of Runx2 +/− and Runx2 +/+ mice. We further established the calvarial bone critical‐size defect (CSD) mouse model. The bone marrow stromal cells (BMSCs) transfected with siRNA targeting DICER were combined with silk scaffolds and transplanted into calvarial bone CSDs. Five weeks post‐surgery, micro‐CT analysis revealed impaired bone formation, and repairing in calvarial defects with the siRNA targeting DICER group. In conclusion, our results suggest that DICER is specifically regulated by osteogenic master gene Runx2 that binds to the DICER promoter. Consequently, DICER cleaves precursors of miR‐335‐5p and miR‐17‐92 cluster to form mature miRNAs, which target and decrease the Dickkopf‐related protein 1 (DKK1), and proapoptotic factor BIM levels, respectively, leading to an enhanced Wnt/β‐catenin signaling pathway. These intriguing results reveal a central mechanism underlying lineage‐specific regulation by a Runx2/DICER/miRNAs cascade during osteogenic differentiation and bone development. Our study, also suggests a potential application of modulating DICER expression for bone tissue repair and regeneration. J. Cell. Physiol. 232: 182–191, 2017. © 2016 Wiley Periodicals, Inc.These intriguing results reveal a central mechanism underlying lineage‐specific regulation by a Runx2/DICER/miRNAs cascade during osteogenic differentiation and bone development. This cell‐ and development‐specific regulation is essential and mandatory for the initiation and progression of osteogenic differentiation. Furthermore, our study also suggests a potential application of specifically regulating DICER for bone tissue repair and regeneration.
      PubDate: 2016-04-26T03:36:35.324038-05:
      DOI: 10.1002/jcp.25406
  • Low‐Dose Farnesyltransferase Inhibitor Suppresses HIF‐1α and Snail
           Expression in Triple‐Negative Breast Cancer MDA‐MB‐231 Cells In
    • Authors: Tomokazu Tanaka; Yuichi Ikegami, Harumasa Nakazawa, Naohide Kuriyama, Miwa Oki, Jun‐ichi Hanai, Vikas P. Sukhatme, Masao Kaneki
      First page: 192
      Abstract: The aggressiveness of triple‐negative breast cancer (TNBC), which lacks estrogen receptor, progesterone receptor and epidermal growth factor receptor 2 (HER2), represents a major challenge in breast cancer. Migratory and self‐renewal capabilities are integral components of invasion, metastasis and recurrence of TNBC. Elevated hypoxia‐inducible factor‐1α (HIF‐1α) expression is associated with aggressiveness of cancer. Nonetheless, how HIF‐1α expression is regulated and how HIF‐1α induces aggressive phenotype are not completely understood in TNBC. The cytotoxic effects of farnesyltransferase (FTase) inhibitors (FTIs) have been studied in cancer and leukemia cells. In contrast, the effect of FTIs on HIF‐1α expression has not yet been studied. Here, we show that clinically relevant low‐dose FTI, tipifarnib (300 nM), decreased HIF‐1α expression, migration and tumorsphere formation in human MDA‐MB‐231 TNBC cells under a normoxic condition. In contrast, the low‐dose FTIs did not inhibit cell growth and activity of the Ras pathway in MDA‐MB 231 cells. Tipifarnib‐induced decrease in HIF‐1α expression was associated with amelioration of the Warburg effect, hypermetabolic state, increases in Snail expression and ATP release, and suppressed E‐cadherin expression, major contributors to invasion, metastasis and recurrence of TBNC. These data suggest that FTIs may be capable of ameliorating the aggressive phenotype of TNBC by suppressing the HIF‐1α‐Snail pathway. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:28.989913-05:
      DOI: 10.1002/jcp.25411
  • Prostaglandin E2‐Dependent Phosphorylation of RAS Inhibition 1 (RIN1) at
           Ser 291 and 292 Inhibits Transforming Growth Factor‐β‐Induced RAS
           Activation Pathway in Human Synovial Fibroblasts: Role in Cell Migration
    • Authors: Casimiro Gerarduzzi; QingWen He, Beibei Zhai, John Antoniou, John A. Di Battista
      Pages: 202 - 215
      Abstract: Prostaglandin E2 (PGE2)‐stimulated G‐protein–coupled receptor (GPCR) activation inhibits pro‐fibrotic TGFβ‐dependent stimulation of human fibroblast to myofibroblast transition (FMT), though the precise molecular mechanisms are not fully understood. In the present study, we describe the PGE2‐dependent suppression and reversal of TGFβ‐induced events such as α‐sma expression, stress fiber formation, and Ras/Raf/ERK/MAPK pathway‐dependent activation of myofibroblast migration. In order to elucidate post‐ligand‐receptor signaling pathways, we identified a predominant PKA phosphorylation motif profile in human primary fibroblasts after treatment with exogenous PGE2 (EC50 30 nM, Vmax 100 nM), mimicked by the adenyl cyclase activator forskolin (EC50 5 μM, Vmax 10 μM). We used a global phosphoproteomic approach to identify a 2.5‐fold difference in PGE2‐induced phosphorylation of proteins containing the PKA motif. Deducing the signaling pathway of our migration data, we identified Ras inhibitor 1 (RIN1) as