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Journal Cover Journal of Cellular Physiology
  [SJR: 1.842]   [H-I: 139]   [6 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  [1605 journals]
  • Targeted Stroma in Pancreatic Cancer: Promises and Failures of Target
    • Authors: Afsane Bahrami; Majid Khazaei, Fariba Bagherieh, Majid Ghayour-Mobarhan, Mina Maftouh, Seyed Mahdi Hassanian, Amir Avan
      Abstract: Desmoplasia or abundant fibrotic stroma is a typical property of most malignancies, which has a great effect on tumorigenesis, angiogenesis, and resistance to therapy. The activated stroma cells comprises several cell types including endothelial cells, nerve cells, inflammatory/macrophages cells, stellate cells, and extracellular matrix. In other word, the interactions of cancer–stroma modulate tumorigenesis, therapy resistance and poor delivery of drugs. Therefore, targeting the tumor stroma in combination with conventional chemotherapeutic agents could provide a promising approach in the treatment of pancreatic cancer. This review summarizes the current knowledge about pancreatic stellate cells, targeting stroma compartments with particular emphasis on preclinical and clinical trials on targeting of stroma as an option in pancreatic cancer treatment. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-12T22:45:24.281697-05:
      DOI: 10.1002/jcp.25798
  • Epithelial-Mesenchymal Transition (EMT): A Biological Process in the
           Development, Stem Cell Differentiation and Tumorigenesis
    • Authors: Tong Chen; Yanan You, Hua Jiang, Zack Z. Wang
      Abstract: The lineage transition between epithelium and mesenchyme is a process known as epithelial-mesenchymal transition (EMT), by which polarized epithelial cells lose their adhesion property and obtain mesenchymal cell phenotypes. EMT is a biological process that is often involved in embryogenesis and diseases, such as cancer invasion and metastasis. The EMT and the reverse process, mesenchymal-epithelial transition (MET), also play important roles in stem cell differentiation and de-differentiation (or reprogramming). In this review, we will discuss current research progress of EMT in embryonic development, cellular differentiation and reprogramming, and cancer progression, all of which are representative models for researches of stem cell biology in normal and in diseases. Understanding of EMT and MET may help to identify specific markers to distinguish normal stem cells from cancer stem cells in future. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-12T07:35:28.855414-05:
      DOI: 10.1002/jcp.25797
  • Cancer Stem Cells (CSCs) in Melanoma: There's Smoke, But Is There
    • Authors: Constance E. Brinckerhoff
      Abstract: Cancer stem cells (CSCs), also called Tumor Initiating Cells (TICs), can be defined as cancer cells that are present within solid tumors or hematological cancers, which have characteristics associated with normal stem cells, but which can give rise to all cell types found in a particular cancer sample. CSCs, therefore, are transformed stem cells, which can self-renew, differentiate into diverse progenies, and drive continuous tumor growth. (Villani et al. 2015) (Zhou et al., 2009)(Kreso & Dick, 2014)(Schatton et al., 2008) (Figure 1). This article is protected by copyright. All rights reserved
      PubDate: 2017-01-11T23:50:35.845685-05:
      DOI: 10.1002/jcp.25796
  • Gating Modulation of the Tumor-Related Kv10.1 Channel by Mibefradil
    • Authors: Froylán Gómez-Lagunas; Elisa Carrillo, Luis A. Pardo, Walter Stühmer
      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. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.Several reports credit mibefradil, a T-type calcium channel inhibitor, with tumor suppressing properties. Given that mibefradil also inhibits some potassium channels, we studied the interaction of this compound with the tumor related Kv10.1 channel. We found that mibefradil induces an apparent inactivation both from the open and early closed states, and accelerates current activation in a manner that depends on the holding potential. We hypothesize that mibefradil binds to the voltage-sensor module altering the gating of the channels.
      PubDate: 2017-01-11T14:50:36.957889-05:
      DOI: 10.1002/jcp.25448
  • Knockdown of REV3 Synergizes with ATR Inhibition to Promote Apoptosis
           Induced by Cisplatin in Lung Cancer Cells
    • Authors: He-Guo Jiang; Ping Chen, Jin-Yu Su, Ming Wu, Hai Qian, Yi Wang, Jian Li
      Abstract: It has been demonstrated that REV3, the catalytic subunit of the translesion synthesis (TLS) polymerase ζ, play an important role in DNA damage response (DDR) induced by cisplatin, and Ataxia telangietasia mutated and Rad-3-related (ATR) knase is a central player in activating cell cycle checkpoint, stabilizing replication forks, regulating DDR, and promoting repair of DNA damage caused by cisplatin. Cancer cells deficient in either one of REV3 and ATR are more sensitive to cisplatin. However, whether co-inhibition of REV3 and ATR can further increase sensitivity of non-small cell lung cancer (NSCLC) cells to cisplatin is not clear. In this study, we show that REV3 knockdown combined with ATR inhibition further enhance cytotoxicity of cisplatin in NSCLC cells, including cisplatin-sensitive and -resistant cell lines, compared to individual knockdown of REV3 or ATR, which are accompanied by markedly caspase-dependent apoptosis response, pronounced DNA damage accumulation and severe impediment of interstrand crosslink (ICL) and double strand break (DSB) repair. Our results suggest that REV3 knockdown synergize strongly with ATR inhibition to significantly increase sensitivity of cisplatin in NSCLC cells by inhibiting ICL and DSB repair. Thus simultaneously targeting REV3 and ATR may represent one approach to overcome cisplatin resistance and improve chemotherapeutic efficacy in NSCLC treatment. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-11T06:50:38.598198-05:
      DOI: 10.1002/jcp.25792
  • Modulation of Long-Term Endothelial-Barrier Integrity Is Conditional to
           the Cross-Talk between Akt and Src Signaling
    • Authors: Fei Gao; Harika Sabbineni, Sandeep Artham, Payaningal R. Somanath
      Abstract: Although numerous studies have implicated Akt and Src kinases in vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1)-induced endothelial-barrier regulation, a link between these two pathways has never been demonstrated. We determined the long-term effects of Akt inhibition on Src activity and vice versa, and in turn, on the human microvascular endothelial cell (HMEC) barrier integrity at the basal level, and in response to growth factors. Our data showed that Akt1 gene knockdown increases gap formation in HMEC monolayer at the basal level. Pharmacological inhibition of Akt, but not Src resulted in exacerbated VEGF-induced vascular leakage and impaired Ang-1-induced HMEC-barrier protection in-vitro at 24 hours. Whereas inhibition of Akt had no effect on VEGF-induced HMEC gap formation in the short term, inhibition of Src blunted this process. In contrast, inhibition of Akt disrupted the VEGF and Ang-1 stabilized barrier integrity in the long-term while inhibition of Src did not. Interestingly, both long-term Akt inhibition and Akt1 gene knockdown in HMECs resulted in increased Tyr416 phosphorylation of Src. Treatment of HMECs with transforming growth factor-β1 (TGFβ1) that inhibited Akt Ser473 phosphorylation in the long-term, activated Src through increased Tyr416 phosphorylation and decreased HMEC-barrier resistance. The effect of TGFβ1 on endothelial-barrier breakdown was blunted in Akt1 deficient HMEC monolayers, where endothelial-barrier resistance was already impaired compared to the control. To our knowledge, this is the first report demonstrating a direct cross-talk between Akt and Src in endothelial-barrier regulation. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-11T06:40:34.87694-05:0
      DOI: 10.1002/jcp.25791
  • Stimulatory Actions of a Novel Thiourea Derivative on Large-Conductance,
           Calcium-Activated Potassium Channels
    • Authors: Sheng-Nan Wu; Jyh-Haur Chern, Santai Shen, Hwei-Hisen Chen, Ying-Ting Hsu, Chih-Chin Lee, Ming-Huan Chan, Ming-Chi Lai, Feng-Shiun Shie
      Abstract: AimIn this study, we examine whether an anti-inflammatory thiourea derivative, compound #326, actions on ion channels.MethodsThe effects of compound #326 on Ca2+-activated K+ channels were evaluated by patch-clamp recordings obtained in cell-attached, inside-out or whole-cell configuration.ResultsIn pituitary GH3 cells, compound #326 increased the amplitude of Ca2+-activated K+ currents (IK(Ca)) with an EC50 value of 11.6 μM, which was reversed by verruculogen, but not tolbutamide or TRAM-34. Under inside-out configuration, a bath application of compound #326 raised the probability of large-conductance Ca2+-activated K+ (BKCa) channels. The activation curve of BKCa channels was shifted to less depolarised potential with no modification of the gating charge of the curve; consequently, the difference of free energy was reduced in the presence of this compound. Compound #326–stimulated activity of BKCa channels is explained by a shortening of mean closed time, despite its inability to alter single-channel conductance. Neither delayed-rectifier nor erg-mediated K+ currents was modified. Compound #326 decreased the peak amplitude of voltage-gated Na+ current with no clear change in the overall current–voltage relationship of this current. In HEK293T cells expressing α-hSlo, compound #326 enhanced BKCa channels effectively. Intriguingly, the inhibitory actions of compound #326 on interleukin 1β in lipopolysaccharide-activated microglia were significantly reversed by verruculogen, whereas BKCa channel inhibitors suppressed the expressions of inducible nitric oxide synthase.ConclusionThe BKCa channels could be an important target for compound #326 if similar in vivo results occur, and the multi-functionality of BKCa channels in modulating microglial immunity merit further investigation. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-11T06:40:31.450218-05:
      DOI: 10.1002/jcp.25788
  • Promising Anti-tumor properties of Bisdemethoxycurcumin: A Naturally
           Occurring Curcumin Analogue
    • Authors: Mahin Ramezani; Mahdi Hatamipour, Amirhosein Sahebkar
      Abstract: Curcuminoids are turmeric-extracted phytochemicals with documented chemopreventive and anti-tumor activities against several types of malignancies. Curcuminoids can modulate several molecular pathways and cellular targets involved in different stages of tumor initiation, growth and metastasis. Bisdemethoxycurcumin (BDMC) is a minor constituent (approximately 3%) of curcuminoids that has been shown to be more stable than the other two main curcuminoids i.e. curcumin and demthoxycurcumin. Recent studies have revealed that BDMC has anti-tumor effects exerted through a multimechanistic mode of action involving inhibition of cell proliferation, invasion and migration, metastasis and tumour growth, and induction of apoptotic death in cancer cells. The present review discusses the findings on the anti-tumor effects of BDMC, underlying mechanisms and the relevance of finding for translational studies in human. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-11T06:20:20.47525-05:0
      DOI: 10.1002/jcp.25795
  • C-Met as a Potential Target for the Treatment of Gastrointestinal Cancer:
           Current Status and Future Perspectives
    • Authors: Afsaneh Bahrami; Soodabeh Shahidsales, Majid Khazaei, Majid Ghayour-Mobarhan, Mina Maftouh, Seyed Mahdi Hassanian, Amir Avan
      Abstract: Aberrant activation of the HGF/c-Met signalling pathways is shown to be related with cell proliferation, progression, metastasis and worse prognosis in several tumor types, including gastrointestinal cancers, suggesting its value as a stimulating-target for cancer-therapy. Several approaches have been developed for targeting HGF and/or c-Met, and one of them, crizotinib (dual c-Met/ALK inhibitor), is recently been approved by FDA for lung-cancers with ALK-rearrangement. The main aim of current review is to give an overview on the role of c-Met/HGF pathway in gastrointestinal cancer, in preclinical and clinical trials. Although several important matters is still remained to be elucidated on the molecular pathways underlying the antitumor effects of this therapy in gastrointestinal-cancers. Further investigations are warranted to recognize the main determinants of the activity of c-Met inhibitors, for parallel targeting signalling pathway associated/activated via MET/HGF pathway or in response to the cell resistance to anti-c-Met agents. Additionally, identification of patients that might benefit from therapy could help to increase the selectivity and efficacy of the therapy. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-11T06:15:23.142888-05:
      DOI: 10.1002/jcp.25794
  • HOTAIR Role in Melanoma Progression and its Identification in the Blood of
           Patients with Advanced Disease
    • Authors: Monica Cantile; Giosuè Scognamiglio, Laura Marra, Gabriella Aquino, Chiara Botti, Maria Rosaria Falcone, Maria Gabriella Malzone, Giuseppina Liguori, Maurizio Di Bonito, Renato Franco, Paolo Antonio Ascierto, Gerardo Botti
      Abstract: ObjectivesThe molecular mechanisms responsible for the metastatic progression of melanoma have not been fully defined yet. We have recently shown that an important role in this process is certainly played by HOX genes, whose regulation is under control of particular non-coding RNAs, some of which are present within the HOX locus. HOTAIR is the most studied among them, whose aberrant expression is associated with the metastatic progression of many malignancies.The aim of this study was to verify the role played by HOTAIR in metastatic progression of melanoma and to evaluate the circulating levels of HOTAIR in the blood of patients with metastatic melanoma.MethodsA series of melanocytic lesions were selected to evaluate the potential changes in the expression of HOTAIR during the evolution of the disease through in situ and molecular approaches.ResultsNone of the benign melanocytic lesions showed the presence of HOTAIR. The staining of HOTAIR resulted very weak in the primary pT1 lesions, while it was very strong in all pairs of primary tissues and corresponding metastases. Surprisingly, we found the presence of HOTAIR in some intratumoral lymphocytes, while this positivity decreased in lymphocyte component further away from the tumor. HOTAIR was also detected in the serum of selected metastatic patients.ConclusionThese data allowed us to speculate on the fundamental role played by HOTAIR in tumor evolution of melanoma. Its presence in intratumoral lymphocytes might suggest that its involvement in the modulation of tumor microenvironment and the detection in the serum could be used in the management of melanoma patients. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-09T05:35:22.894177-05:
      DOI: 10.1002/jcp.25789
  • Lkb1 Regulation of Skeletal Muscle Development, Metabolism and Muscle
           Progenitor Cell Homeostasis
    • Authors: Tizhong Shan; Ziye Xu, Jiaqi Liu, Weiche Wu, Yizhen Wang
      Abstract: Liver kinase B1 (Lkb1), also named as Serine/Threonine protein kinase 11 (STK11), is a serine/threonine kinase that plays crucial roles in various cellular processes including cell survival, cell division, cellular polarity, cell growth, cell differentiation, and cell metabolism. In metabolic tissues, Lkb1 regulates glucose homeostasis and energy metabolism through phosphorylating and activating the AMPK subfamily proteins. In skeletal muscle, Lkb1 affects muscle development and postnatal growth, lipid and fatty acid oxidation, glucose metabolism and insulin sensitivity. Recently, the regulatory roles of Lkb1 in regulating division, self-renew, proliferation and differentiation of skeletal muscle progenitor cells have been reported. In this review, we discuss the roles of Lkb1 in regulating skeletal muscle progenitor cells homeostasis and skeletal muscle development and metabolism. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-09T05:30:38.790719-05:
      DOI: 10.1002/jcp.25786
  • MicroRNA: Relevance to Stroke Diagnosis, Prognosis and Therapy
    • Authors: Hamed Mirzaei; Fatemeh Momeni, Leila Saadatpour, Amirhossein Sahebkar, Mohammad Goodarzi, Aria Masoudifar, Shirin Kouhpayeh, Hossein Salehi, Hamid Reza Mirzaei, Mahmoud Reza Jaafari
      Abstract: Stroke is a life-threatening disease that accounts for a considerable burden of mortality in both developing and developed world. Identification of specific biomarkers for stroke and its outcomes can greatly contribute to improved care of patients. MicroRNAs (miRNAs) are known as novel biomarkers that could be used as diagnostic, prognostic and therapeutic biomarkers. Various studies have shown that miRNAs have key roles in the pathogenesis of stroke, and its complications and outcomes. In addition, there is evidence showing that mesenchaymal stromal cell-derived exosomes containing miRNAs can be used for monitoring and treatment of various diseases such as stroke. Here, we summarized various aspects of miRNA applications in different stages of stroke. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-09T05:30:34.141183-05:
      DOI: 10.1002/jcp.25787
  • Crosstalk of Autophagy and Apoptosis: Involvement of the Dual Role of
           Autophagy Under ER Stress
    • Authors: Shuling Song; Jin Tan, Yuyang Miao, Mengmeng Li, Qiang Zhang
      Abstract: Endoplasmic reticulum (ER) stress is a common cellular stress response that is triggered by a variety of conditions that disturb cellular homeostasis, and induces cell apoptosis. Autophagy, an important and evolutionarily conserved mechanism for maintaining cellular homeostasis, is closely related to the apoptosis induced by ER stress. There are common upstream signaling pathways between autophagy and apoptosis induced by ER stress, including PERK/ATF4, IRE1α, ATF6 and Ca2+. Autophagy can not only block the induction of apoptosis by inhibiting the activation of apoptosis-associated caspase which could reduce cellular injury, but also help to induce apoptosis. In addition, the activation of apoptosis-related proteins can also inhibit autophagy by degrading autophagy-related proteins, such as Beclin-1, Atg4D, Atg3 and Atg5. Although the interactions of different autophagy- and apoptosis-related proteins and also common upstream signaling pathways have been found, the potential regulatory mechanisms have not been clearly understood. In this review, we summarize the dual role of autophagy, and the interplay and potential regulatory mechanisms between autophagy and apoptosis under ER stress condition. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-09T05:30:28.441947-05:
      DOI: 10.1002/jcp.25785
  • Biological Roles of Glial Fibrillary Acidic Protein as a Biomarker in
           Cartilage Regenerative Medicine
    • Authors: Sanshiro Kanazawa; Satoru Nishizawa, Tsuyoshi Takato, Kazuto Hoshi
      Abstract: Glial fibrillary acidic protein (GFAP) is an intermediate filament that is expressed in specifically expressed auricular chondrocytes, which are good cell sources of cartilage regenerative medicine. Although our group uses GFAP as a biomarker of matrix production in the cultured auricular chondrocytes, the biological roles of GFAP in auricular chondrocytes has remained unknown. In this study, we demonstrated the biological functions of GFAP in the human and mouse derived auricles to clarify the significance and role with the chondrocytes of GFAP in order to provide useful information for reliable and safe regenerative medicine. We examined the cell responses to stretch stress for these chondrocytes and completed a nuclear morphological analysis. Based on these results, GFAP seems to support the resistance to severe mechanical stress in the tissue which physiologically suffers from a stretch overload, and plays pivotal roles in the conservation of cell structures and functions through the maintenance of nuclear morphology. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:30:39.421383-05:
      DOI: 10.1002/jcp.25771
  • Cyclic-Glycine-Proline Accelerates Mammary Involution by Promoting
           Apoptosis and Inhibiting IGF-1 Function
    • Authors: Gagandeep Singh-Mallah; Christopher D McMahon, Jian Guan, Kuljeet Singh
      Abstract: In rodents, post-lactational involution of mammary glands is characterized by the loss of mammary epithelial cells via apoptosis, which is associated with a decline in the expression of insulin-like growth factor-1 (IGF-1). Overexpression of IGF-1 delays involution by inhibiting apoptosis of epithelial cells and preserving the remaining secretory alveoli. Cyclic-glycine-proline (cGP), a metabolite of IGF-1, normalizes IGF-1 function under pathological conditions by regulating the bioavailability of IGF-1. The present study investigated the effect of cGP on the physiological decline in IGF-1 function during post-lactational mammary involution. Rat dams were gavaged with either cGP (3 mg/kg) or saline once per day from post-natal d8-22. Before collecting tissue on post-natal d23, a pair of mammary glands were sealed on d20 (72h-engorgement, thus representative of late-involution) and d22 (24h-engorgement, thus representative of mid-involution), while the remaining glands were allowed to involute naturally (early-involution). During early-involution, cGP accelerated the loss of mammary cells through apoptosis, resulting in an earlier clearance of intact secretory alveoli compared with the control group. This coincided with an earlier up-regulation of the cell survival factors, Bcl-xl and IGF-1R, in the early-involution cGP glands compared with the control glands. During late-involution, cGP reduced the bioactivity of IGF-1, which was evident through decreased phosphorylation of IGF-1R in the regressed alveoli. Maternal administration of cGP did not alter milk production and composition during early-, peak- or late- stage of lactation. These data show that cGP accelerates post-lactational involution by promoting apoptosis and the physiological decline in IGF-1 function. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:30:29.705539-05:
      DOI: 10.1002/jcp.25782
  • TRIM33 is Essential for Osteoblast Proliferation and Differentiation via
           BMP Pathway
    • Authors: Jia Guo; Wei Qin, Quan Xing, Manman Gao, Fuxin Wei, Zhi Song, Lingling Chen, Ying Lin, Xianling Gao, Zhengmei Lin
      Abstract: Tripartite motif containing 33 (TRIM33) functions both as a positive and negative regulator of the TGF-β/BMP pathway in tumors; however, its effect and mechanism during osteoblast proliferation and differentiation, which involves the TGF-β/BMP pathway is not defined. In this study, we used mouse C3H10T1/2 mesenchymal stem cell line and MC3T3-E1 preosteoblasts to investigate the role of TRIM33 during this process. The results demonstrated that the expression of TRIM33 increased during the differentiation. Moreover, the overexpression or knockdown of TRIM33 resulted in both an augmentation or decrease in osteoblast differentiation, which were measured by the expression of alkaline phosphatase (ALP) at the mRNA level, both Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) at the protein level, and the formation of mineral modules. To further demonstrate the mechanism of TRIM33 in this process, we found that TRIM33 could positively mediate the BMP pathway by forming TRIM33-Smad1/5 complex. This interaction between TRIM33 and Smad1/5 triggered the phosphorylation of Smad1/5. In addition, the essential role of TRIM33 in osteoblast proliferation was determined in this study by Cell Counting Kit (CCK) -8 and cell cycle assays. In summary, we establish the function of TRIM33 as a positive regulator of osteoblast differentiation in BMP pathway, which mediates its effect through its interaction with and activation of Smad1/5. In addition, the results clearly demonstrate that TRIM33 is necessary for osteoblast proliferation by regulating cell cycle. These results suggest that TRIM33 can be a positive target of osteoblast proliferation and differentiation through BMP pathway. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:30:26.149274-05:
      DOI: 10.1002/jcp.25769
  • Current Status and Prospective Regarding the Therapeutic Potential of
           Natural Autoantibodies in Cancer Therapy
    • Authors: Salimeh Ebrahimnezhad; MirHadi Jazayeri, Seyed Mahdi Hassanian, Amir Avan
      Abstract: The presence of natural auto-antibodies (NAbs) in normal range/activity in healthy individuals is essential for body to maintain hemostasis, which are directed to self and altered self-components, while abnormal activity of this system can be associated with several health related diseases. It has been shown that NAbs regulate immune system, and can be changed during the individual's life. In other word, the level and pattern of Nabs is among the main factors to define the state of the body, suggesting their prognostic values as markers of immune system impairment such as autoimmunity and cancer. Such NAbs have gained substantial attention because several of them, including their recombinant forms, have therapeutic potential (e.g., programmed cell death-1 (PD-1, Pdcd1), which some of its inhibitors have been approved by FDA for cancer therapy).Whereas a large number of IgM and IgG NAbs have a key role in tissue homeostasis, while others modulate cellular and enzyme properties. The aim of current review is to give an overview about some of these NAbs and how these low-titer/affinity interact with Ag in homeostasis, with particular emphasis on related diseases such as systemic inflammatory response syndrome and cancer, and their application as potential therapeutic target for cancer therapy. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:30:22.188017-05:
      DOI: 10.1002/jcp.25765
  • Establishment of a New Conditionally Immortalized Human Skeletal Muscle
           Microvascular Endothelial Cell Line
    • Authors: Hironori Sano; Yasuteru Sano, Eri Ishiguchi, Fumitaka Shimizu, Masatoshi Omoto, Toshihiko Maeda, Hideaki Nishihara, Yukio Takeshita, Shiori Takahashi, Mariko Oishi, Takashi Kanda
      Abstract: In skeletal muscle, the capillaries have tight junctions (TJs) that are structurally similar to those in the blood-brain barrier (BBB) and blood-nerve barrier (BNB). Although many findings have been clarified in the territory of BBB and BNB, few have so far examined the TJs of capillaries in the skeletal muscle. In addition, no in vitro human skeletal muscle microvasculature models have been reported thus far. We newly established a new human skeletal muscle microvascular endothelial cell (HSMMEC) line. HSMMECs were isolated from human skeletal muscle and were infected with retroviruses harboring temperature-sensitive SV40 T antigen and telomerase genes. This cell line, termed TSM15, showed a spindle fiber-shaped morphology, an immunoreactivity to anti-factor VIII and anti-VE-cadherin antibodies, and a temperature-sensitive growth. TSM15 cells grew stably for more than 40 passages when they were cultured at 33°C, thereby retaining their spindle fiber-shaped morphology and contact inhibition at confluence. The cells expressed tight junctional molecules such as claudin-5, occludin, and zonula occludens-1, as well as transporters such as a glucose transporter 1. The transendothelial electrical resistance of TSM15 was as high as those of the human brain microvascular endothelial cell line. This novel cell line might facilitate the analyses of the pathophysiology of inflammatory myopathy, such as dermatomyositis, and can improve our understanding of the physiological and biochemical properties of the microvasculature in human skeletal muscle. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:25:44.960381-05:
      DOI: 10.1002/jcp.25772
  • Exosomes: Nanoparticulate Tools for RNA Interference and Drug Delivery
    • Authors: Fahimeh Shahabipour; Nastaran Barati, Thomas P. Johnston, Giuseppe Derosa, Pamela Maffioli, Amirhossein Sahebkar
      Abstract: Exosomes are naturally occurring extracellular vesicles released by most mammalian cells in all body fluids. Exosomes are known as key mediators in cell-cell communication and facilitate the transfer of genetic and biochemical information between distant cells. Structurally, exosomes are composed of lipids, proteins, and also several types of RNAs which enable these vesicles to serve as important disease biomarkers. Moreover, exosomes have emerged as novel drug and gene delivery tools owing to their multiple advantages over conventional delivery systems. Recently, increasing attention has been focused on exosomes for the delivery of drugs, including therapeutic recombinant proteins, to various target tissues. Exosomes are also promising vehicles for the delivery of microRNAs and small interfering RNAs, which is usually hampered by rapid degradation of these RNAs, as well as inefficient tissue specificity of currently available delivery strategies. This review highlights the most recent accomplishments and trends in the use of exosomes for the delivery of drugs and therapeutic RNA molecules. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:25:40.879746-05:
