for Journals by Title or ISSN
for Articles by Keywords

Publisher: John Wiley and Sons   (Total: 1598 journals)

 A  B  C  D  E  F  G  H  I  J  K  L  M  N  O  P  Q  R  S  T  U  V  W  X  Y  Z  

The end of the list has been reached or no journals were found for your choice.
Journal Cover Journal of Cellular Physiology
  [SJR: 1.948]   [H-I: 125]   [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  [1598 journals]
  • 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
  • Cover Image, Volume 231, Number 10, October 2016
    • Abstract: Cover: The cover image, by Sudha K. Shenoy et al., is based on the Mini‐Review Ubiquitin‐Related Roles of β‐Arrestins in Endocytic Trafficking and Signal Transduction,
      DOI : 10.1002/jcp.25317.
      PubDate: 2016-06-17T09:29:13.804455-05:
  • DGKζ Downregulation Enhances Osteoclast Differentiation and Bone
           Resorption Activity Under Inflammatory Conditions
    • Authors: Kiyoshi Iwazaki; Toshiaki Tanaka, Yasukazu Hozumi, Masashi Okada, Rieko Tsuchiya, Ken Iseki, Matthew K. Topham, Kaneyuki Kawamae, Michiaki Takagi, Kaoru Goto
      Abstract: Bone homeostasis is maintained by a balance between resorption of the bone matrix and its replacement by new bone. Osteoclasts play a crucially important role in bone metabolism. They are responsible for bone resorption under pathophysiological conditions. Differentiation of these cells, which are derived from bone marrow cells, depends on receptor activator of NF‐κB ligand (RANKL). RANKL‐induced osteoclastogenesis is regulated by the phosphoinositide (PI) signaling pathway, in which diacylglycerol (DG) serves as a second messenger in signal transduction. In this study, we examined the functional implications of DG kinase (DGK), an enzyme family responsible for DG metabolism, for osteoclast differentiation and activity. Of DGKs, DGKζ is most abundantly expressed in osteoclast precursors such as bone marrow‐derived monocytes/macrophages. During osteoclast differentiation from precursor cells, DGKζ is downregulated at the protein level. In this regard, we found that DGKζ deletion enhances osteoclast differentiation and bone resorption activity under inflammatory conditions in an animal model of osteolysis. Furthermore, DGKζ deficiency upregulates RANKL expression in response to TNFα stimulation. Collectively, results suggest that DGKζ is silent under normal conditions, but it serves as a negative regulator in osteoclast function under inflammatory conditions. Downregulation of DGKζ might be one factor predisposing a person to osteolytic bone destruction in pathological conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-17T05:50:26.946779-05:
      DOI: 10.1002/jcp.25461
  • Pancreatic Endoderm‐Derived from Diabetic Patient‐Specific
           Induced Pluripotent Stem Cell Generates Glucose‐Responsive
           Insulin‐Secreting Cells
    • Authors: Bahareh Rajaei; Mehdi Shamsara, Masume Fakhr Taha, Mohammad Massumi, Mohammad Hossein Sanati
      Abstract: Human induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient‐ and disease‐specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin‐producing pancreatic β‐cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five‐step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose‐responsive insulin‐producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut‐tube endoderm, posterior foregut, pancreatic endoderm and endocrine precursor. Each stage of differentiation were characterized by stage‐specific markers. The produced cells exhibited many properties of functional β‐cells, including expression of critical β‐cells transcription factors, the potency to secrete C‐peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin‐secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs‐derived glucose‐responsive insulin‐secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-16T06:20:33.930205-05:
      DOI: 10.1002/jcp.25459
  • FGF23 Neutralizing Antibody Ameliorates Hypophosphatemia and Impaired FGF
           Receptor Signaling in Kidneys of HMWFGF2 Transgenic Mice
    • Authors: E. Du; L. Xiao, M. M. Hurley
      Abstract: High molecular weight FGF2 transgenic mice (HMWTg) phenocopy the Hyp mouse, homolog of human X‐linked hypophosphatemic rickets with phosphate wasting and abnormal fibroblast growth factor (FGF23), fibroblast growth factor receptor (FGFR), Klotho and mitogen activated protein kinases (MAPK) signaling in kidney. In this study, we assessed whether short term (24 hour) in vivo administration of FGF23 neutralizing antibody (FGF23Ab) could rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg male mice. Bone mineral density and bone mineral content in one‐month‐old HMWTg mice were significantly reduced compared with Control/VectorTg mice. Serum FGF23 was significantly increased in HMWTg compared with VectorTg. Serum phosphate was significantly reduced in HMWTg and was rescued by FGF23Ab. Serum parathyroid hormone (PTH) was significantly increased in HMWTg but was not reduced by FGF23Ab. 1, 25(OH)2 D was inappropriately normal in serum of HMWTg and was significantly increased in both Vector and HMWTg by FGF23Ab. Analysis of HMWTg kidneys revealed significantly increased mRNA expression of the FGF23 co‐receptor Klotho, transcription factor mRNAs for early growth response‐1 transcription factor (Egr‐1) and c‐fos were all significantly decreased by FGF23Ab. A significant reduction in the phosphate transporter Npt2a mRNA was also observed in HMWTg kidneys, which was increased by FGF23Ab. FGF23Ab reduced p‐FGFR1, p‐FGFR3, KLOTHO, p‐ERK1/2, C‐FOS and increased NPT2A protein in HMWTg kidneys. We conclude that FGF23 blockade rescued hypophosphatemia by regulating FGF23/FGFR downstream signaling in HMWTg kidneys. Furthermore, HMWFGF2 isoforms regulate PTH expression independent of FGF23/FGFR signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-16T06:15:25.784878-05:
      DOI: 10.1002/jcp.25458
  • Ethanol Extract of Cissus quadrangularis Enhances Osteoblast
           Differentiation and Mineralization of Murine Pre‐Osteoblastic
           MC3T3‐E1 Cells
    • Abstract: Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti‐osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre‐osteoblast cell line, MC3T3‐E1. Ethanolic extract of CQ (CQ‐E) was found to affect growth kinetics of MC3T3‐E1 cells in a dosage dependent manner. High concentrations of CQ‐E (more than 10 µg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non‐toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ‐E. CQ‐E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose‐dependent effect of CQ‐E with lower concentrations exhibiting anabolic and osteogenic properties. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:47.466708-05:
      DOI: 10.1002/jcp.25449
  • Transcriptome‐Based Analysis of Molecular Pathways for Clusterin
           Functions in Kidney Cells
    • Abstract: Clusterin (CLU) is a chaperone‐like protein and plays a protective role against renal ischemia‐reperfusion injury (IRI); however, the molecular pathways for its functions in the kidney are not fully understood. This study was designed to investigate CLU‐mediating pathways in kidney cells by using bioinformatics analysis. CLU null renal tubular epithelial cells (TECs) expressing human CLU cDNA (TEC‐CLUhCLU) or empty vector (TEC‐CLU‐/‐) were exposed to normoxia or hypoxia (1% O2). Transcriptome profiling with a significant 2‐fold change was performed using SurePrint G3 Mouse Gene Expression 8 × 60K microarray, and the signaling pathways was ranked by using Ingenuity pathway analysis. Here, we showed that compared to CLU null controls, ectopic expression of human CLU in CLU null kidney cells promoted cell growth but inhibited migration in normoxia, and enhanced cell survival in hypoxia. CLU expression affected expression of 3864 transcripts (1893 up‐regulated) in normoxia and 3670 transcripts (1925 up‐regulated) in hypoxia. CLU functions in normoxia were associated mostly with AKT2/PPP2R2B‐dependent PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling and as well with GSK3B‐mediated cell cycle progression. In addition to unfolded protein response (UPR) and/or endoplasmic reticulum (ER) stress, CLU‐enhanced cell survival in hypoxia was also associated with PIK3CD/MAPK1‐dependent PI3K/AKT, HIF‐α, PTEN, VEGF and ERK/MAPK signaling. In conclusion, our data showed that CLU functions in kidney cells were mainly mediated in a cascade manner by PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling, and specifically by activation of UPR/ER stress in hypoxia, providing new insights into the protective role of CLU in the kidney. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:40.582976-05:
      DOI: 10.1002/jcp.25415
  • MicroRNA‐590‐5p stabilizes Runx2 by targeting Smad7 during
           osteoblast differentiation
    • Authors: M. Vishal; S. Vimalraj, R. Ajeetha, M. Gokulnath, R. Keerthana, Z. He, N.C. Partridge, N. Selvamurugan
      Abstract: Mesenchymal stem cells (MSCs) are multipotent cells and their differentiation into the osteoblastic lineage is strictly controlled by several regulators, including microRNAs (miRNAs). Runx2 is a bone transcription factor required for osteoblast differentiation. Here, we used in silico analysis to identify a number of miRNAs that putatively target Runx2 and its co‐factors to mediate both positive and negative regulation of osteoblast differentiation. Among these miRNAs, miR‐590‐5p was selected and its expression was found to be increased during osteoblast differentiation. When mouse MSCs (mMSCs) were transiently transfected with a miR‐590‐5p mimic, we detected an increase in both calcium deposition and the mRNA expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP) and type I collagen genes. Smad7 was found to be among the putative target genes of miR‐590‐5p and its mRNA and protein expression decreased after miR‐590‐5p mimic transfection in human osteoblast‐like cells (MG63). Our analysis indicated that Runx2 was not a putative target of miR‐590‐5p. However, Runx2 protein, but not mRNA expression, increased after miR‐590‐5p mimic transfection in MG63 cells. Runx2 protein expression was increased with knockdown of Smad7 expression by Smad7 siRNA in these cells. We further identified that the 3′‐untranslated region of Smad7 was directly targeted by miR‐590‐5p; this was done using the luciferase reporter gene system. It is known that Smad7 inhibits osteoblast differentiation via Smurf2‐mediated Runx2 degradation. Hence, based on our results, we suggest that miR‐590‐5p promotes osteoblast differentiation by indirectly protecting and stabilizing the Runx2 protein by targeting Smad7 gene expression. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:34.725034-05:
      DOI: 10.1002/jcp.25434
  • Low‐Dose Farnesyltransferase Inhibitor Suppresses HIF‐1α
           and Snail Expression in Triple‐Negative Breast Cancer
           MDA‐MB‐231 Cells In Vitro
    • 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
  • Inhibition of mTOR Signaling Pathway Delays Follicle Formation in Mice
    • Authors: Jing Zhang; Wenwen Liu, Xinhui Sun, Feifei Kong, Ye Zhu, Yue Lei, Youqiang Su, Yiping Su, Jing Li
      Abstract: In mammalian ovaries, follicle assembly requires proper germ cell cyst breakdown and the invasion of somatic cells to encapsulate individual oocytes. Abnormalities in this process lead to a number of pathologies such as premature ovarian failure and infertility. As a conserved pathway regulating cell growth and metabolism in response to growth factors and nutrients, the roles of mTOR signaling in follicular development have been extensively studied in recent years. However, its functions during follicle formation remain unknown. In this study, the expression of p‐rpS6 (phospho‐ribosomal proteinS6), a downstream marker of mTORC1, showed dynamic changes in perinatal ovaries. When E18.5 ovaries, which mainly contained germ cell nests, were incubated with the mTOR inhibitors Rapamycin and Torin1 for 24 h, follicle assembly was delayed with differential somatic cell invasion into germ cell cyst among the groups. After transplanting treated or untreated ovaries into kidney capsules of recipient ovariectomized mice, follicular development was blocked in treated ovaries, as shown by fewer antral follicles and a higher proportion of primordial follicles. Further studies showed a significant decrease in somatic cell proliferation and the expression of marker genes related to follicular development (Kitl, Kit, Gdf9, Bmp15, Zp3, and Amhr2) in treated ovaries. Moreover, the addition of KITL, a growth factor that is mainly produced by pregranulosa cells during germ cell nest breakdown, rescued the extension of follicle formation induced by mTOR inhibitors. These results suggest that KITL functions downstream of mTOR in somatic cells to regulate their communication with oocytes during follicle formation. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T07:15:34.10707-05:0
      DOI: 10.1002/jcp.25456
  • Cholesterol and its Derivatives that Are Components of an Immune
           Stimulatory Drug Reversibly Inhibit Proteinase K
    • Authors: Namrata Singh; Debasish Bhattacharyya
      Abstract: Microorganisms express a variety of proteases that degrade many proteins of the host body and subvert host immune response. While elucidating the mechanism/s of an immune stimulatory drug that contains bile lipid, regulation of proteolytic activity was investigated. The drug and bile lipids both stabilize Proteinase K, an aggressive protease of fungal origin against auto‐digestion. Among the components of bile lipids, only cholesterol and its derivatives stabilize the enzyme. Biophysical evidences such as scattering of light, intrinsic and extrinsic fluorescence emission spectra, circular dichroism spectra, atomic force microscopy and transmission electron microscopy images indicated that cholesterol and its derivatives interact with Proteinase K. Inhibition kinetics using esterolysis of ATEE revealed non‐competitive inhibition by cholesterol. Surface Plasmon Resonance and mass spectrometric analysis indicated 1:1 stoichiometry of binding and with dissociation constant in the µM range. Further, the presence of four cholesterol recognition amino acid consensus motifs (CRAC motifs I‐IV) was identified in Proteinase K. Bioinformatics analysis revealed that the whole stretch of cholesterol interacts very well with the hydrophobic groove of motif II only among the four CRAC motifs. Variation of cholesterol content of HepG2 human liver carcinoma cells showed positive correlation with binding of fluorescence tagged Proteinase K. Under these conditions, binding of Proteinase K to the cells did not affect their morphology, viability and growth kinetics. Cell bound Proteinase K could be released by an excess of its substrate, thereby restoring reversibly its proteolytic activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T07:15:28.51616-05:0
      DOI: 10.1002/jcp.25457
  • Post‐Transcriptional Regulation of the Human Mu‐Opioid
           Receptor (MOR) by Morphine‐Induced RNA Binding Proteins hnRNP K and
    • Abstract: Expression of the mu‐opioid receptor (MOR) protein is controlled by extensive transcriptional and post‐transcriptional processing. MOR gene expression has previously been shown to be altered by a post‐transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)‐binding protein (hnRNP) K and poly(C)‐binding protein 1 (PCBP1) as post‐transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5‘‐untranslated region of human MOR mRNA. In addition, we also found that morphine‐induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine‐induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). This article is protected by copyright. All rights reserved
