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Journal Cover   Journal of Cellular Physiology
  [SJR: 1.948]   [H-I: 125]   [3 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  [1602 journals]
  • Grp78 is Critical for Amelogenin‐Induced Cell Migration in a
           Multipotent Clonal Human Periodontal Ligament Cell Line
    • Authors: Kyosuke Toyoda; Takao Fukuda, Terukazu Sanui, Urara Tanaka, Kensuke Yamamichi, Ryo Atomura, Hidefumi Maeda, Atsushi Tomokiyo, Takaharu Taketomi, Takeshi Uchiumi, Fusanori Nishimura
      Abstract: Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose‐regulated protein 78 (Grp78) as a new amelogenin‐binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1‐17, which possesses the most typical characteristics of PDLSCs. Confocal co‐localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1‐17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration‐associated genes in 1‐17 cells. Moreover, Grp78 overexpression enhanced rM180‐induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin‐induced migration in 1‐17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin‐induced periodontal tissue regeneration. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-03T17:49:10.870409-05:
      DOI: 10.1002/jcp.25087
  • Alterations in Skeletal Muscle Oxidative Phenotype in Mice Exposed to
           Three Weeks of Normobaric Hypoxia
    • Authors: Ilse G.M. Slot; Annemie M.W.J. Schols, Chiel C. de Theije, Frank J.M. Snepvangers, Harry R. Gosker
      Abstract: Skeletal muscle of patients with chronic respiratory failure is prone to loss of muscle mass and oxidative phenotype. Tissue hypoxia has been associated with cachexia and emphysema in humans. Experimental research on the role of hypoxia in loss of muscle oxidative phenotype however has yielded inconsistent results. Animal studies are frequently performed in young animals, which may hinder translation to generally older aged patients. Therefore in this study we tested the hypothesis that hypoxia induces loss of skeletal muscle oxidative phenotype in a model of aged (52 weeks) mice exposed to three weeks of hypoxia. Additional groups of young (4 weeks) and adult (12 weeks) mice were included to examine age effects. To verify hypoxia‐induced cachexia, fat pad and muscle weights as well as muscle fiber cross‐sectional areas were determined. Muscle oxidative phenotype was assessed by expression and activity of markers of mitochondrial metabolism and fiber‐type distribution. A profound loss of muscle and fat was indeed accompanied by a slightly lower expression of markers of muscle oxidative capacity in the aged hypoxic mice. In contrast, hypoxia‐associated changes of fiber‐type composition were more prominent in the young mice. The differential response of the muscle of young, adult, and aged mice to hypoxia suggests that age matters and that the aged mouse is a better model for translation of findings to elderly patients with chronic respiratory disease. Furthermore, the findings warrant further mechanistic research into putative accelerating effects of hypoxia‐induced loss of oxidative phenotype on the cachexia process in chronic respiratory disease. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:54:30.354054-05:
      DOI: 10.1002/jcp.25083
  • TRPV3 Channel Negatively Regulates Cell Cycle Progression and Safeguards
           the Pluripotency of Embryonic Stem Cells
    • Authors: Iek Chi Lo; Hing Chung Chan, Zenghua Qi, Kwun Lam Ng, Chun So, Suk Ying Tsang
      Abstract: Embryonic stem cells (ESCs) have tremendous potential for research and future therapeutic purposes. However, the calcium handling mechanism in ESCs is not fully elucidated. Aims of this study are 1) to investigate if transient receptor potential vanilloid‐3 (TRPV3) channels are present in mouse ESCs (mESCs) and their subcellular localization; 2) to investigate the role of TRPV3 in maintaining the characteristics of mESCs. Western blot and immunocytochemistry showed that TRPV3 was present at the endoplasmic reticulum (ER) of mESCs. Calcium imaging showed that, in the absence of extracellular calcium, TRPV3 activators camphor and 6‐tert‐butyl‐m‐cresol increased the cytosolic calcium. However, depleting the ER store in advance of activator addition abolished the calcium increase, suggesting that TRPV3 released calcium from the ER. To dissect the functional role of TRPV3, TRPV3 was activated and mESC proliferation was measured by trypan blue exclusion and MTT assays. The results showed that TRPV3 activation led to a decrease in mESC proliferation. Cell cycle analysis revealed that TRPV3 activation increased the percentage of cells in G2/M phase; consistently, Western blot also revealed a concomitant increase in the expression of inactive form of cyclin‐dependent kinase 1, suggesting that TRPV3 activation arrested mESCs at G2/M phase. TRPV3 activation did not alter the expression of pluripotency markers Oct‐4, Klf4 and c‐Myc, suggesting that the pluripotency was preserved. Our study is the first study to show the presence of TRPV3 at ER. Our study also reveals the novel role of TRPV3 in controlling the cell cycle and preserving the pluripotency of ESCs. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:54:08.77255-05:0
      DOI: 10.1002/jcp.25086
  • Potential Role of Glycogen Synthase Kinase‐3β in Regulation of
           Myocardin Activity in Human Vascular Smooth Muscle Cells
    • Authors: Yi‐xia Zhou; Zhan Shi, Pavneet Singh, Hao Yin, Yan‐ni Yu, Long Li, Michael P. Walsh, Yu Gui, Xi‐Long Zheng
      Abstract: Glycogen synthase kinase (GSK)‐3β, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK‐3β results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co‐activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study therefore aimed to examine whether and how inhibition of GSK‐3β regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK‐3β inhibitors AR‐A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22) and ACTA2 (SM α‐actin). In human SMCs overexpressing myocardin through the T‐REx system, treatment with either GSK‐3β inhibitor also inhibited the expression of CNN1, TAGLN1 and ACTA2. These effects of GSK‐3β inhibitors were mimicked by transfection with GSK‐3β siRNA. Notably, both AR‐A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR‐A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant‐negative GSK‐3β mutant in myocardin‐overexpressing SMCs reduced the expression of calponin, SM22 and SM α‐actin. As expected, overexpression of constitutively‐active or wild‐type GSK‐3β in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK‐3β reduces myocardin transcriptional activity, suggesting a role for GSK‐3β in myocardin transcriptional activity and smooth muscle differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:53:47.238546-05:
      DOI: 10.1002/jcp.25084
  • Agents Which Inhibit NF‐κB Signaling Block Spontaneous
           Contractile Activity and Negatively Influence Survival of Developing
    • Authors: C. George Carlson; Lauren Stein, Elizabeth Dole, Ross M. Potter, David Bayless
      Abstract: Inhibiting the NF‐κB signaling pathway provides morphological and functional benefits for the mdx mouse, a model for Duchenne muscular dystrophy characterized by chronic elevations in the nuclear expression of p65, the transactivating component of the NF‐κB complex. The purpose of this study was to examine p65 expression in nondystrophic and mdx myotubes using confocal immunofluorescence, and determine whether inhibitors of the NF‐κB pathway alter myotube development. Primary cultures of nondystrophic and mdx myotubes had identical levels of nuclear and cytosolic p65 expression and exhibited equivalent responses to TNF‐α, thus excluding the hypothesis that the lack of dystrophin is sufficient to induce increases in NF‐κB signaling. The NF‐κB inhibitors pyrrolidine dithiocarbamate (PDTC) and sulfasalazine decreased spontaneous contractile activity and reduced myotube viability in a dose‐ and time‐dependent manner. Similarly, a vivo‐morpholino designed to block translation of murine p65 (m‐p65tb‐vivomorph1) rapidly abolished spontaneous contractile activity, reduced p65 expression measured by confocal immunofluorescence, and induced cell death in primary cultures of nondystrophic and mdx myotubes. Similar effects on p65 immunofluorescence and cell viability were observed following m‐p65tb‐vivomorph1 exposure to spontaneously inactive C2C12 myotubes, while exposure to a control scrambled vivo morpholino had no effect. These results indicate a direct role of the NF‐κB pathway in myotube development and identify a potential therapeutic limitation to the use of NF‐κB inhibitors in treating Duchenne and related muscular dystrophies. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:53:25.163707-05:
      DOI: 10.1002/jcp.25085
  • Pathophysiology of Corneal Dystrophies: From Cellular Genetic Alteration
           to Clinical Findings
    • Authors: Marta Sacchetti; Ilaria Macchi, Alessandro Tiezzi, Maurizio La Cava, Giacomina Massaro‐Giordano, Alessandro Lambiase
      Abstract: Corneal dystrophies are a heterogeneous group of bilateral, inherited, rare diseases characterized by slowly progressive corneal opacities, that lead to visual impairment. Most of them have an autosomal dominant pattern of inheritance with variable expressivity, but new mutations have been described. Many corneal dystrophies have been genetically characterized and the specific gene mutations identified, such as for the epithelial–stromal TGFBI dystrophies. Current classification systems identified four main groups of corneal dystrophies based on clinical, histologic and genetic information. Diagnosis is performed during a routine ophthalmic examination that shows typical cellular abnormalities of the corneal epithelium, stroma or endothelium. Disease progression should be carefully monitored to decide the proper clinical management. The treatment of corneal dystrophies is variable, depending on symptoms, clinical course, severity and type of dystrophy. Management aimed to reduce symptoms and to improve vision, includes different surgical approaches. Novel cellular and genetic therapeutic approaches are under evaluation. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-23T18:46:57.198342-05:
      DOI: 10.1002/jcp.25082
  • Is the Reg3α (HIP/PAP) Protein Really an Obesogenic Factor?