      DOI: 10.1002/jcp.25766
  • Novel Circulating Biomarkers for Non-Alcoholic Fatty Liver Disease: A
           Systematic Review
    • Authors: Amirhossein Sahebkar; Elena Sancho, David Abelló, Jordi Camps, Jorge Joven
      Abstract: Currently, a liver biopsy remains the only reliable way to precisely diagnose non-alcoholic fatty liver disease (NAFLD) and establish the severity of liver injury, presence of fibrosis and architecture remodeling. However, the cost and the intrinsic invasive procedure of a liver biopsy rules it out as a gold standard diagnostic test, and the imaging test are not the best choice due to the price, and currently is being refined. The lack of a biomarker of NAFLD pushes to develop this new line of research. The aim of the present systematic review is to clarify and update all the NAFLD biomarkers described in the literature until recently. We highlight α-ketoglutarate and CK18-F as currently the best potential biomarker of NAFLD. However, due to methodological differences, we propose the implementation of international, multicenter, multiethnic studies with larger population size and biopsy proven NAFLD diagnosis to analyze and compare α-ketoglutarate and CK18-F as potential biomarkers of the silent evolution of NAFLD. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:25:37.869762-05:
      DOI: 10.1002/jcp.25779
  • MicroRNA-1 overexpression blunts cardiomyocyte hypertrophy elicited by
           thyroid hormone
    • Authors: Gabriela Placoná Diniz; Caroline Antunes Lino, Camila Rodrigues Moreno, Nathalia Senger, Maria Luiza Morais Barreto-Chaves
      Abstract: It is well known that increased thyroid hormone (TH) levels induce cardiomyocyte growth. MicroRNAs (miRNAs) have been identified as key players in cardiomyocyte hypertrophy, which is associated with increased risk of heart failure. In this study, we evaluated the miR-1 expression in TH-induced cardiac hypertrophy, as well as the potential involvement of miR-1 in cardiomyocyte hypertrophy elicited by TH in vitro. The possible role of Type 1 angiotensin II receptor (AT1R) in the effect promoted by TH in miR-1 expression was also evaluated. Neonatal rat cardiac myocytes (NRCMs) were treated with T3 for 24 hours and Wistar rats were subjected to hyperthyroidism for 14 days combined or not with AT1R blocker. Real Time RT-PCR analysis indicated that miR-1 expression was decreased in cardiac hypertrophy in response to TH in vitro and in vivo, and this effect was unchanged by AT1R blocker. In addition, HDAC4, which is target of miR-1, was increased in NRCMs after T3 treatment. A gain-of-function study revealed that overexpression of miR-1 prevented T3-induced cardiomyocyte hypertrophy and reduced HADC4 mRNA levels in NRCMs. In vivo experiments confirmed the downregulation of miR-1 in cardiac tissue from hyperthyroid animals, which was accompanied by increased HDAC4 mRNA levels. In addition, HDAC inhibitor prevented T3-induced cardiomyocyte hypertrophy. Our data reveal a new mechanistic insight into cardiomyocyte growth in response to TH, suggesting that miR-1 plays a role in cardiomyocyte hypertrophy induced by TH potentially via targeting HADC4. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:25:35.198587-05:
      DOI: 10.1002/jcp.25781
  • Purification of Small Molecule-induced Cardiomyocytes from Human Induced
           Pluripotent Stem Cells Using a Reporter System
    • Authors: Geun Hye Hwang; So Mi Park, Ho Jae Han, Joong Sun Kim, Seung Pil Yun, Jung Min Ryu, Ho Jin Lee, Woochul Chang, Su-Jin Lee, Jeong-Hee Choi, Jin-Sung Choi, Min Young Lee
      Abstract: In order to realize the practical use of human pluripotent stem cell (hPSC)-derived cardiomyocytes for the purpose of clinical use or cardiovascular research, the generation of large numbers of highly purified cardiomyocytes should be achieved. Here, we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC-derived cardiomyocytes using a reporter system. Regulation of the Wnt/β-catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939, a tankyrase inhibitor and IWP2, a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%), it was difficult to suppress differentiation into non-cardiac cells, Therefore, we applied a lentiviral reporter system, wherein green fluorescence protein (GFP) and Zeocin-resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT), a cardiac-specific protein, to exclude non-cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC-derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re-plated after single-cell dissociation. Purified cells specifically expressed GFP, cTnT, displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC-derived cardiomyocytes for application in regenerative medicine and biomedical research. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:25:34.089387-05:
      DOI: 10.1002/jcp.25783
  • Reciprocal Regulation Between βTrCP and Smurf1 Suppresses Proliferative
           Capacity of Liver Cancer Cells
    • Authors: Yue Zhang; Wenhua Wang, Si Cai, Yan Chen, Qinwan Wang, Qiuhui Pan, Fenyong Sun, Jiayi Wang
      Abstract: We previously reported that both the ubiquitin E3 ligases βTrCP (beta-transducin repeat-containing E3 ubiquitin protein ligase) and Smurf1 (SMAD-specific E3 ubiquitin protein ligase 1) play similar antitumorigenic roles in liver cancer cells. However, whether and how they are reciprocally regulated remains elusive. Here, we show that βTrCP interacts with Smurf1 through the 7 × tryptophan (W) aspartic acid (D)(WD) 40 and the region homologous to the E6-AP carboxyl terminus (HECT) domains, which are the E3 ligase domains of βTrCP and Smurf1, respectively. The E3 ligase domains of βTrCP and Smurf1 are also critical for maintaining the protein expressions of Smurf1 and βTrCP. Moreover, a positive correlation between βTrCP and Smurf1 was also revealed by tissue microarray analysis, indicating that this relationship might be important in liver cancer. Further, we found that Smurf1 increases the protein stability of βTrCP, possibly by reducing autoubiquitination of βTrCP, and vice versa. Interestingly, such effects depended on the presence of E3 ligase domains. Importantly, depletion of Smurf1- or βTrCP-enhanced proliferative capacity of liver cancer cells could be partially reversed by overexpression of wild-type βTrCP or Smurf1 but not their E3 ligase-dead mutants. Collectively, a reciprocal post-translational regulation between βTrCP and Smurf1 has been uncovered in this study. Simultaneous enhancement of βTrCP and Smurf1 functions might be helpful in the treatment of liver cancer. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:25:29.006446-05:
      DOI: 10.1002/jcp.25780
  • Regulation of Chondrocyte Functions by Transient Receptor Potential Cation
           Channel V6 in Osteoarthritis
    • Authors: Tengfei Song; Jun Ma, Lei Guo, Peng Yang, Xuhui Zhou, Tianwen Ye
      Abstract: Transient receptor potential vanilloid (TRPV) channels function to maintain the dynamic balance of calcium signaling and calcium metabolism in bones. The goal of this study was to determine the potential role of TRPV6 in regulation of chondrocytes. The level of TRPV6 expression was analyzed by western blot in articular cartilage derived from the knee joints of osteoarthritis (OA) rat models and OA patients. Bone structure and osteoarthritic changes in the knee joints of TRPV6 knockout mice were examined using micro-computed and histological analysis at the age of 6 and 12 months old. Furthermore, to investigate the effects of TRPV6 on chondrocyte extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation and apoptosis, we decreased and increased TRPV6 expression in chondrocytes with lentiviral constructs encoding shRNA targeting TRPV6 and encoding TRPV6, respectively. The results showed that the level of TRPV6 expression in an OA rat model was markedly down-regulated. TRPV6 knockout mice showed severe osteoarthritis changes, including cartilage fibrillation, eburnation and loss of proteoglycans. In addition, deficiency of TRPV6 clearly affected chondrocyte function, such as extracellular matrix secretion, the release of matrix degrading enzymes, cell proliferation and apoptosis. Taken together, our results implicated that TRPV6 channel, as a chondro-protective factor, was involved in the pathogenesis of OA. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:25:25.443373-05:
      DOI: 10.1002/jcp.25770
  • LIPUS suppressed LPS-induced IL-1α through the inhibition of NF-κB
           nuclear translocation via AT1-PLCβ pathway in MC3T3-E1
    • Authors: Mayu Nagao; Natsuko Tanabe, Soichiro Manaka, Masako Naito, Kumiko Nakai, Tadahiro Takayama, Takayuki Kawato, Go Torigoe, Jumpei Sekino, Naoya Tsukune, Masao Maeno, Naoto Suzuki, Shuichi Sato
      Abstract: Inflammatory cytokines, interleukin (IL)-1, IL-6, and TNF-a, are involved in inflammatory bone diseases such as rheumatoid osteoarthritis and periodontal disease. Particularly, periodontal disease, which destroys alveolar bone, is stimulated by lipopolysaccharide (LPS). Low-intensity pulsed ultrasound (LIPUS) is used for bone healing in orthopedics and dental treatments. However,the mechanism underlying effects of LIPUS on LPS-induced inflammatory cytokine are not well understood. We therefore aimed to investigate the role of LIPUS on LPS-induced IL-1a production. Mouse calvaria osteoblast-like cells MC3T3-E1 were incubated in the presence or absence of LPS (P. gingivalis), and then stimulated with LIPUS for 30 min/day. To investigate the role of LIPUS, we determined the expression of IL-1a stimulated with LIPUS and treated with an angiotensin II receptor type 1 (AT1) antagonist, Losartan. We also investigate to clarify the pathway of LIPUS, we transfected siRNA silencing AT1 (siAT1) in MC3T3-E1. LIPUS inhibited mRNA and protein expression of LPS-induced IL-1a. LIPUS also reduced the nuclear translocation of NF-B by LPS-induced IL-1a. Losartan and siAT1 blocked all the stimulatory effects of LIPUS on IL-1a production and IL-1a-mediated NF-κB translocation induced by LPS. Furthermore, PLCß inhibitor U73122 recovered NF-κB translocation. These results suggest that LIPUS inhibits LPS-induced IL-1a via AT1-PLCß in osteoblasts. We exhibit that these findings are in part of the signaling pathway of LIPUS on the anti-inflammatory effects of IL-1a expression. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:25:23.805513-05:
      DOI: 10.1002/jcp.25777
  • PCSK9 at the Crossroad of Cholesterol Metabolism and Immune Function
           During Infections
    • Authors: Francesco Paciullo; Francesca Fallarino, Vanessa Bianconi, Massimo R. Mannarino, Amirhossein Sahebkar, Matteo Pirro
      Abstract: Sepsis, a complex and dynamic syndrome resulting from microbial invasion and immune system dysregulation, is associated with an increased mortality, reaching up to 35% worldwide. Cholesterol metabolism is often disturbed during sepsis, with low plasma cholesterol levels being associated with poor prognosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of the low-density lipoprotein receptor (LDLR), thus regulating intracellular and plasma cholesterol levels. PCSK9 is often upregulated during sepsis and might have a detrimental effect on immune host response and survival. Accordingly, PCSK9 reduces lipopolysaccharide uptake and clearance by human hepatocytes. Moreover, PCSK9 upregulation exacerbates organ dysfunction and tissue inflammation during sepsis, whereas a protective effect of PCSK9 deficiency has been documented in septic patients. Although a possible detrimental impact of PCSK9 on survival has been described, some beneficial effects of PCSK9 on immune response may be hypothesized. First, PCSK9 is associated with increased plasma cholesterol levels, which might be protective during sepsis. Second, PCSK9, by stimulating LDLR degradation and inhibiting reverse cholesterol transport, might promote preferential cholesterol accumulation in macrophages and other immune cells; these events might improve lipid raft composition and augment toll-like receptor function thus supporting inflammatory response. Hence, a more clear definition of the role of PCSK9 in septic states might provide additional insight in the understanding of the sepsis-associated immune dysregulation and enhance therapeutic outcomes. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:20:28.457716-05:
      DOI: 10.1002/jcp.25767
  • A Critical Role for Adiponectin-Mediated Development of Endometrial
    • Authors: Whasun Lim; Myung Jin Choi, Hyocheol Bae, Fuller W. Bazer, Gwonhwa Song
      Abstract: Adiponectin is one of the adipokines in the collagen superfamily. It is secreted primarily by white adipocytes and influences reproductive processes including ovarian and uterine functions. Adiponectin regulates energy homeostasis, insulin sensitivity and is anti-inflammatory in various tissues. Its receptors (ADIPOR1 and ADIPOR2) are widely expressed in mammalian tissues, including porcine conceptuses and endometrial during the estrous cycle and peri-implantation period of pregnancy. However, regulatory effects of adiponectin on endometrial epithelial cells are unknown. Therefore, we investigated the effects of parity on expression of ADIPOR1 and ADIPOR2 and the effects of adiponectin in the porcine endometrium during early pregnancy. Results of this study revealed robust expression of ADIPOR1 and ADIPOR2 in uterine luminal (LE) and glandular (GE) epithelia during early pregnancy and expression decreased as with increasing parity. For porcine luminal epithelial (pLE) cells, adiponectin enhanced proliferation and increased phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, P38 and P90RSK in a time-dependent manner. Moreover, the abundance of adiponectin-activated signaling molecules were suppressed by pharmacological inhibitors including wortmannin, U0126, SP600125 and SB203580, respectively in pLE cells. Furthermore, inhibition of each targeted signal transduction molecule influenced proliferation of adiponectin-stimulated pLE cells. In addition, adiponectin inhibited tunicamycin-induced endoplasmic reticulum (ER)-stress through effects on ER stress regulated proteins in pLE cells. Collectively, these results suggest that adiponectin affects development of porcine uterine epithelia and reproductive performance through modulation of PI3K/AKT and MAPK cell signaling pathways. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-07T05:20:27.021222-05:
      DOI: 10.1002/jcp.25768
  • Cyclic Tensile Strain Reduces TNF-α Induced Expression of MMP-13 by
           Condylar Temporomandibular Joint Cells
    • Authors: Hessam Tabeian; Astrid D. Bakker, Beatriz F. Betti, Frank Lobbezoo, Vincent Everts, Teun J. de Vries
      Abstract: To investigate whether the disproportionate degradation of mandibular condyle cartilage in arthritic juvenile temporomandibular joint (TMJ) is related to distinctive responses of TMJ-derived cells to tumor necrosis factor-α (TNF-α), and whether mechanical loading affects this response. The effect of TNF-α (0.1–10 ng/ml) was tested on juvenile porcine TMJ cells isolated from the condyle, fossa, and disc, grown in 3D agarose gels. Expression of anabolic and catabolic factors was quantified by RT-qPCR and/or immunohistochemistry. Condylar cells were stimulated for 12 h with TNF-α (10 ng/ml), followed by 8 h of 6% cyclic tensile strain, and gene expression of MMPs was quantified. TNF-α (10 ng/ml) reduced the expression of the matrix proteins collagen types I and II after 6 h of incubation. Aggrecan gene expression was increased in the presence of 0.1 ng/ml TNF-α. The fossa and disc cells responded to TNF-α with an increased expression of the aggrecanase ADAMTS4. TNF-α enhanced MMP-13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect. Cells isolated from the different cartilaginous structures reacted differently to TNF-α. Since the disc and fossa contain a very low level of proteoglycans in comparison to the condyle, the role played by ADAMTS4 in degradation of the fossa and disc might be limited. TNF-α induced MMP-13 expression by condylar cells might be involved in the degradation of the juvenile condyle. Since this expression was reduced by mechanical loading, functional loading with oral physiotherapy or orthodontic activators may help to reduce the catabolic effect of TNF-α. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.Cells isolated from the different cartilaginous structures reacted differently to TNF-α. TNF-α enhanced MMP-13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect.
      PubDate: 2017-01-06T10:30:23.595916-05:
      DOI: 10.1002/jcp.25593
  • The Emerging Role of TRAF7 in Tumor Development
    • Authors: Tiziana Zotti; Ivan Scudiero, Pasquale Vito, Romania Stilo
      Abstract: The seven members of the tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally discovered and characterized as signaling adaptor molecules coupled to the cytoplasmic regions of receptors of the TNF-R superfamily. Functionally, TRAFs act both as a scaffold and/or enzymatic proteins to regulate activation of mitogen-activated protein kinases (MAPKs) and transcription factors of nuclear factor-κB family (NF-κB). Given the wide variety of stimuli intracellularly conveyed by TRAF proteins, they are physiologically involved in multiple biological processes, including embryonic development, tissue homeostasis, and regulation of innate and adaptive immune responses. In the last few years, it has become increasingly evident the involvement of TRAF7, the last member of the TRAF family to be discovered, in the genesis and progression of several human cancers, placing TRAF7 in the spotlight as a novel tumor suppressor protein. In this paper, we review and discuss the literature recently produced on this subject. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals, Inc.TRAF proteins are signaling adaptor molecules that regulate activation of mitogen-activated protein kinases (MAPKs) and transcription factors of nuclear factor-κB family (NF-κB). In this paper, we review and discuss the literature recently produced pointing to an involvement of TRAF7 in the genesis and progression of several human cancers.
      PubDate: 2017-01-06T10:15:36.42382-05:0
      DOI: 10.1002/jcp.25676
  • A Novel Skin Splint for Accurately Mapping Dermal Remodeling and
           Epithelialization During Wound Healing
    • Authors: Shiro Jimi; Francesco De Francesco, Giuseppe A. Ferraro, Michele Riccio, Shuuji Hara
      Abstract: The mouse excisional dorsal full-thickness wound model with a silicon splint fixed on the skin has been widely used to mimic human wound healing. However, the method cannot accurately quantify dermal remodeling, since the initial point of epithelialization on the wound surface is unclear. To overcome this limitation, we have developed a novel mouse excisional wound model to assess the degree of epithelial extension and regeneration, using a plastic ring-shaped splint fixed beneath the surrounding epidermal tissue. At the end of the experiment, tissue samples were fixed in formalin, the splint was excised, and paraffin sections were prepared. Splint holes, corresponding to the prior location of the splint, were evident on the tissue cross-sections, and the epidermis above the holes was considered the initial excision site. The epidermal contraction and epithelial regeneration, as independent essential tissue alterations in wound healing, could be distinguishable and quantified. Compared with previous splint models, this method provides an accurate evaluation of epidermal processes in wound healing, and can be a platform to assess the effects of various wound healing factors. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.The new method established in this manuscript makes possible the accurate assessment and measurement of epidermal changes—dermal remodeling and regeneration—in the wound healing process in mice.
      PubDate: 2017-01-06T10:15:32.764507-05:
      DOI: 10.1002/jcp.25595
  • Therapeutic Effects of Curcumin in Inflammatory and Immune-Mediated
           Diseases: A Nature-Made Jack-of-All-Trades?
    • Authors: Elham Abdollahi; Amir Abbas Momtazi, Thomas P. Johnston, Amirhosein Sahebkar
      Abstract: Curcumin is a dietary polyphenol from turmeric with numerous pharmacological activities. Novel animal and human studies indicate that curcumin can affect different immune cells, such as various T lymphocyte subsets, macrophages, dendritic cells, B lymphocytes and natural killer cells, which results in decreasing severity of various diseases with immunological etiology. The present review provides a comprehensive overview of the effects of curcumin on different immune cells and immune system-related diseases. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-06T06:45:42.996704-05:
      DOI: 10.1002/jcp.25778
  • TNF-α Promotes Osteoclastogenesis through JNK Signaling-Dependent
           Induction of Semaphorin3D Expression in Estrogen-Deficiency Induced
    • Authors: Chenglin Sang; Jiefeng Zhang, Yongxian Zhang, Fangjing Chen, Xuecheng Cao, Lei Guo
      Abstract: Tumor necrosis factor α (TNF-α)-induced osteoclast formation have been demonstrated to play an important role in the pathogenesis of estrogen deficiency-mediated bone loss, but the exact mechanisms by which TNF-α enhanced osteoclast differentiation were not fully elucidated. The class III semaphorins members were critical to regulate bone homeostasis. Here, we identified a novel mechanism whereby TNF-α increasing Semaphorin3D expression contributes to estrogen deficiency-induced osteoporosis. In this study, we found that Semaphorin3D expression was upregulated by TNF-α during the process of RANKL-induced osteoclast differentiation. Inhibition of Semaphorin3D in pre-osteoclasts could attenuate the stimulatory effects of TNF-α on osteoclast proliferation and differentiation. Mechanistically, blocking of the Jun N-terminal kinase (JNK) signaling markedly rescued TNF-α-induced Semaphorin3D expression, suggesting that JNK signaling was involved in the regulation of Semaphorin3D expression by TNF-α. In addition, silencing of Semaphorin3D in vivo could alleviate estrogen deficiency-induced osteoporosis. Our results revealed a novel function for Semaphorin3D and suggested that increased Semaphorin3D may contribute to enhanced bone loss by increased TNF-α in estrogen deficiency-induced osteoporosis. Thus, Semaphorin3D may provide a potential therapeutic target for the treatment of estrogen-deficiency induced osteoporosis. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-06T06:45:40.856792-05:
      DOI: 10.1002/jcp.25784
  • Irx3 and Bmp2 Regulate Mouse Mesenchymal Cell Chondrogenic Differentiation
           in Both a Sox9-Dependent and -Independent Manner
    • Authors: Yoshihiro Tamamura; Kenichi Katsube, Hisashi Mera, Maki Itokazu, Shigeyuki Wakitani
      Abstract: Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-06T06:45:28.56798-05:0
      DOI: 10.1002/jcp.25776
  • KCNQ1 Variants Associate with Hypertension in Type 2 Diabetes and Affect
           Smooth Muscle Contractility In Vitro
    • Authors: Kuo-Chin Huang; Te-Mao Li, Xiang Liu, Jin-Hua Chen, Wen-Kuei Chien, Yi-Tzone Shiao, Hsinyi Tsang, Ting-Hsu Lin, Chiu-Chu Liao, Shao-Mei Huang, Ju-Pi Li, Cheng-Wen Lin, Jung-Chun Lin, Chih-Chien Lin, Chih-Ho Lai, Chi-Fung Cheng, Wen-Miin Liang, Chien-Hui Hung, Ching-Chu Chen, Ying-Ju Lin, Fuu Jen-Tsai
      Abstract: KCNQ1 encodes a potassium voltage-gated channel and represents a susceptibility locus for type 2 diabetes mellitus (T2DM). Here, we explored the association between KCNQ1 polymorphisms and hypertension risk in individuals with T2DM, as well as the role of KCNQ1 in vascular smooth muscle cell contraction in vitro. To investigate the relationship between KCNQ1 and the risk of developing hypertension in patients with T2DM, we divided the T2DM cohort into hypertension (n = 452) and non-hypertension (n = 541) groups. The Mann-Whitney U test, chi-square test, and multivariate regression analyses were used to assess the clinical characteristics and genotypic frequencies. In vitro studies utilized the rat aortic smooth muscle A10 cell line. Patients in the hypertension group were significantly older at the time of enrollment and had higher levels of body mass index, waist-to-hip ratio, and triglyceride than those in the non-hypertension group. The KCNQ1 rs3864884 and rs12576239 genetic variants were associated with hypertension in T2DM. KCNQ1 expression was lower in the individuals with the CC versus the CT and TT genotypes. Smooth muscle cell contractility was inhibited by treatment with a KCNQ1 inhibitor. These results suggest that KCNQ1 might be associated with hypertension in individuals with T2DM. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-06T06:40:42.667888-05:
      DOI: 10.1002/jcp.25775
  • Acetoacetate Induces Hepatocytes Apoptosis by the ROS-Mediated MAPKs
           Pathway in Ketotic Cows
    • Authors: Xiliang Du; Zhen Shi, Zhicheng Peng, Chenxu Zhao, Yuming Zhang, Zhe Wang, Xiaobing Li, Guowen Liu, Xinwei Li
      Abstract: Dairy cows with ketosis are characterized by oxidative stress, hepatic damage and hyperketonemia. Acetoacetate (AA) is the main component of ketone bodies in ketotic cows, and is associated with the above pathological process. However, the potential mechanism was not illuminated. Therefore, the aim of this study was to investigate the mechanism of AA-induced hepatic oxidative damage in ketotic cows. Compared with healthy cows, ketotic cows exhibited severe oxidative stress and hepatic damage. Moreover, the extent of hepatic damage and oxidative stress had a positive relationship with the AA levels. In vitro, AA treatment increased reactive oxygen species (ROS) content and further induced oxidative stress and apoptosis of bovine hepatocytes. In this process, AA treatment increased the phosphorylation levels of JNK and p38MAPK and decreased the phosphorylation level of ERK, which could increase p53 and inhibit nuclear factor E2-related factor 2 (Nrf2) expression, nuclear localization and DNA-binding affinity, thereby inducing the overexpression of pro-apoptotic molecules Bax, Caspase 3, Caspase 9, PARP and inhibition of anti-apoptotic molecule Bcl-2. Antioxidant N-acetylcysteine (NAC) treatment or interference of MAPKs pathway could attenuate the hepatocytes apoptosis induced by AA. Collectively, these results indicate that AA triggers hepatocytes apoptosis via the ROS-mediated MAPKs pathway in ketotic cows. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-06T06:40:30.112685-05:
      DOI: 10.1002/jcp.25773
  • Optimized Method for Isolating Highly Purified and Functional Porcine
           Aortic Endothelial and Smooth Muscle Cells
    • Authors: Farideh Beigi; Mitalben Patel, Marco A. Morales-Garza, Caitlin Winebrenner, Andrea S. Gobin, Eric Chau, Luiz C. Sampaio, Doris A. Taylor
      Abstract: BackgroundNumerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells.MethodsThe lumen of a porcine aorta was filled with 25 U/mL dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution.ResultsThe isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early- and late-passage endothelial cells had a population doubling time of 38 hours and maintained a capacity to take up DiI-Ac-LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha-smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hours at passages 3 and 7; and the cells positively responded to endothelin-1, as shown by a 66% increase in the intracellular calcium level.ConclusionsThis simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-06T05:55:32.09884-05:0
      DOI: 10.1002/jcp.25764
  • Roles of Multifunctional COP9 Signalosome Complex in Cell Fate and
           Implications for Drug Discovery
    • Authors: Ping Li; Longxiang Xie, Yinzhong Gu, Jiang Li, Jianping Xie
      Abstract: The eight subunits containing COP9 signalosome (CSN) complex, is highly conserved among eukaryotes. CSN, identified as a negative regulator of photomorphogenesis, has also been demonstrated to be important in proteolysis, cellular signal transduction and cell cycle regulation in various eukaryotic organisms. This review mainly summarizes the roles of CSN in cell cycle regulation, signal transduction and apoptosis, and its potential as diagnostic biomarkers, drug targets for cancer and infectious diseases. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.This review mainly summarizes the roles of CSN in cell cycle regulation, signal transduction and apoptosis, and its potential as diagnostic biomarkers, drug targets for cancer and infectious diseases.