      PubDate: 2016-06-13T06:56:20.948968-05:
      DOI: 10.1002/jcp.25455
  • Affinity Selection of FGF2‐Binding Heparan Sulfates for Ex Vivo
           Expansion of Human Mesenchymal Stem Cells
    • Authors: Sampath Jeewantha Wijesinghe; Ling Ling, Sadasivam Murali, Hui Qing Yeong, Simon F.R. Hinkley, Susan M. Carnachan, Tracey J. Bell, Kunchithapadam Swaminathan, James H. Hui, Andre J. van Wijnen, Victor Nurcombe, Simon M. Cool
      Abstract: The future of human mesenchymal stem cells (hMSCs) as a successful cell therapy relies on bioprocessing strategies to improve the scalability of these cells without compromising their therapeutic ability. The culture‐expansion of hMSCs can be enhanced by supplementation with growth factors, particularly fibroblast growth factor 2 (FGF2). The biological activity of FGF2 is controlled through interactions with heparan sulfate (HS) that facilitates ligand‐receptor complex formation. We previously reported on an FGF2‐interacting HS variant (termed HS2) isolated in microgram amounts from embryonic tissue by anionic exchange chromatography that increased the proliferation and potency of hMSCs. Here we detail the isolation of gram amounts of an FGF2 affinity‐purified HS variant (HS8) using a scalable platform technology previously employed to generate HS variants with increased affinity for BMP‐2 or VEGF165. This process used a peptide sequence derived from the heparin‐binding domain of FGF2 as a substrate to affinity‐isolate HS8 from a commercially available source of porcine mucosal HS. Our data show that HS8 binds to FGF2 with higher affinity than to FGF1, FGF7, BMP2, PDGF‐BB, or VEGF165. Also, HS8 protects FGF2 from thermal destabilization and increases FGF signaling and hMSC proliferation through FGF receptor 1. Long‐term supplementation of cultures with HS8 increased both hMSC numbers and their colony‐forming efficiency without adversely affecting the expression of hMSC‐related surface antigens. This strategy further exemplifies the utility of affinity‐purifying HS variants against particular ligands important to the stem cell microenvironment and advocates for their addition as adjuvants for the culture‐expansion of hMSCs destined for cellular therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-13T06:56:16.469175-05:
      DOI: 10.1002/jcp.25454
  • Combination of Rapamycin and Resveratrol for Treatment of Bladder Cancer
    • Authors: Anya Alayev; Rachel S. Salamon, Naomi S. Schwartz, Adi Y. Berman, Sara L. Wiener, Marina K. Holz
      Abstract: Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin‐induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1‐/‐ MEF cells, and not in wild‐type MEFs. Similarly, resveratrol alone or in combination with rapamycin induced cell death in human bladder cancer cell lines. These data indicate that administration of resveratrol together with rapamycin may be a promising therapeutic option for treatment of bladder cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T10:01:48.493314-05:
      DOI: 10.1002/jcp.25443
  • Non‐Metabolic Role of PKM2 in Regulation of the HIV‐1 LTR
    • Authors: Satarupa Sen; Satish L. Deshmane, Rafal Kaminski, Shohreh Amini, Prasun K. Datta
      Abstract: Identification of cellular proteins, in addition to already known transcription factors such as NF‐κB, Sp1, C‐EBPβ, NFAT, ATF/CREB, and LEF‐1, which interact with the HIV‐1 LTR is critical in understanding the mechanism of HIV‐1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV‐1SF162 infection of monocyte/macrophages and reactivation of HIV‐1 in U1 cells, a macrophage model of latency. We observed that HIV‐1SF162 infection of monocyte/macrophages and reactivation of HIV‐1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA‐induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA‐induced U1 cells in comparison to PMA‐induced U937 cells. We focused on understanding the potential role of PKM2 in HIV‐1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA‐activated U1 and TZM‐ bl cells demonstrated the interaction of PKM2 with the HIV‐1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV‐1 LTR‐luciferase reporter in U937, U‐ 87 MG, and TZM‐bl cells. Using various truncated constructs of the HIV‐1 LTR, we mapped the region spanning ‐120 bp to ‐80 bp to be essential for PKM2‐mediated transactivation. This region contains the NF‐κB binding site and deletion of this site attenuated PKM2 mediated activation of HIV‐1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF‐κB. These observations demonstrate for the first time that PKM2 is a transcriptional co‐activator of HIV‐1 LTR. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T10:01:42.21665-05:0
      DOI: 10.1002/jcp.25445
  • Light Sheet Microscopy to Measure Protein Dynamics
    • Authors: Matthias Rieckher
      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
  • Leucine‐Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast
           Requires Flotillin‐1 Mediated Endocytosis
    • Abstract: Basic, pre‐clinical and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine‐Rich Amelogenin Peptide ‐ LRAP), which is supposed to act as a signaling molecule in epithelial‐mesenchymal interactions. LRAP‐binding protein complexes from immortalized murine cementoblasts (OCCM‐30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin‐1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co‐precipitation and co‐localization assays as a protein‐binding partner for LRAP in OCCM‐30 cells. In addition, we found that exogenously added GST‐LRAP recombinant protein was internalized by OCCM‐30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1‐dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM‐30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp) and runt‐related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin‐assisted endocytosis, which suggests an involvement of LRAP in lipid‐raft‐dependent signaling pathways which are mediated by flotillin‐1. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:25:20.31246-05:0
      DOI: 10.1002/jcp.25453
  • Browning of White Fat: Novel Insight into Factors, Mechanisms and
    • Authors: Nevena Jeremic; Pankaj Chatuverdi, Suresh C. Tyagi
      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
  • In Vitro and In Vivo Differentiation of Progenitor Stem Cells Obtained
           after Mechanical Digestion of Human Dental Pulp
    • Authors: Manuela Monti; Antonio Graziano, Silvana Rizzo, Cesare Perotti, Claudia Del Fante, Riccardo d'Aquino, CarloAlberto Redi, Ruggero Rodriguez y Baena
      Abstract: Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity. The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical‐grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate towards different cell types and to generate new bone in patients. We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio‐maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:23:35.810575-05:
      DOI: 10.1002/jcp.25452
  • Effects of sex and notch signaling on the osteocyte cell pool
    • Authors: Ernesto Canalis; Lauren Schilling, Stefano Zanotti
      Abstract: Osteocytes play a fundamental role in mechanotransduction and skeletal remodeling. Sex is a determinant of skeletal structure, and female C57BL/6J mice have increased osteoblast number in cancellous bone when compared to male mice. Activation of Notch in the skeleton causes profound cell‐context dependent changes in skeletal physiology. To determine the impact of sex and of Notch signaling on the osteocyte cell pool, we analyzed cancellous and cortical bone of 1 to 6 month old C57BL/6J or 129SvJ/C57BL/6J mice and determined the osteocyte number/area. There was an age‐dependent decline in osteocyte number in cancellous bone of male but not female mice, so that 6 month old female mice had a greater number of osteocytes than male littermates. Although differences between male and female mice were modest, female mice had ∼10‐15% greater number of osteocytes/area. RNA sequence analysis of osteocyte‐rich preparations did not reveal differences between sexes in the expression of genes known to influence bone homeostasis. Neither the activation of Notch1 nor the concomitant inactivation of Notch1 and Notch2 in Osterix (Sp7) or Dentin matrix protein 1 (Dmp1) expressing cells had a pronounced and consistent effect on cancellous or cortical bone osteocyte number in either sex. Moreover, inactivation of Notch1 and Notch2 in Dmp1 expressing cells did not influence the bone loss in a muscle immobilization model of skeletal unloading. In conclusion, cancellous bone osteocytes decline with age in male mice, cortical osteocytes are influenced by sex in younger mice, but osteocyte cell density is not affected substantially by Notch signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-07T11:50:40.061135-05:
      DOI: 10.1002/jcp.25433
  • Early Treatment with Enalapril and Later Renal Injury in Programmed Obese
           Adult Rats
    • Authors: Hyung Eun Yim; Kee Hwan Yoo, In Sun Bae, Young Sook Hong
      Abstract: Obesity‐related kidney disease should be prevented or retarded. We aimed to investigate whether early treatment with enalapril ameliorates later renal injury induced by early postnatal overnutrition. Three or ten male pups per mother were assigned to either the Obese or Lean group during the first 21 days of life. These pups were treated with enalapril (Obese enalapril, OE; Lean enalapril, LE) or vehicle (Obese control, OC; Lean control, LC) for 15–28 days. Body weight, blood pressure (BP), and renal alterations were determined at 3 months. Enalapril decreased body weight only in the Lean group at 3 months (P 
      PubDate: 2016-06-06T10:15:29.747504-05:
      DOI: 10.1002/jcp.25444
  • Gating Modulation of the Tumor‐Related Kv10.1 Channel by Mibefradil
    • Abstract: Several reports credit mibefradil with tumor suppressing properties arising from its known inhibition of Ca2+ currents. Given that mibefradil (Mb) is also known to inhibit K+ channels, we decided to study the interaction between this organic compound and the tumor‐related Kv10.1 channel. Here we report that Mb modulates the gating of Kv10.1. Mb induces an apparent inactivation from both open and early closed states where the channels dwell at hyperpolarized potentials. Additionally Mb accelerates the kinetics of current activation, in a manner that depends on initial conditions. Our observations suggest that Mb binds to the voltage sensor domain of Kv10.1 channels, thereby modifying the gating of the channels in a way that in some, but not all, aspects opposes to the gating effects exerted by divalent cations. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-03T06:58:46.738119-05:
      DOI: 10.1002/jcp.25448
  • T11TS Treatment Augments Apoptosis of Glioma Associated Brain Endothelial
           Cells, Hint towards Anti‐Angiogenic Action in Glioma
    • Authors: Debanjan Bhattacharya; Manoj Kumar Singh, Suhnrita Chaudhuri, Ankur Datta, Swapna Chaudhuri
      Abstract: Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11‐target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma associated brain endothelial cells. In the present study we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma associated brain endothelial cells. Annexin‐V/PI staining showed that T11TS treatment in glioma induced rats increases apoptosis of glioma associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC‐1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma associated brain endothelial cells. Flowcytometry, immunoblotting and in‐situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment upmodulated expression of p53, Bax, Fas, FasL and FADD in glioma associated endothelial cells and downregulated Bcl‐2 protein. T11TS therapy induced cytochrome‐c release into cytosol, activated caspase ‐9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas dependent extrinsic pathway. The pro‐apoptotic action of T11TS on glioma associated endothelial cells provides crucial insight into how T11TS exerts its anti‐angiogenic function in glioma. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-03T06:51:42.586441-05:
      DOI: 10.1002/jcp.25447
  • Cartilage‐specific and Cre‐dependent Nkx3.2 overexpression in
           vivo causes skeletal dwarfism by delaying cartilage hypertrophy
    • 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
  • Generation of neural crest‐like cells from human periodontal
           ligament cell‐derived induced pluripotent stem cells
    • Authors: Atsushi Tomokiyo; Kim Hynes, Jia Ng, Danijela Menicanin, Esther Camp, Agnes Arthur, Stan Gronthos, Peter Mark Bartold
      Abstract: Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue‐derived periodontal ligament (PDL) iPSC to differentiate into neural crest‐like cells (NCLC) relative to iPSC generated from a non‐neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC‐ and FF iPSC‐derived NCLC, which highly expressed HNK1. A high proportion of the HNK1‐positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1‐positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell‐associated markers than the PDL and FF HNK1‐negative populations. Interestingly, the HNK1‐positive cells derived from the PDL‐iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1‐positive cells derived from the FF‐iPSC. Our work suggests that HNK1‐enriched NCLC from neural crest tissue‐derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1‐low population and non‐neural crest iPSC‐derived NCLC. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:43:14.571204-05:
      DOI: 10.1002/jcp.25437
  • Neoadjuvant sequential docetaxel followed by high‐dose epirubicin in
           combination with cyclophosphamide administered concurrently with
    • Abstract: To report the results of the DECT trial, a phase II study of locally advanced or operable HER2‐positive breast cancer (BC) treated with taxanes and concurrent anthracyclines and trastuzumab. Eligible patients (stage IIA‐IIIB HER2‐positive BC),18‐75 years, normal organ functions, ECOG ≤1, and left ventricular ejection fraction (LVEF) ≥55% received 4 cycles of neoadjuvant docetaxel, 100 mg/m2 intravenously, plus trastuzumab 6 mg/kg (loading dose 8mg/kg) every 3 weeks, followed by four 3‐weekly cycles of epirubicin 120 mg/m2 and cyclophosphamide, 600 mg/m2, plus trastuzumab. Primary objective was pathologic complete response (pCR) rate, defined as ypT0/is ypN0 at definitive surgery. We enrolled 45 consecutive patients. All but 6 patients (13.3%) completed chemotherapy and all underwent surgery. pCR was observed in 28 patients (62.2%) overall and in 6 (66.7%) from the inflammatory subgroup. The classification and regression tree analysis showed a 100% pCR rate in patients with BMI≥25 and with hormone responsive disease. The median follow up was 46 months (8‐78). Four‐year recurrence‐free survival was 74.7% (95%CI, 58.2‐91.2). Seven patients (15.6%) recurred and one died. Treatment was well tolerated, with limiting toxicity being neutropenia. No clinical cardiotoxicity was observed. Six patients (13.4%) showed a transient LVEF decrease (
      PubDate: 2016-06-02T10:32:35.767155-05:
      DOI: 10.1002/jcp.25432
  • Microbial community metabolic modeling: A community data‐driven