    • Authors: Patrick Gonzalez; Nicolas Moniaux, Christian Bréchot, Jamila Faivre
      Abstract: We would like to point out an internal inconsistency in the data presented by V. Secq et al. in the article entitled “PAP/HIP protein is an obesogenic factor” published in the February 2014 issue of “Journal of Cellular Physiology”, which cast serious doubt on the validity of the statement serving as the title of the article. We, and others, including J. Iovanna and his collaborators, have long shown that the C‐type lectin PAP/HIP (also known as HIP/PAP or Reg3α) has anti‐inflammatory and antioxidant activities in a number of cell types, among which pancreatic cells, hepatocytes and neurons (Gironella et al., 2005; Closa et al., 2007; Moniaux et al., 2011; Kapur et al., 2012; Haldipur et al., 2014) and that, therefore, it might help cure, or prevent, inflammatory and metabolic diseases. Given its envisaged clinical uses, the question of whether PAP/HIP significantly modifies metabolism toward weight increase is essential and must be subjected to rigorous scrutiny. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-19T17:57:14.333248-05:
      DOI: 10.1002/jcp.25046
  • Critical Role of AMPK/FoxO3A Axis in Globular Adiponectin‐Induced
           Cell Cycle Arrest and Apoptosis in Cancer Cells
    • Authors: Anup Shrestha; Saroj Nepal, Mi Jin Kim, Jae Hoon Chang, Sang‐Hyun Kim, Gil‐Saeng Jeong, Chul‐Ho Jeong, Gyu Hwan Park, Sunghee Jung, Jaecheong Lim, Eunha Cho, Soyoung Lee, Pil‐Hoon Park
      Abstract: Adiponectin predominantly secreted from adipose tissue has exhibited potent anti‐proliferative properties in cancer cells via modulating cell cycle and apoptosis. FoxO3A, a Forkhead box O member of the transcription factor, plays a critical role in modulating expression of genes involved in cell death and/or survival. In this study, we investigated the role of FoxO3A signaling in anti‐cancer activities of adiponectin. Herein, we have shown that treatment with globular adiponectin (gAcrp) increases p27 but decreases cyclinD1 expression in human hepatoma (HepG2) and breast (MCF‐7) cancer cells. Gene ablation of FoxO3A prevented gAcrp‐induced increase in p27 and decreased in cyclin D1 expression, and further ameliorated cell cycle arrest by gAcrp, indicating a critical role of FoxO3A in gAcrp‐induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase‐3/7 activation and increased Fas ligand (FasL) expression in both HepG2 and MCF‐7 cells. Transfection with FoxO3A siRNA inhibited gAcrp‐induced caspase‐3/7 activation and FasL expression, suggesting that FoxO3A signaling also plays an important role in gAcrp‐induced apoptosis of cancer cells. We also found that gene silencing of AMPK prevented gAcrp‐induced nuclear translocation of FoxO3A in HepG2 and MCF‐7 cells. In addition, suppression of AMPK also blocked gAcrp‐induced cell cycle arrest and further attenuated gAcrp‐induced caspase‐3/7 activation, indicating that AMPK signaling plays a pivotal role in both gAcrp‐induced cell cycle arrest and apoptosis via acting as an upstream signaling of FoxO3A. Taken together, our findings demonstrated that AMPK/FoxO3A axis plays a cardinal role in anti‐proliferative effect of adiponectin in cancer cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-18T18:54:36.875656-05:
      DOI: 10.1002/jcp.25080
  • Platelet‐Derived CCL5 Regulates CXC Chemokine Formation and
           Neutrophil Recruitment in Acute Experimental Colitis
    • Authors: Changhui Yu; Songen Zhang, Yongzhi Wang, Su Zhang, Lingtao Luo, Henrik Thorlacius
      Abstract: Accumulating data suggest that platelets not only regulate thrombosis and haemostasis but also inflammatory processes. Platelets contain numerous potent pro‐inflammatory compounds, including the chemokines CCL5 and CXCL4 although their role in acute colitis remains elusive. The aim of this study was to examine the role of platelets and platelet‐derived chemokines in acute colitis. Acute colitis was induced in female Balb/c mice by administration of 5% dextran sodium sulphate (DSS) for five days. Animals received a platelet‐depleting, anti‐CCL5, anti‐CXCL4 or a control antibody prior to DSS challenge. Colonic tissue was collected for quantification of myeloperoxidase (MPO) activity, CXCL5, CXCL2, interleukin‐6 (IL‐6) and CCL5 levels as well as morphological analyses. Platelet depletion reduced tissue damage and clinical disease activity index in DSS‐exposed animals. Platelet depletion not only reduced levels of CXCL2 and CXCL5 but also levels of CCL5 in the inflamed colon. Immunoneutralization of CCL5 but not CXCL4 reduced tissue damage, CXC chemokine expression and neutrophil recruitment in DSS‐treated animals. These findings show that platelets play a key role in acute colitis by regulating CXC chemokine generation, neutrophil infiltration and tissue damage in the colon. Moreover, our results suggest that platelet‐derived CCL5 is an important link between platelet activation and neutrophil recruitment in acute colitis. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-18T18:54:10.466808-05:
      DOI: 10.1002/jcp.25081
  • Genetic and Functional Analysis of Polymorphisms in the Human Dopamine
           Receptor and Transporter Genes in Small Cell Lung Cancer
    • Authors: Emanuela Cherubini; Arianna Di Napoli, Alessia Noto, Giorgia Amira Osman, Maria Cristina Esposito, Salvatore Mariotta, Rossella Sellitri, Luigi Ruco, Giuseppe Cardillo, Gennaro Ciliberto, Rita Mancini, Alberto Ricci
      Abstract: The regulatory role of dopamine (DA) in endocrine, cardiovascular and renal functions has been extensively studied and used for clinical purposes. More recently DA has been indicated as a regulatory molecule for immune cells and malignant cell proliferation. We assessed the expression and the functional role DA, DA receptors and transporters in primary small cell lung cancer (SCLC). By HPLC DA plasma levels were more elevated in SCLC patients in comparison with NSCLC patients and healthy controls. SCLC cell expressed DA D1‐ and D2‐like receptors and membrane and vesicular transporters at protein and mRNA levels. We also investigated the effects of independent D1‐ or D2‐like receptor stimulation on SCLC cell cultures. DA D1 receptor agonist SKF38393 induced the increase of cAMP levels and DARPP‐32 protein expression without affecting SCLC growth rate. Cell treatment with the DA D1 receptor antagonist SCH23390 inhibited SKF38393 effects. In contrast, the DA D2 receptor agonist quinpirole (10μM) counteracted, in a dose and time dependent way, SCLC cell proliferation, it did not affect cAMP levels and decreased phosphorylated AKT that was induced by DA D2 receptor antagonist sulpiride. However, in only one SCLC line, stimulation of DA D2 receptor failed to inhibit cell proliferation in vitro. This effect was associated to the existence of rs6275 and rs6277 polymorphisms in the D2 gene. These results gave more insight into DA control of lung cancer cell behavior and suggested the existence of different SCLC phenotypes. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-16T17:46:14.085232-05:
      DOI: 10.1002/jcp.25079
  • Aquaporin‐1 Plays Important Role in Proliferation by Affecting Cell
           Cycle Progression
    • Authors: Ana Galán‐Cobo; Reposo Ramírez‐Lorca, Juan José Toledo‐Aral, Miriam Echevarría
      Abstract: Aquaporin‐1 (AQP1) has been associated with tumor development. Here, we investigated how AQP1 may affect cell proliferation. The proliferative rate of adult carotid body (CB) cells, known to proliferate under chronic hypoxia, was analyzed in wild‐type (AQP1 +/ +) and knock out (AQP1 ‐/‐) mice, maintained in normoxia or exposed to hypoxia while BrdU was administered. Fewer numbers of total BrdU+ and TH‐BrdU+ cells were observed in AQP1 ‐/‐ mice, indicating a role for AQP1 in CB proliferation. Then, by flow cytometry, cell cycle state and proliferation of cells overexpressing AQP1 were compared to those of wild‐type cells. In the AQP1‐overexpressing cells, we observed higher cell proliferation and percentages of cells in phases S and G2/M and fewer apoptotic cells after nocodazole treatment were detected by annexin V staining. Also in these cells, proteomic assays showed higher expression of cyclin D1 and E1 and microarray analysis revealed changes in many cell proliferation‐related molecules, including, Zeb 2, Jun, NF‐kβ, Cxcl9, Cxcl10, TNF, and the TNF receptor. Overall, our results indicate that the presence of AQP1 modifies the expression of key cell cycle proteins apparently related to increases in cell proliferation. This contributes to explaining the presence of AQP1 in many different tumors. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-16T17:43:07.293339-05:
      DOI: 10.1002/jcp.25078
  • Dexamethasone Downregulates SLC7A5 Expression and Promotes Cell Cycle
           Arrest, Autophagy and Apoptosis in BeWo Cells
    • Authors: Bin He; Nana Zhang, Ruqian Zhao
      Abstract: Synthetic glucocorticoids (GCs) such as dexamethasone (Dex) are widely given to pregnant women to induce maturation and improve viability of preterm infants. Despite the beneficial effects, synthetic GCs have adverse effects on placental growth and nutrient transport system. However, the molecular mechanisms involved in these events remain unknown. Here we use a human placental choriocarcinoma cell line (BeWo) as model to explore the pathway linking amino acids transport with cell viability under Dex challenge. BeWo cells treated with Dex (100 nM) for 24 h demonstrated G1/S cell cycle arrest together with enhanced autophagy and apoptosis. Concurrently, the amino acid carrier SLC7A5 was down‐regulated in association with impaired cellular amino acids uptake and inhibition of mammalian target of rapamycin (mTOR) signaling. Similar cellular responses were observed in BeWo cells treated with BCH, a classical System L inhibitor which inactivates SLC7A5. The glucocorticoid receptor (GR) antagonist RU486 was able to diminish Dex‐induced translocation of GR into nucleus and to abolish these effects. Furthermore, Dex treatment significantly promoted the binding of GR to the proximal promoter sequence of SLC7A5 gene. Taken together, our results show that Dex downregulates SLC7A5 expression via GR‐mediated transrepression. The impaired amino acids uptake leads to inhibition of mTOR signaling which in turn causes inhibited proliferation and enhanced autophagy and apoptosis in BeWo cells. These findings indicate that SLC7A5 mediates the effect of Dex on cell viability, thus providing a novel molecular target for the prevention and treatment of Dex‐induced cell cycle arrest and apoptosis in placental cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:47:13.684676-05:
      DOI: 10.1002/jcp.25076
  • G‐CSF Contributes at the Healing of Tunica Media of
           Arteriotomy‐Injured Rat Carotids by Promoting Differentiation of
           Vascular Smooth Muscle Cells
    • Authors: Barbara Rinaldi; Mauro Finicelli, Maria Donniacuo, Giovanni Di Bernardo, Giulia Gritti, Stefania Del Gaudio, Amalia Forte, Gianfranco Peluso, Marilena Cipollaro, Francesco Rossi, Umberto Galderisi
      Abstract: Restenosis is a complex pathophysiological disease whose causative mechanisms are not fully understood. Previous studies allowed us to demonstrate the efficacy of bone marrow mesenchymal stromal cells (MSCs) transplantation in limiting the pathophysiological remodeling in a model of arteriotomy‐induced (re)stenosis. In the current research we studied the effectiveness of G‐CSF treatment on male rate rats that were subjected carotid arteriotomy in order to evaluate a potentially effective non‐invasive strategy that recapitulates the MSC‐mediated recovery of injured vessels. WKY male rats were subjected carotid arteriotomy and given a nine day treatment (3 days pre‐ to 6 days post‐arteriotomy) with G‐CSF or saline. Carotids were harvested 7 and 30 days following arteriotomy (early‐ and late‐phase, respectively). Although morphometrical analysis did not reveal differences in lumen narrowing between G‐CSF‐ and PBS‐carotids 30 days following arteriotomy, we detected a noticeable conservative effect of G‐CSF treatment on vascular wall morphology. Histological and molecular analysis revealed an increase in cellularity within the tunica media with a concomitant increase of the VSMCs differentiation markers both at early‐ and late‐phases of (re)stenotic response in G‐CSF‐treated carotids (Sm22‐alpha, Myocd and Smtn). These findings were accompanied by the downregulation of oxidative stress‐related genes in G‐CSF‐injured rats. The effect exerted by G‐CSF in our model of arteriotomy‐induced (re)stenosis seemed support the recovery of the architecture of the tunica media of injured vessels by: i) inducing VSMCs differentiation; and ii) limiting the oxidative‐stress response induced by arteriotomy. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:04:03.48673-05:0
      DOI: 10.1002/jcp.25074
  • Pathways Implicated in Tadalafil Amelioration of Duchenne Muscular