      PubDate: 2017-01-05T08:45:23.116809-05:
      DOI: 10.1002/jcp.25696
  • A MicroRNA Cluster miR-23–24–27 Is Upregulated by Aldosterone in the
           Distal Kidney Nephron Where it Alters Sodium Transport
    • Authors: Xiaoning Liu; Robert S. Edinger, Christine A. Klemens, Yu L. Phua, Andrew J. Bodnar, William A. LaFramboise, Jacqueline Ho, Michael B. Butterworth
      Abstract: The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules, and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23–24–27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3′-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR-27 in aldosterone's action via a novel target. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.The steroid hormone aldosterone alters the expression of microRNAs in distal kidney nephron epithelial cells, and coordinately upregulates the miR-23–24–27 cluster. MiR-27 targets intersectin 2 to reduce its expression. Decreased intersectin 2 levels results in an increase in the activity of the epithelial sodium channel to augment sodium transport.
      PubDate: 2017-01-05T08:35:27.33984-05:0
      DOI: 10.1002/jcp.25599
  • Identifying Nuclear Matrix-Attached DNA Across the Genome
    • Authors: Jason R. Dobson; Deli Hong, A. Rasim Barutcu, Hai Wu, Anthony N. Imbalzano, Jane B. Lian, Janet L. Stein, Andre J. van Wijnen, Jeffrey A. Nickerson, Gary S. Stein
      Abstract: Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix-attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix-associated genome is highly cell-context dependent. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.MAR-Seq is a next-generation sequencing strategy to identify matrix attachment regions (MARs) that is based on crosslinking chromatin to its nuclear attachment sites. Comparison of MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells showed that nuclear matrix-attached DNA in normal-like MCF-10A cells is enriched in expressed genes, while in the breast cancer cells, it is enriched in non-expressed genes. This new MAR-Seq approach reveals that the nuclear matrix-associated genome is highly cell-context dependent.
      PubDate: 2017-01-05T08:35:24.082065-05:
      DOI: 10.1002/jcp.25596
  • Fibroblast Growth Factor 21 (FGF21) Promotes Formation of Aerobic
           Myofibers via the FGF21-SIRT1-AMPK-PGC1α Pathway
    • Authors: Xinyi Liu; Yongliang Wang, Liming Hou, Yuanzhu Xiong, Shuhong Zhao
      Abstract: Fibroblast growth factor 21(FGF21) is a pivotal regulator of energy metabolism, which is currently being assessed as a potential drug target for the treatment of insulin-resistant conditions. However, the cellular mechanisms by which FGF21 affects myogenesis remain unclear. In this study, we explored the function of FGF21 in myogenesis both in vitro and in vivo. Our experiments showed for the first time that FGF21 promotes myoblast differentiation and serves as a switch of molecular transformation from anaerobic myofibers to aerobic myofibers via the FGF21-SIRT1-AMPK-PGC1α axis. Furthermore, we employed the Dual-Luciferase Reporter Assay System and Electrophoretic Mobility Shift Assay (EMSA) and demonstrated that MYOD, a major myogenic transcription factor, binds directly to the promoter region of Fgf21, leading to the activation of Fgf21 expression in mouse C2C12 myoblasts. Our study revealed a novel mechanism of myogenesis and muscle fiber transformation and indicated that FGF21 serves as a vital regulator of muscle development and important contributor to the pathogenesis of myopathy. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-05T08:30:37.504911-05:
      DOI: 10.1002/jcp.25735
  • Calretinin Immunoreactivity in the Human Testis Throughout Fetal Life
    • Authors: Giovanna G. Altobelli; Francesca Pentimalli, Mariarosaria D'Armiento, Susan Van Noorden, Vincenzo Cimini
      Abstract: The main functions of the testis are sex hormone and sperm cell production. Steroidogenesis occurs in the Leydig interstitial cells and spermatogenesis in the seminiferous tubules. Male gonad morphogenesis is a finely orchestrated process, mainly coordinated by hormones, whose actions can significantly affect post-pubertal testicular function. Calcium is a key intracellular messenger, which regulates many signal transduction pathways, and is also implicated in steroidogenesis. Calcium homeostasis and signaling rely on many calcium-binding proteins including calretinin, of the "EF-hand" protein family. Calretinin is a highly conserved protein mainly expressed in the nervous system but also detected in rat and human adult and fetal testis as well as in pathological conditions. Calretinin expression in the fetal testis, however, has not been thoroughly analyzed probably owing to limited availability and paucity of tissues. Here we examined by immunocytochemistry the expression of calretinin in human fetal testis specimens, obtained from natural and therapeutic abortions, at various developmental ages. We found that calretinin-immunoreactive Leydig cells were visible throughout the timeframe studied (14th to 27th week). Immunoreactivity was also observed in Sertoli cells and in the germ cells of the immature seminiferous tubules. Overall our data indicate that calretinin expression parallels the decline in Leydig cell number, suggesting that its presence is indeed correlated to their steroidogenic activity. They also suggest that the intratubular positivity of calretinin could be linked to the ability of Sertoli cells to produce locally acting hormones contributing to the histodifferentiation of the male genital tract. This article is protected by copyright. All rights reserved
      PubDate: 2017-01-05T08:25:28.973764-05:
      DOI: 10.1002/jcp.25727
  • Early Subchondral Bone Loss at Arthritis Onset Predicted Late Arthritis
           Severity in a Rat Arthritis Model
    • Authors: Guillaume Courbon; Damien Cleret, Marie-Thérèse Linossier, Laurence Vico, Hubert Marotte
      Abstract: Synovitis is usually observed before loss of articular function in rheumatoid arthritis (RA). In addition to the synovium and according to the “Inside–Outside” theory, bone compartment is also involved in RA pathogenesis. Then, we investigated time dependent articular bone loss and prediction of early bone loss to late arthritis severity on the rat adjuvant-induced arthritis (AIA) model. Lewis female rats were longitudinally monitored from arthritis induction (day 0), with early (day 10) and late (day 17) steps. Trabecular and cortical microarchitecture parameters of four ankle bones were assessed by microcomputed tomography. Gene expression was determined at sacrifice. Arthritis occurred at day 10 in AIA rats. At this time, bone erosions were detected on four ankle bones, with cortical porosity increase (+67%) and trabecular alterations including bone volume fraction (BV/TV: −13%), and trabecular thickness decrease. Navicular bone assessment was the most reproducible and sensitive. Furthermore, strong correlations were observed between bone alterations at day 10 and arthritis severity or bone loss at day 17, including predictability of day 10 BV/TV to day 17 articular index (R2 = 0.76). Finally, gene expression at day 17 confirmed massive osteoclast activation and interestingly provided insights on strong activation of bone formation inhibitor markers at the joint level. In rat AIA, bone loss was already observed at synovitis onset and was predicted late arthritis severity. Our results reinforced the key role of subchondral bone in arthritis pathogenesis, in favour to the “Inside–Outside” theory. Mechanisms of bone loss in rat AIA involved resorption activation and formation inhibition changes. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.We explored time dependent articular bone loss and prediction of early bone loss to late arthritis severity on the rat adjuvant-induced arthritis (AIA) model. At clinical synovitis onset, bone porosity and microarchitecture alterations were recorded in the ankle bones. These early modifications were predictive of arthritis outcome, underlining the importance of early bone loss investigation.
      PubDate: 2017-01-05T08:15:26.639024-05:
      DOI: 10.1002/jcp.25601
  • HMGB1 Down-Regulation Mediates Terameprocol Vascular Anti-Proliferative
           Effect in Experimental Pulmonary Hypertension
    • Authors: Rita Nogueira-Ferreira; Manuel J. Ferreira-Pinto, Ana Filipa Silva, Rui Vitorino, Joana Justino, Raquel Costa, Daniel Moreira-Gonçalves, Jean-François Quignard, Thomas Ducret, Jean-Pierre Savineau, Adelino F. Leite-Moreira, Rita Ferreira, Tiago Henriques-Coelho
      Abstract: Pulmonary arterial hypertension (PAH) is a progressive disease with a poor prognosis. Pulmonary artery smooth muscle cells (PASMCs) play a crucial role in PAH pathophysiology, displaying a hyperproliferative and apoptotic-resistant phenotype. In the present study, we evaluated the potential therapeutic role of terameprocol (TMP), an inhibitor of cellular proliferation and promoter of apoptosis, in a well-established pre-clinical model of PAH induced by monocrotaline (MCT) and studied the biological pathways modulated by TMP in PASMCs. Wistar rats injected with MCT or saline (SHAM group) were treated with TMP or vehicle. On day 21 after injection, we assessed bi-ventricular hemodynamics and cardiac and pulmonary morphometry. The effects of TMP on PASMCs were studied in a primary culture isolated from SHAM and MCT-treated rats, using an iTRAQ-based proteomic approach to investigate the molecular pathways modulated by this drug. In vivo, TMP significantly reduced pulmonary and cardiac remodeling and improved cardiac function in PAH. In vitro, TMP inhibited proliferation and induced apoptosis of PASMCs. A total of 65 proteins were differentially expressed in PASMCs from MCT rats treated with TMP, some of which involved in the modulation of transforming growth factor beta pathway and DNA transcription. Anti-proliferative effect of TMP seems to be explained, at least in part, by the down-regulation of the transcription factor HMGB1. Our findings support the beneficial role of TMP in PAH and suggest that it may be an effective therapeutic option to be considered in the clinical management of PAH. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-30T11:05:31.857793-05:
      DOI: 10.1002/jcp.25763
  • Primary Cilium-Regulated EG-VEGF Signaling Facilitates Trophoblast
    • Authors: Chia-Yih Wang; Hui-Ling Tsai, Jhih-Siang Syu, Ting-Yu Chen, Mei-Tsz Su
      Abstract: Trophoblast invasion is an important event in embryo implantation and placental development. During these processes, endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is the key regulator mediating the crosstalk at the feto-maternal interface. The primary cilium is a cellular antenna receiving environmental signals and is crucial for proper development. However, little is known regarding the role of the primary cilium in early human pregnancy. Here, we demonstrate that EG-VEGF regulates trophoblast cell invasion via primary cilia. We found that EG-VEGF activated ERK1/2 signaling and subsequent upregulation of MMP2 and MMP9, thereby facilitating cell invasion in human trophoblast HTR-8/SVneo cells. Inhibition of ERK1/2 alleviated the expression of MMPs and trophoblast cell invasion after EG-VEGF treatment. In addition, primary cilia were observed in all the trophoblast cell lines tested and, more importantly, in human first-trimester placental tissue. The receptor of EG-VEGF, PROKR1, was detected in primary cilia. Depletion of IFT88, the intraflagellar transporter required for ciliogenesis, inhibited primary cilium growth, thereby ameliorating ERK1/2 activation, MMP upregulation, and trophoblast cell invasion promoted by EG-VEGF. These findings demonstrate a novel function of primary cilia in controlling EG-VEGF-regulated trophoblast invasion and reveal the underlying molecular mechanism. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Primary cilium is a cellular antenna that conveys extracellular signals into a cellular response. EG-VEGF binds to PROKR1 locating on the cilium, and activates ERK1/2 signaling followed by upregulating MMP2 and MMP9 for facilitating cell invasion. We demonstrated that EG-VEGF regulated trophoblast cell invasion via primary cilium in the present study.
      PubDate: 2016-12-29T05:40:59.355062-05:
      DOI: 10.1002/jcp.25649
  • Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced
           Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells
    • Authors: Bahareh Rajaei; Mehdi Shamsara, Masoumeh Fakhr Taha, Leila Mohammadi Amirabad, 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. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This high efficient differentiation protocol, 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.
      PubDate: 2016-12-29T05:37:08.821584-05:
      DOI: 10.1002/jcp.25459
  • Exploring the Role of PGC-1α in Defining Nuclear Organisation in
           Skeletal Muscle Fibres
    • Authors: Jacob Alexander Ross; Adam Pearson, Yotam Levy, Bettina Cardel, Christoph Handschin, Julien Ochala
      Abstract: Muscle fibres are multinucleated cells, with each nucleus controlling the protein synthesis in a finite volume of cytoplasm termed the myonuclear domain (MND). What determines MND size remains unclear. In the present study, we aimed to test the hypothesis that the level of expression of the transcriptional coactivator PGC-1α and subsequent activation of the mitochondrial biogenesis are major contributors. Hence, we used two transgenic mouse models with varying expression of PGC-1α in skeletal muscles. We isolated myofibres from the fast twitch extensor digitorum longus (EDL) and slow twitch diaphragm muscles. We then membrane-permeabilised them and analysed the 3D spatial arrangements of myonuclei. In EDL muscles, when PGC-1α is over-expressed, MND volume decreases; whereas, when PGC-1α is lacking, no change occurs. In the diaphragm, no clear difference was noted. This indicates that PGC-1α and the related mitochondrial biogenesis programme are determinants of MND size. PGC-1α may facilitate the addition of new myonuclei in order to reach MND volumes that can support an increased mitochondrial density. J. Cell. Physiol. 9999: 1–5, 2016. © 2016 Wiley Periodicals, Inc.Muscle fibres are multinucleated cells, with individual nuclei controlling the protein synthesis in a finite volume of cytoplasm termed myonuclear domain. The present study indicates that one important regulator of myonuclear domain size is PGC-1α and related mitochondrial biogenesis programme.
      PubDate: 2016-12-29T05:37:05.80091-05:0
      DOI: 10.1002/jcp.25678
  • Lgr4 Expression in Osteoblastic Cells Is Suppressed by Hydrogen Peroxide
    • Authors: Chantida Pawaputanon Na Mahasarakham; Yayoi Izu, Katsuhiko Nishimori, Yuichi Izumi, Masaki Noda, Yoichi Ezura
      Abstract: LGR4 is expressed in bone and has been shown to be involved in bone metabolism. Oxidative stress is one of the key issues in pathophysiology of osteoporosis. However, the link between Lgr4 and oxidative stress has not been known. Therefore, effects of hydrogen peroxide on Lgr4 expression in osteoblasts were examined. Hydrogen peroxide treatment suppressed the levels of Lgr4 mRNA expression in an osteoblastic cell line, MC3T3-E1. The suppressive effects were not obvious at 0.1 mM, while 1 mM hydrogen peroxide suppressed Lgr4 expression by more than 50%. Hydrogen peroxide treatment suppressed Lgr4 expression within 12 h and this suppression lasted at least up to 48 h. Hydrogen peroxide suppression of Lgr4 expression was still observed in the presence of a transcription inhibitor but was no longer observed in the presence of a protein synthesis inhibitor. Although Lgr4 expression in osteoblasts is enhanced by BMP2 treatment as reported before, hydrogen peroxide treatment suppressed Lgr4 even in the presence of BMP2. Finally, hydrogen peroxide suppressed Lgr4 expression in primary cultures of osteoblasts similarly to MC3T3-E1 cells. These date indicate that hydrogen peroxide suppresses Lgr4 expression in osteoblastic cells. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-12-29T05:37:03.982426-05:
      DOI: 10.1002/jcp.25684
  • Identification of a Novel Human E-Cadherin Splice Variant and Assessment
           of Its Effects Upon EMT-Related Events
    • Authors: María Laura Matos; Lara Lapyckyj, Marina Rosso, María José Besso, María Victoria Mencucci, Clara Isabel Marín Briggiler, Silvina Giustina, Laura Inés Furlong, Mónica Hebe Vazquez-Levin
      Abstract: Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell–cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05–11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them acquisition of a fibroblastic-like cell phenotype, increased expression of Twist, Snail, Zeb1, and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell–cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding the molecular basis of tumor progression and improve cancer diagnosis. J. Cell. Physiol. 9999: 1–19, 2016. © 2016 Wiley Periodicals, Inc.This report describes the identification and partial characterization of a novel human E-cadherin variant mRNA produced by alternative splicing. Stable transfection of the E-cadherin variant mRNA in MCF-7 cells (MCF7Ecadvar cells) resulted in downregulation of wild type E-cadherin and EMT-related changes, that is, acquisition of a fibroblast-like cellular phenotype, increased expression of E-cadherin transcriptional repressors, vimentin and N-cadherin, and loss of cytokeratins. MCF7Ecadvar cells depicted dramatic changes in cellular behavior, showing reduced cell–cell adhesion and increased cell migration and invasiveness.
      PubDate: 2016-12-29T05:36:52.820603-05:
      DOI: 10.1002/jcp.25622
  • Mactosylceramide Prevents Glial Cell Overgrowth by Inhibiting Insulin and
           Fibroblast Growth Factor Receptor Signaling
    • Authors: Stine Gerdøe-Kristensen; Viktor K. Lund, Hans H. Wandall, Ole Kjaerulff
      Abstract: Receptor Tyrosine Kinase (RTK) signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Deregulated RTK signaling also underlies many cancers. Glycosphingolipids (GSL) are essential elements of the plasma membrane. By affecting clustering and activity of membrane receptors, GSL modulate signal transduction, including that mediated by the RTK. GSL are abundant in the nervous system, and glial development in Drosophila is emerging as a useful model for studying how GSL modulate RTK signaling. Drosophila has a simple GSL biosynthetic pathway, in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what extent this effect involves changes in upstream signaling events is unresolved. We show here that glial overgrowth in egh is strongly linked to increased activation of Insulin and Fibroblast Growth Factor receptors (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants of the Drosophila Insulin Receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. We conclude that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of Insulin and Fibroblast Growth Factor Receptors in Drosophila glia. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-26T06:40:24.131594-05:
      DOI: 10.1002/jcp.25762
  • Isolation, Identification, and Characterization of Cancer Stem Cells: A
    • Authors: Vahid Bagheri; Mahya Shariat Razavi, Amir Abbas Momtazi, Amirhossein Sahebkar, Mohammad Reza Abbaszadegan, Mehran Gholamin
      Abstract: Cancer stem cells (CSCs) or tumor-initiating cells (TICs) as a small subset of neoplastic cells are able to produce a tumor (tumorigenesis), maintain the population of tumorigenic cells (self-renewal), and generate the heterogeneous cells constructing the entire tumor (pluripotency). The research on stationary and circulating CSCs due to resistance to conventional therapies and inability in complete eradication of cancer is critical for developing novel therapeutic strategies for a more effective reduction in the risk of tumor metastasis and cancer recurrence. This review compiles information about different methods of detection and dissociation, side population, cellular markers and establishment culture of CSCs, as well as characteristics of CSCs such as tumorigenicity, and signaling pathways associated with self-renewal and the capability of the same histological tumor regeneration in various cancers. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-26T06:20:36.460473-05:
      DOI: 10.1002/jcp.25759
  • Na+/H+ Exchanger NHE1 and NHE2 have Opposite Effects on Migration Velocity
           in the Rat Gastric Surface Cells
    • Authors: Anja Paehler vor der Nolte; Giriprakash Chodisetti, Zhenglin Yuan, Florian Busch, Brigitte Riederer, Min Luo, Yan Yu, Manoj B. Menon, Andreas Schneider, Renata Stripecke, Katerina Nikolovska, Sunil Yeruva, Ursula Seidler
      Abstract: Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+/H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and transport activity, NHE2 was introduced by lentiviral gene transfer. At medium pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1-dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1-dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼ 10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased steady-state pHi and reduced restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore play a role in the initiation and completion of the epithelial restitution process. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-26T06:20:35.178852-05:
      DOI: 10.1002/jcp.25758
  • Smoothened-Antagonists Reverse Homogentisic Acid-Induced Alterations of
           Hedgehog Signalling and Primary Cilium Length in Alkaptonuria
    • Authors: Silvia Gambassi; Michela Geminiani, Stephen D Thorpe, Giulia Bernardini, Lia Millucci, Daniela Braconi, Maurizio Orlandini, Clare L Thompson, Elena Petricci, Fabrizio Manetti, Maurizio Taddei, Martin M Knight, Annalisa Santucci
      Abstract: Alkaptonuria (AKU) is an ultra-rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignalling and Hedgehog signalling pathways. Hedgehog signalling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia hedgehog signalling may also be altered in AKU.Thus, we characterised an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the hedgehog signalling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the hedgehog pathway. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-26T06:20:33.633574-05:
      DOI: 10.1002/jcp.25761
  • Pyk2 Inhibition Promotes Contractile Differentiation in Arterial Smooth
    • Authors: Mario Grossi; Anirban Bhattachariya, Ina Nordström, Karolina M. Turczyńska, Daniel Svensson, Sebastian Albinsson, Bengt-Olof Nilsson, Per Hellstrand
      Abstract: Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent towards voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24 h to 96 h) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-26T06:20:22.227742-05:
      DOI: 10.1002/jcp.25760
  • Curcumin as a Potential Candidate for Treating Hyperlipidemia: A Review of
           Cellular and Metabolic Mechanisms
    • Authors: Yunes Panahi; Yasin Ahmadi, Manouchehr Teymouri, Thomas P. Johnston, Amirhossein Sahebkar
      Abstract: Curcumin is an herbal polyphenol extensively investigated for antioxidant, anti-inflammatory and hypolipidaemic properties. In the present review, the efficacy of curcumin for improving a plasma lipid profile has been evaluated and compared with statins, a well-known class of medicines for treating hypercholesterolemia and hyperlipidaemia. Curcumin is presumably most effective in reducing triglyceride (TG), while statins are most efficient in lowering low-density lipoproteins-cholesterol (LDL-C). Additionally, various molecular and metabolic mediators of cholesterol and plasma lipid homeostasis are discussed in relation to how they are modulated by curcumin or statins. Overall, curcumin influences the same mediators of plasma lipid alteration as statins do. Almost all the pathways through which cholesterol trafficking takes place are affected by these agents. These include gastrointestinal absorption of dietary cholesterol, hepatocellular removal of plasma cholesterol, the mediators of reverse cholesterol transport and removal of cholesterol from peripheral tissues. Moreover, the reactive oxygen species (ROS) scavenging potential of curcumin limits the risk of lipid peroxidation that triggers inflammatory responses causing cardiovascular diseases (CVD) and atherosclerosis. Taken together, curcumin could be used as a safe and well-tolerated adjunct to statins to control hyperlipidaemia more effectively than statins alone. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-24T03:25:31.303795-05:
      DOI: 10.1002/jcp.25756
  • Diverse Functions and Signal Transduction of the Exocyst Complex in Tumor
    • Authors: Toshiaki Tanaka; Kaoru Goto, Mitsuyoshi Iino
      Abstract: The exocyst complex is a large conserved hetero-oligomeric complex that consists of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 subunits. It has been implicated in the targeting of vesicles for regulated exocytosis in various cell types, and is also important for targeted exocytosis of post-Golgi transport vesicles to the plasma membrane. The exocyst complex is essential for membrane growth, secretion, and function during exocytosis and endocytosis. Moreover, the individual components of the complex are thought to act on specific biological processes, such as cytokinesis, ciliogenesis, apoptosis, autophagy, and epithelial-mesenchymal transition (EMT). As a result, recent studies suggest that the exocyst complex may be involved in several diseases such as kidney disease, neuropathogenesis, diabetes, and cancer. In this review, we focus on the diverse functions and cellular signaling pathways of the exocyst complex in various tumors. J. Cell. Physiol. 9999: 1–19, 2016. © 2016 Wiley Periodicals, Inc.Exo70 interacts strongly with the Sec10–Sec15 subcomplex and Exo84 interacts with the Sec10–Sec15–Exo70 subcomplex. Sec3, Sec5, Sec6, and Sec8 form a tight quaternary subcomplex and Sec8 interacts with Sec10. Each component of the exocyst complex contributes to cancer progression and survival in diverse ways such as break of cell–cell contact, EMT, cell cycle progression, DNA repair, migration, autophagy, and anti-apoptosis.