           network reconstruction
    • Abstract: Metabolic network modeling of microbial communities provides an in‐depth understanding of community‐wide metabolic and regulatory processes. Compared to single organism analyses, community metabolic network modeling is more complex because it needs to account for interspecies interactions. To date, most approaches focus on reconstruction of high‐quality individual networks so that, when combined, they can predict community behaviors as a result of interspecies interactions. However, this conventional method becomes ineffective for communities whose members are not well characterized and cannot be experimentally interrogated in isolation. Here, we tested a new approach that uses community‐level data as a critical input for the network reconstruction process. This method focuses on directly predicting interspecies metabolic interactions in a community, when axenic information is insufficient. We validated our method through the case study of a bacterial photoautotroph‐heterotroph consortium that was used to provide data needed for a community‐level metabolic network reconstruction. Resulting simulations provided experimentally validated predictions of how a photoautotrophic cyanobacterium supports the growth of an obligate heterotrophic species by providing organic carbon and nitrogen sources. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:25:22.790673-05:
      DOI: 10.1002/jcp.25428
  • Anti‐diabetic agent sodium tungstate induces the secretion of
           pro‐ and anti‐inflammatory cytokines by human kidney cells
    • Abstract: Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti‐diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type‐2‐diabetic (D‐RPTEC) and non‐diabetic (N‐RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development and repair, all processes that are dysregulated during DKD. In basal conditions, D‐RPTEC cells secreted higher levels of prototypical pro‐inflammatory IL‐6, IL‐8 and MCP‐1 than N‐RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL‐6, IL‐8 and MCP‐1 secretion in both N‐ and D‐RPTEC, together with lower levels of IL‐1 RA, IL‐4, IL‐10 and GM‐CSF, suggesting that it may contribute to the extent of renal damage/repair during DKD. Besides, NaW induced the accumulation of IκBα, the main inhibitor protein of one major pathway involved in cytokine production, suggesting further anti‐inflammatory effect in the long‐term. A better understanding of the mechanisms involved in the interplay between the anti‐diabetic and immunomodulatory properties of NaW will facilitate future studies about its clinical relevance. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:25:19.149981-05:
      DOI: 10.1002/jcp.25429
  • Ginkgolic Acid Inhibits Invasion and Migration and
           TGF‐β‐Induced EMT of Lung Cancer Cells through
           PI3K/Akt/mTOR Inactivation
    • Abstract: Epithelial‐to‐mesenchymal transition (EMT) is a critical cellular phenomenon regulating tumor metastases. In the present study, we investigated whether ginkgolic acid can affect EMT in lung cancer cells and the related underlying mechanism(s) of its actions. We found that ginkgolic acid C15:1 (GA C15:1) inhibited cell proliferation, invasion, and migration in both A549 and H1299 lung cancer cells. GA C15:1 also suppressed the expression of EMT related genes (Fibronectin, Vimentin, N‐cadherin, MMP‐9, MMP‐2, Twist, and Snail) and suppressed TGF‐β‐induced EMT as assessed by reduced expression of mesenchymal markers (Fibronectin, Vimentin, N‐cadherin), MMP‐9, MMP‐2, Twist, and Snail. However, GA C15:1 did not affect the expression of various epithelial marker proteins (Occludin and E‐cadherin) in both A549 and H1299 cells. TGF‐β‐induced morphologic changes from epithelial to mesenchymal cells and induction of invasion and migration were reversed by GA C15:1. Finally, GA C15:1 not only abrogated basal PI3K/Akt/mTOR signaling cascade, but also reduced TGF‐β‐induced phosphorylation of PI3K/Akt/mTOR pathway in lung cancer cells. Overall, these findings suggest that GA C15:1 suppresses lung cancer invasion and migration through the inhibition of PI3K/Akt/mTOR signaling pathway and provide a source of potential therapeutic compounds to control the metastatic dissemination of tumor cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:13:27.95026-05:0
      DOI: 10.1002/jcp.25426
  • Animal models of congenital cardiomyopathies associated with mutations in
           Z‐line proteins
    • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:06:33.346872-05:
      DOI: 10.1002/jcp.25424
  • Sex differences in estrogen receptor α and β levels and
           activation status in LPS‐stimulated human macrophages
    • Authors: Ilaria Campesi; Maria Marino, Andrea Montella, Sara Pais, Flavia Franconi
      Abstract: Immune function, inflammation and atherosclerosis display sex differences and are influenced by 17β‐estradiol through estrogen receptors subtypes ERα and ERβ. Male tissues express active ERs, but their possible involvement in inflammation in males has never been assessed. Macrophages express both ERα and ERβ and offer the opportunity to evaluate the role of ER levels and activation in inflammation. We assessed the ability of lipopolysaccharide (LPS) to modulate, in a sex‐specific way, the expression and the activation status of ERα and ERβ in blood monocytes‐derived macrophages (MDMs) from men and women. MDMs were incubated with 100 ng/ml LPS for 24 h and used to evaluate ERα, ERβ, P‐ ERα, p38 and P‐p38 expression by Western Blotting. In basal conditions, ERα and ERβ were significantly higher in female MDMs than in male MDMs. LPS up‐regulated ERα and ERα phosphorylation in both sexes, with a significantly higher effect observed in male MDMs, and down‐regulated ERβ level only in female MDMs. p38 and P‐p38 proteins, indicative of ERβ activity, did not show sex differences both in basal conditions and after LPS treatment. Finally, ERα/ERβ and P‐ERα/ERα ratios were significantly higher in male MDMs than in female ones. Our data indicate, for the first time, that LPS affects ERα but not ERβ activation status. We identify a significant role of ERα in LPS‐mediated inflammatory responses in MDMs, which represents an initial step in understanding the influence of sex in the relationship between LPS and ERα. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:06:28.352424-05:
      DOI: 10.1002/jcp.25425
  • Chrysophanol induces apoptosis of choriocarcinoma by through regulation of
           ROS and the AKT and ERK1/2 pathways
    • Authors: Whasun Lim; Changwon Yang, Fuller W. Bazer, Gwonhwa Song
      Abstract: Chrysophanol is an anthraquinone compound, mainly isolated from rhubarb, with anti‐cancer effects on some types of cancer cells. However, effects of chrysophanol on human choriocarcinoma cells are not known. Therefore, the objective of this study was to determine effects of chrysophanol on choriocarcinoma cells (JAR and JEG‐3) and identify signal transduction cascades activated by chrysophanol. Results of present study showed that chrysophanol decreased cell viability and induced apoptosis of JEG‐3, but not JAR cells, in a dose‐dependent manner. Chrysophanol also increased oxidative stress in JEG‐3 cells by inducing ROS generation followed by mitochondrial dysfunction including depolarization of mitochondrial inner membrane potential. Western blot analysis revealed that ERK1/2, P90RSK, AKT and P70S6K were increased significantly in JEG‐3 cells by chrysophanol. Next, we investigated chrysophanol‐mediated effects on proliferation of JEG‐3 cells using pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126). Inhibition of AKT and ERK1/2 prevented chrysophanol‐induced stimulation of proliferation of JEG‐3 cells. In addition, the phosphorylation of AKT and ERK1/2 was suppressed by LY294002 and U0126 in JEG‐3 cells treated with chrysophanol, whereas the AKT protein was activated by pre‐treatment of JEG‐3 cells with U0126. Furthermore, we compared therapeutic effects of chrysophanol with cisplatin and paclitaxel which are conventional salvage regimens for choriocarcinoma. Our results verified that chrysophanol has synergistic effects with traditional therapy to increase apoptosis of JEG‐3 cells. Collectively, these results indicate that chrysophanol is a potential effective chometherapeutic agent for treatment of choriocarcinoma therapy and minimizing side effects of conventional treatment regimens. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:03:58.124871-05:
      DOI: 10.1002/jcp.25423
  • Repression of primitive erythroid program is critical for the initiation
           of multi‐lineage hematopoiesis in mouse development
    • Authors: Toshiyuki Yamane; Chie Ito, Aya Washino, Kana Isono, Hidetoshi Yamazaki
      Abstract: Formation of the hematopoietic cells occurs in multiple steps. The first hematopoietic cells observed during ontogeny are primitive erythrocytes, which are produced in the early yolk sac within a limited temporal window. Multi‐lineage hematopoiesis, which supplies almost the entire repertoire of blood cell lineages, lags behind primitive erythropoiesis in the tissue. However, molecular mechanisms regulating sequential generation of primitive erythrocytes and multipotent hematopoietic progenitors in the yolk sac are largely unknown. In this study, the transcription factors involved in the development of hematopoietic cells were examined in purified progenitor cell populations from pluripotent stem cell cultures and from the yolk sac of developing embryos. We found that the earliest committed hematopoietic progenitors highly expressed Gata1, Scl/tal1, and Klf1 genes. Expression of these transcription factors, which is known to form a core erythroid transcriptional network, explained the prompt generation of primitive erythrocytes from these earliest progenitors. Importantly, the multipotent hematopoietic cells, which lack the differentiation potential into primitive erythroid cells, down‐regulated these genes during a transition from the earliest committed progenitors. In addition, we showed that Pu.1 is involved in the multipotent cell differentiation through the suppression of erythroid transcription program. We propose that these molecular mechanisms governed by transcription factors form sequential waves of primitive erythropoiesis and multi‐lineage hematopoiesis in the early yolk sac of developing embryos. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T09:57:07.656034-05:
      DOI: 10.1002/jcp.25422
  • Flavopiridol: An old drug with new perspectives' Implication for
           development of new drugs
    • Authors: Annamaria Cimini; Michele d'Angelo, Elisabetta Benedetti, Barbara D'Angelo, Giulio Laurenti, Andrea Antonosante, Loredana Cristiano, Antonella Di Mambro, Marcella Barbarino, Vanessa Castelli, Benedetta Cinque, Maria Grazia Cifone, Rodolfo Ippoliti, Francesca Pentimalli, Antonio Giordano
      Abstract: Glioblastoma, the most common brain tumor, is characterized by high proliferation rate, invasion, angiogenesis and chemo‐ and radio‐resistance. One of most remarkable feature of glioblastoma is the switch toward a glycolytic energetic metabolism that leads to high glucose uptake and consumption and a strong production of lactate. Activation of several oncogene pathways like Akt, c‐myc and ras, induces glycolysis and angiogenesis and acts to assure glycolysis prosecution, tumor proliferation and resistance to therapy. Therefore, the high glycolytic flux depends on the overexpression of glycolysis‐related genes resulting in an overproduction of pyruvate and lactate. Metabolism of glioblastoma thus represents a key issue for cancer research. Flavopiridol is a synthetic flavonoid that inhibits a wide range of Cyclin‐dependent kinase, that has been demonstrate to inactivate glycogen phosphorylase, decreasing glucose availability for glycolysis. In this work is reported the study of glucose metabolism upon flavopiridol treatment in the two different glioblastoma cell lines. The results obtained point towards an effect of flavopiridol in glycolytic cells, thus suggesting a possible new use of this compound or flavopiridol‐derived formulations in combination with anti‐proliferative agents in glioblastoma patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T09:52:49.630745-05:
      DOI: 10.1002/jcp.25421
  • Probing Micromechanical Properties of the Extracellular Matrix of Soft
           Tissues by Atomic Force Microscopy
    • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T06:06:07.36028-05:0
      DOI: 10.1002/jcp.25420
  • How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy
    • Authors: Albert T. Gacerez; Benjamine Arellano, Charles L. Sentman
      Abstract: Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor‐ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T05:58:42.372515-05:
      DOI: 10.1002/jcp.25419
  • 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
      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 100,000g pellet (P100), which contains nanoparticles (
      PubDate: 2016-06-02T05:51:51.12926-05:0
      DOI: 10.1002/jcp.25414
  • Targeting the breast cancer kinome
    • Authors: Samantha M. Miller; Daniel R. Goulet, Gary L. Johnson
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-31T07:45:26.494401-05:
      DOI: 10.1002/jcp.25427
  • Epithelial‐to‐mesenchymal transition in
           paclitaxel‐resistant ovarian cancer cells is downregulated by
    • Authors: Vermont P Dia; Philipus Pangloli
      Abstract: Ovarian cancer (OVCA) is the deadliest of all gynecological cancers which is attributed to late presentation, persistence and development of chemoresistance. The objectives were to evaluate the association between OVCA paclitaxel‐resistance and epithelial‐to‐mesenchymal transition (EMT) and to determine the capability of luteolin to chemosensitize OVCA cells. X10 and X22 cells were 11.8–25.3‐fold and 7.8–8.6‐fold resistant to paclitaxel than 1AP cells. X10 and X22 cells exhibited a mesenchymal phenotype while 1AP has an epithelial characteristics. Furthermore, the expression of the epithelial marker E‐cadherin was downregulated while mesenchymal markers Vimentin and N‐cadherin were upregulated in X10 and X22 cells when compared to 1AP cells. Transcription factors Snail, Slug and Twist1 were upregulated in X10 cells while Twist1 was highly expressed in X22 cells. Luteolin treatment caused cytotoxicity being most potent to X10 OVCA cells. Treatment of non‐cytotoxic dose of luteolin at 15.625 µM chemosensitized X10 and X22 OVCA cells to paclitaxel as evidenced by reduced ED50 values from 11.8 to 0.2 µM and 8.6 to 3.6 µM for X10 and X22 cells, respectively. Moreover, luteolin treatment led to a more epithelial phenotype of X10 and X22 cells and modification of EMT markers indicating reversal of EMT. The mechanism involved is through reduction of phosphorylation of FAK and ERK leading to reduced nuclear translocation of p65. Our results highlight the significance of EMT in OVCA resistance to paclitaxel and warrant the investigation of luteolin as a potential therapeutic agent in chemoresistant OVCA. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-27T08:25:26.002642-05:
      DOI: 10.1002/jcp.25436
  • LPS induces hyper‐permeability of intestinal epithelial cells
    • Authors: Amir Bein; Alexander Zilbershtein, Michael Golosovsky, Dan Davidov, Betty Schwartz