    • Authors: Valeria De Arcangelis; Georgios Strimpakos, Francesca Gabanella, Nicoletta Corbi, Siro Luvisetto, Armando Magrelli, Annalisa Onori, Claudio Passananti, Cinzia Pisani, Sophie Rome, Cinzia Severini, Fabio Naro, Elisabetta Mattei, Maria Grazia Di Certo, Lucia Monaco
      Abstract: Numerous therapeutic approaches for Duchenne and Becker Muscular Dystrophy (DMD and BMD), the most common X‐linked muscle degenerative disease, have been proposed. So far, the only one showing a clear beneficial effect is the use of corticosteroids. Recent evidence indicates an improvement of dystrophic cardiac and skeletal muscles in the presence of sustained cGMP levels secondary to a blocking of their degradation by phosphodiesterase 5 (PDE5). Due to these data, we performed a study to investigate the effect of the specific PDE5 inhibitor, tadalafil, on dystrophic skeletal muscle function. Chronic pharmacological treatment with tadalafil has been carried out in mdx mice. Behavioral and physiological tests, as well as histological and biochemical analyses, confirmed the efficacy of the therapy. We then performed a microarray‐based genomic analysis to assess the pattern of gene expression in muscle samples obtained from the different cohorts of animals treated with tadalafil. This scrutiny allowed us to identify several classes of modulated genes. Our results show that PDE5 inhibition can ameliorate dystrophy by acting at different levels. Tadalafil can lead to 1) increased lipid metabolism; 2) a switch towards slow oxidative fibers driven by the up‐regulation of PGC‐1α; 3) an increased protein synthesis efficiency; 4) a better actin network organization at Z‐disk. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:03:22.713185-05:
      DOI: 10.1002/jcp.25075
  • Tamoxifen Inhibits TGF‐β‐Mediated Activation of
           Myofibroblasts by Blocking Non‐Smad Signaling through ERK1/2
    • Authors: Jon M. Carthy; Anders Sundqvist, Angelos Heldin, Hans van Dam, Dimitris Kletsas, Carl‐Henrik Heldin, Aristidis Moustakas
      Abstract: TGF‐β is a multifunctional cytokine which stimulates the differentiation of fibroblasts into myofibroblasts. Myofibroblasts are critical for normal wound healing, but also accumulate pathologically in a number of chronic inflammatory conditions where they are key contributors to aberrant tissue remodeling and fibrosis, and in cancer stroma. In the current study, we identified a role for tamoxifen as a potent inhibitor of the TGF‐β‐mediated activation of primary human skin and breast fibroblasts. Our data indicate that tamoxifen does not interfere with canonical Smad signaling downstream of TGF‐β but rather blocks non‐Smad signaling through ERK1/2 MAP‐kinase and the AP‐1 transcription factor FRA2. We further demonstrate by siRNA‐mediated knockdown that FRA2 is critical for the induced expression of myogenic proteins in response to TGF‐β. Functionally, TGF‐β‐stimulated fibroblast‐mediated contraction of collagen gels was impaired in the presence of tamoxifen. Altogether, these data demonstrate that tamoxifen prevents myofibroblast differentiation and therefore may provide therapeutic benefits to patients suffering from chronic inflammatory conditions or cancer. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:02:58.161846-05:
      DOI: 10.1002/jcp.25049
  • Translating Regenerative Biomaterials into Clinical Practice
    • Authors: Edward T Stace; Stephanie G Dakin, Pierre‐Alexis Mouthuy, Prof Andrew J Carr
      Abstract: Globally health care spending is increasing unsustainably. This is especially true of the treatment of Musculoskeletal (MSK) disease where in the USA the MSK disease burden has doubled over the last 15 years. With an ageing and increasingly obese population, the surge in MSK related spending is only set to worsen. Despite increased funding, research and attention to this pressing health need, little progress has been made towards novel therapies. Tissue Engineering and Regenerative Medicine (TERM) strategies could provide the solutions required to mitigate this mounting burden. Biomaterial based treatments in particular present a promising field of potentially cost‐effective therapies. However, the translation of a scientific development to a successful treatment is fraught with difficulties. These barriers have so far limited translation of TERM science into clinical treatments. It is crucial for primary researchers to be aware of the barriers currently restricting the progression of science to treatments. Researchers need to act prospectively to ensure the clinical, financial and regulatory hurdles which seem so far removed from laboratory science do not stall or prevent the subsequent translation of their idea into a treatment. The aim of this review is to explore the development and translation of new treatments. Increasing the understanding of these complexities and barriers amongst primary researchers could enhance the efficiency of biomaterial translation. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-09T02:41:27.983892-05:
      DOI: 10.1002/jcp.25071
  • Core Binding Factorβ Plays a Criticalrole During Chondrocyte
    • Authors: Na‐Rae Park; Kyung‐Eun Lim, Min‐Su Han, Xiangguo Che, Clara YongjooPark, Jung‐Eun Kim, Ichiro Taniuchi, Suk‐Chul Bae, Je‐Yong Choi
      Abstract: Core binding factorβ (Cbfβ) is a partner protein of Runx family transcription factors with minimally characterized function in cartilage. Here we address the role of Cbfβ in cartilage by generating chondrocyte‐specific Cbfβ‐deficient mice (CbfbΔch/Δch) from Cbfb‐floxed mice crossed with mice expressing Cre from the Col2a1 promoter. Cbfb▵ch/▵ch mice diedsoon after birth and exhibiteddelayed endochondral bone formation,shorter appendicular skeleton length with increased proliferative chondrocytes,and nearly absent hypertrophic chondrocyte zones. Immunohistochemicaland quantitative real‐time PCR analyses showed that the proliferative chondrocytes increasedand the expression of markers of chondrocyte maturation at the growth plates, including Runx2, osterix, and osteopontin, were significantlydiminishedin Cbfb▵ch/▵ch mice compared to wild type mice. With regard to signaling pathways, both PTHrP‐Ihh and BMP signaling were compromised inCbfb▵ch/▵ch mice. Mechanistically, Cbfβdeficiency in chondrocytes caused a decrease of protein levels of Runxtranscription factors by accelerating polyubiquitination‐mediated proteosomal degradation in vitro. Indeed, Runx2 and Runx3, but notRunx1,decreased in Cbfb▵ch/▵ch mice. Collectively, these findings indicate that Cbfβplays a critical role for chondrocyte differentiationthrough stabilizingRunx2 and Runx3protein in cartilage This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:19:12.920884-05:
      DOI: 10.1002/jcp.25068
  • Characterization of Human Dermal Fibroblasts in Fabry Disease
    • Authors: J. Lakomá; V. Donadio, R. Liguori, M. Caprini
      Abstract: Fabry disease (FD) is a hereditary X‐linked metabolic lysosomal storage disorder due to insufficient amounts or a complete lack of the lysosomal enzyme α‐galactosidase A (α − GalA). The loss of α‐GalA activity leads to an abnormal accumulation of globotriaosylceramide (Gb3) in lysosomes and other cellular components of different tissues and cell types, affecting the cell function. However, whether these biochemical alterations also modify functional processes associated to the cell mitotic ability is still unknown. The goal of the present study was to characterize lineages of human dermal fibroblasts (HDFs) of FD patients and healthy controls focusing on Gb3 accumulation, expression of chloride channels that regulate proliferation, and proliferative activity. The biochemical and functional analyses indicate the existence of quantitative differences in some but not all the parameters of cytoskeletal organization, proliferation and differentiation processes. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:18:30.179422-05:
      DOI: 10.1002/jcp.25072
  • Participation of TNF‐α in Inhibitory Effects of Adipocytes on
           Osteoblast Differentiation
    • Authors: Robrigo P. F. Abuna; Fabiola S. de Oliveira, Thiago De S. Santos, Thais R. Guerra, Adalberto L. Rosa, Marcio M. Beloti
      Abstract: Mesenchymal stem cells from bone marrow (BM‐MSCs) and adipose tissue (AT‐MSCs) are attractive tools for cell‐based therapies to repair bone tissue. In this study, we investigated the osteogenic and adipogenic potential of BM‐MSCs and AT‐MSCs as well as the effect of crosstalk between osteoblasts and adipocytes on cell phenotype expression. Rat BM‐MSCs and AT‐MSCs were cultured either in growth, osteogenic or adipogenic medium to evaluate osteoblast and adipocyte differentiation. Additionally, osteoblasts and adipocytes were indirectly co‐cultured to investigate the effect of adipocytes on osteoblast differentiation and vice versa. BM‐MSCs and AT‐MSCs exhibit osteogenic and adipogenic potential under non‐differentiation‐inducing conditions. When exposed to osteogenic medium, BM‐MSCs exhibited higher expression of bone markers compared with AT‐MSCs. Conversely, under adipogenic conditions, AT‐MSCs displayed higher expression of adipose tissue markers compared with BM‐MSCs. The presence of adipocytes as indirect co‐culture repressed the expression of the osteoblast phenotype, whereas osteoblasts did not exert remarkable effect on adipocytes. The inhibitory effect of adipocytes on osteoblasts was due to the release of tumor necrosis factor alpha (TNF‐α) in culture medium by adipocytes. Indeed, the addition of exogenous TNF‐α in culture medium repressed the differentiation of BM‐MSCs into osteoblasts mimicking the indirect co‐culture effect. In conclusion, our study showed that BM‐MSCs are more osteogenic while AT‐MSCs are more adipogenic. Additionally, we demonstrated the key role of TNF‐α secreted by adipocytes on the inhibition of osteoblast differentiation. Thus, we postulate that the higher osteogenic potential of BM‐MSCs makes them the first choice for inducing bone repair in cell‐based therapies. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:17:38.647095-05:
      DOI: 10.1002/jcp.25073
  • ATP release and P2Y Receptor Signalling are Essential for Keratinocyte
    • Authors: Aimie Riding; Christine E. Pullar
      Abstract: Repair to damaged tissue requires directional cell migration to heal the wound. Immediately upon wounding an electrical guidance cue is created with the cathode of the electric field (EF) located at the center of the wound. Previous research has demonstrated directional migration of keratinocytes towards the cathode when an EF of physiological strength (100–150 mV/mm) is applied in vitro, but the “sensor” by which keratinocytes sense the EF remains elusive. Here we use a customised chamber design to facilitate the application ofa direct current (DC) EF of physiological strength (100 mV/mm) to keratinocytes whilst pharmacologically modulating the activation ofboth connexin hemichannels and purinergic receptorsto determine their role inEF‐mediated directional keratinocyte migration, galvanotaxis. In addition, keratinocytes were exposed to DiSCAC2(3) dye to visualize membrane potential changes within the cell upon exposure to the applied DC EF. Here we unveil ATP‐medicated mechanisms that underpin the initiation of keratinocyte galvanotaxis. The application of a DC EF of 100 mV/mm releases ATP via hemichannels activatinga subset of purinergic P2Y receptors, locally, to initiate the directional migration of keratinocytes towards the cathodein vitro, the center of the woundin vivo. The delineation of the mechanisms underpinning galvanotaxis extends our understanding of this endogenous cue and will facilitate the optimization and wider use of EF devices for chronic wound treatment. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:16:51.910134-05:
      DOI: 10.1002/jcp.25070
  • Different Effects of p52SHC1 and p52SHC3 on the Cell Cycle of Neurons and
           Neural Stem Cells
    • Authors: Ning Tang; Dan Lyu, Tao Liu, Fangjin Chen, Shuqian Jing, Tianyu Hao, Shaojun Liu
      Abstract: SHC3 is exclusively expressed in postmitotic neurons, while SHC1 is found in neural stem cells and neural precursor cells but absent in mature neurons. In this study, we discovered that suppression of p52SHC1 expression by RNA interference resulted in proliferation defects in neural stem cells, along with significantly reduced protein levels of cyclin E and cyclin A. At the same time, p52SHC3 RNAi caused cell cycle re‐entry (9.54% in S phase and 5.70% in G2‐M phase) in primary neurons with significantly up‐regulated expression of cyclin D1, cyclin E, cyclin A, CDK2 and phosphorylated CDK2. When p52SHC3 was overexpressed, the cell cycle of neural stem cells was arrested with reduced protein levels of cyclin D1, cyclin E and cyclin A, while overexpression of p52SHC1 did not result in significant changes in postmitotic neurons. Our results indicate that p52SHC3 plays an important role in maintaining the mitotic quiescence of neurons, while p52SHC1 regulates the proliferation of neural stem cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:09:34.439338-05:
      DOI: 10.1002/jcp.25069