      PubDate: 2016-12-22T06:30:54.740663-05:
      DOI: 10.1002/jcp.25619
  • Proinflammatory Signaling Functions of Thrombin in Cancer
    • Authors: Safieh Ebrahimi; Farzad Rahmani, Reihane Behnam-Rassouli, Fatemeh Hoseinkhani, Mohammad Reza Parizadeh, Mohammad Reza Keramati, Majid Khazaie, Amir Avan, Seyed Mahdi Hassanian
      Abstract: Thrombin-induced activation of protease-activated receptors (PARs) represents a link between inflammation and cancer. Proinflammatory signaling functions of thrombin are associated with several inflammatory diseases including neurodegenerative, cardiovascular, and of special interest in this review cancer. Thrombin-induced inflammatory responses up-regulates expression of cytokines, adhesion molecules, angiogenic factors and matrix-degrading proteases that facilitate tumor cells proliferation, angiogenesis, invasion and metastasis. This review summarizes the current knowledge about the mechanisms of thrombin-mediated proinflammatory responses in cancer pathology for a better understanding and hence a better management of this disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-22T03:45:46.762515-05:
      DOI: 10.1002/jcp.25753
  • LCN2 Overexpression in Bone Enhances the Hematopoietic Compartment via
           Modulation of the Bone Marrow Microenvironment
    • Authors: Delfina Costa; Elisa Principi, Edoardo Lazzarini, Fiorella Descalzi, Ranieri Cancedda, Patrizio Castagnola, Sara Tavella
      Abstract: Lipocalin-2 (LCN2) is a member of the lipocalin family whose expression is modulated in several conditions, including cell differentiation, innate immunity, stress and cancer. Although it is known that it is expressed in bone, its function in this tissue remains poorly studied. To this end, we took advantage of transgenic mice lines that expressed LCN2 driven by a bone specific type I collagen (LCN2-Tg).In the bone marrow (BM) of LCN2-Tg mice we observed an increased number of phenotypically long-term hematopoietic stem cells (LT-HSC) that also displayed a higher proliferation rate compared to wild type controls (Wt). Furthermore, hematopoietic progenitor cells, obtained from LCN2-Tg BM showed an increased clonogenic capacity compared to those obtained from LCN2-Tg spleen, a higher concentration of serum erythropoietin and a higher number of mature erythrocytes in the peripheral blood of old LCN2-Tg animals compared to aged-matched wt. The findings of a combined increase in the BM of the LCN2-Tg mice of SDF-1, SCF and TIMP-1 levels along with the reduction of both MMP-9 activity and cathepsin K concentration may explain the observed effects on the HSC compartment.This study shows that LCN2 overexpression in bones modifies the BM microenvironment via modulation of the expression of key secreted factors and cytokines, which in turn regulate the HSC niche behavior enhancing both HSC homing in young mice and erythrocytes production in older mice. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-22T03:45:40.02211-05:0
      DOI: 10.1002/jcp.25755
  • Deregulated ALG-2/HEBP2 Axis Alters Microtubule Dynamics and Mitotic
           Spindle Behavior to Stimulate Cancer Development
    • Authors: Juan Qin; Yang Yang, Siqi Gao, Yang Liu, Fan Yu, Yunqiang Zhou, Rui Lyu, Min Liu, Xinqi Liu, Dengwen Li, Jun Zhou
      Abstract: Cancer cells are characterized by genomic instability, resulting in the accumulation of mutations that promote cancer progression. One way that genomic instability can arise is through improper regulation of the microtubule cytoskeleton that impacts the function of the mitotic spindle. In this study, we have identified a critical role for the interaction between apoptosis-linked gene 2 (ALG-2) and heme-binding protein 2 (HEBP2) in the above processes. Our data show that the gene copy numbers and mRNA levels for both ALG-2 and HEBP2 are significantly upregulated in breast and lung cancer. Coexpression of ALG-2 and HEBP2 markedly increases the cytoplasmic pool of ALG-2 and alters the subcellular distribution of HEBP2. Our data further reveal that abnormality in the ALG-2/HEBP2 interaction impairs spindle orientation and positioning during mitosis. In addition, this complex appears to modulate the dynamic properties of microtubules in cancer cells. These finding thus uncover an important function for deregulated ALG-2/HEBP2 axis in cancer development by influencing microtubule dynamics and spindle behavior, providing novel insight into the etiology and pathogenesis of cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-22T03:45:31.812432-05:
      DOI: 10.1002/jcp.25754
  • Differential Expression of miR-4520a Associated With Pyrin Mutations in
           Familial Mediterranean Fever (FMF)
    • Authors: Helen Latsoudis; Mir-Farzin Mashreghi, Joachim R. Grün, Hyun-Dong Chang, Bruno Stuhlmüller, Argyro Repa, Irini Gergiannaki, Eleni Kabouraki, George S. Vlachos, Thomas Häupl, Andreas Radbruch, Prodromos Sidiropoulos, Kimon Doukoumetzidis, Dimitris Kardassis, Timothy B. Niewold, Dimitrios T. Boumpas, George N. Goulielmos
      Abstract: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute and self-limiting attacks of fever. In the present study we presented for the first time results supporting a role of deregulated autophagy in the pathogenesis of FMF.
      PubDate: 2016-12-20T08:30:57.563042-05:
      DOI: 10.1002/jcp.25602
  • RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease
           Modeling and Therapy
    • Authors: James Kehler; Marianna Greco, Valentina Martino, Manickam Pachiappan, Hiroko Yokoe, Alice Chen, Miranda Yang, Jonathan Auerbach, Joel Jessee, Martin Gotte, Luciano Milanesi, Alberto Albertini, Gianfranco Bellipanni, Ileana Zucchi, Rolland A. Reinbold, Antonio Giordano
      Abstract: Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogrammed iPSCs lines may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedures for making and editing iPSCs. These two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic and translational research.
      PubDate: 2016-12-20T08:30:50.328732-05:
      DOI: 10.1002/jcp.25597
  • Regulation of Mitotic Spindle Orientation during Epidermal Stratification
    • Authors: Wei Xie; Jun Zhou
      Abstract: The epidermis is a stratified epithelium that serves as a barrier to infection from environmental pathogens and prevents water loss. Epidermal stratification is tightly controlled during embryogenesis. Progenitor cells in the developing epidermis undergo both symmetric and asymmetric cell divisions to balance the growth of the skin surface area against the generation of differentiated cell layers. Therefore, understanding the relationship between oriented divisions of progenitor cells and the development and stratification of the epidermis is of paramount importance in the field of skin biology and pathology. We provide here an integrated view of recent studies implicating that improper orientation of the mitotic spindle contributes to disorders associated with abnormal epidermal stratification and suggesting that spindle orientation could serve as a potential therapeutic target in skin diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-20T07:10:48.890982-05:
      DOI: 10.1002/jcp.25750
  • Super-Resolution Structure of DNA Significantly Differs in Buccal Cells of
           Controls and Alzheimer's Patients
    • Authors: Angeles Garcia; David Huang, Amanda Righolt, Christiaan Righolt, Maria Carmela Kalaw, Shubha Mathur, Elizabeth McAvoy, James Anderson, Angela Luedke, Justine Itorralba, Sabine Mai
      Abstract: The advent of super-resolution microscopy allowed for new insights into cellular and physiological processes of normal and diseased cells. In this study, we report for the first time on the super-resolved DNA structure of buccal cells from patients with Alzheimer's disease (AD) vs. age- and gender-matched healthy, non-caregiver controls. In this super-resolution study cohort of 74 participants, buccal cells were collected and their spatial DNA organization in the nucleus examined by 3D Structured Illumination Microscopy (3D-SIM). Quantitation of the super-resolution DNA structure revealed that the nuclear super-resolution DNA structure of individuals with AD significantly differs from that of their controls (p 
      PubDate: 2016-12-20T07:10:47.868614-05:
      DOI: 10.1002/jcp.25751
  • Tolerogenic Probiotics: Potential Immunoregulators in Systemic Lupus
    • Authors: Seyed Alirezae Esmaeili; Mahmoud Mahmoudi, Amir Abbas Momtazi-borojeni, Amirhossein Sahebkar, Hassan Doulabi, Maryam Rastin
      Abstract: Probiotics are commensal or nonpathogenic microbes that colonize the gastrointestinal tract and confer beneficial effects on the host through several mechanisms such as competitive exclusion, anti-bacterial effects, and modulation of immune responses. There is growing evidence supporting the immunomodulatory ability of some probiotics. Several experimental and clinical studies have been shown beneficial effect of some probiotic bacteria, particularly Lactobacillus and Bifidobacteria strains, on inflammatory and autoimmune diseases. Systemic lupus erythematosus (SLE) is an autoimmune disease that is mainly characterized by immune intolerance towards self-antigens. Some immunomodulatory probiotics have been found to regulate immune responses via tolerogenic mechanisms. Dendritic and T regulatory (Treg) cells, IL-6, IFN-γ, IL-17, and IL-23 can be considered as the most determinant dysregulated mediators in tolerogenic status. As demonstrated by documented experimental and clinical trials on inflammatory and autoimmune diseases, a number of probiotic bacterial strains can restore tolerance in host through modification of such dysregulated mediators. Since there are limited reports regarding to impact of probiotic supplementation in SLE patients, the preset review was aimed to suggest a number of probiotics bacteria, mainly from Bifidobacteria and Lactobacillus strains that are able to ameliorate immune responses. The aim was followed through literature survey on immunoregulatory probiotics that can restore tolerance and also modulate the important dysregulated pro/anti-inflammatory cytokines contributing to the pathogenesis of SLE. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-20T07:10:45.457475-05:
      DOI: 10.1002/jcp.25748
  • Biological and Pharmacological Evaluation of Dimethoxycurcumin: A
           Metabolically Stable Curcumin Analogue with a Promising Therapeutic
    • Authors: Manouchehr Teymouri; Nastaran Barati, Matteo Pirro, Amirhosein Sahebkar
      Abstract: Dimethoxycurcumin (DiMC) is a synthetic analogue of curcumin with superior inter-related pro-oxidant and anti-cancer activity, and metabolic stability. Numerous studies have shown that DiMC reserves the biologically beneficial features, including anti-inflammatory, anti-carcinogenic and cytoprotective properties, almost to the same extent as curcumin exhibits. DiMC lacks the phenolic-OH groups as opposed to curcumin, dimethoxycurcumin, and bis-demethoxycurcumin that all vary in the number of methoxy groups per molecule, and has drawn the attentions of researchers who attempted to discover the structure-activity relationship (SAR) of curcumin. In this regard, tetrahydrocurcumin (THC), the reduced and biologically inert metabolite of curcumin, denotes the significance of the conjugated α,β diketone moiety for the curcumin activity. DiMC exerts unique molecular activities compared to curcumin, including induction of androgen receptor (AR) degradation and suppression of the transcription factor activator protein-1 (AP-1). The enhanced AR degradation on DiMC treatment suggests it as a novel anticancer agent against resistant tumors with androgenic etiology. Further, DiMC might be a potential treatment for acne vulgaris. DiMC induces epigenetic alteration more effectively than curcumin, although both showed no direct DNA hypomethylating activity. Given the metabolic stability, nanoparticulation of DiMC is more promising for in vivo effectiveness. However, studies in this regard are still in its infancy. In the current review, we portray the various molecular and biological functions of DiMC reported so far. Whenever possible, the efficiency is compared with curcumin and the reasons for DiMC being more metabolically stable are elaborated. We also provide future perspective investigations with respect to varying DiMC-nanoparticles. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-20T07:10:43.061371-05:
      DOI: 10.1002/jcp.25749
  • Activin-SMAD Signaling Is Required for Maintenance of Porcine iPS Cell
           Self-Renewal through Upregulation of NANOG and OCT4 Expression
    • Authors: Fan Yang; Ning Wang, Yaxian Wang, Tong Yu, Huayan Wang
      Abstract: Porcine induced pluripotent stem cells (piPSCs) retain the enormous potential for farm animal reproduction and translational medicine, and have been reported by many laboratories worldwide. Some piPSC lines were bFGF-dependence and showed mouse EpiSC-like morphology; other lines were LIF-dependence and showed mouse ESC-like morphology. Metastable state of piPSC line that required both LIF and bFGF was also reported. Because bona fide pig embryonic stem cells were not available, uncovering piPSC state-specific regulatory circuitries was the most important task. In this study, we explored the function of Activin-SMAD signaling pathway and its downstream activated target genes in piPSCs. Transcriptome analysis showed that genes involved in Activin-SMAD signaling pathway were evidently activated during porcine somatic cell reprogramming, regardless piPSCs were LIF- or bFGF-dependent. Addition of Activin A and overexpression of SMAD2/3 significantly promoted expressions of porcine NANOG and OCT4, whereas inhibition of Activin-SMAD signaling by SB431542 and SMAD7 reduced NANOG and OCT4 expressions, and induced piPSCs differentiation exiting from pluripotent state. Our data demonstrate that activation of Activin-SMAD signaling pathway by addition of Activin A in culture medium is necessary for maintenance of self-renewal in porcine pluripotent stem cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-20T07:10:31.452619-05:
      DOI: 10.1002/jcp.25747
  • Dlx3 and GCM-1 Functionally Coordinate the Regulation of Placental Growth
           Factor in Human Trophoblast-Derived Cells
    • Authors: Sha Li; Mark S. Roberson
      Abstract: Placental growth factor (PGF) is abundantly expressed by trophoblast cells within human placentae and is important for trophoblast development and placental vascularization. Circulating maternal serum levels of PGF are dynamically upregulated across gestation in normal pregnancies, whereas low circulating levels and placental production of PGF have been implicated in the pathogenesis of preeclampsia and other gestational diseases. However, the underlying molecular mechanism of regulating PGF expression in the human placenta remains poorly understood. In this study, we demonstrated that transcription factors Distal-less 3 (DLX3) and Glial cell missing-1 (GCM1) were both sufficient and required for PGF expression in human trophoblast-derived cells by overexpression and knockdown approaches. Surprisingly, while DLX3 and GCM1 were both positive regulators of PGF, co-overexpression of DLX3 and GCM1 led to an antagonist effect on PGF expression on the endogenous gene and a luciferase reporter. Further, deletion and site-directed mutagenesis studies identified a novel regulatory element on the PGF promoter mediating both DLX3- and GCM1-dependent PGF expression. This regulatory region was also found to be essential for the basal activity of the PGF promoter. Finally, Chromatin-immunoprecipitation (ChIP) assays revealed colocalization of DLX3 and GCM1 at the identified regulatory region on the PGF promoter. Taken together, our studies provide important insights into intrinsic regulation of human placental PGF expression through the functional coordination of DLX3 and GCM1, and are likely to further the understanding of pathogenesis of PGF dysregulation in preeclampsia and other disease conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-20T07:10:27.300147-05:
      DOI: 10.1002/jcp.25752
  • Overexpression of Gremlin1 in Mesenchymal Stem Cells Improves Hindlimb
           Ischemia in Mice by Enhancing Cell Survival
    • Authors: Qiuling Xiang; Dongxi Hong, Yan Liao, Yong Cao, Muyun Liu, Jun Pang, Junjie Zhou, Guang Wang, Renhao Yang, Maosheng Wang, Andy Peng Xiang
      Abstract: Mesenchymal stem cells (MSCs) are a promising cell resource for the treatment of ischemic diseases, partially through paracrine effects. One of the major obstacles of MSC treatment is the poor survival rate and low efficiency of transplanted stem cells due to ischemic or inflammatory environments. Gremlin1 (GREM1), a regulator of growth, differentiation and development, has been identified as a novel proangiogenic factor. However, the role and mechanism of GREM1 in MSCs remains unclear. Therefore, we assessed the putative beneficial effects of GREM1 on MSC-based therapy for hindlimb ischemia. The lentiviral vector, EF1a-GREM1, was constructed using the Multisite Gateway System and used to transduce MSCs. In vitro studies demonstrated increased survival of GREM1-MSCs exposed to H2O2, which is consistent with the activation of caspase-3. Conditional medium from GREM1-MSCs (GREM1-MSC-CM) increased the anti-apoptotic effects of human umbilical vein endothelial cells (HUVECs), and this effect was attenuated by treatment with the PI3K/Akt pathway inhibitor LY294002. MSCs modified with GREM1 could significantly increase blood perfusion of the ischemic hindlimb in vivo in a mouse model, which was correlated to improved MSC survival. This study demonstrates that overexpression of GREM1 in MSCs have greater therapeutic effects against ischemia compared with wild-type MSCs by enhancing the survival of MSCs and ECs, which may provide new tools for studies investigating the treatment of ischemic diseases. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.This study demonstrates that overexpression of GREM1 in MSCs have greater therapeutic effects against ischemia compared with wild-type MSCs by enhancing the survival of MSCs and ECs, which may provide new tools for studies investigating the treatment of ischemic diseases.
      PubDate: 2016-12-20T05:11:06.645394-05:
      DOI: 10.1002/jcp.25578
  • Cover Image, Volume 232, Number 4, April 2017
    • Authors: Federico Castro-Muñozledo; Diana G. Meza-Aguilar, Rocío Domínguez-Castillo, Veremundo Hernández-Zequinely, Erika Sánchez-Guzmán
      Abstract: Cover: The cover image, by Federico Castro-Muñozledo et al., is based on the Original Research Article Vimentin as a Marker of Early Differentiating, Highly Motile Corneal Epithelial Cells,
      DOI : 10.1002/jcp.25487.
      PubDate: 2016-12-19T15:47:59.871225-05:
  • Acute Ethanol Increases IGF-I-Induced Phosphorylation of ERKs by Enhancing
           Recruitment of p52-Shc to the Grb2/Shc Complex
    • Authors: Matthew Dean; Adam Lassak, Anna Wilk, Adriana Zapata, Luis Marrero, Patricia Molina, Krzysztof Reiss
      Abstract: Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin-like growth factor I receptor (IGF-IR) function. Because the IGF-IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane-associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST-pulldowns following acute (1–24 h) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 h) ethanol (50 mM) enhanced IGF-I-induced phosphorylation of extracellular regulated kinases (ERKs), without affecting IGF-IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF-IR signaling following 3-week ethanol exposure. We then expressed a GFP-tagged IGF-IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently labeled cells, we determined that 50 mM ethanol decreased the half-time of the IGF-IR-associated FRAP, which implied that cell membrane-associated signaling events could be affected. Indeed, co-immunoprecipitation and GST-pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52-Shc to the Grb2-Shc complex, which is known to engage the Ras-Raf-ERKs pathway following IGF-1 stimulation. These experiments indicate that even a short and low-dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.In the current study, we show a novel effect of ethanol on the IGF-I receptor (IGF-IR) signaling, and provided a possible mechanism through which this occurs. We determine that ethanol enhances mobility of the IGF-IR molecules within the cell membrane, and that this stimulatory effect enhances formation of the Grb2-Shc signaling complex leading to the transient increase of the signal toward extracellular regulated kinases (ERKs). Because of the critical role of the IGF-IR signaling in neuronal growth and survival, and the detrimental effects of alcohol on CNS development, we believe that our new data will help in better understanding of the fetal alcohol syndrome.