      Abstract: Background Necrotizing Enterocolitis (NEC) is a severe inflammatory disorder leading to high morbidity and mortality rates. A growing body of evidence demonstrate the key role of the Toll like receptor 4 (TLR4) in NEC. This membranal receptor recognizes lipopolysaccharides (LPS) from the bacterial wall and triggers an inflammatory response. The aim of the present study was to elucidate the effect of LPS on paracellular permeability known to be severely affected in NEC. Methods IEC‐18 cells were treated with LPS and the effects on morphology, paracellular permeability and their associated gene and protein expressions were measured. Results LPS down regulated the expression of occludin and ZO‐1 mRNAs while up regulating Cdkn1a. In addition LPS caused a significant increase in paracellular permeability and epithelial barrier damage. Finally ZO‐1 protein was found to be spatially disarrayed in the intercellular junctions in response to LPS. Conclusion LPS adversely affected the functionality of the intestinal epithelial barrier suggesting a new mechanism by which bacterial infection may contribute to the development of NEC. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-26T09:11:37.61133-05:0
      DOI: 10.1002/jcp.25435
  • 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
      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 30nM, Vmax 100nM), mimicked by the adenyl cyclase activator forskolin (EC50 5µM, Vmax 10µM). 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. Over expression experiments in tandem with pull‐down assays indicated that specific Ser291/292 phosphorylation of RIN1 favoured 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-26T08:46:51.819046-05:
      DOI: 10.1002/jcp.25412
  • 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
  • Pin1, the Master Orchestrator of Bone Cell Differentiation
    • Abstract: Pin1 is an enzyme that specifically recognizes the peptide bond between phosphorylated serine or threonine (pS/pT‐P) and proline. This recognition causes a conformational change of its substrate, which further regulates downstream signaling. Pin1‐/‐ mice show developmental bone defects and reduced mineralization. Pin1 targets RUNX2 (Runt‐Related Transcription Factor 2), SMAD1/5, and β‐catenin in the FGF, BMP, and WNT pathways, respectively. Pin1 has multiple roles in the crosstalk between different anabolic bone signaling pathways. For example, it controls different aspects of osteoblastogenesis and increases the transcriptional activity of Runx2, both directly and indirectly. Pin1 also influences osteoclastogenesis at different stages by targeting PU.1 (Purine‐rich nucleic acid binding protein 1), C‐FOS, and DC‐STAMP. The phenotype of Pin1‐/‐ mice has led to the recent identification of multiple roles of Pin1 in different molecular pathways in bone cells. These roles suggest that Pin1 can be utilized as an efficient drug target in congenital and acquired bone diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-25T17:40:37.258658-05:
      DOI: 10.1002/jcp.25442
  • Association between aerobic exercise and rosiglitazone avoided the NAFLD
           and liver inflammation exacerbated in PPAR‐α knockout mice
    • Abstract: Nonalcoholic fatty liver disease (NAFLD) is one of the main liver diseases today, and may progress to steatohepatitis, cirrhosis and hepatocellular carcinoma. Some studies have shown the beneficial effects of aerobic exercise on reversing NAFLD. Aim: To verify whether chronic aerobic exercise improves the insulin resistance, liver inflammation and steatohepatitis caused by a high fat diet (HF) and whether PPARα is involved in these actions. Methods: C57BL6 wild type (WT) and PPAR‐α knockout (KO) mice were fed with a standard diet (SD) or HF during 12 weeks; the HF mice were trained on a treadmill during the last 8 weeks. Serum glucose and insulin tolerances, serum levels of aspartate aminotransferase, hepatic content of triacylglycerol, cytokines, gene expression and protein expression were evaluated in all animals. Results: Chronic exposure to HF diet increased triacylglycerol accumulation in the liver, leading to NAFLD, increased aminotransferase in the serum, increased peripheral insulin resistance, and higher adiposity index. Exercise reduced all these parameters in both animal genotypes. The liver lipid accumulation was not associated with inflammation; trained KO mice, however, presented a huge inflammatory response that was probably caused by a decrease in PPAR‐γ expression. Conclusion: We conclude that exercise improved the damage caused by a HF independently of PPARα, apparently by a peripheral fatty acid oxidation in the skeletal muscle. We also found that the absence of PPARα together with exercise leads to a decrease in PPAR‐γ and a huge inflammatory response. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-23T16:00:32.884989-05:
      DOI: 10.1002/jcp.25440
  • Ascorbic acid induces necrosis in human laryngeal squamous cell carcinoma
           via ROS, PKC, and calcium signaling
    • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-22T23:40:54.779976-05:
      DOI: 10.1002/jcp.25438
  • Intricate functions of matrix metalloproteinases in physiological and
           pathological conditions
    • Authors: Rahul Mittal; Amit P. Patel, Luca H. Debs, Desiree Nguyen, Kunal Patel, M'hamed Grati, Jeenu Mittal, Denise Yan, Prem Chapagain, Xue Zhong Liu
      Abstract: Matrix metalloproteinases (MMPs) are a diverse group of proteolytic enzymes and play an important role in the degradation and remodeling of the extracellular matrix (ECM). In normal physiological conditions, MMPs are usually minimally expressed. Despite their low expression, MMPs have been implicated in many cellular processes ranging from embryological development to apoptosis. The activity of MMPs is controlled at three different stages: (1) transcription, (2) zymogen activation, and (3) inhibition of active forms by tissue inhibitor metalloproteinases (TIMPs). They can collectively degrade any component of ECM and basement membrane, and their excessive activity has been linked to numerous pathologies mainly including, but not limited to, tumor evasion and metastasis. The lack of information about several MMPs and the steady stream of new discoveries suggest that there is much more to be studied in this field. In particular, there is a need for controlling their expression in disease states. Various studies over the past 30 years have found that each MMP has a specific mode of activation, action, and inhibition. Drugs specifically targeting individual MMPs could revolutionize the treatment of a great number of health conditions and tremendously reduce their burden. In this review article, we have summarized the recent advances in understanding the role of MMPs in physiological and pathological conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-17T16:25:35.052746-05:
      DOI: 10.1002/jcp.25430
  • Prototype of Multiplex Bead Assay for Quantification of Three Serum
           Biomarkers for In Vitro Diagnosis of Endometriosis
    • Authors: Pietro Giulio Signorile; Alfonso Baldi
      Abstract: Endometriosis is a very common disease, affecting 10% of women in the reproductive age. To date, a significant delay between onset of the symptoms and definitive diagnosis is caused by the lack of a reliable non‐invasive diagnostic test. Recently, the potential value as diagnostic markers for endometriosis of three proteins (Zn‐alpha2‐glycoprotein, serum albumin and complement C3 precursor), has been showed. In this article, we have defined the experimental conditions for the development of a multiplex bead array assay for rapid and simultaneous quantification of these three biomarkers in the serum of patients with endometriosis. Finally, pivotal experiments on a small cohort of patients have confirmed the diagnostic value of this assay. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-16T01:25:32.216622-05:
      DOI: 10.1002/jcp.25410
  • The Non‐Conventional Effects of Glucocorticoids in Cancer
    • Authors: Simra Azher; Omid Azami, Caterina Amato, Michael McCullough, Antonio Celentano, Nicola Cirillo
      Abstract: Synthetic corticosteroids are widely used for the treatment of a variety of diseases, including pre‐malignant and malignant conditions. In striking contrast, recent evidence suggests that corticosteroids can bear tumour‐promoting effects in solid tumours of epithelial origin. We have recently shown that epithelial tissues, including the mucosa of the oral cavity and the skin, are able to modulate the local concentration of active corticosteroids and to produce steroids de novo. This has important clinical and physiopathological implications, because tissue‐specific regulation of glucocorticoids plays a key role in the overall effect of these molecules. In the present review of the current English literature, performed using MEDLINE / PubMed / Ovid databases, we collected published evidence to demonstrate that corticosteroids induce effects that are more complex and controversial than previously acknowledged. Published studies clearly demonstrate that this class of molecules influences pathophysiological processes that are strictly related to malignancy, providing the rationale for further investigation. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-08T23:55:54.654891-05:
      DOI: 10.1002/jcp.25408
  • Targeted Modification of Mitochondrial ROS Production Converts High
           Glucose‐Induced Cytotoxicity to Cytoprotection: Effects on
           Anesthetic Preconditioning
    • Authors: Filip Sedlic; Maria Muravyeva, Ana Sepac, Marija Sedlic, Anna Marie Williams, Meiying Yang, Xiaowen Bai, Zeljko J. Bosnjak
      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
  • SHP‐2 Phosphatase Prevents Colonic Inflammation by Controlling
           Secretory Cell Differentiation and Maintaining Host‐Microbiota
    • Abstract: Polymorphisms in the PTPN11 gene encoding for the tyrosine phosphatase SHP‐2 were described in patients with ulcerative colitis. We have recently demonstrated that mice with an intestinal epithelial cell‐specific deletion of SHP‐2 (SHP‐2IEC‐KO) develop severe colitis one month after birth. However, the mechanisms by which SHP‐2 deletion induces colonic inflammation remain to be elucidated. We generated SHP‐2IEC‐KO mice lacking Myd88 exclusively in the intestinal epithelium. The colonic phenotype was histologically analyzed and cell differentiation was determined by electron microscopy and lysozyme or Alcian blue staining. Microbiota composition was analyzed by 16S sequencing. Results show that innate defense genes including those specific to Paneth cells were strongly up‐regulated in SHP‐2‐deficient colons. Expansion of intermediate cells (common progenitors of the Goblet and Paneth cell lineages) was found in the colon of SHP‐2IEC‐KO mice while Goblet cell number was clearly diminished. These alterations in Goblet/intermediate cell ratio were noticed two weeks after birth, before the onset of inflammation and were associated with significant alterations in microbiota composition. Indeed, an increase in Enterobacteriaceae and a decrease in Firmicutes were observed in the colon of these mice, indicating that dysbiosis also occurred prior to inflammation. Importantly, loss of epithelial Myd88 expression inhibited colitis development in SHP‐2IEC‐KO mice, rescued Goblet/intermediate cell ratio and prevented NFκB hyperactivation and inflammation. These data indicate that SHP‐2 is functionally important for the maintenance of appropriate barrier function and host‐microbiota homeostasis in the large intestine. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-21T14:15:45.654798-05:
      DOI: 10.1002/jcp.25407
  • 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, Jane Lian, Gary Stein, Jake Chen
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-11T12:31:05.364501-05:
      DOI: 10.1002/jcp.25406
  • 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
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-11T12:30:53.004404-05:
      DOI: 10.1002/jcp.25405
  • Properties of Adult Lung Stem and Progenitor Cells
    • Authors: Ivan Bertoncello
      Abstract: The last decade has seen significant progress in understanding the organisation of regenerative cells in the adult lung. Cell lineage tracing and in vitro clonogenic assays have enabled the identification and characterisation of endogenous lung epithelial stem and progenitor cells. Selective lung injury models, and genetically engineered mice have revealed highly conserved gene networks, factors, signalling pathways, and cellular interactions important in maintaining lung homeostasis and regulating lung regeneration and repair following injury. This review describes the current models of lung epithelial stem and progenitor cell organisation in adult mice, and the impediments encountered in translational studies aiming to identify and characterise their human homologs. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-07T16:26:52.308325-05:
      DOI: 10.1002/jcp.25404
  • Metabolic Adaptation of the Small Intestine to Short‐ and
           Medium‐Term High‐Fat Diet Exposure
    • Abstract: The small intestine is the main organ involved in the digestion and absorption of nutrients, i.e., 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-07T16:26:19.058388-05:
      DOI: 10.1002/jcp.25402
  • Epigenetics in Intestinal Epithelial Cell Renewal
    • Abstract: A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt‐villus axis. One important check‐point in this process occurs in the transit amplifying zone of the intestinal crypts where different signalling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development and tissue renewal. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-07T16:25:48.709253-05:
      DOI: 10.1002/jcp.25401
  • Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of
           Osteoblast Growth and Differentiation
    • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-06T17:02:53.67624-05:0
      DOI: 10.1002/jcp.25399
  • Differentiation of Keratinocytes Modulates Expression of Epidermal
           Elements of the Cutaneous Analog of
           Hypothalamus‐Pituitary‐Adrenal Axis
    • 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
  • 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
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-02T06:40:50.273009-05:
      DOI: 10.1002/jcp.25397
  • Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and
           Epithelial Factors
    • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-02T06:35:50.209015-05:
      DOI: 10.1002/jcp.25398
  • MG132 Induces Expression of Monocyte Chemotactic Protein‐ Induced
           Protein 1 in Vascular Smooth Muscle Cells
    • 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 thorugh 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-31T09:15:51.140802-05:
      DOI: 10.1002/jcp.25396
  • 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
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-30T06:22:16.134871-05:
      DOI: 10.1002/jcp.25395
  • Quercetin Partially Preserves Development of Osteoblast Phenotype in Fetal
           Rat Calvaria Cells in an Oxidative Stress Environment
    • Authors: Jonathan G. Messer; Stephanie La, Robin G. Hopkins, Deborah E. Kipp
      Abstract: Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up‐regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide‐induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for four days (D5‐9) with 300 µM hydrogen peroxide resulted in fewer alkaline phosphatase‐positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression (p 