  • COMMD7 as a novel NEMO interacting protein involved in the termination of
    • Authors: Elio Esposito; Gennaro Napolitano, Alessandra Pescatore, Giuseppe Calculli, Maria Rosaria Incoronato, Antonio Leonardi, Matilde Valeria Ursini
      Abstract: NEMO/IKKγ is the regulatory subunit of the IκB Kinase (IKK) complex, required for the activation of the NF‐κB pathway, which is involved in a variety of key processes, including immunity, inflammation, differentiation and cell survival. Termination of NF‐κB activity on specific ‐κB responsive genes, which is crucial for the resolution of inflammatory responses, can be achieved by direct degradation of the chromatin‐bound NF‐κB subunit RelA/p65, a process mediated by a protein complex that contains Copper Metabolism Murr1 Domain 1 (COMMD1). In this study, we identify COMMD7, another member of the COMMDs protein family, as a novel NEMO‐interacting protein. We show that COMMD7 exerts an inhibitory effect on NF‐κB activation upon TNFα stimulation. COMMD7 interacts with COMMD1 and together they cooperate to down‐regulate NF‐κB activity. Accordingly, termination of TNFα‐induced NF‐κB activity on the ‐κB responsive gene, Icam1, is defective in cells silenced for COMMD7 expression. Furthermore, this impairment is not greatly increased when we silence the expression of both COMMD7 and COMMD1 indicating that the two proteins participate in the same pathway of termination of TNFα‐induced NF‐κB activity. Importantly, we have demonstrated that COMMD7's binding to NEMO does not interfere with the binding to the IKKs, and that the disruption of the IKK complex through the use of the NBP competitor impairs the termination of NF‐κB activity. We propose that an intact IKK complex is required for the termination of NF‐κB‐dependent transcription and that COMMD7 acts as a scaffold in the IKK‐mediated NF‐κB termination. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:09:11.40997-05:0
      DOI: 10.1002/jcp.25066
  • Potential for Stem Cell‐Based Periodontal Therapy
    • Authors: Seyed Hossein Bassir; Wichaya Wisitrasameewong, Justin Raanan, Sasan Ghaffarigarakani, Jamie Chung, Marcelo Freire, Luciano C. Andrada, Giuseppe Intini
      Abstract: Periodontal diseases are highly prevalent and are linked to several systemic diseases.The goal of periodontal treatment is to halt the progression of the disease and regenerate the damaged tissue. However, achieving complete and functional periodontal regeneration is challenging because the periodontium is a complex apparatus composed of different tissues, including bone, cementum, and periodontal ligament. Stem cell‐based regenerative therapy may represent an effective therapeutic tool for periodontal regeneration due to their plasticity and ability to differentiate into different cell lineages. This review presents and critically analyzesthe available information on stem cell‐based therapyfor the regeneration of periodontal tissues and suggests new avenues for the development of more effective therapeutic protocols. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:08:37.852821-05:
      DOI: 10.1002/jcp.25067
  • The Association between p38 MAPK‐Mediated TNF‐α/TNFR2
           Up‐Regulation and
           Apoptosis in Human Leukemia U937 Cells
    • Authors: Chia‐Hui Huang; Ying‐Jung Chen, Tzu‐Yu Chao, Wen‐Hsin Liu, Jung‐Jung Changchien, Wan‐Ping Hu, Long‐Sen Chang
      Abstract: The primary cause of treatment failures in acute myeloid leukemia is usually associated with defects in the apoptotic pathway. Several studies suggest that 2‐(4‐aminophenyl)‐7‐methoxybenzothiazole (7‐OMe‐APBT) may potentially induce apoptosis of cancer cells. Thus, the present study was conducted to explore the cytotoxic effect of 7‐OMe‐APBT on human leukemia U937 cells. The apoptosis of human leukemia U937 cells induced by 7‐OMe‐APBT was characterized by an increase in mitochondrial membrane depolarization, procaspase‐8 degradation, and tBid production. Down‐regulation of FADD blocked 7‐OMe‐APBT‐induced procaspase‐8 degradation and rescued the viability of 7‐OMe‐APBT‐treated cells, suggesting the involvement of a death receptor‐mediated pathway in 7‐OMe‐APBT‐induced cell death. Increased TNF‐α expression, TNFR2 expression, and p38 MAPK phosphorylation were noted in 7‐OMe‐APBT‐treated cells. Pretreatment with a p38 MAPK inhibitor abolished 7‐OMe‐APBT‐induced TNF‐α and TNFR2 up‐regulation. 7‐OMe‐APBT stimulated p38 MAPK/c‐Jun‐mediated transcriptional up‐regulation of TNFR2, while the increased TNF‐α mRNA stability led to TNF‐α up‐regulation in 7‐OMe‐APBT‐treated cells. Treatment with 7‐OMe‐APBT up‐regulated protein phosphatase 2A catalytic subunit α (PP2Acα) expression via the p38 MAPK/c‐Jun/ATF‐2 pathway, which, in turn, promoted tristetraprolin (TTP) degradation. Pretreatment with a protein phosphatase 2A inhibitor or TTP over‐expression abrogated TNF‐α up‐regulation in 7‐OMe‐APBT‐treated cells. Abolishment of TNF‐α up‐regulation or knock‐down of TNFR1/TNFR2 by siRNA restored the viability of 7‐OMe‐APBT‐treated cells. Taken together, our data indicate a connection between p38 MAPK‐mediated TNF‐α and TNFR2 up‐regulation and 7‐OMe‐APBT‐induced TNF‐α‐mediated death pathway activation in U937 cells. The same pathway also elucidates the mechanism underlying 7‐OMe‐APBT‐induced death of human leukemia HL‐60 cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:01:09.859542-05:
      DOI: 10.1002/jcp.25064
  • Lanthanum Chloride Attenuates Osteoclast Formation and Function via the
           Downregulation of RANKL‐Induced NF‐κB and NFATc1
    • Authors: Chuan Jiang; Jiangyinzi Shang, Zhe Li, An Qin, Zhengxiao Ouyang, Xinhua Qu, Haowei Li, Bo Tian, Wengang Wang, Chuanlong Wu, Jinwu Wang, Min Dai
      Abstract: The biological activities of lanthanum chloride (LaCl3) and the molecular mechanisms of action underlying its anti‐inflammatory, anti‐hyperphosphatemic, and osteoblast‐enhancing effects have been studied previously, but less is known about the effects of LaCl3 on osteoclasts. The present study used in vivo and in vitro approaches to explore the effects of LaCl3 on osteoclasts and osteolysis. The results indicated that LaCl3 concentrations that were non‐cytotoxic to mouse bone marrow‐derived monocytes attenuated receptor activator of nuclear factor‐κB ligand (RANKL)‐stimulated osteoclastogenesis, bone resorption, mRNA expression of osteoclastogenic genes in these cells, including cathepsin K, calcitonin receptor, and tartrate‐resistant acid phosphatase (TRAP). Further, LaCl3inhibited RANKL‐mediated activation of the nuclear factor‐κB (NF‐κB) signalingpathway, and downregulated mRNA and protein levels of nuclear factor of activated T‐cells, cytoplasmic, calcineurin‐dependent 1 (NFATc1) and c‐fos. In vivo, LaCl3attenuated titanium (Ti) particle‐induced bone loss in a murine calvarial osteolysis model. Histological analyses revealed that LaCl3 ameliorated bone destruction and decreased the number of TRAP‐positive osteoclasts in this model. These results demonstrated that LaCl3 inhibitedosteoclast formation, function, and osteoclast‐specific gene expression in vitro, and attenuated Ti particle‐induced mouse calvarial osteolysis in vivo, where the inhibition of NF‐κB signaling and downregulation of NFATc1 and c‐fos played an important role. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:00:26.107893-05:
      DOI: 10.1002/jcp.25065
  • C‐ing the Genome: A Compendium of Chromosome Conformation Capture
           Methods to Study Higher‐Order Chromatin Organization
    • Authors: A. Rasim Barutcu; Andrew J. Fritz, Sayyed K. Zaidi, André J. vanWijnen, Jane B. Lian, Janet L. Stein, Jeffrey A. Nickerson, Anthony N. Imbalzano, Gary S. Stein
      Abstract: Three‐dimensional organization of the chromatin has important roles in transcription, replication, DNA repair, and pathologic events such as translocations. There are two fundamental ways to study higher‐order chromatin organization: microscopic and molecular approaches. In this review, we briefly introduce the molecular approaches, focusing on chromosome conformation capture or“3C” technology and its derivatives, which can be used to probe chromatin folding at resolutions beyond that provided by microscopy techniques. We further discuss the different types of data generated by the 3C‐based methods and how they can be used to answer distinct biological questions. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-08T09:00:16.939204-05:
      DOI: 10.1002/jcp.25062
  • Mechanisms and Consequences of Double‐strand DNA Break Formation in
    • Authors: Wendy J. Cannan ; David S. Pederson
      Abstract: All organisms suffer double‐strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter certain DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that can lead to cell death or cancer. The most common pathway used to repair DSBs in metazoans (non‐homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual's mutation burden and risk of cancer. This review describes radiological, chemical and biological mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiology, cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section will focus on nucleosome‐specific mechanisms that influence DSB production, and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis).This article is protected by copyright. All rights reserved
      PubDate: 2015-06-03T06:06:33.191832-05:
      DOI: 10.1002/jcp.25048
  • Animal Models for Progressive Multifocal Leukoencephalopathy
    • Authors: Martyn K. White ; Jennifer Gordon, Joseph R. Berger, Kamel Khalili
      Abstract: Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the CNS caused by the human polyomavirus JC (JCV). JCV replication occurs only in human cells and investigation of PML has been severely hampered by the lack of an animal model. The common feature of PML is impairment of the immune system. The key to understanding PML is working out the complex mechanisms that underlie viral entry and replication within the CNS and the immunosurveillance that suppresses the virus or allows it to reactivate. Early models involved the simple inoculation of JCV into animals such as monkeys, hamsters, and mice. More recently, mouse models transgenic for the gene encoding the JCV early protein, T‐antigen, a protein thought to be involved in the disruption of myelin seen in PML, have been employed. These animal models resulted in tumorigenesis rather than demyelination. Another approach is to use animal polyomaviruses that are closely related to JCV but able to replicate in the animal such as mouse polyomavirus and SV40. More recently, novel models have been developed that involve the engraftment of human cells into the animal. Here, we review progress that has been made to establish an animal model for PML, the advances and limitations of different models and weigh future prospects.This article is protected by copyright. All rights reserved
      PubDate: 2015-06-03T06:06:06.456436-05:
      DOI: 10.1002/jcp.25047
  • Brg1 Controls the Expression of Pax7 to Promote Viability and
           Proliferation of Mouse Primary Myoblasts
    • Authors: Teresita Padilla‐Benavides ; Brian T. Nasipak, Anthony N. Imbalzano
      Abstract: Brg1 (Brahma‐related gene 1) is a catalytic component of the evolutionarily conserved mammalian SWI/SNF ATP‐dependent chromatin remodeling enzymes that disrupt histone‐DNA contacts on the nucleosome. While the requirement for the SWI/SNF enzymes in cell differentiation has been extensively studied, its role in precursor cell proliferation and survival is not as well defined. Muscle satellite cells constitute the stem cell pool that sustains and regenerates myofibers in adult skeletal muscle. Here, we show that deletion of Brg1 in primary mouse myoblasts derived from muscle satellite cells cultured ex vivo leads to a cell proliferation defect and apoptosis. We determined that Brg1 regulates cell proliferation and survival by controlling chromatin remodeling and activating transcription at the Pax7 promoter, which is expressed during somite development and is required for controlling viability of the satellite cell population. Reintroduction of catalytically active Brg1 or of Pax7 into Brg1‐deficient satellite cells rescued the apoptotic phenotype and restored proliferation. These data demonstrate that Brg1 functions as a positive regulator for cellular proliferation and survival of primary myoblasts. Therefore, the regulation of gene expression through Brg1‐mediated chromatin remodeling is critical not just for skeletal muscle differentiation but for maintaining the myoblast population as well. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-02T08:35:40.093852-05:
      DOI: 10.1002/jcp.25031
  • Establishment of Immortalized Mouse Bmp2 Knock‐out Dental Papilla
           Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and
    • Authors: Lian Wu; Feng Wang, Kevin J. Donly, Chunyan Wan, Daoshu Luo, Stephen E. Harris, Mary MacDougall, Shuo Chen