      PubDate: 2016-12-19T11:50:27.245701-05:
      DOI: 10.1002/jcp.25586
  • Cytoplasmic Localization of RUNX3 Via Histone Deacetylase-Mediated SRC
           Expression in Oxidative-Stressed Colon Cancer Cells
    • Authors: Kyoung Ah Kang; Mei Jing Piao, Yea Seong Ryu, Young Hee Maeng, Jin Won Hyun
      Abstract: Runt domain transcription factor 3 (RUNX3) is a transcription factor that functions as a tumor suppressor. RUNX3 is frequently inactivated by epigenetic silencing or its protein mislocalization (cytoplasmic localization) in many cancer types. This study investigated whether oxidative stress induces redistribution of RUNX3 from the nucleus to the cytoplasm. The cytoplasmic localization of RUNX3 was associated with oxidative stress-induced RUNX3 phosphorylation at tyrosine residues via SRC activation. Moreover, oxidative stress increased expression of histone deacetylases (HDACs). RUNX3 phosphorylation and SRC expression induced by oxidative stress were inhibited by knockdown of HDAC1, restoring the nuclear localization of RUNX3 under oxidative stress. In conclusion, these results demonstrate that HDAC1- and SRC-mediated phosphorylation of RUNX3 induced by oxidative stress is associated with the cytoplasmic localization of RUNX3 and can lead to RUNX3 inactivation and carcinogenesis. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-19T02:30:26.186407-05:
      DOI: 10.1002/jcp.25746
  • Human white adipocytes convert into “rainbow” adipocytes in
    • Authors: Giulia Maurizi; Antonella Poloni, Domenico Mattiucci, Spartaco Santi, Angela Maurizi, Valerio Izzi, Angelica Giuliani, Stefania Mancini, Maria Cristina Zingaretti, Jessica Perugini, Ilenia Severi, Massimo Falconi, Marco Vivarelli, Maria Rita Rippo, Silvia Corvera, Antonio Giordano, Pietro Leoni, Saverio Cinti
      Abstract: White adipocytes are plastic cells able to reversibly transdifferentiate into brown adipocytes and into epithelial glandular cells under physiologic stimuli in vivo. These plastic properties could be used in future for regenerative medicine, but are incompletely explored in their details.Here, we focused on plastic properties of human mature adipocytes (MA) combining gene expression profile through microarray analysis with morphologic data obtained by electron and time lapse microscopy.Primary MA showed the classic morphology and gene expression profile of functional mature adipocytes. Notably, despite their committed status, MA expressed high levels of reprogramming genes.MA from ceiling cultures underwent transdifferentiation towards fibroblast-like cells with a well-differentiated morphology and maintaining stem cell gene signatures. The main morphologic aspect of the transdifferentiation process was the secretion of large lipid droplets and the development of organelles necessary for exocrine secretion further supported the liposecretion process. Of note, electron microscope findings suggesting liposecretion phenomena were found also in explants of human fat and rarely in vivo in fat biopsies from obese patients.In conclusion, both MA and post-liposecretion adipocytes show a well-differentiated phenotype with stem cell properties in line with the extraordinary plasticity of adipocytes in vivo. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-17T10:24:29.960529-05:
      DOI: 10.1002/jcp.25743
  • Association between AXL, Hippo transducers and survival outcomes in male
           breast cancer
    • Authors: Anna Di Benedetto; Marcella Mottolese, Francesca Sperati, Cristiana Ercolani, Luigi Di Lauro, Laura Pizzuti, Patrizia Vici, Irene Terrenato, Abeer M. Shaaban, Matthew P. Humphries, Sreekumar Sundara-Rajan, Maddalena Barba, Valerie Speirs, Ruggero De Maria, Marcello Maugeri-Saccà
      Abstract: Male breast cancer (MBC) is an uncommon malignancy. We have previously reported that the expression of the Hippo transducers TAZ/YAP and their target CTGF was associated with inferior survival in MBC patients. Preclinical evidence demonstrated that Axl is a transcriptional target of TAZ/YAP. Thus, we herein assessed AXL expression to further investigate the significance of active TAZ/YAP-driven transcription in MBC. For this study, 255 MBC samples represented in tissue microarrays were screened for AXL expression, and 116 patients were included. The association between categorical variables was verified by the Pearson's Chi-squared test of independence (2-tailed) or the Fisher Exact test. The relationship between continuous variables was tested with the Pearson's correlation coefficient. The Kaplan-Meier method was used for estimating survival curves, which were compared by log-rank test. Factors potentially impacting 10-year and overall survival were verified in Cox proportional regression models. AXL was positively associated with the TAZ/CTGF and YAP/CTGF phenotypes (p = 0.001 and p = 0.002, respectively). Patients with TAZ/CTGF/AXL- or YAP/CTGF/AXL-expressing tumors had inferior survival compared with non-triple-positive patients (log rank p = 0.042 and p = 0.048, respectively). The variables TAZ/CTGF/AXL and YAP/CTGF/AXL were adverse factors for 10-year survival in the multivariate Cox models (HR 2.31, 95%CI:1.02-5.22, p = 0.045, and HR 2.27, 95%CI:1.00-5.13, p = 0.050). Nearly comparable results were obtained from multivariate analyses of overall survival. The expression pattern of AXL corroborates the idea of the detrimental role of TAZ/YAP activation in MBC. Overall, Hippo-linked biomarkers deserve increased attention in this rare disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-17T10:23:08.604491-05:
      DOI: 10.1002/jcp.25745
  • TUDCA: An Agonist of the Bile Acid Receptor GPBAR1/TGR5 with
           Anti-Inflammatory Effects in Microglial Cells
    • Authors: Natalia Yanguas-Casás; M. Asunción Barreda-Manso, Manuel Nieto-Sampedro, Lorenzo Romero-Ramírez
      Abstract: Bile acids are steroid acids found in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is neuroprotective in different animal models of stroke and neurological diseases. We have previously shown that TUDCA has anti-inflammatory effects on glial cell cultures and in a mouse model of acute neuroinflammation.We show now that microglial cells (central nervous system resident macrophages) express the G protein-coupled bile acid receptor 1/ Takeda G protein-coupled receptor 5 (GPBAR1/TGR5) in vivo and in vitro. TUDCA binding to GPBAR1/TGR5 caused an increase in intracellular cAMP levels in microglia that induced anti-inflammatory markers, while reducing pro-inflammatory ones. This anti-inflammatory effect of TUDCA was inhibited by small interference RNA for GPBAR1/TGR5 receptor, as well as by treatment with a protein kinase A (PKA) inhibitor.In the mouse model of acute neuroinflammation, treating the animals with TUDCA was clearly anti-inflammatory. TUDCA biased the microglial phenotype in vivo and in vitro towards the anti-inflammatory.The bile acid receptor GPBAR1/TGR5 could be a new therapeutic target for pathologies coursing with neuroinflammation and microglia activation, such as traumatic brain injuries, stroke or neurodegenerative diseases. TUDCA and other GPBAR1/TGR5 agonists need to be further investigated, to determine their potential in attenuating the neuropathologies associated with microglia activation. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-17T10:22:46.078782-05:
      DOI: 10.1002/jcp.25742
  • MicroRNA-125a-5p Is a Downstream Effector of Sorafenib in its
           Antiproliferative Activity Toward Human Hepatocellular Carcinoma Cells
    • Authors: Nicoletta Potenza; Nicola Mosca, Silvia Zappavigna, Filomena Castiello, Marta Panella, Carmela Ferri, Daniela Vanacore, Antonio Giordano, Paola Stiuso, Michele Caraglia, Aniello Russo
      Abstract: Sorafenib is an antitumor drug for treatment of advanced hepatocellular carcinoma. It acts as a multikinase inhibitor suppressing cell proliferation and angiogenesis. Human microRNA-125a-5p (miR-125a) is endowed with similar activities and is frequently downregulated in hepatocellular carcinoma. Looking for a potential microRNA-based mechanism of action of the drug, we found that sorafenib increases cellular expression of miR-125a in cultured HuH-7 and HepG2 hepatocellular carcinoma cells. Upregulation of the microRNA inhibited cell proliferation by suppression of sirtuin-7, a NAD(+)-dependent deacetylase, and p21/p27-dependent cell cycle arrest in G1. Later, recruitment of miR-125a in the antiproliferative activity of sorafenib was inquired by modulating its expression in combination with the drug treatment. This analysis showed that intracellular delivery of miR-125a had no additive effect on the antiproliferative activity of sorafenib, whereas a miR-125a inhibitor could counteract it. Finally, evaluation of other oncogenic targets of miR-125a revealed its ability to interfere with the expression of matrix metalloproteinase-11, Zbtb7a proto-oncogene, and c-Raf, possibly contributing to the antiproliferative activity of the drug. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-16T06:50:33.379965-05:
      DOI: 10.1002/jcp.25744
  • Anti-Atherosclerotic Effects of Vitamins D and E in Suppression of
    • Authors: Bahman Rashidi; Zahra Hoseini, Amirhossein Sahebkar, Hamed Mirzaei
      Abstract: Atherosclerosis is a progressive and multifactorial disease which occurs under the influence of various risk factors including endothelial dysfunction (ED), oxidative stress, and low-density lipoprotein (LDL) oxidation.In contract to the initial hypotheses on the usefulness of vitamin E supplementation for cardiovascular disease prevention, large outcome trials showed consumption of vitamin E has no obvious effect on cardiovascular disease and, in some cases, it may even increase the rate of mortality. This seemingly unexpected finding may be due to the opposite effects of vitamin E compounds. Vitamin E is a group of compounds which have different and even opposing effects, yet in most of the studies, the exact consumed component of vitamin E is not determined. It appears that the combined consumption of gamma-tocopherol, vitamin C, vitamin D, and tetrahydrobiopterin (BH4) may be extremely effective in both preventing atherogenesis and suppressing plaque development. In this regard, one of main issues is effect of vitamin E and D deficiency on microRNAs network in atherosclerosis. Various studies have indicated that miRNAs have key roles in atherosclerosis pathogenesis. The deficiency of vitamin E and D could provide a deregulation for miRNAs network and these events could lead to progression of atherosclerosis. Here, we highlighted a variety of mechanisms involve in the progression of atherosclerosis and effects of vitamin D and E on these mechanisms. Moreover, we summarized miRNAs involve in atherosclerosis and their regulation by vitamin E and D deficiency. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-14T06:50:42.945057-05:
      DOI: 10.1002/jcp.25738
  • The Role of Exercise and TFAM in Preventing Skeletal Muscle Atrophy
    • Authors: Nicholas T. Theilen; George H. Kunkel, Suresh C. Tyagi
      Abstract: Skeletal muscle atrophy is the consequence of protein degradation exceeding protein synthesis. This arises for a multitude of reasons including the unloading of muscle during microgravity, post-surgery bedrest, immobilization of a limb after injury, and overall disuse of the musculature. The development of therapies prior to skeletal muscle atrophy settings to diminish protein degradation is scarce. Mitochondrial dysfunction is associated with skeletal muscle atrophy and contributes to the induction of protein degradation and cell apoptosis through increased levels of ROS observed with the loss of organelle function. ROS binds mtDNA, leading to its degradation and decreasing functionality. Mitochondrial transcription factor A (TFAM) will bind and coat mtDNA, protecting it from ROS and degradation while increasing mitochondrial function. Exercise stimulates cell signaling pathways that converge on and increase PGC-1α, a well-known activator of the transcription of TFAM and mitochondrial biogenesis. Therefore, in the present review we are proposing, separately, exercise and TFAM treatments prior to atrophic settings (muscle unloading or disuse) alleviate skeletal muscle atrophy through enhanced mitochondrial adaptations and function. Additionally, we hypothesize the combination of exercise and TFAM leads to a synergistic effect in targeting mitochondrial function to prevent skeletal muscle atrophy. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-14T06:50:39.818122-05:
      DOI: 10.1002/jcp.25737
  • Tumor Necrosis Factor-α Increases Claudin-1, 4 and 7 Expression in
           Tubular Cells: Role in Permeability Changes
    • Authors: Yasaman Amoozadeh; Qinghong Dan, Shaista Anwer, Hsiao Han Huang, Vanessa Barbieri, Faiza Waheed, Mazharul Maishan, Katalin Szászi
      Abstract: Tumor necrosis factor-α (TNFα), is a pathogenic cytokine in kidney disease that alters expression of claudins in tubular cells. Previously we showed that in LLC-PK1 cells TNFα caused a biphasic change in transepithelial resistance (TER) consisting of an early drop and recovery, followed by a late increase. However, the underlying mechanisms and the role of specific claudins in the TER effect remained incompletely understood. Here we sought to define how TNFα affects claudins 1, 4 and 7 in tubular cells and to correlate their changes with the TER effect. We show that TNFα elevates total and surface levels of Cldn-1, 4 and 7, and increases their mRNA expression through the ERK and JNK pathways. Further, JNK is also important for TNFα-induced changes in claudin-2 expression. Continuous monitoring of TER using Electric cell-substrate impedance sensing (ECIS) reveals that the two phases of the TNFα effect are differently regulated. Specifically, inhibition of the ERK or JNK pathways prevent the late TER increase, but not the early TER effect. Silencing experiments also show that Cldn-1 is necessary for the early TNFα-induced TER change, while all three claudins appear to contribute to the late TER increase. In summary, we define a central role for ERK and JNK in TNFα-induced altered claudin expression and barrier tightening. Together, our current and previous works show that the TNFα-induced early TER effect requires claudin-1, while claudin-2 decrease is a significant mediator of the late TER increase, and elevation in claudin-1, 4 and 7 contribute to a smaller extent. This article is protected by copyright. All rights reservedKeywords: tubular epithelial cells; paracellular permeability; claudins; Tumor Necrosis Factor; ECIS
      PubDate: 2016-12-14T06:50:33.341518-05:
      DOI: 10.1002/jcp.25736
  • Diagnostic and Therapeutic Potential of Exosomes in Cancer: The Beginning
           of a New Tale'
    • Authors: Hamed Mirzaei; Amirhossein Sahebkar, Mahmoud Reza Jaafari, Mohammad Goodarzi, Hamid Reza Mirzaei
      Abstract: Exosomes have emerged as one of the main players in intercellular communication. These small nano-sized particles have many roles in various physiological pathways in normal and abnormal cells. Exosomes can carry various cargos such as proteins, mRNAs and miRNAs to recipient cells. Uptake of exosomes and their cargo can induce and/or inhibit different cellular and molecular pathways that lead to the alteration of cell behavior. Multiple lines of evidence have indicated that exosomes released from cancer cells can effect development of cancer in different stages. These particles and their cargo could regulate different processes such as tumor growth, metastasis, drug resistance, angiogenesis and immune system functioning. It has been observed that exosomes can be used as potential diagnostic biomarkers in various cancer types. Moreover, some studies have used these particles as biological vehicles for delivery of various drugs such as doxorubicin, siRNAs and miRNAs. Here, we summarized the findings on the role of exosomes in different pathological processes involved in cancer. Moreover, application of these particles as diagnostic and therapeutic biomarkers in different types of cancers is discussed. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-14T06:50:31.801093-05:
      DOI: 10.1002/jcp.25739
  • Mesenchyme Homeobox 2 Enhances Migration of Endothelial Colony Forming
           Cells Exposed to Intrauterine Diabetes Mellitus
    • Authors: Cassandra R. Gohn; Emily K. Blue, BreAnn M. Sheehan, Kaela M. Varberg, Laura S. Haneline
      Abstract: Diabetes mellitus (DM) during pregnancy has long-lasting implications for the fetus, including cardiovascular morbidity. Previously, we showed that endothelial colony forming cells (ECFCs) from DM human pregnancies have decreased vasculogenic potential. Here, we evaluate whether the molecular mechanism responsible for this phenotype involves the transcription factor, Mesenchyme Homeobox 2 (MEOX2). In human umbilical vein endothelial cells, MEOX2 upregulates cyclin dependent kinase inhibitor expression, resulting in increased senescence and decreased proliferation. We hypothesized that dysregulated MEOX2 expression in neonatal ECFCs from DM pregnancies decreases network formation through increased senescence and altered cell cycle progression. Our studies show that nuclear MEOX2 is increased in ECFCs from DM pregnancies. To determine if MEOX2 is sufficient and/or required to induce impaired network formation, MEOX2 was overexpressed and depleted in ECFCs from control and DM pregnancies, respectively. Surprisingly, MEOX2 overexpression in control ECFCs resulted in increased network formation, altered cell cycle progression, and increased senescence. In contrast, MEOX2 knockdown in ECFCs from DM pregnancies led to decreased network formation, while cell cycle progression and senescence were unaffected. Importantly, migration studies demonstrated that MEOX2 overexpression increased migration, while MEOX2 knockdown decreased migration. Taken together, these data suggest that altered migration may be mediating the impaired vasculogenesis of ECFCs from DM pregnancies. While initially believed to be maladaptive, these data suggest that MEOX2 may serve a protective role, enabling increased vessel formation despite exposure to a DM intrauterine environment. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-14T06:50:29.396814-05:
      DOI: 10.1002/jcp.25734
  • Notch Signaling Participates in TGF-β Induced SOST Expression under
           Intermittent Compressive Stress
    • Authors: Jeeranan Manokawinchoke; Piyamas Sumrejkanchanakij, Prasit Pavasant, Thanaphum Osathanon
      Abstract: Notch signaling is regulated by mechanical stimuli in various cell types. It has previously been reported that intermittent compressive stimuli enhanced sclerostin (SOST) expression in human periodontal ligament cells (hPDLs) by regulating transforming growth factor-β (TGF-β) expression. The aim of the present study was to determine the involvement of Notch signaling in the TGF-β induced SOST expression in hPDLs. Cells were treated with intermittent compressive stress in a computer-controlled apparatus for 24 h. The mRNA and protein expression of the cells were determined by real-time polymerase chain reaction and western blot analysis, respectively. In some experiments, the target signaling pathway was impeded by the addition of a TGF-β receptor kinase inhibitor (SB431542) or a γ-secretase inhibitor (DAPT). The results demonstrated that hPDLs under intermittent compressive stress exhibited significantly higher NOTCH2, NOTCH3, HES1, and HEY1 mRNA expression compared with control, indicating that mechanical stress induced Notch signaling. DAPT pretreatment markedly reduced the intermittent stress-induced SOST expression. The expression of NOTCH2, NOTCH3, HES1, and HEY1 mRNA under compressive stress was significantly reduced after pretreatment with SB431542, coinciding with a reduction in SOST expression. Recombinant human TGF-β1 enhanced SOST, Notch receptor, and target gene expression in hPDLs. Further, DAPT treatment attenuated rhTGF-β1 induced SOST expression. In summary, intermittent compressive stress regulates Notch receptor and target gene expression via the TGF-β signaling pathway. In addition, Notch signaling participates in TGF-β induced SOST expression in hPDLs. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-14T06:50:22.870897-05:
      DOI: 10.1002/jcp.25740
  • Identification of MSX1 and DCLK1as mRNA Biomarkers for Colorectal Cancer
           Detection through DNA Methylation Information
    • Authors: Ai-Jun Sun; Hai-Bo Gao, Gao Liu, Heng-Fa Ge, Zun-Ping Ke, Sen Li
      Abstract: Colorectal cancer is the second most deadly malignancy in the United States. However, the currently screening options had their limitation. Novel biomarkers for colorectal cancer detections are necessary to reduce the mortality. The clinical information, mRNA expression levels and DNA methylation information of colorectal cancer were downloaded from TCGA. The patients were separated into training group and testing group based on their platforms for DNA methylation. Beta values of DNA methylation from tumor tissues and normal tissues were utilized to figure out the position that were differentially methylated. The expression levels of mRNA of thirteen genes, whose CpG islands were differentially methylated, were extracted from the RNA-Seq results from TCGA. The probabilities whether the mRNA was differentially expressed between tumor and normal samples were calculated using Student's t-test. Logistic regression and decision tree were built for cancer detection and their performances were evaluated by the area under the curve (AUC). Twenty-four genomic locations were differentially methylated, which could be mapped to eleven genes. Nine out of eleven genes had differentially expressed mRNA levels, which were used to build the model for cancer detection. The final detection models consisting of mRNA expression levels of these nine genes had great performances on both training group and testing group. The model that constructed in this study suggested MSX1 and DCLK1might be used in colorectal cancer detection or as target of cancer therapies. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-14T06:45:28.983959-05:
      DOI: 10.1002/jcp.25733
  • A Human 3D In Vitro Model to Assess the Relationship between Osteoporosis
           and Dissemination to Bone of Breast Cancer Tumour Cells
    • Authors: F Salamanna; V Borsari, S Brogini, P Torricelli, S Cepollaro, M Cadossi, M Fini
      Abstract: Despite consistent improvements in diagnostic and therapeutic strategies for breast cancer, up to 40% of patients will develop bone metastases. To reduce the morbidity and complications related with bone metastases, it is imperative to reduce their etiological factors. Osteoporosis, being characterized by a sudden estrogen deficiency, may provide a favorable condition for bone metastasis. This work, using a humanized 3D in vitro model, aims at evaluating the relationship between osteoporosis and breast cancer-derived bone metastases. Bone tissue discarded from total hip replacement surgery of healthy and osteoporotic patients was cultured in a rolling apparatus system in hypoxic environment. Protein levels (i.e. vascular endothelial growth factor (VEGF), VEGF receptor 1, VEGF receptor 2, interleukin (IL)-6, IL-1β, IL-8 IL-10, tumor necrosis factor α (TNF-α), osteoprotegerin (OPG), Receptor Activator for Nuclear factor KB Ligand (RANKL)) and histological and immunohistochemical (i.e. cytokeratin 8 and 18) analyses showed a noticeable specificity of breast cancer cells for the colonization of osteoporotic bone. These data are the first to demonstrate that using humanized 3D in vitro systems, which individually model the pre- and postmenopausal bone microenvironment, it is possible to recognize major differences in tumor growth and colonization between healthy and osteoporotic status. Thus, this system might help to develop a shared system between basic and clinical sciences where a personalized diagnosis is associated to a therapeutic strategy designed for a single patient: a model able to achieve a translational research approach in the clinical setting, which may lead to the application and dissemination of personalized medicine. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-07T07:52:39.43569-05:0
      DOI: 10.1002/jcp.25708
  • SV40 Infection of Mesenchymal Stromal Cells from Wharton's Jelly Drives
           the Production of Inflammatory and Tumoral Mediators
    • Authors: Carolina Cason; Giuseppina Campisciano, Nunzia Zanotta, Erica Valencic, Serena Delbue, Ramona Bella, Manola Comar
      Abstract: The Mesenchymal Stromal Cells from umbilical cord Wharton's jelly (WJSCs) are a source of cells with high potentiality for the treatment of human immunological disorders. Footprints of the oncogenic viruses Simian Virus 40 (SV40) and JC Virus (JCPyV) have been recently detected in human WJSCs specimens. The aim of this study is to evaluate if WJSCs can be efficiently infected by these Polyomaviruses and if they can potentially exert tumoral activity. Cell culture experiments indicated that WJSCs could sustain both SV40 and JCPyV infections. A transient and lytic replication was observed for JCPyV, while SV40 persistently infected WJSCs over a long period of time, releasing a viral progeny at low titer without evident cytopathic effect (CPE). Considering the association between SV40 and human tumors and the reported ability of the oncogenic viruses to drive the host innate immune response to cell transformation, the expression profile of a large panel of immune mediators was evaluated in supernatants by the Bioplex platform. RANTES, IL-3, MIG and IL-12p40, involved in chronic inflammation, cells differentiation and transformation, were constantly measured at high concentration comparing to control. These findings represent a new aspect of SV40 biological activity in the humans, highlighting its interaction with specific host cellular pathways. In view of these results, it seems to be increasingly urgent to consider Polyomaviruses in the management of WJSCs for their safely use as promising therapeutic source. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-07T07:50:49.611089-05:
      DOI: 10.1002/jcp.25723
  • Factor Xa Signaling Contributes to the Pathogenesis of Inflammatory
    • Authors: Safieh Ebrahimi; Sara Rezaei, Parvaneh Seiri, Mikhail Ryzhikov, Seyed Issac Hashemy, Seyed Mahdi Hassanian
      Abstract: The coagulation protease Factor Xa (FXa) triggers a variety of signaling pathways through activation of protease-activated receptors (PARs) and non-PAR receptors. FXa-mediated signaling is strongly implicated in the pathogenesis of several inflammatory diseases including fibrosis, cardiovascular diseases and cancer. Thus, targeting of FXa can have great clinical significance in terms of the treatment of these disorders. This review summarizes the current knowledge about the mechanism of FXa signaling in cellular and animal systems under (patho)physiological conditions for a better understanding and hence a better management of FXa–induced disorders. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-07T07:19:19.500821-05:
      DOI: 10.1002/jcp.25714
  • Rabs, Membrane Dynamics and Parkinson's Disease
    • Authors: Bor Luen Tang
      Abstract: Genes encoding cellular membrane trafficking components, namely RAB7L1 and RAB39B, are more recently recognized factors associated with Parkinson's disease (PD). Encoded by a gene within the PARK16 locus, RAB7L1 interacts with Leucine-rich repeat kinase 2 (LRRK2) to act in intracellular transport processes that are likely important in neuronal survival and function. LRRK2 also directly phosphorylates a number of other Rab proteins. On the other hand, nonsense and missense mutations of the X-chromosome localized RAB39B, were shown to underlie X-linked intellectual disability (ID) in male patients with early-onset PD. The cellular or neuronal functions of RAB39B are not yet known with certainty, but it has recently been shown to play a role in glutamate receptor trafficking. Importantly, RAB39B is also functionally connected to components of autophagy regulation, which affects α-synuclein processing and clearance. In this review, we discuss the association of Rabs with PD pathology, and potential etiological mechanisms whereby defects or deficiencies in certain Rab proteins could lead to PD susceptibility. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-07T07:13:28.793959-05:
      DOI: 10.1002/jcp.25713
  • Calprotectin Induces IL-6 and MCP-1 Production via Toll-Like Receptor 4
           Signaling in Human Gingival Fibroblasts
    • Authors: Yasufumi Nishikawa; Yukari Kajiura, Jung Hwan Lew, Jun-ichi Kido, Toshihiko Nagata, Koji Naruishi
      Abstract: Calprotectin, a heterodimer of S100A8 and S100A9 molecules, is associated with inflammatory diseases such as inflammatory bowel disease. We have reported that calprotectin levels in gingival crevicular fluids of periodontitis patients are significantly higher than in healthy subjects. However, the functions of calprotectin in pathophysiology of periodontitis are still unknown. The aim of this study is to investigate the effects of calprotectin on the productivity of inflammatory cytokines in human gingival fibroblasts (HGFs). The HGFs cell line CRL-2014® (ATCC) were cultured, and total RNAs were collected to examine the expression of TLR2/4 and RAGE mRNA using RT-PCR. After the cells were treated with S100A8, S100A9 and calprotectin, supernatants were collected and the levels of IL-6 and MCP-1 were measured using ELISA methods. To examine the intracellular signals involved in calprotectin-induced cytokine production, several chemical inhibitors were used. Furthermore, after the siRNA-mediated TLR4 down-regulated cells were treated with S100A8, S100A9 and calprotectin, the levels of IL-6 and MCP-1 were also measured. HGFs showed greater expression of TLR4 mRNA, but not TLR2 and RAGE mRNA compared with human oral epithelial cells. Calprotectin increased significantly the production of MCP-1 and IL-6 in HGFs, and the cytokine productions were significantly suppressed in the cells treated with MAPKs, NF-κB and TLR4 inhibitors. Furthermore, calprotectin-mediated MCP-1 and IL-6 production were significantly suppressed in TLR4 down-regulated cells. Taken together, calprotectin induces IL-6 and MCP-1 production in HGFs via TLR4 signaling that involves MAPK and NF-κB, resulting in the progression of periodontitis. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-07T07:11:32.759639-05:
      DOI: 10.1002/jcp.25724
  • SIRT1-SIRT3 Axis Regulates Cellular Response to Oxidative Stress and
    • Authors: Ilaria Carnevale; Laura Pellegrini, Patrizia D'Aquila, Serena Saladini, Emanuela Lococo, Lucia Polletta, Enza Vernucci, Eleonora Foglio, Stefano Coppola, Luigi Sansone, Giuseppe Passarino, Dina Bellizzi, Matteo A. Russo, Massimo Fini, Marco Tafani
      Abstract: Sirtuins are conserved NAD+-dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1-SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1-SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1-silenced cell lines. Our results demonstrate that SIRT1-silenced cells are more resistant to H2O2 and etoposide treatment showing decreased ROS accumulation, γ-H2AX phosphorylation, caspase-3 activation and PARP cleavage. Interestingly, we observed that SIRT1-silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1-silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co-immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1-silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription.In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. This article is protected by copyright. All rights reserved
      PubDate: 2016-12-07T07:08:05.755569-05:
      DOI: 10.1002/jcp.25711
  • Association Between Aerobic Exercise and Rosiglitazone Avoided the NAFLD
           and Liver Inflammation Exacerbated in PPAR-α Knockout Mice
    • Authors: Helena A.P. Batatinha; Edson A. Lima, Alexandre A.S. Teixeira, Camila O. Souza, Luana A. Biondo, Loreana S. Silveira, Fabio S. Lira, José C. Rosa Neto
      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. 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. 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. 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. 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. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.Exercise training improved lipids toxicity in the liver; PPAR-alpha KO developed liver inflammation after training protocol Roziglitazone reduced liver inflammation independent of PPAR-alpha.