      PubDate: 2016-03-29T03:41:44.132435-05:
      DOI: 10.1002/jcp.25392
  • Enhancement of Lytic Activity by Leptin Is Independent from Lipid Rafts in
           Murine Primary Splenocytes
    • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-29T03:36:47.50878-05:0
      DOI: 10.1002/jcp.25394
  • 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
  • The Integrin‐Mediated ILK‐Parvin‐αPix Signalling
           Axis Controls Differentiation in Mammary Epithelial Cells
    • Authors: Nicholas Rooney; Pengbo Wang, Keith Brennan, Andrew P Gilmore, Charles H Streuli
      Abstract: Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behaviour and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1‐integrin (β1‐itg) to establish apico‐basal polarity and to differentiate in response to prolactin. Downstream of β1‐itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico‐basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue‐specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA‐mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin‐binding guanine nucleotide exchange factor αPix prevented prolactin‐induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation‐specific bifurcation point in β1‐itg‐ILK adhesive signalling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin‐ILK signalling axis for MEC differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:41:06.985668-05:
      DOI: 10.1002/jcp.25390
  • Atp2c2 Is Transcribed from a Unique Transcriptional Start Site in Mouse
           Pancreatic Acinar Cells
    • Authors: Melissa Fenech; Caitlin M. Sullivan, Lucimar T. Ferreira, Rashid Mehmood, William A. MacDonald, Peter B. Stathopulos, Christopher L. Pin
      Abstract: Regulation of cytosolic Ca2+ is critical for pancreatic acinar cell function. Disruptions in normal Ca2+ concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca2+ homeostasis by promoting rapid Ca2+ movement. Determining how expression of Ca2+ modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas‐specific isoform for Secretory pathway Ca2+ ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA‐seq), chromatin immunoprecipitation for epigenetic modifications and promoter‐reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last 4 exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca2+. RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca2+ levels. Combined, these results suggest SPCA2C affects Ca2+ homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca2+ATPases. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:40:45.708336-05:
      DOI: 10.1002/jcp.25391
  • iPSCs, a Future Tool for Therapeutic Intervention in Mitochondrial
           Disorders: Pros and Cons
    • Abstract: Mitochondrial disorders, although individually are rare, taken together constitute a big group of diseases that share a defect in the oxidative phosphorylation system. Up to now, the development of therapies for these diseases is very slow and ineffective due in part to the lack of appropriate disease models. Therefore, there is an urgent need for the discovery of new therapeutic interventions. Regarding this, the generation of induced pluripotent stem cells (iPSCs) has opened new expectations in the regenerative medicine field. However, special cares and considerations must be taken into account previous to a replacement therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:37:01.892538-05:
      DOI: 10.1002/jcp.25386
  • DC‐STAMP: A Key Regulator in Osteoclast Differentiation
    • Abstract: Bone is a dynamic tissue that continuously undergoes remodeling with diverse functions including locomotion, hemopoiesis, and protection of internal organs. Osteoimmunology has recently become an emerging field of musculoskeletal research, which connects the bone and immune systems. Results from osteoimmunology studies provide a new concept that bone is not only an essential component of the musculoskeletal system, but it also participates in immune regulation. Many important factors involved in osteoimmunology regulation also participate in bone homeostasis. Bone homeostasis is achieved by a coordinated action between bone‐synthesizing osteoblasts and bone‐degrading osteoclasts. An imbalanced action between osteoblasts and osteoclasts often results in pathological bone diseases: osteoporosis is caused by an excessive osteoclast activity over that of osteoblasts, whereas osteopetrosis results from an increased osteoblast activity. This review focus on Dendritic Cell‐Specific Transmembrane Protein (DCSTAMP), an important protein currently considered as the master regulator of osteoclastogenesis. DC‐STAMP is required for cell‐cell fusion during osteoclast differentiation. Intriguingly, the frequency of circulating DCSTAMP+ cells is elevated during the pathogenesis of psoriatic diseases. Current data collectively suggest that DC‐STAMP also plays an imperative role in bone homeostasis by regulating the differentiation of both osteoclasts and osteoblasts. This article summarizes our current knowledge on DC‐STAMP by focusing on its interacting proteins, its regulation on osteoclastogenesis‐related genes, its possible involvement in immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐mediated signaling cascade, and its potential of developing therapeutics for clinical applications. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:36:35.866617-05:
      DOI: 10.1002/jcp.25389
  • Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,
           Differentiation and Myotube Hypertrophy
    • Authors: K. van der Meijden; N. Bravenboer, N.F. Dirks, A.C. Heijboer, M. den Heijer, G.M.J. de Wit, C. Offringa, P. Lips, R.T. Jaspers
      Abstract: An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2D3 and 25(OH)D3 affect myoblast proliferation, differentiation and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2D3. We showed that myoblasts not only respond to 1,25(OH)2D3, but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2D3 as well as 25(OH)D3 stimulate VDR mRNA expression and in myotubes 1,25(OH)2D3 also stimulates MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes express CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2D3. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:36:11.821671-05:
      DOI: 10.1002/jcp.25388
  • Quantification of Exosomes
    • Authors: E. H. Koritzinsky; J. M. Street, R. A. Star, P.S.T. Yuen
      Abstract: Exosomes are released by cells as self‐contained vesicles with an intact lipid bilayer that encapsulates a small portion of the parent cell. Exosomes have been studied widely as information‐rich sources of potential biomarkers that can reveal cellular physiology. We suggest that quantification is essential to understand basic biological relationships between exosomes and their parent cells and hence the underlying interpretation of exosome signals. The number of methods for quantifying exosomes has expanded as interest in exosomes has increased. However, a consensus on proper quantification has not developed, making each study difficult to compare to another. Overcoming this ad hoc approach will require widely available standards that have been adequately characterized, and multiple comparative studies across platforms. We outline the current status of these technical approaches and our view of how they can become more coherent. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:35:45.891505-05:
      DOI: 10.1002/jcp.25387
  • Stiffness of Intact Endothelial Cells from Fresh Aortic Bifurcations of
           Atherosclerotic Rabbits ‐ Atomic Force Microscopic Study
    • Authors: Kozaburo Hayashi; Michitaka Higaki
      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
  • L‐glutamine Improves Skeletal Muscle Cell Differentiation and
           Prevents Myotube Atrophy after Cytokine (TNF‐α) Stress via
           Reduced p38 MAPK Signal Transduction
    • Authors: Matthew Girven; Hannah F. Dugdale, Daniel J. Owens, David C. Hughes, Claire E. Stewart, Adam P. Sharples
      Abstract: Tumour Necrosis Factor‐ Alpha (TNF‐α) is chronically elevated in conditions where skeletal muscle loss occurs. As L‐glutamine can dampen the effects of inflamed environments, we investigated the role of L‐glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF‐α (20−1) ± L‐glutamine (20 mM).TNF‐α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf‐I and Igfbp5. Furthermore, when administered to mature myotubes, TNF‐α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf‐I, Igfbp2 and glutamine synthetase and parallel increases in Fox03, Cfos, p53 and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non‐significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF‐α. Importantly, 20 mM L‐glutamine reduced p38 MAPK activity in TNF‐α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. L‐glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf‐Ir, Myhc2 & 7, Tnfsfr1b, Adra1d and restored atrophic gene expression of Fox03 back to baseline in TNF‐α conditions. In conclusion, L‐glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. L‐glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:42:28.208974-05:
      DOI: 10.1002/jcp.25380
  • XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of
           Telomeres in Tumor Cells
    • Abstract: Previous work has shown that the three‐dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT‐185, KPT‐330/selinexor, and KPT‐8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment‐naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non‐lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:42:03.802445-05:
      DOI: 10.1002/jcp.25378
  • Kynurenine Modulates MMP‐1 and Type‐I Collagen Expression via
           Aryl Hydrocarbon Receptor Activation in Dermal Fibroblasts
    • Abstract: Dermal fibrosis is characterized by a high deposition of extracellular matrix (ECM) and tissue cellularity. Unfortunately all means of treating this condition are unsatisfactory. We have previously reported the anti‐fibrotic effects of Kynurenine (Kyn), a tryptophan metabolite, in fibrotic rabbit ear model. Here, we report the mechanism by which Kyn modulates the expression of key ECM components in dermal fibroblasts. The results showed that Kyn activates aryl hydrocarbon receptor (AHR) nuclear translocation and up‐regulates cytochrome‐P450 (CYP1A‐1) expression, the AHR target gene. A specific AHR antagonist, 6,2′,4′‐trimethoxyflavone, inhibited the Kyn‐dependent modulation of CYP1A‐1, MMP‐1 and type‐I collagen expression. Establishing the anti‐fibrogenic effect of Kyn and its mechanism of action, we then developed nano‐fibrous Kyn slow‐releasing dressings and examined their anti‐fibrotic efficacy in vitro and in a rat model. Our results showed the feasibility of incorporating Kyn into PVA/PLGA nanofibers, prolonging the Kyn release up to 4 days tested. Application of medicated‐dressings significantly improved the dermal fibrosis indicated by MMP‐1 induction, alpha‐smooth muscle actin and type‐I collagen suppression, and reduced tissue cellularity, T‐cells and myofibroblasts. This study clarifies the mechanism by which Kyn modulates ECM expression and reports the development of a new slow‐releasing anti‐fibrogenic dressing. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:40:42.51292-05:0
      DOI: 10.1002/jcp.25383
  • Generation of Human Lens Epithelial‐Like Cells from
           Patient‐Specific Induced Pluripotent Stem Cells
    • Authors: Dan Li; Xiaodi Qiu, Jin Yang, Tianjin Liu, Yi Luo, Yi Lu
      Abstract: Cataractogenesis begins from the dynamic lens epithelial cells (LECs) and adjacent fiber cells. LECs derived from cell lines cannot maintain the crystalline expression as the primary LECs. The current study aimed to efficiently generate large numbers of human LECs from patient‐specific induced pluripotent stem cells (iPSCs). Anterior lens capsules were collected from cataract surgery and were used to culture primary hLECs. iPSCs were induced from these primary hLECs by lentiviral transduction of Oct4, Sox2, Klf4 and c‐Myc. Then, the generated iPSCs were re‐differentiated into hLECs by the 3‐step addition of defined factor combinations (Noggin, BMP4/7, bFGF and EGF) modified from an established method. During the re‐differentiation process, colonies of interest were isolated using a glass picking tool and cloning cylinders based on the colony morphology. After two steps of isolation, populations of LEC‐like cells (LLCs) were generated and identified by the expression of lens marker genes by qPCR, western blot and immunofluorescence staining. The study introduced a modified protocol to isolate LLCs from iPSCs by defined factors in a short time frame. This technique could be useful for mechanistic studies of lens‐related diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-15T02:56:45.612403-05:
      DOI: 10.1002/jcp.25374
  • Serum from Advanced Heart Failure Patients Promotes Angiogenic Sprouting
           and Affects the Notch Pathway in Human Endothelial Cells
    • Authors: M Pannella; C Caliceti, F Fortini, G Aquila, F Vieceli Dalla Sega, A Pannuti, C Fortini, MB Morelli, A Fucili, G Francolini, R Voltan, P Secchiero, G Dinelli, E Leoncini, M Ferracin, S Hrelia, L Miele, P Rizzo
      Abstract: It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined 1) sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel and 2) protein levels of Notch receptors 1, 2, 4 and ligands Jagged1, Delta‐like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT‐proBNP (N‐terminal of the pro‐hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera‐ treated cells (r = ‐0.526 and r = ‐0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-14T08:12:10.748909-05:
      DOI: 10.1002/jcp.25373
  • Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis
           via the PI3K/AKT and ERK1/2 MAPK Pathways
    • Authors: Whasun Lim; Sunwoo Park, Fuller W. Bazer, Gwonhwa Song
      Abstract: Apigenin is a flavonoid found in parsley, onions, oranges, tea, chamomile, wheat and sprouts. It has a variety of biological properties including anti‐oxidant, anti‐mutagenic, anti‐carcinogenic, anti‐inflammatory, anti‐proliferative and anti‐spasmodic effects. Based on epidemiological and case‐control studies, apigenin is regarded as a novel chemotherapeutic agent against various cancer types. However, little is known about the effects of apigenin on choriocarcinoma cells. Therefore, we investigated the anti‐cancer effects of apigenin on choriocarcinoma cells (JAR and JEG3) in the present study. Apigenin reduced viability and migratory properties, increased apoptosis, and suppressed mitochondrial membrane potential in both the JAR and JEG3 cells. In addition, apigenin predominantly decreased phosphorylation of AKT, P70RSK and S6 whereas the phosphorylation of ERK1/2 and P90RSK was increased by apigenin treatment of JAR and JEG3 cells in a dose‐dependent manner. Moreover, treatment of JAR and JEG3 cells with both apigenin and pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126) revealed synergistic anti‐proliferative effects. Collectively, these results indicated that the apigenin is an invaluable chemopreventive agent that inhibits progression and metastasis of choriocarcinoma cells through regulation of PI3K/AKT and ERK1/2 MAPK signal transduction mechanism. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-11T02:43:28.687733-05:
      DOI: 10.1002/jcp.25372
  • The Impact of Epigenetics on Mesenchymal Stem Cell Biology
    • Authors: Yusuf Ozkul; Umberto Galderisi
      Abstract: Changes in epigenetic marks are known to be important regulatory factors in stem cell fate determination and differentiation. In the past years, the investigation of the epigenetic regulation of stem cell biology has largely focused on embryonic stem cells (ESCs). Contrarily, less is known about the epigenetic control of gene expression during differentiation of adult stem cells (AdSCs). Among AdSCs, mesenchymal stem cells (MSCs) are the most investigated stem cell population because of their enormous potential for therapeutic applications in regenerative medicine and tissue engineering. In this review we analyze the main studies addressing the epigenetic changes in MSC landscape during in vitro cultivation and replicative senescence, as well as follow osteocyte, chondrocyte, and adipocyte differentiation. In these studies, histone acetylation, DNA methylation, and miRNA expression are among the most investigated phenomena. We describe also epigenetic changes that are associated with in vitro MSC trans‐differentiation. Although at the at initial stage, the epigenetics of MSCs promise to have profound implications for stem cell basic and applied research. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-09T09:00:56.097947-05:
      DOI: 10.1002/jcp.25371
  • Alternative Start Codon Connects eIF5A to Mitochondria
    • Abstract: Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N‐terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N‐terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co‐purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-08T06:02:49.087117-05:
      DOI: 10.1002/jcp.25370
  • Phospholipidomic Profile Variation on THP‐1 Cells Exposed to Skin or
           Respiratory Sensitizers and Respiratory Irritant
    • Abstract: Occupational exposure to low molecular weight reactive chemicals often leads to development of allergic reactions such as allergic contact dermatitis and respiratory allergies. Further insights into the interaction of these chemicals with physiopathological relevant cellular models might provide the foundations for novel non‐animal approaches to safety assessment. In this work we used the human THP‐1 cell line to determine phospholipidome changes induced by the skin sensitizer 1‐fluoro‐2,4‐dinitrobenzene (DNFB), the respiratory allergen hexamethylene diisocyanate (HDI) and the irritant methyl salicylate (MESA). We detected that these chemicals differently induce lipid peroxidation and modulate THP‐1 IL‐1β, IL‐12B, IL‐8, CD86 and HMOX1 transcription. Decreased phosphatidylethanolamine content was detected in cells exposed to MESA, while profound alterations in the relative abundance of cardiolipin species were observed in cells exposed to DNFB. All chemicals tested induced a decrease in the relative abundance of plasmanyl phosphatidylcholine species PC (O‐16:0e/18:1) and phosphatidylinositol species PI (34:1), while increasing PI (38:4). An increased abundance of oleic acid was observed in the phospholipids of cells exposed to DNFB while a decreased abundance of palmitic acid was detected in cells treated with MESA or DNFB. We conclude that both specific and common alterations at phospholipidome levels are triggered by the different chemicals, while not allowing a complete distinction between them using a Canonical Analysis of Principal Coordinates (CAP). The common effects observed at phospholipids level with all the chemicals tested might be related to unspecific cell cytotoxic mechanisms that nevertheless may contribute to the elicitation of specific immune responses. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-04T09:06:24.071788-05:
      DOI: 10.1002/jcp.25365
  • The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion
           and Fenestrations
    • Abstract: In eukaryotes, the nuclear envelope physically separates nuclear components and activities from rest of the cell. The nuclear envelope also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the nuclear envelope is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the nuclear envelope of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and expands asymmetrically a mitotic delay. Moreover, its nuclear envelope is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast nuclear envelope lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast nuclear envelope have uncovered interesting, and likely conserved, processes that contribute to nuclear envelope dynamics. In particular, we discuss the processes that drive and enable nuclear envelope expansion and the dramatic changes in the nuclear envelope that lead to extensions and fenestrations. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-22T17:34:06.862231-05:
      DOI: 10.1002/jcp.25355
  • Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer
    • Authors: Yuhui Yang; Svetlana Karakhanova, Werner Hartwig, Jan G. D'Haese, Pavel P. Philippov, Jens Werner, Alexandr V. Bazhin
      Abstract: Mitochondria are indispensable for energy metabolism, apoptosis regulation and cell signaling. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and participate actively in metabolic reprogramming. Mitochondria in cancer cells are characterized by reactive oxygen species (ROS) overproduction, which promotes cancer development by inducing genomic instability, modifying gene expression and participating in signaling pathways. Mitochondrial and nuclear DNA mutations caused by oxidative damage that impair the oxidative phosphorylation process will result in further mitochondrial ROS production, completing the “vicious cycle” between mitochondria, ROS, genomic instability and cancer development. The multiple essential roles of mitochondria have been utilized for designing novel mitochondria‐targeted anticancer agents. Selective drug delivery to mitochondria helps to increase specificity and reduce toxicity of these agents. In order to reduce mitochondrial ROS production, mitochondria‐targeted antioxidants can specifically accumulate in mitochondria by affiliating to a lipophilic penetrating cation and prevent mitochondria from oxidative damage. In consistence with the oncogenic role of ROS, mitochondria‐targeted antioxidants are found to be effective in cancer prevention and anticancer therapy. A better understanding of the role played by mitochondria in cancer development will help to reveal more therapeutic targets, and will help to increase the activity and selectivity of mitochondria‐targeted anticancer drugs. In this review we summarized the impact of mitochondria on cancer and gave summary about the possibilities to target mitochondria for anticancer therapies. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-19T20:43:18.622087-05:
      DOI: 10.1002/jcp.25349
  • A Novel Biological Role of α‐Mangostin in Modulating
           Inflammatory Response through the Activation of SIRT‐1 Signaling
    • Authors: Sara Franceschelli; Mirko Pesce, Alessio Ferrone, Antonia Patruno, Pasqualone Livia, Giuseppe Carlucci, Vincenzo Ferrone, Maura Carlucci, Maria Anna De Lutiis, Alfredo Grilli, Mario Felaco, Lorenza Speranza
      Abstract: Several studies have shown that xanthones obtained from Garcinia Mangostana (GM) have remarkable biological activities. α‐mangostin (α‐MG) is the main constituent of the fruit hull of the GM. Several findings have suggested that SIRT‐1, a nuclear histone deacetylase, could influence cellular function by the inhibition of NF‐kB signaling. ROS can inhibit SIRT‐1 activity by initiating oxidative modifications on its cysteine residues, and suppression of SIRT‐1 enhances the NF‐κB signaling resulting in inflammatory responses. The goals of the present study were to evaluate the quantity of α‐MG in the methanolic extract of GM (Vithagroup Spa) and to investigate the activity of this xanthone in U937 cell line and in human monocytes from responsive to inflammatory insult analyzing the possible changes on the activation of SIRT‐1 protein via NF‐Kb. Cells were treated with the methanolic extract of GM and/or LPS. The chromatographic separation of α‐MG was performed by an HPLC analysis. EX 527, a specific SIRT‐1 inhibitor, was used to determine if SIRT‐1/NfkB signaling pathway might be involved in α‐MG action on cells. Our results show that α‐MG inhibits p65 acetylation and down‐regulates the pro‐inflammatory gene products as COX‐2, iNOS via SIRT‐1 activation. Cells treated with EX 527 showed an up‐regulation of NFkB acetylation and an over expression of inducible enzymes and their product of catalysis (NO and PGE2). These results suggest that α‐MG may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-19T20:42:29.518255-05:
      DOI: 10.1002/jcp.25348
  • A New In Vitro Co‐Culture Model Using Magnetic Force‐Based
    • Abstract: Background Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in‐vitro model of myoblast transplantation with “patterned” or “randomly‐mixed” co‐culture of SkMBs and cardiomyocytes enabling subsequent electrophysiological and arrhythmogenic evaluation. Methods SkMBs were magnetically labeled with magnetite nanoparticles and co‐cultured with neonatal rat ventricular myocytes (NRVMs) on multi‐electrode arrays. SkMBs were patterned by a magnet beneath the arrays. Excitation synchronicity was evaluated by Ca2+ imaging using a gene‐encoded Ca2+ indicator, G‐CaMP2. Results In the monoculture of NRVMs (control), conduction was well‐organized. In the randomly‐mixed co‐culture of NRVMs and SkMBs (random group), there was inhomogeneous conduction from multiple origins. In the “patterned” co‐culture where an en bloc SKMB‐layer was inserted into the NRVM‐layer, excitation homogenously propagated although conduction was distorted by the SkMB‐area. The 4‐mm distance conduction time (CT) in the random group was significantly longer (197 ± 126 ms) than in control (17 ± 3 ms). In the patterned group, CT through NRVM‐area did not change (25 ± 3 ms), although CT through the SkMB‐area was significantly longer (132 ± 77 ms). The intervals between spontaneous excitation varied beat‐to‐beat in the random group, while regular beating was recorded in the control and patterned groups. Synchronized Ca2+ transients of NRVMs were observed in the patterned group, whereas those in the random group were asynchronous. Conclusions Patterned alignment of SkMBs is feasible with magnetic nanoparticles. Using the novel in‐vitro model mimicking cell transplantation, it may become possible to predict arrhythmogenicity due to heterogenous cell transplantation. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-12T20:55:31.564092-05:
      DOI: 10.1002/jcp.25342
  • Table of Contents, Editor's Choice, Highlights
    • Pages: 2065 - 2070
      PubDate: 2016-06-17T09:29:19.624234-05:
      DOI: 10.1002/jcp.25418
  • Ubiquitin‐Related Roles of β‐Arrestins in Endocytic
           Trafficking and Signal Transduction
    • Pages: 2071 - 2080
      Abstract: The non‐visual arrestins, β‐arrestin1, and β‐arrestin2 were originally identified as proteins that bind to seven‐transmembrane receptors (7TMRs, also called G protein‐coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non‐receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non‐receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β‐arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β‐arrestins in protein ubiquitination is evolutionarily conserved. β‐arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β‐arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β‐arrestin plays a critical role in stabilizing β‐arrestin‐7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin‐dependent regulation of 7TMRs as well as β‐arrestins and the potential role of reversible ubiquitination as a “biological switch” in signal transduction. J. Cell. Physiol. 231: 2071–2080, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-03T03:46:58.859088-05:
      DOI: 10.1002/jcp.25317
  • Metabolic Modification in Gastrointestinal Cancer Stem Cells:
           Characteristics and Therapeutic Approaches
    • Authors: Angela Maria Di Francesco; Amelia Toesca, Carlo Cenciarelli, Antonio Giordano, Antonio Gasbarrini, Maria Ausiliatrice Puglisi
      Pages: 2081 - 2087
      Abstract: Currently, there is much interest in the characterization of metabolic profiling of cancer stem cells (CSCs), a small subset of tumor cells with self‐renewal capacity. Indeed, ever‐growing evidence indicate that metabolism and stemness are highly intertwined processes in tumor tissue. In this review, we analyze the potential metabolic targeting strategies for eradicating CSCs that could help to develop a more effective therapeutic approach for gastrointestinal cancers. Indeed, the successful elimination of a tumor requires an anticancer therapy that affects both cancer cells and CSCs. The observation that gastrointestinal CSCs possess higher inducible nitric oxide sinthase (iNOS) expression, lower reactive oxygen species (ROS) production, and a different metabolism respect to no‐CSCs tumor cells has paved the way to develop drugs targeting CSC specific signaling. In particular, several studies have highlighted that metformin, aldehyde dehydrogenase 1, and iNOS inhibitors selectively suppressed CSC growth and that combinatorial therapy of them with standard chemotherapeutic drugs had a synergistic effect resulting in reduced tumor burden and delayed tumor recurrence. Thus, the possibility of combining specific CSC metabolism inhibitors with existing therapeutic approaches could have profound anticancer effects, changing the conventional treatment approaches to gastrointestinal cancers. J. Cell. Physiol. 231: 2081–2087, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-02T04:58:40.772889-05:
      DOI: 10.1002/jcp.25318
  • PERK Integrates Oncogenic Signaling and Cell Survival During Cancer
    • Authors: Yiwen Bu; J. Alan Diehl
      Pages: 2088 - 2096
      Abstract: Unfolded protein responses (UPR), consisting of three major transducers PERK, IRE1, and ATF6, occur in the midst of a variety of intracellular and extracellular challenges that perturb protein folding in the endoplasmic reticulum (ER). ER stress occurs and is thought to be a contributing factor to a number of human diseases, including cancer, neurodegenerative disorders, and various metabolic syndromes. In the context of neoplastic growth, oncogenic stress resulting from dysregulation of oncogenes such as c‐Myc, BrafV600E, and HRASG12V trigger the UPR as an adaptive strategy for cancer cell survival. PERK is an ER resident type I protein kinase harboring both pro‐apoptotic and pro‐survival capabilities. PERK, as a coordinator through its downstream substrates, reprograms cancer gene expression to facilitate survival in response to oncogenes and microenvironmental challenges, such as hypoxia, angiogenesis, and metastasis. Herein, we discuss how PERK kinase engages in tumor initiation, transformation, adaption microenvironmental stress, chemoresistance and potential opportunities, and potential opportunities for PERK targeted therapy. J. Cell. Physiol. 231: 2088–2096, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-03-06T21:54:30.97112-05:0
      DOI: 10.1002/jcp.25336
  • MicroRNA Regulation of Acute Lung Injury and Acute Respiratory Distress
    • Authors: Subbiah Rajasekaran; Dhamotharan Pattarayan, P. Rajaguru, P. S. Sudhakar Gandhi, Rajesh K. Thimmulappa