      Abstract: Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long term culture of these primary cells, Availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2ko/ko dp) cell line by introducing Cre recombinase and green fluorescent protein (FGP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2fx/fx dp) cells. iBmp2ko/ko dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth‐related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2ko/ko dp cells as reflected by the decreased Mmp‐9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmpko/ko cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-02T07:45:59.458148-05:
      DOI: 10.1002/jcp.25061
  • EGF‐Induced Connexin43 Negatively Regulates Cell Proliferation in
           Human Ovarian Cancer
    • Authors: Xin Qiu; Jung‐Chien Cheng, Christian Klausen, Hsun‐Ming Chang, Qianlan Fan, Peter C.K. Leung
      Abstract: Connexin43 (Cx43) has been shown to regulate cell proliferation and its down‐regulation is correlated with poor prognosis and survival in several types of human cancer. Cx43 expression levels are frequently down‐regulated in human ovarian cancer, suggesting a potential role for Cx43 in regulating the progression of this disease. Epidermal growth factor (EGF) is a well‐characterized hormone that stimulates ovarian cancer cell proliferation. Although EGF is able to regulate Cx43 expression in other cell types, it is unclear whether EGF can regulate Cx43 expression in ovarian cancer cells. Additionally, it remains unknown whether Cx43 is involved in EGF‐stimulated ovarian cancer cell proliferation. In the present study, we demonstrate that treatment with EGF up‐regulates Cx43 expression in two ovarian cancer cell lines, SKOV3 and OVCAR4. Although treatment with EGF activates both ERK1/2 and Akt signaling pathways, pharmacological inhibition and siRNA‐mediated knockdown suggest that only the activation of Akt1 is required for EGF‐induced Cx43 up‐regulation. Functionally, Cx43 knockdown enhanced basal and EGF‐induced cell proliferation, whereas the proliferative effects of EGF were reduced by Cx43 overexpression. Co‐treatment with the gap junction inhibitor carbenoxolone did not alter the suppressive effects of Cx43 overexpression on EGF‐induced cell proliferation, suggesting a gap junction‐independent mechanism. This study reveals an important role for Cx43 as a negative regulator of EGF‐induced human ovarian cancer cell proliferation. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:44:55.234757-05:
      DOI: 10.1002/jcp.25058
  • Cell‐Penetrating and Endoplasmic Reticulum‐Locating
           TAT‐IL‐24‐KDEL Fusion Protein Induces Tumor Apoptosis
    • Authors: Jian Zhang; Aiyou Sun, Rui Xu, Xinyi Tao, Yuguo Dong, Xinxin Lv, Dongzhi Wei
      Abstract: Interleukin‐24 (IL‐24) is a unique IL‐10 family cytokine that could selectively induce apoptosis in cancer cells without harming normal cells. Previous research demonstrated that intracellular IL‐24 protein induces an endoplasmic reticulum (ER) stress response only in cancer cells, culminating in apoptosis. In this study, we developed a novel recombinant fusion protein to penetrate into cancer cells and locate on ER. It is composed of three distinct functional domains, IL‐24, and the targeting domain of transactivator of transcription (TAT) and an ER retention four‐peptide sequence KDEL (Lys‐Asp‐Glu‐Leu) that link at its NH2 and COOH terminal, respectively. The in vitro results indicated that TAT‐IL‐24‐KDEL inhibited growth in bladder cancer cells, as well as in non‐small cell lung cancer cell line and breast cancer cell line, but the normal human lung fibroblast cell line was not affected, indicating the cancer specificity of TAT‐IL‐24‐KDEL. Western blot analysis showed that apoptosis activation was induced by TAT‐IL‐24‐KDEL through the ER stress‐mediated cell death pathway. Treatment with TAT‐IL‐24‐KDEL significantly inhibited the growth of human H460 xenografts in nude mice, and the tumor growth inhibition was correlated with increased hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) staining. These findings suggest that the artificially designed recombinant fusion protein TAT‐IL‐24‐KDEL may be highly effective in cancer therapy and worthy of further evaluation and development. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:44:35.492967-05:
      DOI: 10.1002/jcp.25054
  • Error‐Prone Repair of DNA Double‐Strand Breaks
    • Authors: Kasey Rodgers; Mitch McVey
      Abstract: Preserving the integrity of the DNA double helix is crucial for the maintenance of genomic stability. Therefore, DNA double‐strand breaks represent a serious threat to cells. In this review, we describe the two major strategies used to repair double strand breaks: non‐homologous end joining and homologous recombination, emphasizing the mutagenic aspects of each. We focus on emerging evidence that homologous recombination, long thought to be an error‐free repair process, can in fact be highly mutagenic, particularly in contexts requiring large amounts of DNA synthesis. Recent investigations have begun to illuminate the molecular mechanisms by which error‐prone double‐strand break repair can create major genomic changes, such as translocations and complex chromosome rearrangements. We highlight these studies and discuss proposed models that may explain some of the more extreme genetic changes observed in human cancers and congenital disorders. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:44:16.245325-05:
      DOI: 10.1002/jcp.25053
  • p53 Modulates Notch Signaling in MCF‐7 Breast Cancer Cells by
           Associating with the Notch Transcriptional Complex via MAML1
    • Authors: Jieun Yun; Ingrid Espinoza, Antonio Pannuti, Damian Romero, Luis Martinez, Mary Caskey, Adina Stanculescu, Maurizio Bocchetta, Paola Rizzo, Vimla Band, Hamid Band, Hwan Mook Kim, Song‐Kyu Park, Keon Wook Kang, Maria Laura Avantaggiati, Christian R. Gomez, Todd Golde, Barbara Osborne, Lucio Miele
      Abstract: p53 and Notch‐1 play important roles in breast cancer biology. Notch‐1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T‐cells but stimulates it in human keratinocytes. Notch co‐activator MAML1 binds p53 and functions as a p53 co‐activator. We studied the regulation of Notch signaling by p53 in MCF‐7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin‐3 inhibits Notch‐dependent transcriptional activity and Notch target expression in a dose‐dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co‐immunoprecipitation experiments readily detected a complex containing p53 and Notch‐1 in MCF‐7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN‐MAML1) and stimulated by wild‐type MAML1. Standard and quantitative far‐Western experiments showed a complex including p53, Notch‐1 and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch‐dependent HEY1 promoter and this association is inhibited by DN‐MAML1 and stimulated by wild‐type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1‐dependent fashion, most likely through a p53‐MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch‐dependent transcription. Our data suggest that p53‐null breast cancers may lack this Notch‐modulatory mechanism, and that therapeutic strategies that activate wild‐type p53 can indirectly cause inhibition of Notch transcriptional activity. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:43:59.77372-05:0
      DOI: 10.1002/jcp.25052
  • microRNA as Biomarkers and Diagnostics
    • Authors: Jin Wang; Jinyun Chen, Subrata Sen
      Abstract: MicroRNAs (miRNAs) are a group of small non‐coding RNAs that are involved in regulating a range of developmental and physiological processes; their dysregulation has been associated with development of diseases including cancer. Circulating miRNAs and exosomal miRNAs have also been proposed as being useful in diagnostics as biomarkers for diseases and different types of cancer. In this review, miRNAs are discussed as biomarkers for cancer and other diseases, including viral infections, nervous system disorders, cardiovascular disorders, and diabetes. We summarize some of the clinical evidence for the use of miRNAs as biomarkers in diagnostics and provide some general perspectives on their use in clinical situations. The analytical challenges in using miRNAs in cancer and disease diagnostics are evaluated and discussed. Validation of specific miRNA signatures as biomarkers is a critical milestone in diagnostics. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:43:44.603437-05:
      DOI: 10.1002/jcp.25056
  • Regulation of Spermatogenic Cell T‐Type Ca2+ Currents by Zn2+:
           Implications in Male Reproductive Physiology
    • Abstract: Zn2+ is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis and prostate contain high Zn2+ concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express CaV3.2‐encoded T‐type Ca2+ currents (ICaT) which are positively or negatively modulated by Zn2+ in other tissues. To explore whether ICaT could be regulated by Zn2+ and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell ICaT in the absence or presence of different Zn2+ concentrations. Zn2+ decreased ICaT in a concentration‐dependent manner (IC50= 2 μM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, ICaT did not show a tonic inhibition by Zn2+. Further analysis showed that Zn2+ did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady‐state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong ICaT reduction. Using non‐stationary fluctuation analysis, we found that Zn2+ mainly reduced the number of available Ca2+ channels without changing the single channel current amplitude. ICaT modulation by Zn2+ could be relevant for spontaneous Ca2+ oscillations during spermatogenesis and in pathophysiological conditions such as diabetes. This article is protected by copyright. All rights reserved
  • IPSCs, a Promising Tool to Restore Muscle Atrophy
    • Abstract: Induced pluripotent stem cells (iPSCs) are a promising tool for regenerative medicine in chronic conditions associated with muscle atrophy since iPSCs are easier to obtain, pose less ethical limitations and can better capture human genetic diversity compared with human embryonic stem cells. We highlight the potentiality of iPSCs for treating muscle‐affecting conditions for which no effective cure is yet available, notably aging sarcopenia and inherited neurometabolic conditions. This article is protected by copyright. All rights reserved
  • Hypoxia Promotes the Inflammatory Response and Stemness Features in
           Visceral Fat Stem Cells from Obese Subjects
    • Abstract: Low‐grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of 2 subpopulations of adipose‐derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of 2 subpopulations of ASC from the same isolation sites of non‐obese individuals. In particular, the behavior of ASC of obese vs non‐obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV‐ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation‐related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue‐specific NF‐kB activation and an NF‐kB‐mediated increase in inflammatory and fibrogenic responses. Moreover, obV‐ASC, which showed a less stem‐like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV‐ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF‐kB inhibitors. These novel findings suggest that the deregulated hyper‐responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low‐grade chronic inflammation, which has been implicated in obesity‐related morbidity. This article is protected by copyright. All rights reserved
  • Hyperosmolarity‐Induced Down‐Regulation of Claudin‐2
           Mediated by Decrease in PKCβ‐Dependent GATA‐2 in MDCK
    • Abstract: Hyperosmolarity decreases claudin‐2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity‐induced decrease in claudin‐2 expression is inhibited by Go6983, a non‐selective protein kinase C (PKC) inhibitor, and PKCβ specific inhibitor in Madin‐Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca2+ concentration and phosphorylated PKCβ level, which were inhibited by RN‐1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12‐myristate 13‐acetate, a PKC activator, decreased claudin‐2 expression. These results indicate hyperosmolarity decreases claudin‐2 expression mediated by the activation of RN‐1734‐sensitive channel and PKCβ. Hyperosmolarity decreased promoter activity of claudin‐2, which was inhibited by Go6983 and PKCβ inhibitor similar to those in real‐time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of −469/−6, a deletion mutant. Claudin‐2 has hyperosmolarity‐sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA‐2, which was inhibited by Go6983 and PKCβ inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity‐induced decrease in reporter activity and claudin‐2 expression were rescued by over‐expression of wild type GATA‐2. Chromatin immunoprecipitation assay showed that GATA‐2 bound to promoter region of claudin‐2. These results suggest that hyperosmolarity decreases the expression level of claudin‐2 via a decrease in PKCβ‐dependent GATA‐2 transcriptional activity in renal tubular epithelial cells. J. Cell. Physiol. 9999: 2776–2787, 2015. © 2015 Wiley Periodicals, Inc.