      PubDate: 2016-11-30T10:40:48.544758-05:
      DOI: 10.1002/jcp.25440
  • Mechanical Stress Regulates Bone Metabolism through microRNAs
    • Authors: Yu Yuan; Lingli Zhang, Xiaoyang Tong, Miao Zhang, Yilong Zhao, Jianming Guo, Le Lei, Xi Chen, Jennifer Tickner, Jiake Xu, Jun Zou
      Abstract: There are many lines of evidence indicating that mechanical stress regulates bone metabolism and promotes bone growth. BMP, Wnt, ERK1/2 and OPG/RANKL are the main molecules thought to regulate the effects of mechanical loading on bone formation. Recently, microRNAs were found to be involved in bone cell proliferation and differentiation, regulating the balance of bone formation and bone resorption. Emerging evidence indicates that microRNAs also participate in mechanical stress-mediated bone metabolism, and is associated with disuse induced osteoporosis or osteopenia. Mechanical stress is able to induce expression of microRNAs that modulate the expression of osteogenic and bone resorption factors, leading to the positive impact of mechanical stress on bone. This review discusses the emerging evidence implicating an important role for microRNAs in the mechanical stress response in bone cells, as well as challenges translating microRNA research into potential treatment. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-28T12:00:24.433488-05:
      DOI: 10.1002/jcp.25688
  • Condensin Regulation of Genome Architecture
    • Authors: Vibhuti Rana; Giovanni Bosco
      Abstract: Progression through the cell cycle for both prokaryotes and eukaryotes is required for cell division. At almost every level of cellular organization structure and function of subcellular compartments and organelles must accommodate the progression of cell division. As cells progress through the cell division cycle, perhaps the most dramatic observable cellular change is the shape of chromosomes. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-26T01:35:40.940435-05:
      DOI: 10.1002/jcp.25702
  • Modulation of Chloride Currents in Human Lung Epithelial Cells Exposed to
           Exogenous Oxidative Stress
    • Authors: Rita Canella; Marta Martini, Roberta Borriello, Carlotta Cavicchio, Ximena M Muresan, Mascia Benedusi, Franco Cervellati, Giuseppe Valacchi
      Abstract: Air pollution continues to be a major public health concern affecting 9 out of 10 individuals living in urban areas worldwide. Respiratory tract is the organ most exposed to gas pollution and ozone has been shown to be one of the most noxious pollutants to which living organisms are exposed. In the present work, we have investigated the effects of 0.1 ppm of ozone on chloride currents in human lung epithelial cells (A549 line) and whether this effect could be modulated by vitamin E pre-treatment.Whole-cell patch clamp technique was applied to not excitable cells in order to obtain information about chloride currents behavior, important for epithelial lung cells homeostasis. Significant alteration of the I-V curve after ozone treatment was observed, with the appearance of a large outward rectifier component decreasing over time and returning to the basal state levels after 24 hours. Statistical analysis indicated a modification of the amount of ions passing the membrane in the unit of time as a possible cause of this difference. RT-qPCR analysis showed an increase in ClC-2 and ORCC mRNA after ozone exposure.In addition, pre-treatment with vitamin E was able to suppress the outward rectifier component induced by ozone, bringing back the current values to the control level and preventing ozone induced chloride channels up regulation.Our data suggest that ozone exposure is able to modify chloride current density and the use of vitamin E can prevent the above mentioned damage. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-25T08:05:22.13479-05:0
      DOI: 10.1002/jcp.25705
  • Supernatants of Adipocytes from Obese vs Normal Weight Women and Breast
           Cancer Cells: In Vitro Impact on Angiogenesis
    • Authors: L. Bougaret; L. Delort, H. Billard, C. Lequeux, N. Goncalves-Mendes, A. Mojallal, O. Damour, M.-P. Vasson, F. Caldefie-Chezet
      Abstract: Breast cancer is correlated with a higher risk of metastasis in obese postmenopausal women. Adipokines, whose plasma concentrations are modulated in obese subjects, surrounds mammary cells, suggesting that adipocyte secretions affect mammary tumorogenesis. We hypothsesis that matures adipocytes secretions from obese women conditioned or not by breast neoplasic cells, increase changes on the stages of angiogenesis.Supernatants of human matures adipocytes differentiated from stem cells of either adipose tissue of normal weight (MA20) or obese (MA30) women or obtained from co-cultures between MA20 or MA30 and breast cancer cell line MCF-7 were collected. The impact of these supernatants was investigated on proliferation, migration and tubes formation by endothelial cells (HUVEC).MA20 and MA30 showed a preservation of their “metabolic memory” (increase of Leptin, ObR, VEGF, CYP19A1 and decrease of Adiponectin expressions in MA30 compared to MA20).Supernatant from obese-adipocytes increased HUVEC proliferation, migration and sprouting like with supernatant obtained from co-cultures of MA/MCF-7 regardless the women's BMI. Additional analyses such as the use of neutralizing antibodies, analysis of supernatants (Milliplex®) and variations in gene expression (qRT-PCR), strongly suggest an implication of IL-6 or a synergistic action among adipokines, probably associated with that of VEGF or IL-6.As a conclusion, supernatant from co-cultures of MA30 and MCF-7 cells increase proliferation, migration and sprouting of HUVEC cells. These results provide insights into the interaction between adipocytes and epithelial cancer cells, particularly in case of obesity. The identification of synergistic action of adipokines would therefore be a great interest in developing preventive strategies. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-25T08:02:08.465987-05:
      DOI: 10.1002/jcp.25701
  • TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell
           Culture Model of the Corneal Epithelium
    • Authors: Jacqueline Martínez-Rendón; Erika Sánchez-Guzmán, Angélica Rueda, James González, Rosario Gulias-Cañizo, Guillermo Aquino-Jarquín, Federico Castro-Muñozledo, Refugio García-Villegas
      Abstract: TRPV4 (Transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-21T07:15:23.334122-05:
      DOI: 10.1002/jcp.25698
  • Roles of Notch Signaling in Adipocyte Progenitor Cells and Mature
    • Authors: Tizhong Shan; Jiaqi Liu, Weiche Wu, Ziye Xu, Yizhen Wang
      Abstract: Adipose tissues, composed with mature adipocytes and preadipocytic stromal/stem cells, play crucial roles in whole body energy metabolism and regenerative medicine. Mature adipocytes are derived and differentiated from mesenchymal stem cells (MSCs) or preadipocytes. This differentiation process, also called adipogenesis, is regulated by several signaling pathways and transcription factors. Notch1 signaling is a highly conserved pathway that is indispensable for stem cell hemostasis and tissue development. In adipocyte progenitor cells, Notch1 signaling regulates the adipogenesis process including proliferation and differentiation of the adipocyte progenitor cells in vitro. Notably, the roles of Notch1 signaling in beige adipocytes formation, adipose development and function, and the whole body energy metabolism have been recently reported. Here, we mainly review and discuss the roles of Notch1 signaling in adipogenesis in vitro as well as in beige adipocytes formation, adipocytes dedifferentiation and function in vivo. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-21T07:10:20.732207-05:
      DOI: 10.1002/jcp.25697
  • Notch Signaling Pathway Regulates Angiogenesis via Endothelial Cell in 3D
           Co-Culture Model
    • Authors: Dan Zhao; Changyue Xue, Shiyu Lin, Sirong Shi, Qianshun Li, Mengting Liu, Xiaoxiao Cai, Yunfeng Lin
      Abstract: ObjectivesThis study aimed to investigate the role of Notch signaling pathway for angiogenesis in a three-dimensional (3D) collagen gel model with co-culture of adipose-derived stromal cells (ASCs) and endothelial cells (ECs).MethodsA 3D collagen gel model was established in vitro by implanting both ASCs from green fluorescent protein-labeled mouse and ECs from red fluorescent protein-labeled mouse, and the phenomena of angiogenesis with Notch signaling inducer Jagged1, inhibitor DAPT and PBS respectively were observed by confocal laser scanning microscopy. Semi-quantitative PCR and immunofluorescent staining were conducted to detect expressions of angiogenesis-related genes and proteins.ResultsAngiogenesis in the co-culture gels was promoted by Jagged1 treatment while attenuated by DAPT treatment, compared to control group. In co-culture system of ASCs and ECs, the gene expressions of VEGFA, VEGFB, Notch1, Notch2, Hes1, Hey1, VEGFR1and the protein expression of VEGFA, VEGFB, Notch1, Hes1, Hey1 were increased by Jagged1 treatment and decreased by DAPT treatment in ECs. And the result of VEGFR3 was the opposite. However, the same results did not appear completely in ASCs.ConclusionsThese results revealed the VEGFA/B-Notch1/2-Hes1/Hey1- VEGFR1/3 signal axis played an important role in angiogenesis when ASCs and ECs were co-cultured in a 3D collagen gel model. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-20T15:45:44.326649-05:
      DOI: 10.1002/jcp.25681
  • Tracing Males from Different Continents by Genotyping JC Polyomavirus in
           DNA from Semen Samples
    • Authors: John Charles Rotondo; Tommaso Candian, Rita Selvatici, Elisa Mazzoni, Gloria Bonaccorsi, Pantaleo Greco, Mauro Tognon, Fernanda Martini
      Abstract: The human JC polyomavirus (JCPyV) is an ubiquitous viral agent infecting approximately 60% of humans. Recently, JCPyV sequences have been detected in semen samples. The aim of this investigation was to test whether semen JCPyV genotyping can be employed to trace the origin continent of males. Semen DNA samples (n = 170) from males of different Continents, were investigated by PCR for the polymorphic JCPyV viral capsid protein 1 (VP1) sequences, followed by DNA sequencing. JCPyV sequences were detected with an overall prevalence of 27.6% (47/170). DNA sequencing revealed that European males carried JCPyV types 1A (71.4%), 4 (11.4%), 2B (2.9%), 2D1 (2.9%) and 3A (2.9%), Asians JCPyV type 2D1 (66.7%) and Africans JCPyV types 3A (33.3%) and 1A (33.3%). In 10.6% of males, two different JCPyV genotypes were detected suggesting that the second JCPyV genotype was acquired in the destination country. This study indicates that the majority of semen samples, found to be JCPyV-positive, were infected with the JCPyV genotype found in the geographic area of male origin. Therefore, semen JCPyV genotyping could be employed to trace the origin continent of males. Our findings could be applied to forensic investigations, in case of, for instance, sexual crimes. Indeed, JCPyV genotyping should enable investigators to make additional detailed profiling of the offender. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-20T15:45:36.45259-05:0
      DOI: 10.1002/jcp.25686
  • Wnt3A Induces GSK-3β Phosphorylation and β-Catenin Accumulation
           Through RhoA/ROCK
    • Authors: Jae-Gyu Kim; Myoung-Ju Kim, Won-Ji Choi, Mi-Young Moon, Hee-Jun Kim, Jae-Yong Lee, Jaebong Kim, Sung-Chan Kim, Seung Goo Kang, Goo-Young Seo, Pyeung-Hyeun Kim, Jae-Bong Park
      Abstract: In canonical pathway, Wnt3A has been known to stabilize β-catenin through the dissociation between β-catenin and glycogen synthase kinase-3β (GSK-3β) that suppresses the phosphorylation and degradation of β-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3β and accumulation of β-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3β and accumulation of β-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3β phosphorylation and β-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3β in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3β phosphorylation and β-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.Wnt3A activates RhoA and ROCK, which induces GSK-3beta phosphorylation and beta-catenin accumulation. RhoA and ROCK are involved in the regulation of cell proliferation and migration upon Wnt3A.
      PubDate: 2016-11-20T15:45:29.212161-05:
      DOI: 10.1002/jcp.25572
  • CD93 as a Potential Target in Neovascular Age-Related Macular Degeneration
    • Authors: Gian Marco Tosi; Elena Caldi, Barbara Parolini, Paolo Toti, Giovanni Neri, Federica Nardi, Claudio Traversi, Gabriele Cevenini, Davide Marigliani, Elisabetta Nuti, Tommaso Bacci, Federico Galvagni, Maurizio Orlandini
      Abstract: In patients with age-related macular degeneration (AMD), choroidal neovascularization is the major cause of severe visual loss. In these patients, the persistence of neovascular growth despite vascular endothelial growth factor-A blockage needs the discovery of new endothelial cell targets. The glycoprotein CD93, highly expressed in activated endothelial cells, has been recently involved in the regulation of the angiogenic process both as transmembrane and soluble protein.Choroidal neovascular membranes from patients affected by AMD were examined by immunofluorescence using anti-CD93 and anti-von Willebrand factor antibodies. Blood vessels within intraocular and extraocular neoplasias were used as controls for CD93 expression. All choroidal neovascular membranes displayed strong CD93 staining in the von Willebrand factor-positive endothelial cells, consistently with the analyses showing a high colocalization coefficient in the blood vessels. Intraocular and extraocular tumor vessels showed similar results, whereas the normal choroid displayed blood vessels with only faint CD93 staining. Additionally, the concentration of soluble CD93 was determined in the aqueous humor of patients affected by naïve neovascular AMD by enzyme-linked immunosorbent assays. Age-matched cataract patients served as controls. Soluble CD93 was significantly increased in the aqueous humor of naïve neovascular AMD patients and tended to decrease after treatment with an antiangiogenic drug. In conclusion, both transmembrane and soluble CD93 are overexpressed in patients with neovascular AMD, indicating that CD93 may represent a potential new antiangiogenic target in the treatment of choroidal neovascularization. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-15T03:31:09.290756-05:
      DOI: 10.1002/jcp.25689
  • Oligodendrocyte Progenitor Cells Directly Utilize Lactate for Promoting
           Cell Cycling and Differentiation
    • Authors: Yoshinori Ichihara; Toru Doi, Youngjae Ryu, Motoshi Nagao, Yasuhiro Sawada, Toru Ogata
      Abstract: Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC-rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU-positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate-mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α-cyano-4-hydroxy-cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-15T03:31:01.611104-05:
      DOI: 10.1002/jcp.25690
  • A Real-World Multicentre Retrospective Study of Paclitaxel-Bevacizumab and
           Maintenance Therapy as First-Line for HER2-Negative Metastatic Breast
    • Authors: T. Gamucci; L. Mentuccia, C. Natoli, I. Sperduti, A. Cassano, A. Michelotti, L. Di Lauro, D. Sergi, A. Fabi, M.G. Sarobba, P. Marchetti, M. Barba, E. Magnolfi, M. Maugeri-Saccà, E. Rossi, V. Sini, A. Grassadonia, D. Pellegrini, A. Astone, C. Nisticò, F. Angelini, A. Vaccaro, A. Pellegrino, C. De Angelis, M. Palleschi, L. Moscetti, I. Bertolini, S. Buglioni, A. Giordano, L. Pizzuti, P. Vici
      Abstract: Bevacizumab in combination with taxanes in HER2-negative metastatic breast cancer (MBC) patients has shown improved progression-free survival (PFS), despite the lack of clear overall survival (OS) benefit. We performed a retrospective analysis to evaluate the impact of paclitaxel-bevacizumab and of maintenance therapy with bevacizumab (BM) and endocrine therapy (ET) in the real-world practice.We identified 314 HER2-negative MBC patients treated in 12 cancer centres.Overall, the median PFS and OS were 14 and 40 months, respectively. Among the 254 patients potentially eligible for BM, 183 received BM after paclitaxel discontinuation until progression/toxicity. PFS and OS were improved in patients who had received BM in comparison with those potentially eligible but who did not receive BM (p 
      PubDate: 2016-11-11T07:15:43.103661-05:
      DOI: 10.1002/jcp.25685
  • The Multifaceted Roles of Primary Cilia in the Regulation of Stem Cell
           Properties and Functions
    • Authors: Rui Lyu; Jun Zhou
      Abstract: Stem cells are a unique class of cells that are capable of self-renewal and differentiation into multiple lineages. An increasing number of studies have suggested that both embryonic and adult stem cells possess primary cilia, antenna-like structures protruding from cell surfaces that are critical for sensing and transducing environmental cues. The primary cilium appears to regulate stem cells in multiple aspects, such as lineage specification and stemness maintenance. Understanding the role of primary cilia in the control of stem cell behavior could lead to the identification of new targets for regenerative therapies. Here, we discuss recent studies investigating the diverse roles of primary cilia in the regulation of stem cell properties and functions. We also propose potential new avenues for exploration in this promising field. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-11T07:05:29.536643-05:
      DOI: 10.1002/jcp.25683
  • S100B + A1 CELISA: A Novel Potency Assay and Screening Tool for
           Redifferentiation Stimuli of Human Articular Chondrocytes
    • Authors: Jose Diaz-Romero; Sibylle Kürsener, Sandro Kohl, Dobrila Nesic
      Abstract: During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell-based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: 1) demonstrate a correlation between S100B + A1-positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; 2) develop an S100B + A1 cell-based ELISA, and 3) prove that S100B + A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high-osmolarity/BMP4 treatment in monolayer) using qRT-PCR, immunocytochemistry or immunohistochemistry. A cell-based ELISA (CELISA) was developed as a 96-well microplate multiplex assay to measure S100B + A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow-derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B + A1-positive HAC in monolayer and their neochondrogenesis in pellets validates S100B + A1 as a marker of chondrogenic potency. The S100B + A1-CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high-throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets - induced with high-osmolarity and BMP4 treatment in monolayer - suggests that cell instruction prior to implantation may improve cartilage repair. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-11T07:05:24.089704-05:
      DOI: 10.1002/jcp.25682
  • E-Cigarette Vapor Induces an Apoptotic Response in Human Gingival
           Epithelial Cells Through the Caspase-3 Pathway
    • Authors: Mahmoud Rouabhia; Hyun Jin Park, Abdelhabib Semlali, Andrew Zakrzewski, Witold Chmielewski, Jamila Chakir
      Abstract: Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-03T05:59:00.705487-05:
      DOI: 10.1002/jcp.25677
  • Calcium Signaling During Meiotic Cell Cycle Regulation and Apoptosis in
           Mammalian Oocytes
    • Authors: Meenakshi Tiwari; Shilpa Prasad, Tulsidas G. Shrivastav, Shail K. Chaube
      Abstract: Calcium (Ca++) is one of the major signal molecules that regulate various aspects of cell functions including cell cycle progression, arrest and apoptosis in wide variety of cells. This review summarizes current knowledge on the differential roles of Ca++ in meiotic cell cycle resumption, arrest and apoptosis in mammalian oocytes. Release of Ca++ from internal stores and/or Ca++ influx from extracellular medium causes moderate increase of intracellular Ca++ ([Ca++]i) level and reactive oxygen species (ROS). Increase of Ca++ as well as ROS levels under physiological range trigger maturation promoting factor (MPF) destabilization thereby meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in oocytes. A sustained increase of [Ca++]i level beyond physiological range induces generation of ROS sufficient enough to cause oxidative stress (OS) in aging oocytes. The increased [Ca++]i triggers Fas ligand-mediated oocyte apoptosis. Further, OS triggers mitochondria-mediated oocyte apoptosis in several mammalian species. Thus, Ca++ exerts differential roles on oocyte physiology depending upon its intracellular concentration. A moderate increase of [Ca++]i as well as ROS mediate spontaneous resumption of meiosis from diplotene as well as M-II arrest, while their high levels cause meiotic cell cycle arrest and apoptosis by operating both mitochondria- as well as Fas ligand-mediated apoptotic pathways. Indeed, Ca++ regulates cellular physiology by modulating meiotic cell cycle and apoptosis in mammalian oocytes. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:53:27.761713-05:
      DOI: 10.1002/jcp.25670
  • The Emerging Role of MORC Family Proteins in Cancer Development and Bone
    • Authors: Guoju Hong; Heng Qiu, Chao Wang, Gaurav Jadhav, Haibin Wang, Jennifer Tickner, Wei He, Jiake Xu
      Abstract: Microrchidia (MORC or MORC family CW-type zinc finger protein), a highly conserved nuclear protein superfamily, is an interesting new player in signaling-dependent chromatin remodeling and epigenetic regulation. MORC family proteins consist of MORC1, MORC2, MORC3 and MORC4 which display common structural determinants such as CW-type zinc finger and coiled-coil domains. They also exhibit unique structural motifs and tissue-specific expression profiles. Morc1 was first discovered as a key regulator for male meiosis and spermatogenesis. Accumulating biochemical and functional analyses unveil MORC proteins as key regulators for cancer development. More recently, using an ENU mutagenesis mouse model, MORC3 was found to play a role in regulating bone and calcium hemostasis. Here we discuss recent research progress on the emerging role of MORC proteins in cancer development and bone metabolism. Unravelling the cellular and molecular mechanisms by which MORC proteins carry out their functions in a tissue specific manner are important subjects for future investigation. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:46:21.015841-05:
      DOI: 10.1002/jcp.25665
  • Extracellular Matrix and Colorectal Cancer: How Surrounding
           Microenvironment Affects Cancer Cell Behavior?