      Pages: 2097 - 2106
      Abstract: The acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI), is a very common condition associated with critically ill patients, which causes substantial morbidity and mortality worldwide. Despite decades of research, effective therapeutic strategies for clinical ALI/ARDS are not available. In recent years, microRNAs (miRNAs), small non‐coding molecules have emerged as a major area of biomedical research as they post‐transcriptionally regulate gene expression in diverse biological and pathological processes, including ALI/ARDS. In this context, this present review summarizes a large body of evidence implicating miRNAs and their target molecules in ALI/ARDS originating largely from studies using animal and cell culture model systems of ALI/ARDS. We have also focused on the involvement of miRNAs in macrophage polarization, which play a critical role in regulating the pathogenesis of ALI/ARDS. Finally, the possible future directions that might lead to novel therapeutic strategies for the treatment of ALI/ARDS are also reviewed. J. Cell. Physiol. 231: 2097–2106, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-04T04:19:00.353386-05:
      DOI: 10.1002/jcp.25316
  • Zebrafish as a Model for the Study of Chaperonopathies
    • Authors: Gianfranco Bellipanni; Francesco Cappello, Federica Scalia, Everly Conway de Macario, Alberto J.L. Macario, Antonio Giordano
      Pages: 2107 - 2114
      Abstract: There is considerable information on the clinical manifestations and mode of inheritance for many genetic chaperonopathies but little is known on the molecular mechanisms underlying the cell and tissue abnormalities that characterize them. This scarcity of knowledge is mostly due to the lack of appropriate animal models that mimic closely the human molecular, cellular, and histological characteristics. In this article we introduce zebrafish as a suitable model to study molecular and cellular mechanisms pertaining to human chaperonopathies. Genetic chaperonopathies manifest themselves from very early in life so it is necessary to examine the impact of mutant chaperone genes during development, starting with fertilization and proceeding throughout the entire ontogenetic process. Zebrafish is amenable to such developmental analysis as well as studies during adulthood. In addition, the zebrafish genome contains a wide range of genes encoding proteins similar to those that form the chaperoning system of humans. This, together with the availability of techniques for genetic manipulations and for examination of all stages of development, makes zebrafish the organism of choice for the analysis of the molecular features and pathogenic mechanisms pertaining to human chaperonopathies. J. Cell. Physiol. 231: 2107–2114, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-16T05:00:47.942532-05:
      DOI: 10.1002/jcp.25319
  • Emerging From the Unknown: Structural and Functional Features of
           Agnoprotein of Polyomaviruses
    • Pages: 2115 - 2127
      Abstract: Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV, and SV40. In the absence of its expression, these viruses are unable to sustain their productive life cycle. It is a highly basic phosphoprotein that localizes mostly to the perinuclear area of infected cells, although a small amount of the protein is also found in nucleus. Much has been learned about the structure and function of this important regulatory protein in recent years. It forms highly stable dimers/oligomers in vitro and in vivo through its Leu/Ile/Phe‐rich domain. Structural NMR studies revealed that this domain adopts an alpha‐helix conformation and plays a critical role in the stability of the protein. It associates with cellular proteins, including YB‐1, p53, Ku70, FEZ1, HP1α, PP2A, AP‐3, PCNA, and α‐SNAP; and viral proteins, including small t antigen, large T antigen, HIV‐1 Tat, and JCV VP1; and significantly contributes the viral transcription and replication. This review summarizes the recent advances in the structural and functional properties of this important regulatory protein. J. Cell. Physiol. 231: 2115–2127, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-24T04:37:38.216046-05:
      DOI: 10.1002/jcp.25329
  • Fatty Acids and Breast Cancer: Make Them on Site or Have Them Delivered
    • Authors: William B. Kinlaw; Paul W. Baures, Leslie E. Lupien, Wilson L. Davis, Nancy B. Kuemmerle
      Pages: 2128 - 2141
      Abstract: Brisk fatty acid (FA) production by cancer cells is accommodated by the Warburg effect. Most breast and other cancer cell types are addicted to fatty acids (FA), which they require for membrane phospholipid synthesis, signaling purposes, and energy production. Expression of the enzymes required for FA synthesis is closely linked to each of the major classes of signaling molecules that stimulate BC cell proliferation. This review focuses on the regulation of FA synthesis in BC cells, and the impact of FA, or the lack thereof, on the tumor cell phenotype. Given growing awareness of the impact of dietary fat and obesity on BC biology, we will also examine the less‐frequently considered notion that, in addition to de novo FA synthesis, the lipolytic uptake of preformed FA may also be an important mechanism of lipid acquisition. Indeed, it appears that cancer cells may exist at different points along a “lipogenic‐lipolytic axis,” and FA uptake could thwart attempts to exploit the strict requirement for FA focused solely on inhibition of de novo FA synthesis. Strategies for clinically targeting FA metabolism will be discussed, and the current status of the medicinal chemistry in this area will be assessed. J. Cell. Physiol. 231: 2128–2141, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-16T04:51:51.623433-05:
      DOI: 10.1002/jcp.25332
  • Polyglucosan Molecules Induce Mitochondrial Impairment and Apoptosis in
           Germ Cells Without Affecting the Integrity and Functionality of Sertoli
    • Pages: 2142 - 2152
      Abstract: Glycogen is the main storage form of glucose; however, the accumulation of glycogen‐like glucose polymers can lead to degeneration and cellular death. Previously, we reported that the accumulation of glycogen in testis of transgenic animals overexpressing a constitutively active form of glycogen synthase enhances the apoptosis of pre‐meiotic male germ cells and a complete disorganization of the seminiferous tubules. Here we sought to further identify the effects of glycogen storage in cells from the seminiferous tubules and the mechanism behind the pro‐apoptotic activity induced by its accumulation. Using an in vitro culture of Sertoli cells (line 42GPA9) and spermatocyte‐like cells (line GC‐1) expressing a superactive form of glycogen synthase or the Protein Targeting to Glycogen (PTG), we found that glycogen synthesized in both cell lines is poorly branched. In addition, the immunodetection of key molecules of apoptotic events suggests that cellular death induced by polyglucosan molecules affects GC‐1 cells, but not 42GPA9 cells by mitochondrial impairment and activation of an intrinsic apoptotic pathway. Furthermore, we analyzed the effects of glycogen deposition during the establishment of an in vitro blood‐testis barrier. The results using a non‐permeable fluorescent molecule showed that, in conditions of over‐synthesis of glycogen, 42GPA9 cells do not lose their capacity to generate an impermeable barrier and the levels of connexin43, occludin, and ZO1 proteins were not affected. These results suggest that the accumulation of polyglucosan molecules has a selective effect—triggered by the intrinsic activation of the apoptotic pathway—in germ cells without directly affecting Sertoli cells. J. Cell. Physiol. 231: 2142–2152, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-11T05:33:36.805914-05:
      DOI: 10.1002/jcp.25315
  • Corticosteroid‐Induced MKP‐1 Represses Pro‐Inflammatory
           Cytokine Secretion by Enhancing Activity of Tristetraprolin (TTP) in ASM
    • Authors: Pavan Prabhala; Kristin Bunge, Qi Ge, Alaina J. Ammit
      Pages: 2153 - 2158
      Abstract: Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti‐inflammatory pharmacotherapies, including corticosteroids, are front‐line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their actions are not fully understood. The corticosteroid‐inducible gene, mitogen‐activated protein kinase (MAPK) phosphatase 1 (MKP‐1, DUSP1) has emerged as a key molecule responsible for the repressive effects of steroids. MKP‐1 is known to deactivate p38 MAPK phosphorylation and can control the expression and activity of the mRNA destabilizing protein—tristetraprolin (TTP). But whether corticosteroid‐induced MKP‐1 acts via p38 MAPK‐mediated modulation of TTP function in a pivotal airway cell type, airway smooth muscle (ASM), was unknown. While pretreatment of ASM cells with the corticosteroid dexamethasone (preventative protocol) is known to reduce ASM synthetic function in vitro, the impact of adding dexamethasone after stimulation (therapeutic protocol) had not been explored. Whether dexamethasone modulates TTP in a p38 MAPK‐dependent manner in this cell type was also unknown. We address this herein and utilize an in vitro model of asthmatic inflammation where ASM cells were stimulated with the pro‐asthmatic cytokine tumor necrosis factor (TNF) and the impact of adding dexamethasone 1 h after stimulation assessed. IL‐6 mRNA expression and protein secretion was significantly repressed by dexamethasone acting in a temporally distinct manner to increase MKP‐1, deactivate p38 MAPK, and modulate TTP phosphorylation status. In this way, dexamethasone‐induced MKP‐1 acts via p38 MAPK to switch on the mRNA destabilizing function of TTP to repress pro‐inflammatory cytokine secretion from ASM cells. J. Cell. Physiol. 231: 2153–2158, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-16T04:53:36.954813-05:
      DOI: 10.1002/jcp.25327
  • Bee Venom Accelerates Wound Healing in Diabetic Mice by Suppressing
           Activating Transcription Factor‐3 (ATF‐3) and Inducible Nitric
           Oxide Synthase (iNOS)‐Mediated Oxidative Stress and Recruiting Bone
           Marrow‐Derived Endothelial Progenitor Cells
    • Authors: Gamal Badr; Wael N. Hozzein, Badr M. Badr, Ahmad Al Ghamdi, Heba M. Saad Eldien, Olivier Garraud
      Pages: 2159 - 2171
      Abstract: Multiple mechanisms contribute to impaired diabetic wound healing including impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Bee venom (BV) has been used as an anti‐inflammatory agent for the treatment of several diseases. Nevertheless, the effect of BV on the healing of diabetic wounds has not been studied. Therefore, in this study, we investigated the impact of BV on diabetic wound closure in a type I diabetic mouse model. Three experimental groups were used: group 1, non‐diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice treated with BV. We found that the diabetic mice exhibited delayed wound closure characterized by a significant decrease in collagen production and prolonged elevation of inflammatory cytokines levels in wounded tissue compared to control non‐diabetic mice. Additionally, wounded tissue in diabetic mice revealed aberrantly up‐regulated expression of ATF‐3 and iNOS followed by a marked elevation in free radical levels. Impaired diabetic wound healing was also characterized by a significant elevation in caspase‐3, ‐8, and ‐9 activity and a marked reduction in the expression of TGF‐β and VEGF, which led to decreased neovascularization and angiogenesis of the injured tissue by impairing EPC mobilization. Interestingly, BV treatment significantly enhanced wound closure in diabetic mice by increasing collagen production and restoring the levels of inflammatory cytokines, free radical, TGF‐β, and VEGF. Most importantly, BV‐treated diabetic mice exhibited mobilized long‐lived EPCs by inhibiting caspase activity in the wounded tissue. Our findings reveal the molecular mechanisms underlying improved diabetic wound healing and closure following BV treatment. J. Cell. Physiol. 231: 2159–2171, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-02-15T05:49:23.11395-05:0
      DOI: 10.1002/jcp.25328
  • First Evidence of DAAM1 Localization During the Post‐Natal
           Development of Rat Testis and in Mammalian Sperm
    • Authors: Paolo Pariante; Raffaele Dotolo, Massimo Venditti, Diana Ferrara, Aldo Donizetti, Francesco Aniello, Sergio Minucci
      Pages: 2172 - 2184
      Abstract: Dishevelled‐associated activator of morphogenesis 1 (DAAM1) is a formin‐family protein involved in nucleation of unbranched actin filaments and in cytoskeletal organization through Wnt‐Dishevelled PCP pathway, which participates in essential biological processes, such as cell polarity, movement, and adhesion during morphogenesis and organogenesis. While its role has been investigated during development and in somatic cells, its potential association with the germinal compartment and reproduction is still unexplored. In this work, we assessed the possible association of DAAM1 with the morphogenesis of rat testis. We studied its expression and profiled its localization versus actin and tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, DAAM1 shares its localization with actin in Sertoli cells, gonocytes, and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, while only actin is detectable at the forming blood‐testis barrier. DAAM1, then, follows the development of the acrosome system throughout spermiogenesis, and it is finally retained inside the cytoplasmic droplet in mature gametes, as corroborated by additional immunolocalization data on both rat and human sperm. Unlike the DAAM1, actin keeps its localization in Sertoli cells, and tubulin is associated with their protruding cytoplasm during the process. Our data support, for the first time, the hypothesis of a role for DAAM1 in cytoskeletal organization during Mammalian testis morphogenesis and gamete progression, while also hinting at its possible investigation as a morphological marker of germ cell and sperm physiology. J. Cell. Physiol. 231: 2172–2184, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-03-06T21:52:12.075052-05:
      DOI: 10.1002/jcp.25330
  • Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound
           Healing Process
    • Authors: Cristina La Torre; Benedetta Cinque, Francesca Lombardi, Gianfranca Miconi, Paola Palumbo, Zoran Evtoski, Giuseppe Placidi, Donatella Fanini, Anna Maria Cimini, Elisabetta Benedetti, Maurizio Giuliani, Maria Grazia Cifone
      Pages: 2185 - 2195
      Abstract: An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S‐nitroso‐N‐acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analyzed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0–28 h) and the wound area was analyzed through software included in Matlab®. As early as 15 min, SNAP induced significant cytoskeletal remodeling, as shown by immunostaining (phalloidin‐labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki‐67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP‐induced migration and proliferation were antagonized by the NO‐sensitive GC inhibitor 1H‐[1,2,4]oxadiazolo[‐4,3‐a]quinoxalin‐1‐one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP‐induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR‐β antagonist (GSK0660) may interfere with NO‐mediated wound healing process. J. Cell. Physiol. 231: 2185–2195, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-03-06T21:55:44.451025-05:
      DOI: 10.1002/jcp.25331
  • Dynamic Histone Acetylation of H3K4me3 Nucleosome Regulates MCL1
           Pre‐mRNA Splicing
    • Authors: Dilshad H. Khan; Carolina Gonzalez, Nikesh Tailor, Mohammad K. Hamedani, Etienne Leygue, James R. Davie
      Pages: 2196 - 2204
      Abstract: Pre‐mRNA splicing is a cotranscriptional process affected by the chromatin architecture along the body of coding genes. Recruited to the pre‐mRNA by splicing factors, histone deacetylases (HDACs) and K‐acetyltransferases (KATs) catalyze dynamic histone acetylation along the gene. In colon carcinoma HCT 116 cells, HDAC inhibition specifically increased KAT2B occupancy as well as H3 and H4 acetylation of the H3K4 trimethylated (H3K4me3) nucleosome positioned over alternative exon 2 of the MCL1 gene, an event paralleled with the exclusion of exon 2. These results were reproduced in MDA‐MB‐231, but not in MCF7 breast adenocarcinoma cells. These later cells have much higher levels of demethylase KDM5B than either HCT 116 or MDA‐MB‐231 cells. We show that H3K4me3 steady‐state levels and H3K4me3 occupancy at the end of exon 1 and over exon 2 of the MCL1 gene were lower in MCF7 than in MDA‐MB‐231 cells. Furthermore, in MCF7 cells, there was minimal effect of HDAC inhibition on H3/H4 acetylation and H3K4me3 levels along the MCL1 gene and no change in pre‐mRNA splicing choice. These results show that, upon HDAC inhibition, the H3K4me3 mark plays a critical role in the exclusion of exon 2 from the MCL1 pre‐mRNA. J. Cell. Physiol. 231: 2196–2204, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-03-06T21:52:32.32712-05:0
      DOI: 10.1002/jcp.25337
  • Phosphorylation Regulates Functions of ZEB1 Transcription Factor
    • Authors: M. Candelaria Llorens; Guadalupe Lorenzatti, Natalia L. Cavallo, Maria V. Vaglienti, Ana P. Perrone, Anne L. Carenbauer, Douglas S. Darling, Ana M. Cabanillas
      Pages: 2205 - 2217
      Abstract: ZEB1 transcription factor is important in both development and disease, including many TGFβ‐induced responses, and the epithelial‐to‐mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA‐binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF‐1) can inhibit transcriptional repression by either a ZEB1‐ZD2 domain clone, or full‐length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA‐binding activity of ZEB1. Immunoprecipitation with anti‐ZEB1 antibodies followed by western analysis with a phospho‐Threonine‐Proline‐specific antibody indicates that the ERK consensus site at Thr‐867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA‐binding measured by EMSA, IGF‐1‐induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP‐ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF‐1. J. Cell. Physiol. 231: 2205–2217, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-03-10T04:15:51.849458-05:
      DOI: 10.1002/jcp.25338
  • Lack of Dystrophin Affects Bronchial Epithelium in mdx Mice
    • Pages: 2218 - 2223
      Abstract: Mild exercise training may positively affect the course of Duchenne Muscular Dystrophy (DMD). Training causes mild bronchial epithelial injury in both humans and mice, but no study assessed the effects of exercise in mdx mice, a well known model of DMD. The airway epithelium was examined in mdx (C57BL/10ScSn‐Dmdmdx) mice, and in wild type (WT, C57BL/10ScSc) mice either under sedentary conditions (mdx‐SD, WT‐SD) or during mild exercise training (mdx‐EX, WT‐EX). At baseline, and after 30 and 45 days of training (5 d/wk for 6 weeks), epithelial morphology and markers of regeneration, apoptosis, and cellular stress were assessed. The number of goblet cells in bronchial epithelium was much lower in mdx than in WT mice under all conditions. At 30 days, epithelial regeneration (PCNA positive cells) was higher in EX than SD animals in both groups; however, at 45 days, epithelial regeneration decreased in mdx mice irrespective of training, and the percentage of apoptotic (TUNEL positive) cells was higher in mdx‐EX than in WT‐EX mice. Epithelial expression of HSP60 (marker of stress) progressively decreased, and inversely correlated with epithelial apoptosis (r = −0.66, P = 0.01) only in mdx mice. Lack of dystrophin in mdx mice appears associated with defective epithelial differentiation, and transient epithelial regeneration during mild exercise training. Hence, lack of dystrophin might impair repair in bronchial epithelium, with potential clinical consequences in DMD patients. J. Cell. Physiol. 231: 2218–2223, 2016. © 2016 Wiley Periodicals, Inc. Airway epithelium was studied in mdx and wild‐type mice at rest and during exercise training. The major finding of the study was that mdx mice showed very few goblet cells at all times, irrespective of training. In addition, epithelial regeneration sharply decreased, and apoptosis increased, late in the course of the study only in mdx mice, together with decreased expression of HSP60.
      PubDate: 2016-03-10T04:16:15.820589-05:
      DOI: 10.1002/jcp.25339
  • Absence of K‐Ras Reduces Proliferation and Migration But Increases
           Extracellular Matrix Synthesis in Fibroblasts
    • Pages: 2224 - 2235
      Abstract: The involvement of Ras‐GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H‐ and N‐Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K‐Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K‐Ras knockout (KO) mouse embryonic fibroblasts (K‐ras−/−) stimulated with transforming growth factor‐β1 (TGF‐β1) exhibited reduced proliferation and impaired mobility than wild‐type fibroblasts. Moreover, an increase on ECM production was observed in K‐Ras KO fibroblasts in basal conditions. The absence of K‐Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF‐β1‐induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K‐ras but reduced migration and ECM proteins expression only in wild‐type fibroblasts, while the PI3K‐AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K‐Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K‐Ras may be a crucial mediator in TGF‐β1‐mediated effects in this cell type. J. Cell. Physiol. 231: 2224–2235, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-03-06T21:53:43.792793-05:
      DOI: 10.1002/jcp.25340
  • miR‐19a, ‐19b, and ‐26b Mediate CTGF Expression and
           Pulmonary Fibroblast Differentiation
    • Pages: 2236 - 2248
      Abstract: Although microRNA (miRNA) dysregulation with intracellular signaling cascade disruption has been demonstrated in the pathophysiology of pulmonary fibrosis, the relationship between miRNAs and intracellular signaling cascades in pulmonary fibrosis remains unclear. Using the human embryonic lung fibroblast cell line WI‐38, we observed endothelin‐1 (ET‐1)‐ and thrombin‐induced expression of the differentiation markers α‐smooth muscle actin (α‐SMA) and vimentin along with increased connective tissue growth factor (CTGF) protein expression. Decreased CTGF protein expression by CTGF siRNA significantly blocked ET‐1‐ and thrombin‐induced α‐SMA and vimentin expression in WI‐38 cells. Activation of the mitogen‐activated protein kinases (MAPKs) extracellular signal‐regulated kinase ERK, c‐Jun N‐terminal kinase (JNK), and p38 contributed to ET‐1‐ and thrombin‐induced CTGF, α‐SMA, and vimentin expression in WI‐38 cells. TargetScan Human, miRanda, and PicTar prediction algorithms were used to predict miRNAs with binding sites in the 3′ untranslated region (UTR) of CTGF mRNA. miR‐19a, ‐19b, and ‐26b were candidate miRNAs of CTGF. Direct binding of the candidate miRNAs to the 3′‐UTR of CTGF mRNA was verified through luciferase assay by using SV40‐promoter‐IRES‐driven luciferase containing the 3′‐UTR of CTGF mRNA as a reporter plasmid. ET‐1 and thrombin reduced candidate miRNA levels. Candidate miRNA overexpression significantly suppressed ET‐1‐ and thrombin‐induced CTGF expression and reduced α‐SMA and vimentin expression in the WI‐38 cells. Furthermore, candidate miRNA levels were decreased in the lung tissues of mice with bleomycin‐induced pulmonary fibrosis, and intratracheal application of miR‐19a, ‐19b, and 26b reduced the pulmonary fibrotic severity induced by bleomycin. This study is the first to demonstrate crosstalk between MAPK activation and reduction in miR‐19a, ‐19b, and ‐26b expression leading to lung fibroblast differentiation. J. Cell. Physiol. 231: 2236–2248, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-03-14T07:37:23.22443-05:0
      DOI: 10.1002/jcp.25341
  • Differential Response of Human Hepatocyte Chromatin to HDAC Inhibitors as
           a Function of Microenvironmental Glucose Level
    • Authors: Marina Barreto Felisbino; Thiago Alves da Costa, Maria Silvia Viccari Gatti, Maria Luiza Silveira Mello
      Pages: 2257 - 2265
      Abstract: Diabetes is a complex multifactorial disorder characterized by chronic hyperglycemia due to impaired insulin secretion. Recent observations suggest that the complexity of the disease cannot be entirely accounted for genetic predisposition and a compelling argument for an epigenetic component is rapidly emerging. The use of histone deacetylase inhibitor (HDACi) in clinical setting is an emerging area of investigation. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of HDACi treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. More striking changes in the cell cycle progression and mitotic ratios required drastic treatment. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite the absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production. These observations allow us to conclude that the glucose level to which the hepatocytes are subjected affects how chromatin responds to HDACi and their action under high‐glucose environment might not reflect on chromatin remodeling. J. Cell. Physiol. 231: 2257–2265, 2016. © 2016 Wiley Periodicals, Inc. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of histone deacetylase inhibitors (HDACi) treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production.
      PubDate: 2016-03-06T21:56:30.869903-05:
      DOI: 10.1002/jcp.25343
  • Alteration in Phospholipidome Profile of Myoblast H9c2 Cell Line in a
           Model of Myocardium Starvation and Ischemia
    • First page: 2266
      Abstract: Myocardium infarction is one of the most deathly cardiovascular diseases. It is characterized by myocardium ischemia as a result of nutrients depletion and hypoxia. The cell can respond to this injury by autophagy or apoptosis, which determines the evolution and recovery of the myocardium infarction. Lipids play an important role in cardiovascular disease. However reports stating lipidome variations in cardiovascular disease are scarce and the role that lipids play in this pathological condition is not completely understood. The aim of this work was to identify changes in lipid profile of a myoblast H9c2 cell line under starvation and ischemia, to better understand and recognize new biomarkers for myocardial infarction. Lipidomic profile was evaluated by HILIC‐LC‐MS and GC‐MS. Cardiac cells showed alterations in phosphatidylcholines PC (34:1) and PC (36:2), lysophosphatidylcholines lyso PC(16:0), lysoPC(18:1) and lysoPC(18:0), phosphatidylethanolamine PE (34:1), phosphatidylserine PS (36:1), phosphatidylinositol PI (36:2), PI (38:3) and PI (38:5), sphingomyelin SM (34:1) and cardiolipins CL(68:4), CL(72:5) and CL(74:7) in ischemia and/or starvation, in comparison with control. Specific differences observed only in starvation were decrease of SM (34:1) and FA (20:4), and increase of PS (36:1). Differences observed only in ischemia were decrease of PC (36:2), lyso PC (16:0) and FA (18:1) and simultaneous increase of FA (16:0), FA (18:0). PC (34:1) increased in ischemia and decreased in starvation. In conclusion, our work suggests that lipids are potential markers for evaluation of cell fate, either cell death or recovery, which will be useful to improve diagnosis and prognostic of cardiovascular diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-16T04:47:07.267837-05:
      DOI: 10.1002/jcp.25344
  • Involvement of Transient Receptor Potential Cation Channel Vanilloid 1
           (TRPV1) in Myoblast Fusion
    • Authors: Mitsutoshi Kurosaka; Yuji Ogura, Toshiya Funabashi, Tatsuo Akema
      Pages: 2275 - 2285
      Abstract: The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)‐induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX‐induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin‐4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275–2285, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-03-08T04:58:27.133158-05:
      DOI: 10.1002/jcp.25345
  • AR‐12 inhibits multiple chaperones concomitant with stimulating
           autophagosome formation collectively preventing virus replication
    • First page: 2286
      Abstract: We have recently demonstrated that AR‐12 (OSU‐03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR‐12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR‐12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α –dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over‐expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response and autophagosome formation. AR‐12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR‐12–stimulated the co‐localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70 and GRP78. Knock down of Beclin1 suppressed drug‐induced autophagosome formation and reduced the anti‐viral protection afforded by AR‐12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR‐12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation / viral protein degradation, AR‐12 acts as a broad‐specificity anti‐viral drug in vitro and in vivo. We argue future patient studies with AR‐12 are warranted. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-06T10:10:36.196345-05:
      DOI: 10.1002/jcp.25431
  • Retraction: “Inactivation of Ink4a/Arf Leads to Deregulated
           Expression of miRNAs in K‐Ras Transgenic Mouse Model of Pancreatic
           Cancer” by Ali et al.
    • Pages: 2303 - 2303
      Abstract: The above article, published online on June 21, 2012 in Wiley Online Library (, has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 5A to be inappropriately manipulated. Literature Cited Ali S, Banerjee S, Logna F, Bao B, Philip PA, Korc M, Sarkar FH. 2012. Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K‐Ras transgenic mouse model of pancreatic cancer. J Cell Physiol 227:3373–3380; doi: 10.1002/jcp.24036
      PubDate: 2016-06-17T09:29:18.994512-05:
      DOI: 10.1002/jcp.25416
  • Retraction: “Activated K‐Ras and INK4a/Arf Deficiency Promote
           Aggressiveness of Pancreatic Cancer by Induction of EMT Consistent With
           Cancer Stem Cell Phenotype” by Wang et al.
    • Pages: 2304 - 2304
      Abstract: The above article, published online on November 23, 2012 in Wiley Online Library (, has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 4B and C to be inappropriately manipulated and re‐labeled. Literature Cited Wang Z, Ali S, Banerjee S, Bao B, Li Y, Azmi AS, Korc M, Sarkar FH. 2013. Activated K‐Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype. J Cell Physiol 228:556–562; doi: 10.1002/jcp.24162
      PubDate: 2016-06-17T09:29:18.189735-05:
      DOI: 10.1002/jcp.25417
School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
Tel: +00 44 (0)131 4513762
Fax: +00 44 (0)131 4513327
Home (Search)
Subjects A-Z
Publishers A-Z
Your IP address:
About JournalTOCs
News (blog, publications)
JournalTOCs on Twitter   JournalTOCs on Facebook

JournalTOCs © 2009-2015