  • Angiogenic Role of MMP‐2/9 Expressed on the Cell Surface of Early
           Endothelial Progenitor Cells/Myeloid Angiogenic Cells
    • Abstract: Since the introduction of angiogenic cell therapy using early endothelial progenitor cells (EPCs), myeloid angiogenic cells (MACs) have been expected to be useful in treating ischemic diseases. In order to elucidate the angiogenic properties of MACs/EPCs, we clarified the characteristics of MACs as compared to M2 macrophages (Mϕs). Comparison of the gene expression profiles of MACs and late EPCs revealed that MACs expressed greater amounts of metalloproteinase (MMP)‐9. It should be noted that the profile of MMP‐2/9 expression on the cell surface of MACs was similar to that of M2 Mϕs, and that cell surface MMP‐2/9 might be an active form based on molecular size. In addition, the invasion of MACs was prohibited not only by MMP‐2/9 inhibitor, but also by the hyaluronidase treatment that caused the down‐regulation of MMP‐9 on the cell surface of MACs and inhibited their invasion activity. These results indicate that cell surface MMP‐2/9 plays an important role in the high invasion ability of MACs. The conditioned medium of both MACs and M2 Mϕs stimulated tube formation of endothelial cells in vitro. MACs caused an increase in vessel formation in in vivo models through the production of IL‐8. We propose that the role of MACs with cell surfaces expressing MMP‐2/9 is rapidly invading ischemic tissue. J. Cell. Physiol. 9999: 2763–2775, 2015. © 2015 Wiley Periodicals, Inc.
  • Lactogenic hormone stimulation and epigenetic control of L‐amino
           acid oxidase expression in lactating mammary glands
    • Abstract: L‐amino acid oxidase (LAO), a classic flavoprotein, shows antibacterial activity by producing hydrogen peroxide. LAO exists in many tissues such as salivary gland, thymus, spleen, small intestine and testis. In particular, LAO was highly expressed in mice milk and plays an important factor in innate immunity of mammary glands. However, the mechanism which LAO expression is regulated spatially and temporally in lactating mammary glands has been unclear. In this study, we showed the contribution of lactogenic hormone and epigenetic control on LAO gene expression. In monolayer of mammary epithelial cells, treatment of lactogenic hormone mixture, dexamethasone, insulin and prolactin, did not induce LAO mRNA expression and its promoter activity, even though one of milk protein β‐casein expression was stimulated. However, increase of LAO expression was observed when the cells were treated with lactogenic hormones in a 3‐dimensional culture. The results of chromatin immunoprecipitation analysis revealed that histone H3K18 acetylation increased and histone H3K27 tri‐methylation decreased with lactation, which is associated with a period of high LAO expression. Moreover, the treatment of histone methylation inhibitor (DZNep) as well as histone deacetylation inhibitor (Trichostatine A) induced LAO expression in monolayer of mammary cells. Taken together, this is the first demonstration showing that LAO expression is induced in cell culture, and stimulation of lactogenic hormone and change of histone modification are promising signals to show highly expression of LAO in lactating mammary glands. J. Cell. Physiol. 9999: 2755–2762, 2015. © 2015 Wiley Periodicals, Inc.
  • Nuclear Translocation of Calpain‐2 Mediates Apoptosis of
           Hypertrophied Cardiomyocytes in Transverse Aortic Constriction Rat
    • Abstract: Apoptosis of cardiomyocytes plays an important role in the transition from cardiac hypertrophy to heart failure. Hypertrophied cardiomyocytes show enhanced susceptibility to apoptosis. Therefore, the aim of this study was to determine the susceptibility to apoptosis and its mechanism in hypertrophied cardiomyocytes using a rat model of transverse abdominal aortic constriction (TAC). Sixteen weeks of TAC showed compensatory and pathological hypertrophy in the left ventricle. TUNEL‐positive nuclei were significantly increased in TAC with angiotensin II (Ang II) treatment. Calpain inhibitor, PD150606, effectively inhibited Ang II‐induced apoptosis of hypertrophied cardiomyocytes. Ang II increased nuclear translocation of intracellular Ca2+ activated calpain‐2 in hypertrophied cardiomyocytes. Ang II enhanced the interaction between activated calpain‐2 and Ca2+/calmodulin‐dependent protein kinase II δB (CaMKIIδB), and promoted the degradation of CaMKIIδB by calpain‐2 in the nuclei of hypertrophied cardiomyocytes. Consequently, the depressed CaMKIIδB downregulated the expression of antiapoptotic Bcl‐2 leading to mitochondrial depolarization and release of cytochrome c led to apoptosis of hypertrophied cardiomyocytes. In conclusion, hypertrophied cardiomyocytes show increased susceptibility to apoptosis during Ang II stimulation via nuclear calpain‐2 and CaMKIIδB pathway. J. Cell. Physiol. 9999: 2743–2754, 2015. © 2015 Wiley Periodicals, Inc.
  • VEGF Enhances the Migration of MSCs in Neural Differentiation by
           Regulating Focal Adhesion Turnover
    • Abstract: Mesenchymal stem cells (MSCs) hold great promise in neural regeneration, due to their intrinsic neuronal potential and migratory tropism to damaged nervous tissues. However, the chemotactic signals mediating the migration of MSCs remain poorly understood. Here, we investigated the regulatory roles for focal adhesion kinase (FAK) and Rac1 in vascular endothelial growth factor (VEGF)‐stimulated migration of MSCs in neural differentiation. We found that MSCs in various differentiation states show significant different chemotactic responses to VEGF and cells in 24‐h preinduction state possess the highest migration speed and efficiency. FAK, as the downstream signaling molecule, is involved in the VEGF‐induced migration by regulating the assembly and distribution of focal adhesions (FAs) and reorganization of F‐actin. The features of FAs and cytoskeletons and the ability of lamellipodia formation are closely related to the neural differentiation states of MSCs. VEGF promotes FA formation with an asymmetric distribution of FAs and induces the activation of Y397‐FAK and Y31/118‐paxillin of undifferentiated and 24‐h preinduced MSCs in a time‐dependent manner. Inhibition of FAK by PF‐228 or expressing FAK‐Y397F mutant impairs the dynamics of FAs in MSCs during VEGF‐induced migration. Furthermore, Rac1 regulates FA formation in a FAK‐dependent manner. Overexpression of constitutive activated mutants of Rac1 increases the number of FAs in undifferentiated and 24‐h preinduced MSCs, while VEGF‐induced increase of FA formation is decreased by inhibiting FAK by PF‐228. Collectively, these results demonstrate that FAK and Rac1 signalings coordinately regulate the dynamics of FAs during VEGF‐induced migration of MSCs in varying neural differentiation states. J. Cell. Physiol. 9999: 2728–2742, 2015. © 2015 Wiley Periodicals, Inc.
  • Establishment of Four New Human Primary Cell Cultures from
           Chemo‐Naïve Italian Osteosarcoma Patients
    • Abstract: Osteosarcoma (OS) is a primary highly malignant tumor of bone, affecting predominately adolescents and young adults between 10 and 20 years of age. OS is characterized by an extremely aggressive clinical course, with a rapid development of metastasis to the lung and distant bones. J. Cell. Physiol. 9999: 2718–2727, 2015. © 2015 Wiley Periodicals, Inc.
  • TGF‐β Suppresses Ift88 Expression in Chondrocytic ATDC5 Cells
    • Abstract: Ift88 is an intraflagella transport protein, critical for the cilium, and has been shown to be required for the maintenance of chondrocytes and cartilage. However, how Ift88 is controlled by cytokines that play a role in osteoarthritis is not well understood. Therefore, we examined the effects of TGF‐β on the expression of Ift88. We used ATDC5 cells as chondrocytes and analyzed the effects of TGF‐β on gene expression. TGF‐β treatment suppresses the levels of Ift88 mRNA in a dose‐dependent manner starting from as low as 0.5 ng/mL and reaching the nadir at around 2 ng/mL. TGF‐β treatment also suppresses the protein levels of Ift88. TGF‐β suppression of Ift88 is still observed when the cells are cultured in the presence of a transcriptional inhibitor while the TGF‐β suppression is weakened in the presence of a protein synthesis inhibitor, cycloheximide. TGF‐β treatment suppresses the levels of Ift88 mRNA stability suggesting the presence of posttranscriptional regulation. TGF‐β treatment reduces the number of cilia positive cells and suppresses average length of cilia. Knockdown of Ift88 by siRNA enhances TGF‐β‐induced increase in type II collagen mRNA expression in ATDC5 cells revealing the suppressive role of Ift88 on TGF‐β‐induced regulation of extracellular matrix protein expression. TGF‐β also suppresses Ift88 mRNA expression in primary culture of rib chondrocytes. These data indicate that TGF‐β regulates Ift88 gene expression at least in part via posttrascriptional manner. J. Cell. Physiol. 9999: 2788–2795, 2015. © 2015 Wiley Periodicals, Inc.