    • Authors: Sara Crotti; Martina Piccoli, Flavio Rizzolio, Antonio Giordano, Donato Nitti, Marco Agostini
      Abstract: Colorectal cancer (CRC) whit more than a million of new cases per year is one of the most common registered cancers worldwide with few treatment options especially for advanced and metastatic patients.The tumor microenvironment is composed by extracellular matrix (ECM), cells and interstitial fluids. Among all these constituents, in the last years an increased interest around the ECM and its potential role in cancer tumorigenesis is arisen. During cancer progression the ECM structure and composition became disorganized, allowing cellular transformation and metastasis.Up to now, the focus has mainly been on the characterization of CRC microenvironment analyzing separately structural ECM components or cell secretome modifications. A more extensive view that interconnects these aspects should be addressed. In this review, biochemical (secretome) and biomechanical (structure and architecture) changes of tumor microenvironment will be discussed, giving suggestions on how these changes can affect cancer cell behavior. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-24T05:15:47.729256-05:
      DOI: 10.1002/jcp.25658
  • Functional Roles of Eph A-Ephrin A1 System in Endometrial Luminal
           Epithelial Cells During Early Pregnancy
    • Authors: Whasun Lim; Hyocheol Bae, Fuller W. Bazer, Gwonhwa Song
      Abstract: Eph and ephrin regulate diverse biological events such as proliferation, adhesion, migration, and angiogenesis through cell-to-cell interactions. However, little is known of their functional role and mechanisms of action in uterine endometrial cells. In the present study, we demonstrated the effects of the Eph and ephrin on interactions between blastocysts and endometrial luminal epithelial (pLE) cells in the pig that is regarded as an excellent biomedical animal model for research on the peri-implantation period of pregnancy. Results of this study indicated that among eight members of the Eph A family, expression of Eph A1, A2, A4 and A7 was strongly detected in endometrial epithelial cells during early pregnancy. Of these, for identification of signal transduction pathways induced by ephrin A1, a major ligand for Eph A, cell proliferation assays, and immunofluorescence and cell cycle regulation were analyzed following treatment of pLE cells with ephrin A1. Ephrin A1 stimulated proliferation of pLE cells as evidenced by abundant PCNA expression and an increase in the G2/M phase. Western blot analysis showed that ephrin A1 activated PI3K and MAPK signaling proteins in a time-dependent manner. Moreover, phosphorylation of AKT, ERK1/2, P38 and JNK proteins were suppressed by their inhibitors wortmannin, U0126, SB203580 and SP600125, respectively. Also, phosphor-AKT was reduced by ERK1/2 and P38 inhibitors. Ephrin A1-induced proliferation and migration of pLE cells was also blocked by those inhibitors. Collectively, these results suggest that ephrin A1 enhances interactions between porcine blastocysts and endometrial luminal epithelial cells by activating PI3K and MAPK signal transduction pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-24T05:15:41.367406-05:
      DOI: 10.1002/jcp.25659
  • Dermal-Epidermal Cross-Talk: Differential Interactions With Microvascular
           Endothelial Cells
    • Authors: Eleonora Bassino; Edoardo Vallariello, Franco Gasparri, Luca Munaron
      Abstract: The biological importance of circulatory blood supply and angiogenesis for hair growth is now well recognized, but the their regulatory mechanisms require more mechanistic investigation. In vitro cocultures and tricultures can be successfully employed to greatly improve our knowledge on paracrine crosstalk between cell types that populate the dermal-epidermal interface and cutaneous vasculature. Here we report that human dermal fibroblasts (NHDF) promote viability and proliferation of microvascular endothelial cells (HMVEC), while HMVEC are not mitogenic for NHDF. In triculture setup, conditioned media obtained by cocultures (HMVEC/NHDF or HMVEC/follicle fibroblasts) differently modulate growth and proliferation of keratinocytes and alter the expression of metabolic and pro-inflammatory markers. In conclusion, tricultures were successfully employed to characterize in vitro dermal-epithelial and endothelial interactions and could integrate ex vivo and in vivo approaches by the use of high-throughput and standardized protocols in controlled conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-20T18:36:10.658839-05:
      DOI: 10.1002/jcp.25657
  • ADP-Induced Ca2+ Signalling and Proliferation of Rat Ventricular
           Myofibroblasts Depend on Phospholipase C-Linked TRP Channels Activation
           Within Lipid Rafts
    • Authors: Mariana Certal; Adriana Vinhas, Aurora Barros-Barbosa, Fátima Ferreirinha, Maria Adelina Costa, Paulo Correia-de-Sá
      Abstract: Nucleotides released during heart injury affect myocardium electrophysiology and remodelling through P2 purinoceptors activation in cardiac myofibroblasts. ATP and UTP endorse [Ca2+]i accumulation and growth of DDR-2/a-SMA-expressing myofibroblasts from adult rat ventricles via P2Y4 and P2Y2 receptors activation, respectively. Ventricular myofibroblasts also express ADP-sensitive P2Yi, P2Yi2 and P2Yi3 receptors as demonstrated by immunofluorescence confocal microscopy and western blot analysis, but little information exists on ADP effects in these cells. ADP (0.003-3mM) and its stable analogue, ADPBS (100uM), caused fast [Ca2+]i transients originated from thapsigargin-sensitive internal stores, which partially declined to a plateau sustained by capacitative Ca2+ entry through transient receptor potential (TRP) channels inhibited by 2-APB (50uM) and flufenamic acid (100uM). Hydrophobic interactions between Gq/11-coupled P2Y purinoceptors and TRP channels were suggested by prevention of the ADP-induced [Ca2+]i plateau following PIP2 depletion with LiCl (10mM) and cholesterol removal from lipid rafts with methyl-B-cyclodextrin (2mM). ADP [Ca2+]i transients were insensitive to P2Y1, P2Y12 and P2Y13 receptor antagonists, MRS2179 (10uM), AR-C66096 (0.1uM) and MRS2211 (10uM), respectively, but were attenuated by suramin and reactive blue-2 (100uM) which also blocked P2Y4 receptors activation by UTP. Cardiac myofibroblasts growth and type I collagen production were favoured upon activation of MRS2179-sensitive P2Y1 receptors with ADP or ADPBS (30uM). In conclusion, ADP exerts a dual role on ventricular myofibroblasts: [Ca2+]i transients are mediated by fast-desensitizing P2Y4 receptors, whereas the pro-fibrotic effect of ADP involves the P2Y1 receptor activation. Data also show that ADP-induced capacitative Ca2+ influx depends on phospholipase C-linked TRP channels opening in lipid raft microdomains. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-18T11:55:22.363946-05:
      DOI: 10.1002/jcp.25656
  • Neuroprotection and Blood-Brain Barrier Restoration by Salubrinal After a
           Cortical Stab Injury
    • Authors: M. Asunción Barreda-Manso; Natalia Yanguas-Casás, Manuel Nieto-Sampedro, Lorenzo Romero-Ramírez
      Abstract: Following a central nervous system (CNS) injury, restoration of the blood-brain barrier (BBB) integrity is essential for recovering homeostasis. When this process is delayed or impeded, blood substances and cells enter the CNS parenchyma, initiating an additional inflammatory process that extends the initial injury and causes so-called secondary neuronal loss. Astrocytes and profibrotic mesenchymal cells react to the injury and migrate to the lesion site, creating a new glia limitans that restores the BBB. This process is beneficial for the resolution of the inflammation, neuronal survival and the initiation of the healing process. Salubrinal is a small molecule with neuroprotective properties in different animal models of stroke and trauma to the CNS. Here, we show that salubrinal increased neuronal survival in the neighbourhood of a cerebral cortex stab injury. Moreover, salubrinal reduced cortical blood leakage into the parenchyma of injured animals compared with injured controls. Adjacent to the site of injury, salubrinal induced immunoreactivity for platelet-derived growth factor subunit B (PDGF-B), a specific mitogenic factor for mesenchymal cells. This effect might be responsible for the increased immunoreactivity for fibronectin and the decreased activation of microglia and macrophages in injured mice treated with salubrinal, compared with injured controls. The immunoreactivity for PDGF-B colocalized with neuronal nuclei (NeuN), suggesting that cortical neurons in the proximity of the injury were the main source of PDGF-B. Our results suggest that after an injury, neurons play an important role in both, the healing process and the restoration of the BBB integrity. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-18T06:40:27.39664-05:0
      DOI: 10.1002/jcp.25655
  • Src Family Kinase Links Insulin Signaling to Short Term Regulation of
           Na,K-ATPase in Nonpigmented Ciliary Epithelium
    • Authors: Mohammad Shahidullah; Amritlal Mandala, Nicholas A. Delamere
      Abstract: Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2 fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2 and p38 activation, all of which were prevented by a PI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates PI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-17T06:44:20.795008-05:
      DOI: 10.1002/jcp.25654
  • The Natural cAMP Elevating Compound Forskolin in Cancer Therapy: Is it
    • Authors: Luigi Sapio; Monica Gallo, Michela Illiano, Emilio Chiosi, Daniele Naviglio, Annamaria Spina, Silvio Naviglio
      Abstract: Cancer is a major public health problem and the second leading cause of mortality around the world. Although continuous advances in the science of oncology and cancer research are now leading to improved outcomes for many cancer patients, novel cancer treatment options are strongly demanded. Naturally occurring compounds from a variety of vegetables, fruits and medicinal plants have been shown to exhibit various anticancer properties in a number of in vitro and in vivo studies and represent an attractive research area for the development of new therapeutic strategies to fight cancer.Forskolin is a diterpene produced by the roots of the Indian plant Coleus forskohlii. The natural compound forskolin has been used for centuries in traditional medicine and its safety has also been documented in conventional modern medicine. Forskolin directly activates the adenylate cyclase enzyme, that generates cAMP from ATP, thus raising intracellular cAMP levels.Notably, cAMP signaling, through the PKA-dependent and/or independent pathways, is very relevant to cancer and its targeting has shown a number of antitumor effects, including the induction of mesenchymal-to-epithelial transition, inhibition of cell growth and migration and enhancement of sensitivity to conventional antitumor drugs in cancer cells.Here, we describe some features of cAMP signaling that are relevant to cancer biology and address the state of the art concerning the natural cAMP elevating compound forskolin and its perspectives as an effective anticancer agent. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-14T03:21:46.56839-05:0
      DOI: 10.1002/jcp.25650
  • Increased Chondrogenic Potential of Mesenchymal Cells from Adipose Tissue
           Versus Bone Marrow Derived Cells in Osteoarthritic In Vitro Models
    • Authors: Stefania Pagani; Veronica Borsari, Francesca Veronesi, Andrea Ferrari, Simona Cepollaro, Paola Torricelli, Giuseppe Filardo, Milena Fini
      Abstract: ObjectivesPrimarily, to compare the behavior of human mesenchymal stem cells (MSCs) derived from bone marrow (hBMSCs) and adipose tissue (hADSCs) in an osteoarthritic (OA) microenvironment; secondly, to investigate the reaction of these cell types in two alternative in vitro culture systems, obtained by using TNFα and/or IL1β as inflammation mediators, or by using synovial fluid harvested by OA patients (OSF) to simulate the complex inflamed knee microenvironment.Methods3D micromass cultures of hBMSCs or hADSCs were grown in chondrogenic medium (CTR), in the presence of TNFα and/or IL1β, or synovial fluid from OA patients. After 1 month of culture, the chondrogenic differentiation of micromasses was evaluated by gene expression, matrix composition and organization.ResultsBoth hMSCs types formed mature micromasses in CTR, but a better response of hADSCs to the inflammatory environment was documented by micromass area and Bern score evaluations. The addition of OSF elicited a milder reaction than with TNFα and/or IL1β by both cell types, probably due to the presence of both catabolic and protective factors. In particular, SOX9 and ACAN gene expression and GAG synthesis were more abundant in hADSCs than hBMSCs when cultured in OSF. The expression of MMP1 was increased for both hMSCs in inflammatory conditions, but in particular by hBMSCs.ConclusionhADSCs showed an increased chondrogenic potential in inflammatory culture systems, suggesting a better response of hADSCs in the OA environment, thus underlining the importance of appropriate in vitro models to study MSCs and potential advantages of using these cells for future clinical applications. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-14T03:21:41.163181-05:
      DOI: 10.1002/jcp.25651
  • PPARα Antagonist AA452 Triggers Metabolic Reprogramming and Increases
           Sensitivity to Radiation Therapy in Human Glioblastoma Primary Cells
    • Authors: E Benedetti; M d'Angelo, A Ammazzalorso, G Gravina, C Laezza, A Antonosante, G Panella, B Cinque, L Cristiano, AC Dhez, C Astarita, R Galzio, MG Cifone, R Ippoliti, R Amoroso, E Di Cesare, A Giordano, A Cimini
      Abstract: Glioblastoma is the most-common cancer in the brain and with an increasing incidence. Despite major advances in the field, there is no curative therapy for GB to date. Many solid tumors, including GB, experienced metabolic reprogramming in order to sustain uncontrolled proliferation, hypoxic conditions and angiogenesis. PPARs, member of the steroid hormone receptor superfamily, are particularly involved in the control of energetic metabolism, particularly lipid metabolism, which has been reported deregulated in gliomas. PPARα was previously indicated by us as a potential therapeutic target for this neoplasm, due to the malignancy grade dependency of its expression, being particularly abundant in GB. In this work we used a new PPARα antagonist on patient-derived GB primary cells with particular focus on the effects on lipid metabolism and response to radiotherapy. The results obtained demonstrated that blocking PPARα results in cell death induction, increase of radiosensitivity and decrease of migration. Therefore, AA452 arises as a potent drug adjuvanting the gold standard therapies for GB, opening the possibility for pre-clinical and clinical trials for this class of compounds. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:47:07.048024-05:
      DOI: 10.1002/jcp.25648
  • Osteogenic Differentiation of Periosteal Cells during Fracture Healing
    • Authors: Tao Wang; Xinping Zhang, Daniel D. Bikle
      Abstract: 5-10% of fractures fail to heal normally leading to additional surgery, morbidity, and altered quality of life. Fracture healing involves the coordinated action of stem cells primarily coming from the periosteum which differentiate into the chondrocytes and osteoblasts, forming first the soft (cartilage) callus followed by the hard (bone) callus. These stem cells are accompanied by a vascular invasion that appears critical for the differentiation process and which may enable the entry of osteoclasts necessary for the remodeling of the callus into mature bone. However, more research is needed to clarify the signaling events that activate the osteochondroprogenitor cells of periosteum and stimulate their differentiation into chondrocytes and osteoblasts. Ultimately a thorough understanding of the mechanisms for differential regulation of these osteochondroprogenitors will aid in the treatment of bone healing and the prevention of delayed union and nonunion of fractures. In this review, evidence supporting the concept that the periosteal cells are the major cell sources of skeletal progenitors for the fracture callus will be discussed. The osteogenic differentiation of periosteal cells manipulated by Wnt/β-catenin, TGF/BMP, Ihh/PTHrP, and IGF-1/PI3K–Akt signaling in fracture repair will be examined. The effect of physical (hypoxia and hyperoxia) and chemical factors (reactive oxygen species) as well as the potential coordinated regulatory mechanisms in the periosteal progenitor cells promoting osteogenic differentiation will also be discussed. Understanding the regulation of periosteal osteochondroprogenitors during fracture healing could provide insight into possible therapeutic targets and thereby help to enhance future fracture healing and bone tissue engineering approaches. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:40:55.646886-05:
      DOI: 10.1002/jcp.25641
  • Low Shear Stress Attenuates COX-2 Expression Induced by Resistin in Human
           Osteoarthritic Chondrocytes
    • Authors: Yu-Ping Su; Cheng-Nan Chen, Hsin-I Chang, Kuo-Chin Huang, Chin-Chang Cheng, Fang-Yao Chiu, Ko-Chao Lee, Chun-Min Lo, Shun-Fu Chang
      Abstract: Low shear stress has been proposed to play a reparative role in modulating cartilage homeostasis. Recently, epidemiological studies have found a positive correlation between the resistin level in serum and synovial fluid and osteoarthritis (OA) severity in patients. However, the effect of moderate shear stress on the catabolic stimulation of resistin in OA chondrocytes remains unclear. Hence, this study was to investigate whether low shear stress could regulate resistin-induced catabolic cyclooxygenase (COX)-2 expression in human OA chondrocytes and the underlying mechanism. Human OA chondrocytes and SW1353 chondrosarcoma cells were used in this study. Two modes of low shear stress (2 dyn/cm2), pre-shear and post-shear, were applied to the chondrocytes. A specific activator and siRNAs were used to investigate the mechanism of low shear stress-regulated COX-2 expression of resistin induction. We found that human OA chondrocytes exposed to different modes of low shear stress elicit an opposite effect on resistin-induced COX-2 expression: pre-shear for a short duration attenuates the resistin effect by inhibiting the transcription factor nuclear factor (NF)-κB-p65 subunit and the cAMP response element binding protein; however, post-shear over a longer duration enhances the resistin effect by activating only the NF-κB-p65 subunit. Moreover, our results demonstrated that the regulation of both shear modes in resistin-stimulated COX-2 expression occurs through increasing AMP-activated protein kinase activation and then sirtuin 1 expression. This study elucidates the detailed mechanism of low shear stress regulating the resistin-induced catabolic COX-2 expression and indicates a possible reparative role of moderate shear force in resistin-stimulated OA development. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:50.402155-05:
      DOI: 10.1002/jcp.25644
  • Peripheral Blood Mononuclear Cells Infiltration Downregulates Decidual
           FAAH Activity in an LPS-Induced Embryo Resorption Model
    • Authors: Manuel Luis Wolfson; Julieta Aisemberg, Fernando Correa, Ana María Franchi
      Abstract: Background and PurposeMaternal infections with Gram-negative bacteria are associated with miscarriage and are one of the most common complications during pregnancy. Previous studies from our group have shown that lipopolysaccharide (LPS)-activated infiltrating peripheral blood mononuclear cells (PBMC) into decidual tissue play an important role on the establishment of a local inflammatory process that result in embryo cytotoxicity and early embryo resorption. Moreover, we have also shown that an increased endocannabinoid tone mediates LPS-induced deleterious effects during early pregnancy loss. Here we sought to investigate whether the infiltrating PBMC modulates the decidual endocannabinoid tone and the molecular mechanisms involved.Experimental approachPBMC isolated from 7-days pregnant mice subjected to different treatments were co-cultured in a transwell system with decidual tissue from control 7-days pregnant mice. Decidual fatty acid amide hydrolase (FAAH) activity was measure by radioconvertion, total decidual protein nitration by western blot (WB) and decidual FAAH nitration by immuneprecipitation followed by WB.Key ResultsWe found that co-culture of PBMC obtained from LPS-treated mice increased the level of nitration of decidual FAAH, which resulted in a negative modulation of decidual FAAH activity. Interestingly, co-treatment with progesterone or aminoguanidine prevented this effect.Conclusions & ImplicationsWe found that LPS-treated PBMC release high amounts of nitric oxide (NO) which causes tyrosine nitration of decidual FAAH, diminishing its enzymatic activity. Inactivation of FAAH, the main degrading enzyme of anandamide and similar endocannabinoids, could lead to an increased decidual endocannabinoid tone with embryotoxic effects. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:34.357677-05:
      DOI: 10.1002/jcp.25640
  • Inhibitory Effects of Quercetin on Progression of Human Choriocarcinoma
    • Authors: Whasun Lim; Changwon Yang, Sunwoo Park, Fuller W. Bazer, Gwonhwa Song
      Abstract: As a major dietary flavonol, quercetin mitigates proliferation and progression of cancer due to its anti-angiogenic, anti-inflammatory, anti-oxidant and apoptotic biological effects on cells. Although its apoptotic effects have been reported for various cancers, little is known of the functional role of quercetin in gestational choriocarcinoma. Results of the present study indicated that quercetin reduced proliferation and induced cell death in two choriocarcinoma cell lines, JAR and JEG3 cells, with an increase in the sub-G1 phase of the cell cycle. In addition, quercetin induced mitochondrial dysfunction significantly reduced mitochondrial membrane potential (MMP) and increased production of reactive oxygen species (ROS) in both JAR and JEG3 cells. Further, quercetin inhibited phosphorylation of AKT, P70S6K and S6 proteins whereas it increased phosphorylation of ERK1/2, P38, JNK and P90RSK proteins in JAR and JEG3 cells. The decrease in viability of choriocarcinoma cells treated with quercetin was confirmed by using combinations of quercetin and pharmacological inhibitors of the PI3K and MAPK signaling pathways. Classical chemotherapeutic agents, cisplatin (a platinum-based drug) and paclitaxel (a taxene-based drug), inhibited proliferation of JAR and JEG3 cells and when combined with quercetin, the antiproliferative effects of cisplatin and paclitaxel were enhanced for both choriocarcinoma cell lines. Collectively, these results suggest that quercetin prevents development of choriocarcinoma and may be a valuable therapeutic agent for treatment of choriocarcinoma through its regulation of PI3K and MAPK signal transduction pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:49:16.88812-05:0
      DOI: 10.1002/jcp.25637
  • Essential Role of CFTR in PKA-Dependent Phosphorylation, Alkalinization
           and Hyperpolarization During Human Sperm Capacitation
    • Authors: Lis C. Puga Molina; Nicolás A. Pinto, Paulina Torres Rodríguez, Ana Romarowski, Alberto Vicens Sanchez, Pablo E. Visconti, Alberto Darszon, Claudia L. Treviño, Mariano G. Buffone
      Abstract: Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3- is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm and membrane hyperpolarization. However, how HCO3- is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3--entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:36:13.303942-05:
      DOI: 10.1002/jcp.25634
  • Indispensable Role of Ion Channels and Transporters in the Auditory System
    • Authors: Rahul Mittal; Mayank Aranke, Luca H. Debs, Desiree Nguyen, Amit P. Patel, M'hamed Grati, Jeenu Mittal, Denise Yan, Prem Chapagain, Adrien A. Eshraghi, Xue Zhong Liu
      Abstract: Ear is a complex system where appropriate ionic composition is essential for maintaining the tissue homeostasis and hearing function. Ion transporters and channels present in the auditory system plays a crucial role in maintaining proper ionic composition in the ear. The extracellular fluid, called endolymph, found in the cochlea of the mammalian inner ear is particularly unique due to its electrochemical properties. At an endocochlear potential of about +80 mV, signaling initiated by acoustic stimuli at the level of the hair cells is dependent on the unusually high potassium (K+) concentration of endolymph. There are ion channels and transporters that exists in the ear to ensure that K+ is continually being cycled into the stria media endolymph. This review is focused on the discussion of the molecular and genetic basis of previously and newly recognized ion channels and transporters that support sensory hair cell excitation based on recent knock-in and knock-out studies of these channels. This article also addresses the molecular and genetic defects and the pathophysiology behind Meniere's disease as well as how the dysregulation of these ion transporters can result in severe defects in hearing or even deafness. Understanding the role of ion channels and transporters in the auditory system will facilitate in designing effective treatment modalities against ear disorders including Meniere's disease and hearing loss. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-05T05:11:04.350582-05:
      DOI: 10.1002/jcp.25631
  • Role of Protein Kinase G and Reactive Oxygen Species in the Regulation of
           Podocyte Function in Health and Disease
    • Authors: Agnieszka Piwkowska
      Abstract: Podocytes and their foot processes form an important cellular layer of the glomerular barrier involved in the regulation of glomerular permeability. Disturbing the function of podocytes plays a central role in the development of proteinuria in diabetic nephropathy. Retraction of the podocyte foot processes that form slit diaphragms is a common feature of proteinuria; although, the correlation between these events in not well understood. Notably, it is unclear whether podocyte foot processes are able to regulate slit diaphragm permeability and glomerular ultrafiltration. The occurrence of reactive oxygen species generation, insulin resistance, and hyperglycemia characterizes early stages of type 2 diabetes. Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. It is activated in both cGMP-dependent and cGMP-independent manners. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI-dependent signaling pathways in podocytes. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI-dependent signaling pathways in podocytes.
      PubDate: 2016-09-30T08:58:57.845176-05:
      DOI: 10.1002/jcp.25613
  • Basic Signaling in Cardiac Fibroblasts
    • Authors: Stéphanie Chacar; Nassim Farès, Patrick Bois, Jean-François Faivre
      Abstract: Cardiac fibroblasts are commonly known as supporting cells of the cardiac network and exert many essential functions that are fundamental for normal cardiac growth as well as for cardiac remodeling process during pathological conditions. This review focuses on the roles of cardiac fibroblasts in the formation and regulation of the extracellular matrix components, and in maintaining structural, biochemical and mechanical properties of the heart. Additionally, though considered as non-excitable cells, we review the functional expression in cardiac fibroblasts of a wide variety of transmembrane ion channels which activity may contribute to key regulation of cardiac physiological processes. All together, cardiac fibroblasts which actively participate to fundamental regulation of cardiac physiology and physiopathology processes may represent pertinent targets for pharmacological approaches of cardiac diseases and lead to new tracks of therapeutic strategies. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-29T14:05:11.464802-05:
      DOI: 10.1002/jcp.25624
  • New Insights on the Regulation of Ca2+-Activated Chloride Channel TMEM16A
    • Authors: Ke Ma; Hui Wang, Jiankun Yu, Minjie Wei, Qinghuan Xiao
      Abstract: TMEM16A, also known as anoctamin 1, is a recently identified Ca2+-activated chloride channel and the first member of a 10-member TMEM16 family. TMEM16A dysfunction is implicated in many diseases such as cancer, hypertension, and cystic fibrosis. TMEM16A channels are well known to be dually regulated by voltage and Ca2+. In addition, recent studies have revealed that TMEM16A channels are regulated by many molecules such as calmodulin, protons, cholesterol, and phosphoinositides, and a diverse range of stimuli such as thermal and mechanical stimuli. A better understanding of the regulatory mechanisms of TMEM16A is important to understand its physiological and pathological role. Recently, the crystal structure of a TMEM16 family member from the fungus Nectria haematococcaten (nhTMEM16) is discovered, and provides valuable information for studying the structure and function of TMEM16A. In this review, we discuss the structure and function of TMEM16A channels based on the crystal structure of nhTMEM16A and focus on the regulatory mechanisms of TMEM16A channels. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:13.785451-05:
      DOI: 10.1002/jcp.25621
  • Understanding the Pathogenesis of Neurotrophic Keratitis: The Role of
           Corneal Nerves
    • Authors: Leonardo Mastropasqua; Giacomina Massaro-Giordano, Mario Nubile, Marta Sacchetti
      Abstract: Neurotrophic keratitis (NK) is a rare degenerative disease of the cornea caused by trigeminal nerve damage, which leads to loss of corneal sensitivity, corneal epithelium breakdown, and poor healing. Though extremely uncommon, NK is increasingly recognized for its characteristics as a distinct and well-defined clinical entity rather than a rare complication of various diseases that can disrupt trigeminal innervation. Indeed, the defining feature of NK is loss of corneal sensitivity, and its clinical findings do not correlate with the wide range of systemic or ocular conditions that underlie trigeminal nerve damage. Despite increasing awareness of NK as a distinct condition, its management continues to be challenged by the lack of treatments that target nerve regeneration. This review focuses on the role of corneal nerves in maintaining ocular surface homeostasis, the consequences (such as alterations in neuromediators and corneal cell morphology/function) of impaired innervation, and advances in NK diagnosis and management. Novel therapeutic strategies should aim to improve corneal innervation in order support corneal renewal and healing. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:09.942628-05:
      DOI: 10.1002/jcp.25623
  • p120-Catenin Is Required for Dietary Calcium Suppression of Oral
           Carcinogenesis in Mice
    • Authors: Zhongjian Xie; Yuan Yuan, Yi Jiang, Chandrama Shrestha, Ying Chen, Liyan Liao, Shangli Ji, Xiaoge Deng, Eryuan Liao, Daniel D. Bikle
      Abstract: Previous studies have shown that dietary calcium suppresses oral carcinogenesis, but the mechanism is unclear. p120-catenin (p120) is a cytoplasmic protein closely associated with E-cadherin to form the E-cadherin-β-catenin complex and may function as a tumor suppressor in the oral epithelium. To determine whether p120 is involved in the mechanism by which dietary calcium suppresses oral carcinogenesis, The normal, low or high calcium diet was fed control mice (designated as floxed p120 mice) or mice in which p120 was specifically deleted in the oral squamous epithelium during the adult stage (designated as p120cKO mice). All mice were exposed to a low dose of oral cancer carcinogen 4-nitroquinoline 1-oxide and rates of oral squamous cell carcinoma (OSCC) and proliferation and differentiation in the cancerous and non-cancerous oral epithelium of these mice were examined. The results showed that the low calcium diet increased rates of OSCC and proliferation of the non-cancerous oral epithelium and decreased differentiation of the non-cancerous oral epithelium, but had no effect on cancerous oral epithelium. In contrast, the high calcium diet had opposite effects. However, the effect of the dietary calcium on the rates of OSCC, proliferation and differentiation of the non-cancerous epithelium were not seen in p120cKO mice. Based on these results, we conclude that p120 is required for dietary calcium suppression of oral carcinogenesis and oral epithelial proliferation and dietary calcium induction of oral epithelial differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:02.467485-05:
      DOI: 10.1002/jcp.25620
  • Nephroprotective Effects of Metformin in Diabetic Nephropathy
    • Authors: Sreenithya Ravindran; Vinitha Kuruvilla, Kerry Wilbur, Shankar Munusamy
      Abstract: Metformin, a well-known anti-diabetic agent, is very effective in lowering blood glucose in patients with type 2 diabetes with minimal side-effects. Metformin is also being recommended in the treatment of obesity and polycystic ovary syndrome. Metformin elicits its therapeutic effects mainly via activation of AMP-activated kinase (AMPK) pathway. Renal cells under hyperglycemic or proteinuric conditions exhibit inactivation of cell defense mechanisms such as AMPK and autophagy, and activation of pathologic pathways such as mammalian target of rapamycin (mTOR), endoplasmic reticulum (ER) stress, epithelial-to-mesenchymal transition (EMT), oxidative stress, and hypoxia. As these pathologic pathways are intertwined with AMPK signaling, the potential benefits of metformin therapy in patients with type 2 diabetes would extend beyond its anti-hyperglycemic effects. However, since metformin is eliminated unchanged through the kidneys and some studies have shown the incidence of lactic acidosis with its use during severe renal dysfunction, the use of metformin was contraindicated in patients with renal disease until recently. With more studies indicating the relatively low incidence of lactic acidosis and revealing the additional benefits with metformin therapy, the US FDA has now approved metformin to be administered in patients with established renal disease based on their renal function. The purpose of this review is to highlight the various mechanisms by which metformin protects renal cells that have lost its functionality in a diabetic or non-diabetic setting and to enlighten the advantages and therapeutic potential of metformin as a nephroprotectant for patients with diabetic nephropathy and other non-diabetic forms of chronic kidney disease. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.Metformin, a popular anti-diabetic drug and an AMPK activator, modulates various signaling pathways altered in diabetic nephropathy and exhibits nephroprotective properties in various animal models of diabetes. Additional studies in humans are needed to ascertain its therapeutic potential as a nephroprotectant to treat nephropathy in patients with diabetes.