a substrate, whereby PGE2 induced its phosphorylation at Ser291 and at Ser292 by a 5.4‐ and 4.8‐fold increase, respectively. In a series of transient and stable over expression studies in HEK293T and HeLa cells using wild‐type (wt) and mutant RIN1 (Ser291/292Ala) or Ras constructs and siRNA knock‐down experiments, we showed that PGE2‐dependent phosphorylation of RIN1 resulted in the abrogation of TGFβ‐induced Ras/Raf signaling activation and subsequent downstream blockade of cellular migration, emphasizing the importance of such phosphosites in PGE2 suppression of wound closure. Overexpression experiments in tandem with pull‐down assays indicated that specific Ser291/292 phosphorylation of RIN1 favored binding to activated Ras. In principal, understanding PGE2‐GPCR activated signaling pathways mitigating TGFβ‐induced fibrosis may lead to more evidence‐based treatments against the disease. J. Cell. Physiol. 232: 202–215, 2017. © 2016 Wiley Periodicals, Inc.Background: PGE2 is an antifibrotic agent with a limited molecular mechanism of action against myofibroblasts. Results: PGE2 induces the phosphorylation of RIN1 at Ser291/292 to inhibit the TGFβ‐activated Ras/Raf pathway of myofibroblast migration. Conclusion: An antifibrotic mechanism of PGE2 blocks myofibroblast migration through RIN1. Significance: Understanding the endogenous mechanisms of antifibrosis could be extrapolated for therapeutic means.
      PubDate: 2016-05-26T08:46:51.819046-05:
      DOI: 10.1002/jcp.25412
  • Targeted Modification of Mitochondrial ROS Production Converts High
           Glucose‐Induced Cytotoxicity to Cytoprotection: Effects on Anesthetic
    • Authors: Filip Sedlic; Maria Muravyeva, Ana Sepac, Marija Sedlic, Anna Marie Williams, Meiying Yang, Xiaowen Bai, Zeljko J. Bosnjak
      First page: 216
      Abstract: Contradictory reports on the effects of diabetes and hyperglycemia on myocardial infarction range from cytotoxicity to cytoprotection. The study was designed to investigate acute effects of high glucose‐driven changes in mitochondrial metabolism and osmolarity on adaptive mechanisms and resistance to oxidative stress of isolated rat cardiomyocytes.We examined the effects of high glucose on several parameters of mitochondrial bioenergetics, including changes in oxygen consumption, mitochondrial membrane potential and NAD(P)H fluorometry. Effects of high glucose on the endogenous cytoprotective mechanisms elicited by anesthetic preconditioning (APC) and the mediators of cell injury were also tested. These experiments included real‐time measurements of reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) opening in single cells by laser scanning fluorescence confocal microscopy, and cell survival assay.High glucose rapidly enhanced mitochondrial energy metabolism, observed by increase in NAD(P)H fluorescence intensity, oxygen consumption and mitochondrial membrane potential. This substantially elevated production of ROS, accelerated opening of the mPTP and decreased survival of cells exposed to oxidative stress. Abrogation of high glucose‐induced mitochondrial hyperpolarization with 2,4 dinitrophenol (DNP) significantly, but not completely, attenuated ROS production to a level similar to hyperosmotic mannitol control. DNP treatment reversed high glucose‐induced cytotoxicity to cytoprotection. Hyperosmotic mannitol treatment also induced cytoprotection. High glucose abrogated APC‐induced mitochondrial depolarization, delay in mPTP opening and cytoprotection.In conclusion, high glucose‐induced mitochondrial hyperpolarization abolishes APC and augments cell injury. Attenuation of high glucose‐induced ROS production by eliminating mitochondrial hyperpolarization protects cardiomyocytes. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-02T13:22:40.120836-05:
      DOI: 10.1002/jcp.25413
  • Milk‐Derived Nanoparticle Fraction Promotes the Formation of Small
           Osteoclasts But Reduces Bone Resorption
    • Authors: Marina C. Oliveira; Irene Di Ceglie, Onno J. Arntz, Wim B. van den Berg, Frank H.J. van den Hoogen, Adaliene V.M. Ferreira, Peter L.E.M. van Lent, Fons A.J. van de Loo
      Pages: 225 - 233
      Abstract: The general consensus is that milk promotes bone growth and density because is a source of calcium and contains components that enhance intestinal calcium uptake or directly affect bone metabolism. In this study, we investigated the effect of bovine‐derived milk 100,000 g pellet (P100), which contains nanoparticles (
      PubDate: 2016-06-02T05:51:51.12926-05:0
      DOI: 10.1002/jcp.25414
  • Erratum: ATP‐Sensitive Potassium Channel: A Novel Target for Protection
           Against UV‐Induced Human Skin Cell Damage by Cong Cao, Sarah Healey,
           Ashley Amaral, Avery Lee‐Couture, Shu Wan, Nicola Kouttab, Wenming Chu
           and Yinsheng Wan
    • First page: 236
      PubDate: 2016-07-14T10:30:44.777224-05:
      DOI: 10.1002/jcp.25480
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