  • Increased linear bone growth by GH in the absence of SOCS2 is independent
           of IGF‐1
    • Abstract: Growth hormone (GH) signaling is essential for postnatal linear bone growth, but the relative importance of GHs actions on the liver and/or growth plate cartilage remains unclear. The importance of liver derived insulin like‐growth factor‐1 (IGF‐1) for endochondral growth has recently been challenged. Here, we investigate linear growth in Suppressor of Cytokine Signaling‐2 (SOCS2) knockout mice, which have enhanced growth despite normal systemic GH/IGF‐1 levels. Wild‐type embryonic ex vivo metatarsals failed to exhibit increased linear growth in response to GH, but displayed increased Socs2 transcript levels (P 
  • Loss of MicroRNA‐101 Promotes Epithelial to Mesenchymal Transition
           in Hepatocytes
    • Abstract: Epithelial‐to‐mesenchymal transition (EMT) has been implicated in embryonic development and various pathological events. However, the involvement of microRNA in the process of EMT remains to be fully defined in hepatocyte. ZEB1 is a well‐known transcriptional repressor of E‐cadherin and plays a major role in triggering EMT during organ fibrosis and cancer cell metastasis. Computational microRNA target predictions detect a conserved sequence matching to miR‐101 in the 3′UTR of ZEB1 mRNA. Our results confirm that miR‐101 suppresses ZEB1 expression by targeting the predicted site of ZEB1 3′UTR. Subsequent investigations show that miR‐101 is significantly downregulated in the cultured hepatocytes undergoing EMT and in the hepatocytes isolated from fibrotic liver. Along with the loss of miR‐101, the ZEB1 expression increases simultaneously in hepatocytes. In addition, miR‐101 levels in HCC cell lines are negatively associated with the ZEB1 productions and the metastatic potentials of tumor cells. Mechanistically, we demonstrate that miR‐101 significantly inhibits the TGF‐β1‐induced EMT in hepatocytes, whereas inhibition of miR‐101 promotes the EMT process as indicated by the changes of morphology, cell migration, and the expression profiles of EMT markers. In the fibrotic liver, ectopic expression of miR‐101 can significantly downregulate ZEB1 in the hepatocyte and thereby reduces the mesenchymal marker expression. Moreover, miR‐101 significantly inhibits the proliferation and migration of HCC cell. Our results demonstrate that miR‐101 regulates HCC cell phenotype by upregulating the epithelial marker genes and suppressing the mesenchymal ones. J. Cell. Physiol. 9999: 2706–2717, 2015. © 2015 Wiley Periodicals, Inc.
  • The Role of Maternal Gestational Diabetes in Inducing Fetal Endothelial
    • Abstract: Gestational diabetes mellitus (GDM) is known to be associated with fetal endothelial dysfunction, however, the mechanisms are not fully understood. This study examines the effect of maternal diabetes on fetal endothelial function and gene expression under physiological glucose conditions (5 mM). Human umbilical vein endothelial cell (HUVEC) isolated from diabetic mothers (d.HUVEC) grew more slowly than HUVEC isolated from healthy mothers (c.HUVEC) and had delayed doubling time despite increased levels of total vascular endothelial growth factor (VEGF) expression and protein production as determined by real‐time PCR and ELISA respectively. Using western blot, the levels of antiproliferative VEGF165b isoform were increased in d.HUVEC relative to c.HUVEC. Successful VEGF165b knockdown by small interfering RNA (siRNA) resulted in increased proliferation of d.HUVEC measured by MTT, compared with negative siRNA control, to similar levels measured in c.HUVEC. In addition, d.HUVEC generated excess levels of ROS as revealed by 2′,7′ Dichlorodihydrofluorescein Diacetate (DCFH‐DA) and Nitrotetrazolium blue (NBT). Using microarray, 102 genes were differentially overexpressed between d.HUVEC versus c.HUVEC (>1.5‐fold change; P 
  • TRPC1 regulates calcium‐activated chloride channels in salivary
           gland cells
    • Abstract: Calcium‐activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca2+ influx that activates ion channels such as CaCC to initiate Cl− efflux. However direct evidence as well as the molecular identity of the Ca2+ channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl− current was activated by increasing [Ca2+]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh‐A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store‐depletion and activates TRPC1‐mediated Ca2+ entry, potentiated the Cl− current, which was inhibited by the addition of a non‐specific TRPC channel blocker SKF96365 or removal of external Ca2+. Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca2+ entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl− currents upon increasing [Ca2+]i. These Cl− currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh‐A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl− currents without decreasing TMEM16a expression. Together the data suggests that Ca2+ entry via the TRPC1 channels is essential for the activation of CaCC. J. Cell. Physiol. 9999: 2848–2856, 2015. © 2015 Wiley Periodicals, Inc.
  • Development of an Experimental Animal Model for Lower Back Pain by
           Percutaneous Injury‐Induced Lumbar Facet Joint Osteoarthritis
    • Abstract: We report generation and characterization of pain‐related behavior in a minimally invasive facet joint degeneration (FJD) animal model in rats. FJD was produced by a non‐open percutaneous puncture‐induced injury on the right lumbar FJs at three consecutive levels. Pressure hyperalgesia in the lower back was assessed by measuring the vocalization response to pressure from a force transducer. After hyperalgesia was established, pathological changes in lumbar FJs and alterations of intervertebral foramen size were assessed by histological and imaging analyses. To investigate treatment options for lumber FJ osteoarthritis‐induced pain, animals with established hyperalgesia were administered with analgesic drugs, such as morphine, a selective COX‐2 inhibitor, a non‐steroidal anti‐inflammatory drug (NSAID) (ketorolac), or pregabalin. Effects were assessed by behavioral pain responses. One week after percutaneous puncture‐induced injury of the lumbar FJs, ipsilateral primary pressure hyperalgesia developed and was maintained for at least 12 weeks without foraminal stenosis. Animals showed decreased spontaneous activity, but no secondary hyperalgesia in the hind paws. Histopathological and microfocus X‐ray computed tomography analyses demonstrated that the percutaneous puncture injury resulted in osteoarthritis‐like structural changes in the FJs cartilage and subchondral bone. Pressure hyperalgesia was completely reversed by morphine. The administration of celecoxib produced moderate pain reduction with no statistical significance while the administration of ketorolac and pregabalin produced no analgesic effect on FJ osteoarthritis‐induced back pain. Our animal model of non‐open percutanous puncture‐induced injury of the lumbar FJs in rats shows similar characteristics of low back pain produced by human facet arthropathy. J. Cell. Physiol. 9999: 2837–2847, 2015. © 2015 Wiley Periodicals, Inc.
  • The SWI/SNF ATPases Are Required for Triple Negative Breast Cancer Cell
    • Abstract: The Brahma (BRM) and Brahma‐related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi‐subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells. J. Cell. Physiol. 9999: 2683–2694, 2015. © 2015 Wiley Periodicals, Inc.
  • Cross‐Talk Between VEGF and BMP‐6 Pathways Accelerates
           Osteogenic Differentiation of Human Adipose‐Derived Stem Cells
    • Abstract: Deficiency in vascular endothelial growth factor (VEGF) or bone morphogenetic proteins (BMPs) results in fracture non‐unions. Therefore, it is indispensable to comprehend the combined effect of VEGF and BMPs on the osteogenic differentiation of osteoprogenitor mesenchymal stem cells (MSCs) that are either naturally occurring at the fracture repair site or exogenously added to enhance the bone repair. We found that the combination of VEGF and BMP‐6 enhanced COL1A2 expression, which correlated with upregulated expression of osterix, Dlx5, and Msx2 in human adipose‐derived stem cells (hADSCs). Cross‐talk between VEGF and BMP‐6 pathways upregulated activation of p38 mitogen‐activated kinase (p38 MAPK) and inhibited activation of protein kinase B (PKB, also known as Akt), whereas phosphorylation of “mothers against decapentaplegic” homologs 1/5/8 (Smads 1/5/8) and extracellular signal‐regulated kinases 1 and 2 (ERK 1/2) was not affected. Consistent with these findings, p38 inhibitor SB203580, or siRNA knockdown of osterix, abrogated crosstalk between the VEGF and BMP‐6 pathways and significantly reduced the observed upregulation of COL1A2. Nuclear translocation of the phosphorylated form of osterix was also inhibited by SB203580. Although crosstalk between the VEGF‐BMP‐6 pathways did not show an effect on the extent of mineralization, inhibition of any one of the three components that were upregulated through the cross‐talk, i.e., osterix, Dlx5, and p38 activation, led to a complete inhibition of mineralization. Inhibition of PKB/Akt activation, which is attenuated through the cross‐talk, significantly enhanced ALP gene expression. These observations imply that crosstalk between the VEGF and BMP‐6 signaling pathways enhances osteogenic differentiation of MSCs. J. Cell. Physiol. 9999: 2671–2682, 2015. © 2015 Wiley Periodicals, Inc.
  • Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells
           Through TGFβ1‐Mediated Upregulation of Periostin Expression
    • Abstract: Wnt5a, a member of the noncanonical Wnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this study, we examined the localization and potential function of Wnt5a in PDL tissue. Immunohistochemical analysis revealed that Wnt5a was expressed predominantly in rat PDL tissue. Semi‐quantitative reverse‐transcription polymerase chain reaction and Western blotting analysis demonstrated that human PDL cells (HPDLCs) expressed Wnt5a and its receptors (Ror2, Fzd2, Fzd4, and Fzd5). Removal of occlusal pressure by extraction of opposing teeth decreased Wnt5a expression in rat PDL tissue, and the expression of Wnt5a and its receptors in HPDLCs was upregulated by exposure to mechanical stress. Stimulation with Wnt5a significantly enhanced the proliferation and migration of HPDLCs. Furthermore, Wnt5a suppressed osteoblastic differentiation of HPDLCs cultivated in osteogenic induction medium, while it significantly enhanced the expression of PDL‐related genes, such as periostin, type‐I collagen, and fibrillin‐1 genes, and the production of collagen in HPDLCs cultivated in normal medium. Both knockdown of periostin gene expression by siRNA and inhibition of TGFβ1 function by neutralizing antibody suppressed the Wnt5a‐induced PDL‐related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFβ1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFβ1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFβ1‐mediated upregulation of periostin expression. J. Cell. Physiol. 9999: 2647–2660, 2015. © 2015 Wiley Periodicals, Inc.
  • Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of
           Breast Cancer Cells
    • Abstract: The tumor‐suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho‐MAPK, and phospho‐STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation. J. Cell. Physiol. 9999: 2661–2670, 2015. © 2015 Wiley Periodicals, Inc.