      PubDate: 2016-09-26T11:05:22.881493-05:
      DOI: 10.1002/jcp.25598
  • G Protein-Coupled Receptor Endosomal Signaling and Regulation of Neuronal
           Excitability and Stress Responses: Signaling Options and Lessons from the
           PAC1 Receptor
    • Authors: Victor May; Rodney L. Parsons
      Abstract: Our understanding of G protein coupled receptor (GPCR) mechanisms and functions have evolved considerably. Among the many conceptual realignments, GPCRs can exist in an ensemble of active microstates that have the potential to differentially engage specific downstream signaling events. Furthermore, among GPCR dynamics, GPCR internalization and vesicular trafficking are no longer solely mechanisms for desensitization, but now appreciated to form intricate endosomal signaling complexes that can potentially target second messengers to intracellular compartments with high temporal and spatial resolution. The PACAPergic system is important in the maintenance of physiological homeostasis in the central and peripheral nervous systems and activation of the PACAP-selective PAC1 receptor can generate differential but coordinate plasma membrane and endosomal signals for cellular responses. The integration of these signals can modulate PACAP-induced changes in ionic conductances that gate neuronal excitability. PACAP/PAC1 receptor generation of endosomal ERK signals participate in chronic pain and anxiety-like responses which can be attenuated with endocytosis inhibitors. From the abilities of ligands to stabilize the different GPCR microstates for biased downstream signaling, the development of biased PAC1 receptor agonists and antagonists may provide opportunities to dissociate the homeostatic regulatory signals of PACAP from the maladaptive effects. In particular, the development of biased antagonists to PAC1 receptor-mediated endosomal signaling may offer therapeutic options for chronic pain and stress-related disorders. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-23T17:41:35.805915-05:
      DOI: 10.1002/jcp.25615
  • Valproic Acid and Lithium Meditate Anti-Inflammatory Effects by
           Differentially Modulating Dendritic Cell Differentiation and Function
    • Authors: Sy-Jye Leu; Yi-Yuan Yang, Hsing-Cheng Liu, Chieh-Yu Cheng, Yu-Chen Wu, Ming-Chyi Huang, Yuen-Lun Lee, Chi-Ching Chen, Winston W. Shen, Ko-Jiunn Liu
      Abstract: Valproic acid (VPA), with inhibition activity mainly towards histone deacetylase (HDAC) and Glycogen Synthase Kinase (GSK)-3, and lithium, with inhibition activity mainly toward GSK-3, are both prescribed in clinical as mood-stabilizers and anticonvulsants for the control of bipolar disorder. This study aims to compare the immuno-modulation activities of VPA and lithium, especially on the differentiation and functions of dendritic cells (DC). Our data show that treatment with VPA or lithium effectively alleviated the severity of collagen-induced arthritis triggered by LPS in mice. Both agents reduced the serum level of IL-6 and IL-10 after LPS challenge in mice. VPA and lithium both induce significant down-regulation of group I CD1 expression and secretion of IL-6 during differentiation of human monocyte-derived immature DC, while they differ in the induction of CD83 and CD86 expression, secretion of IL-8, IL-10, and TNF-α. Upon stimulation of immature DC with LPS, VPA and lithium both reduced the secretion of IL-6 and TNF-α. However, only lithium significantly increased the production of IL-10, while VPA increased the production of IL-8 but substantially reduce the secretion of IL-10 and IL-23. Treatment with VPA resulted in a reduced capacity of LPS-stimulated DC to promote the differentiation of T helper 17 cells that are critical in the promotion of inflammatory responses. Taken together, our results suggest that VPA and lithium may differentially modulate inflammation through regulating the capacity of DC to mediate distinct T cell responses, and they may provide a complementary immunomodulatory effects for the treatment of inflammation-related diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:30:50.294937-05:
      DOI: 10.1002/jcp.25604
  • Human Brain Malignant Glioma (BMG-1) 3D Aggregate Morphology and Screening
           for Cytotoxicity and Anti-Proliferative Effects
    • Authors: Maddaly Ravi; Aarthi Ramesh, Aishwarya Pattabhi
      Abstract: Two interesting aspects of cell lines grown in 3D conditions are their distinct morphology and production of extracellular matrix (ECM). Also, it is known that 3D aggregates have different susceptibilities to damage-inducing agents compared to their 2D monolayer counterparts. We describe the effect of ECM on 3D aggregate morphology, the effect of cisplatin, bleomycin and UV on the 3D aggregates and 2D monolayers of the BMG-1 cell line. We also present a rapid method for analyzing cytotoxicity and anti-proliferative effects of 3D aggregates in 96-well plates. We utilized a single-step protocol using the dye resazurin.BMG-1 cells formed floating aggregates on 1% agarose hydrogels. The extent of ECM formed by them was dependent on number of cells seeded irrespective of the seeding density, which in turn directed the 3D aggregates compactness. The 3D aggregates were less susceptible to cisplatin and UV-induced cytotoxicity compared to 2D counterparts. The IC 50 value of cisplatin was elevated at 210 µg/ml for the aggregates compared to 170µg/ml for the monolayers. Exposure to UV for 10, 20 and 30 minutes gave inhibition values of 2.98%, 8%, 22.99% and 31.8% for the aggregates as compared to 3.06%, 7.5%, 39.4% and 46.7% for the monolayers. While bleomycin-induced effects were unapparent when analyzed by vital staining for the doses used, the rapid, single-step method in 96-well plates was able to provide a dose-response for cytotoxicity and anti-proliferative effects. Also, comparative analysis of results obtained from vital staining and the single-step method demonstrates the reliability of the assay described. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:26:20.762476-05:
      DOI: 10.1002/jcp.25603
  • Table of Contents, Editor's Choice, Highlights
    • First page: 679
      PubDate: 2016-12-19T15:47:59.781016-05:
      DOI: 10.1002/jcp.25707
  • Integrated Transcriptome Map Highlights Structural and Functional Aspects
           of the Normal Human Heart
    • Authors: Maria Caracausi; Allison Piovesan, Lorenza Vitale, Maria Chiara Pelleri
      First page: 759
      Abstract: A systematic meta-analysis of the available gene expression profiling datasets for the whole normal human heart generated a quantitative transcriptome reference map of this organ. Transcriptome Mapper (TRAM) software integrated 32 gene expression profile datasets from different sources returning a reference value of expression for each of the 43,360 known, mapped transcripts assayed by any of the experimental platforms used in this regard. Main findings include the visualization at the gene and chromosomal levels of the classical description of the basic histology and physiology of the heart, the identification of suitable housekeeping reference genes, the analysis of stoichiometry of gene products, and the focusing on chromosome 21 genes, which are present in one excess copy in Down syndrome subjects, presenting cardiovascular defects in 30–40% of cases. Independent in vitro validation showed an excellent correlation coefficient (r = 0.98) with the in silico data. Remarkably, heart/non-cardiac tissue expression ratio may also be used to anticipate that effects of mutations will most probably affect or not the heart. The quantitative reference global portrait of gene expression in the whole normal human heart illustrates the structural and functional aspects of the whole organ and is a general model to understand the mechanisms underlying heart pathophysiology. J. Cell. Physiol. 232: 759–770, 2017. © 2016 Wiley Periodicals, Inc.A systematic meta-analysis of the available gene expression profiling datasets for the whole normal human heart retrieved 32 microarray experiments performed on the whole organ. These data were integrated through the Transcriptome Mapper (TRAM) software to obtain a reference typical value of expression for each of the 43,360 known, mapped transcripts. In vitro validation through real-time RT-PCR showed an excellent correlation coefficient (r = 0.98) with the in silico data.
      PubDate: 2016-07-21T11:10:38.808796-05:
      DOI: 10.1002/jcp.25471
  • Transcriptomic Analyses of Adipocyte Differentiation From Human
           Mesenchymal Stromal-Cells (MSC)
    • Authors: Antonio Casado-Díaz; Jaouad Anter, Sören Müller, Peter Winter, José Manuel Quesada-Gómez, Gabriel Dorado
      First page: 771
      Abstract: Adipogenesis is a physiological process required for fat-tissue development, mainly involved in regulating the organism energetic-state. Abnormal distribution-changes and dysfunctions in such tissue are associated to different pathologies. Adipocytes are generated from progenitor cells, via a complex differentiating process not yet well understood. Therefore, we investigated differential mRNA and miRNA expression patterns of human mesenchymal stromal-cells (MSC) induced and not induced to differentiate into adipocytes by next (second)-generation sequencing. A total of 2,866 differentially expressed genes (101 encoding miRNA) were identified, with 705 (46 encoding miRNA) being upregulated in adipogenesis. They were related to different pathways, including PPARG, lipid, carbohydrate and energy metabolism, redox, membrane-organelle biosynthesis, and endocrine system. Downregulated genes were related to extracellular matrix and cell migration, proliferation, and differentiation. Analyses of mRNA-miRNA interaction showed that repressed miRNA-encoding genes can act downregulating PPARG-related genes; mostly the PPARG activator (PPARGC1A). Induced miRNA-encoding genes regulate downregulated genes related to TGFB1. These results shed new light to understand adipose-tissue differentiation and physiology, increasing our knowledge about pathologies like obesity, type-2 diabetes and osteoporosis. J. Cell. Physiol. 232: 771–784, 2017. © 2016 Wiley Periodicals, Inc.Adipogenesis transcriptomics was studied in mesenchymal stem-cells (MSC) and 659 mRNA and 46 miRNA were upregulated in adipocyte differentiation. Metabolic, redox, and endocrinology process were upregulated in adipogenesis and Migration, proliferation, and differentiation to others cell types were downregulated. miRNA that can interact with PPARG and TGFB1 pathways were both downregulated and upregulated.
      PubDate: 2016-07-12T10:43:02.696974-05:
      DOI: 10.1002/jcp.25472
  • Parathyroid Hormone-Related Protein Protects Osteoblastic Cells From
           Oxidative Stress by Activation of MKP1 Phosphatase
    • Authors: Juan A. Ardura; Sergio Portal-Núñez, Irantzu Castelbón-Calvo, Irene Martínez de Toda, Mónica De la Fuente, Pedro Esbrit
      First page: 785
      Abstract: Oxidative damage is an important contributor to the morphological and functional changes in osteoporotic bone. Aging increases the levels of reactive oxygen species (ROS) that cause oxidative stress and induce osteoblast apoptosis. ROS modify several signaling responses, including mitogen-activated protein kinase (MAPK) activation, related to cell survival. Both parathyroid hormone (PTH) and its bone counterpart, PTH-related protein (PTHrP), can regulate MAPK activation by modulating MAPK phosphatase-1 (MKP1). Thus, we hypothesized that PTHrP might protect osteoblasts from ROS-induced apoptosis by targeting MKP1. In osteoblastic MC3T3-E1 and MG-63 cells, H2O2 triggered p38, JNK, ERK and p66Shc phosphorylation, and cell apoptosis. Meanwhile, PTHrP (1-37) rapidly but transiently increased ERK and Akt phosphorylation without affecting p38, JNK, or p66Shc activation. H2O2-induced p38 and ERK phosphorylation and apoptosis were both decreased by pre-treatment with specific kinase inhibitors or PTHrP (1-37) in both osteoblastic cell types. These dephosphorylating and prosurvival actions of PTHrP (1-37) were prevented by a phosphatase inhibitor cocktail, the phosphatase MKP1 inhibitor sanguinarine or a MKP1 siRNA. PTHrP (1-37) promptly enhanced MKP1 protein and gene expression and MKP1-dependent catalase activity in osteoblastic cells. Furthermore, exposure to PTHrP (1-37) adsorbed in an implanted hydroxyapatite-based ceramic into a tibial defect in aging rats increased MKP1 and catalase gene expression in the healing bone area. Our findings demonstrate that PTHrP counteracts the pro-apoptotic actions of ROS by a mechanism dependent on MKP1-induced dephosphorylation of MAPKs in osteoblasts. J. Cell. Physiol. 232: 785–796, 2017. © 2016 Wiley Periodicals, Inc.PTHrP counteracts the pro-apoptotic actions of ROS by a mechanism dependent on MKP1 dephosphorylation of MAP kinases and decreasing reactive oxygen species accumulation.
      PubDate: 2016-07-12T10:32:04.676822-05:
      DOI: 10.1002/jcp.25473
  • Evidence for the Role of BAG3 in Mitochondrial Quality Control in
    • Authors: Farzaneh G. Tahrir; Tijana Knezevic, Manish K. Gupta, Jennifer Gordon, Joseph Y. Cheung, Arthur M. Feldman, Kamel Khalili
      First page: 797
      Abstract: Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co-chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates clearance of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. J. Cell. Physiol. 232: 797–805, 2017. © 2016 Wiley Periodicals, Inc.BAG3 is critical for homeostasis of skeletal and cardiac tissues. Here we demonstrate that knockdown of BAG3 expression increased the level of Parkin, a key regulator of autophagy in neonatal rat ventricular cardiomyocytes. Furthermore, a decrease in the level of BAG3 promotes translocation of Parkin to the depolarized mitochondria and its degradation. BAG3 suppression significantly decreased the clearance of Tom20, an essential import receptor for mitochondria protein, after treatment with electron uncoupler, suggesting that mitophagy is impaired in the absence of BAG3. These observations ascribe a critical role for BAG3 in the maintenance of mitochondrial function under stress conditions.
      PubDate: 2016-07-19T09:00:53.700909-05:
      DOI: 10.1002/jcp.25476
  • Atrogin-1 Increases Smooth Muscle Contractility Through Myocardin
    • Authors: Pavneet Singh; Dong Li, Yu Gui, Xi-Long Zheng
      First page: 806
      Abstract: Atrogin-1, an E3 ligase present in skeletal, cardiac and smooth muscle, down-regulates myocardin protein during skeletal muscle differentiation. Myocardin, the master regulator of smooth muscle cell (SMC) differentiation, induces expression of smooth muscle marker genes through its association with serum response factor (SRF), which binds to the CArG box in the promoter. Myocardin undergoes ubiquitylation and proteasomal degradation. Evidence suggests that proteasomal degradation of myocardin is critical for myocardin to exert its transcriptional activity, but there is no report about the E3 ligase responsible for myocardin ubiquitylation and subsequent transactivation. Here, we showed that overexpression of atrogin-1 increased contractility of cultured SMCs and mouse aortic tissues in organ culture. Overexpression of dominant-negative myocardin attenuated the increase in SMC contractility induced by atrogin-1. Atrogin-1 overexpression increased expression of the SM contractile markers while downregulated expression of myocardin protein but not mRNA. Atrogin-1 also ubiquitylated myocardin for proteasomal degradation in vascular SMCs. Deletion studies showed that atrogin-1 directly interacted with myocardin through its amino acids 284–345. Immunostaining studies showed nuclear localization of atrogin-1, myocardin, and the Rpt6 subunit of the 26S proteasome. Atrogin-1 overexpression not only resulted in degradation of myocardin but also increased recruitment of RNA Polymerase II onto the promoters of myocardin target genes. In summary, our results have revealed the roles for atrogin-1 in the regulation of smooth muscle contractility through enhancement of myocardin ubiquitylation/degradation and its transcriptional activity. J. Cell. Physiol. 232: 806–817, 2017. © 2016 Wiley Periodicals, Inc.Atrogin-1 is an E3 ligase present in skeletal, cardiac and smooth muscle. Our study has shown that atrogin-1 down-regulates myocardin protein in human vascular smooth muscle cells, but increases transcriptional activity of myocardin on its target genes.
      PubDate: 2016-07-21T11:20:36.573109-05:
      DOI: 10.1002/jcp.25485
  • Vimentin as a Marker of Early Differentiating, Highly Motile Corneal
           Epithelial Cells
    • Authors: Federico Castro-Muñozledo; Diana G. Meza-Aguilar, Rocío Domínguez-Castillo, Veremundo Hernández-Zequinely, Erika Sánchez-Guzmán
      First page: 818
      Abstract: Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial-Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal-like derivatives. Based on previous results showing that epithelial cells co-express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co-expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+/Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4–5 layered epithelium, while there was a concomitant increase of both Pax-6 expression and Krt+/Vim− cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co-linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+/Krt+/ΔNp63α+/Pax-6low/α6 integrin+ phenotype. J. Cell. Physiol. 232: 818–830, 2017. © 2016 Wiley Periodicals, Inc.During growth and wound healing, highly motile epithelial cells require the expression of a specific cytoskeletal network to sustain organelle translocation during cell movement. Such requirements might be fulfilled by the copolymerization of Vim and Krt at the migratory edges of cells, and its interaction with Extracellular Matrix, a task achieved by relocation of alpha6beta4 integrin into focal adhesion plaques, and its association with plectin as an integrator molecule.
      PubDate: 2016-07-20T12:01:04.868486-05:
      DOI: 10.1002/jcp.25487
  • Tissue Inhibitor of Metalloproteinase 1 Influences Vascular Adaptations to
           Chronic Alterations in Blood Flow
    • Authors: Erin R. Mandel; Cassandra Uchida, Emmanuel Nwadozi, Armin Makki, Tara L. Haas
      First page: 831
      Abstract: Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). We hypothesized that the loss of TIMP1 would enhance both ischemia and flow-induced vascular remodeling by increasing MMP activity. TIMP1 deficient (Timp1−/−) and wild-type (WT) C57BL/6 mice underwent unilateral femoral artery (FA) ligation or were treated with prazosin, an alpha-1 adrenergic receptor antagonist, in order to investigate vascular remodeling to altered flow. Under basal conditions, Timp1−/− mice had reduced microvascular content as compared to WT mice. Furthermore, vascular remodeling was impaired in Timp1−/− mice. Timp1−/− mice displayed reduced blood flow recovery in response to FA ligation and no arteriogenic response to prazosin treatment. Timp1−/− mice failed to undergo angiogenesis in response to ischemia or prazosin, despite maintaining the capacity to increase VEGF-A and eNOS mRNA. Vascular permeability was increased in muscles of Timp1−/− mice in response to both prazosin treatment and FA ligation, but this was not accompanied by greater MMP activity. This study highlights a previously undescribed integral role for TIMP1 in both vascular network maturation and adaptations to ischemia or alterations in flow. J. Cell. Physiol. 232: 831–841, 2017. © 2016 Wiley Periodicals, Inc.Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). This study highlights a previously undescribed integral role for TIMP1 in vascular network maturation and in adaptations to altered blood flow.
      PubDate: 2016-07-28T05:45:36.851462-05:
      DOI: 10.1002/jcp.25491
  • Extracellular Proteasomes Are Deficient in 19S Subunits as Revealed by
           iTRAQ Quantitative Proteomics
    • Authors: Anna S. Tsimokha; Julia J. Zaykova, Andrew Bottrill, Nikolai A. Barlev
      First page: 842
      Abstract: Proteasome-mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well-documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ-based quantitative proteomics (iTRAQ LC/MS-MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. Sixty-two of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP-PIPs were associated with protein biosynthesis and, unlike CP-PIPs, were under represented by chaperon/ATP-binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. J. Cell. Physiol. 232: 842–851, 2017. © 2016 Wiley Periodicals, Inc.To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ-based quantitative proteomics (iTRAQ LC/MS-MS). Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes, which implies that the two species of proteasomes may have different biological functions.
      PubDate: 2016-08-16T02:05:30.033441-05:
      DOI: 10.1002/jcp.25492
  • Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb
    • Authors: Ahn R. Lee; Wayne Hung, Ning Xie, Liangliang Liu, Leye He, Xuesen Dong
      First page: 852
      Abstract: The non-POU-domain-containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site-directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co-immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein–protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non-specific binding affinity to anti-phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co-repression and RNA splicing in gene context-dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co-regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. J. Cell. Physiol. 232: 852–861, 2017. © 2016 Wiley Periodicals, Inc.P54nrb (aka NONO) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although Tyr phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. Our studies demonstrate that the Tyr residues of p54nrb are not phosphorylated, but nevertheless significantly regulate p54nrb function by means of potential temporal and spatial regulation of p54nrb, resulting in altered interaction of p54nrb with various co-regulatory splicing or transcriptional proteins.
      PubDate: 2016-07-27T10:45:26.08248-05:0
      DOI: 10.1002/jcp.25493
  • Coumestrol Inhibits Proliferation and Migration of Prostate Cancer Cells
           by Regulating AKT, ERK1/2, and JNK MAPK Cell Signaling Cascades
    • Authors: Whasun Lim; Muhah Jeong, Fuller W. Bazer, Gwonhwa Song
      First page: 862
      Abstract: Coumestrol is the one of the major phytoestrogens which is abundant in soybeans, legumes, brussel sprouts, and spinach. The beneficial effects of coumestrol are well known in various biological processes including; neuroprotective effects on the nervous system, function of the female reproductive system, anti-bacterial properties, and anti-cancer effects. Although the anti-tumor activity of coumestrol has been demonstrated for ovarian, breast, lung, and cervical cancers, little is known of its effects on prostate cancer. Therefore, in the present study, we investigated the chemotherapeutic effects of coumestrol on two prostate cancer cell lines, PC3 and LNCaP. Our results showed that coumestrol decreased proliferation and migration and induced apoptosis in both PC3 and LNCaP cells. Moreover, effects of coumestrol on cell signaling pathways were investigated and it increased phosphorylation of ERK1/2, JNK, P90RSK, and P53 proteins in a dose- and time-dependent manner whereas phosphorylation of AKT was reduced by coumestrol under the same conditions for culture of PC3 and LNCaP cells. In addition, mitochondrial dysfunction was induced by coumestrol as evidenced by a significant loss of mitochondrial membrane potential. Furthermore, cleavage of caspase-3 and caspase-9, the apoptotic proteins associated with mitochondria, also changed in response to coumestrol. Coumestrol also caused mitochondrial dysfunction resulting in an increase in ROS production in PC3 and LNCaP cells. These results suggest that coumestrol can inhibit progression of prostate cancer and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signaling pathways. J. Cell. Physiol. 232: 862–871, 2017. © 2016 Wiley Periodicals, Inc.Our results indicated that coumestrol can inhibit progression of prostate cancer cells by affecting their viability and mitochondrial functions, and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signal transduction cascades.
      PubDate: 2016-07-28T05:51:13.440339-05:
      DOI: 10.1002/jcp.25494
  • Late Endosomal Recycling of Open MHC-I Conformers
    • Authors: Hana Mahmutefendić; Gordana Blagojević Zagorac, Kristina Grabušić, Ljerka Karleuša, Senka Maćešić, Frank Momburg, Pero Lučin
      First page: 872
      Abstract: With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty Ld (eLd) molecules, an open form of murine MHC-I allele, in fibroblast-like cells. Pulse labeling of cell surface eLd with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC-I conformers. Kinetic modeling, using in-house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eLd demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eLd distributed into pre-degradative multivesicular bodies (MVBs), these LE subsets were not a source for eLd recycling. The LE recycling of eLd did not require Rab7 membrane domains, as demonstrated by Rab7-silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eLd in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. J. Cell. Physiol. 232: 872–887, 2017. © 2016 Wiley Periodicals, Inc.Study of endosomal trafficking of open MHC-I conformers reveals that endosomal recycling may occur from late endosomes, suggesting that the late endosomal system should not be simply considered as a feeder for loading of the degradative tract of the cell.
      PubDate: 2016-07-27T10:50:44.218757-05:
      DOI: 10.1002/jcp.25495
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