  • β2‐Adrenoreceptor‐Mediated Proliferation Inhibition of
           Embryonic Pluripotent Stem Cells
    • Abstract: Adrenoreceptors (ARs) are widely expressed and play essential roles throughout the body. Different subtype adrenoceptors elicit distinct effects on cell proliferation, but knowledge remains scarce about the subtype‐specific effects of β2‐ARs on the proliferation of embryonic pluripotent stem (PS) cells that represent different characteristics of proliferation and cell cycle regulation with the somatic cells. Herein, we identified a β2‐AR/AC/cAMP/PKA signaling pathway in embryonic PS cells and found that the pathway stimulation inhibited proliferation and cell cycle progression involving modulating the stem cell growth and cycle regulatory machinery. Embryonic stem (ES) cells and embryonal carcinoma stem (ECS) cells expressed functional β‐ARs coupled to AC/cAMP/PKA signaling. Agonistic activation of β‐ARs led to embryonic PS cell cycle arrest and proliferation inhibition. Pharmacological and genetic analyzes using receptor subtype blocking and RNA interference approaches revealed that this effect selectively depended on β2‐AR signaling involving the regulation of AKT, ERK, Rb, and Cyclin E molecules. Better understanding of the effects of β2‐ARs on embryonic PS cell proliferation and cycle progression may provide new insights into stem cell biology and afford the opportunity for exploiting more selective ligands targeting the receptor subtype for the modulation of stem cells. J. Cell. Physiol. 9999: 2640–2646, 2015. © 2015 Wiley Periodicals, Inc.
  • BMP‐2 Induced Expression of PLCβ1 That Is a Positive Regulator
           of Osteoblast Differentiation
    • Abstract: Bone morphogenetic protein 2 (BMP‐2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide‐phospholipase Cβ 1 (PLCβ1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCβ1. Here, the function of PLCβ1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP‐2 we assist to a remarkable increase in PLCβ1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCβ1 promotes osteogenic differentiation by up‐regulating alkaline phosphatase (ALP). Moreover, PLCβ1 is present in the nuclear compartment of these cells and overexpression of a cytosolic‐PLCβ1mutant (cyt‐PLCβ1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR‐214 is a regulator of Osterix (Osx) which is an osteoblast‐specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCβ1 could be a potential target of miR‐214 in the control of osteogenic differentiation by gain‐ and loss‐ of function experiment. The results indicated that inhibition of miR‐214 in C2C12 cells significantly enhances the protein level of PLCβ1 and promotes C2C12 BMP‐2‐induced osteogenesis by targeting PLCβ1. This article is protected by copyright. All rights reserved
  • The Other Side of the RAAS: Aldosterone Improves Migration of Cardiac
           Progenitor Cells
    • Abstract: Stem cell therapy is a promising new option for patients suffering from heart failure. Though many clinical studies show encouraging results, little is known about the signals which cause stem cells to home to diseased but not to healthy hearts. We hypothesized that aldosterone as one of the main players of heart failure functions as an attractant for progenitor cells and stimulates their migration. Stem cell antigen‐1 (Sca‐1) positive cells were isolated from the hearts of wild type FVB mice via magnetic cell sorting. The migration rate of the cells was determined using aldosterone as an attractant in a modified Boyden chamber (n = 5). Aldosterone led to a dose dependent increase in migration rate and this effect could be prevented by adding its blocker eplerenone. The mineralocorticoid receptor could be detected on Sca‐1+ cells via western blot and immunofluorescence. Therefore, aldosterone seems to play a role in stem cell migration and there the effect is most likely mediated by the mineralocorticoid receptor. J. Cell. Physiol. 9999: 2829–2836, 2015. © 2015 Wiley Periodicals, Inc.
  • Myeloid Zinc Finger 1 (MZF‐1) Regulates Expression of the CCN2/CTGF
           and CCN3/NOV Genes in the Hematopoietic Compartment
    • Abstract: Connective Tissue Growth Factor (CCN2/CTGF) and Nephroblastoma Overexpressed (CCN3/NOV) execute key functions within the hematopoietic compartment. Both are abundant in the bone marrow stroma, which is a niche for hematopoiesis and supports marrow function. Roles for 1,25‐dihydroxyvitamin D3 (calcitriol) and all‐trans retinoic acid in the bone marrow have also been elucidated. Interestingly, some of the annotated roles of these vitamins overlap with established functions of CCN2 and CCN3. Yet, no factor has been identified that unifies these observations. In this study, we report the regulation of the CTGF and NOV genes by Myeloid Zinc Finger‐1 (MZF‐1), a hematopoietic transcription factor. We show the interaction of MZF‐1 with the CTGF and NOV promoters in several cell types. Up‐regulation of MZF‐1 via calcitriol and vitamin A induces expression of CTGF and NOV, implicating a role for these vitamins in the functions of these two genes. Lastly, knockdown of MZF1 reduces levels of CTGF and NOV. Collectively, our results argue that MZF‐1 regulates the CTGF and NOV genes in the hematopoietic compartment, and may be involved in their respective functions in the stroma. J. Cell. Physiol. 9999: 2634–2639, 2015. © 2015 Wiley Periodicals, Inc.
  • Determinants of Muscle and Bone Aging
    • Abstract: Loss of bone and muscle with advancing age represent a huge threat to loss of independence in later life. Osteoporosis represents a major public health problem through its association with fragility fractures, primarily of the hip, spine and distal forearm. Sarcopenia, the age related loss of muscle mass and function, may add to fracture risk by increasing falls risk. In the context of muscle aging, it is important to remember that it is not just a decline in muscle mass which contributes to the deterioration of muscle function. Other factors underpinning muscle quality come into play, including muscle composition, aerobic capacity and metabolism, fatty infiltration, insulin resistance, fibrosis and neural activation. Genetic, developmental, endocrine and lifestyle factors, such as physical activity, smoking and poor diet have dual effects on both muscle and bone mass in later life and these will be reviewed here. Recent work has highlighted a possible role for the early environment. Inflammaging is an exciting emerging research field that is likely to prove relevant to future work, including interventions designed to retard to reverse bone and muscle loss with age. J. Cell. Physiol. 9999: 2618–2625, 2015. © 2015 Wiley Periodicals, Inc.
  • Melanoma Treatments: Advances and Mechanisms
    • Abstract: Advances in the understanding of the molecular pathogenesis of melanoma and in cancer immunology have led to the rational design and recent clinical implementation of a variety of novel therapies for metastatic melanoma. BRAF and MEK inhibitors that target the MAPK pathway have high rates of clinical response in BRAF‐mutant melanoma. Therapies that modulate the immune response to melanoma, including monoclonal antibodies that interfere with pathways that inhibit T‐cell function, have been shown to be effective in melanoma. Lessons learned from these encouraging results are driving the development of new treatments for melanoma and other cancers. This review will focus on the science and clinical findings related to targeted therapies that inhibit BRAF or MEK as well as the immunotherapies that block the CTLA‐4 or PD‐1 pathways. Other experimental and combinatorial therapeutic approaches will also be discussed. J. Cell. Physiol. 9999: 2626–2633, 2015. © 2015 Wiley Periodicals, Inc.
  • Effect of Diabetes Mellitus on Adipocyte‐Derived Stem Cells in Rat
    • Abstract: Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein 4 (BMP4) is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte‐derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR, and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co‐localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes. J. Cell. Physiol. 9999: 2821–2828, 2015. © 2015 Wiley Periodicals, Inc.
  • Treg(s) in Cancer: Friends or Foe'
    • Abstract: Immune escape is a hallmark of cancer. Regulatory T cells (Treg) have been described to maintain peripheral tolerance. The role of Treg in cancer is ambiguous, as they are central inhibitory regulators in solid tumors, whereas during inflammation‐driven tumorigenesis they prevent cancer initiation by restraining inflammation. As a consequence, under conditions with chronic inflammation that may initiate malignant transformation, application rather than depletion of Treg may be helpful. In solid tumors, however, the success story of immune‐activating antibodies targeting checkpoint molecules of T cell activation fuels the hope that Treg inactivation or depletion may additionally boost anti‐tumor immune response. In this review we summarize important aspects on the dual role of Treg in cancer to provide a rationale for future Treg targeting attempts. J. Cell. Physiol. 9999: 2598–2605, 2015. © 2015 Wiley Periodicals, Inc.
  • The Immunomodulatory and Therapeutic Effects of Mesenchymal Stromal Cells
           for Acute Lung Injury and Sepsis
    • Abstract: It is increasingly recognized that immunomodulation represents an important mechanism underlying the benefits of many stem cell therapies, rather than the classical paradigm of transdifferentiation and cell replacement. In the former paradigm, the beneficial effects of cell therapy result from paracrine mechanism(s) and/or cell–cell interaction as opposed to direct engraftment and repair of diseased tissue and/or dysfunctional organs. Depending on the cell type used, components of the secretome, including microRNA (miRNA) and extracellular vesicles, may be able to either activate or suppress the immune system even without direct immune cell contact. Mesenchymal stromal cells (MSCs), also referred to as mesenchymal stem cells, are found not only in the bone marrow, but also in a wide variety of organs and tissues. In addition to any direct stem cell activities, MSCs were the first stem cells recognized to modulate immune response, and therefore they will be the focus of this review. Specifically, MSCs appear to be able to effectively attenuate acute and protracted inflammation via interactions with components of both innate and adaptive immune systems. To date, this capacity has been exploited in a large number of preclinical studies and MSC immunomodulatory therapy has been attempted with various degrees of success in a relatively large number of clinical trials. Here, we will explore the various mechanism employed by MSCs to effect immunosuppression as well as review the current status of its use to treat excessive inflammation in the context of acute lung injury (ALI) and sepsis in both preclinical and clinical settings. J. Cell. Physiol. 9999: 2606–2617, 2015. © 2015 Wiley Periodicals, Inc.
  • Reflections on Cellular Physiology as a Catalytic Framework for Medicine
    • Abstract: These are the reflections of a retired physician who was introduced to cellular physiology in 1947 at the University of Pennsylvania. I majored in Zoology. I took the required major courses, which were interesting but not exciting. I was a premedical student at the time. Fortunately I was introduced to cellular physiology in 1947. The teacher was Professor Louis Heilbrunn, an amazing man dedicated to teaching and the love of his students. He had a number of graduate students at the time. The undergraduates were well integrated with these advanced students. We used his textbook of physiology and we were all proud of being his students. He gave us a new way of thinking about biology. It was exciting and we were treated as mature students. J. Cell. Physiol. 9999: 2596–2597, 2015. © 2015 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
  • Journal of Cellular Physiology: Volume 230, Number 11, November 2015
    • Abstract: Cover: Triple negative primary breast tumors stained for BRG1. See article by Wu et al. on pages 2683–2694.
  • Table of Contents: Volume 230, Number 11
  • Editor's Choice
  • Highlights: Volume 230, Number 11
  • Immortalized Mouse Floxed Fam20c Dental Papillar Mesenchymal and
           Osteoblast Cell Lines Retain Their Primary Characteristics
    • Abstract: Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T‐antigen (SV40 T‐Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long‐term passages and genomic integration of SV40 T‐Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. J. Cell. Physiol. 9999: 2581–2587, 2015. © 2015 Wiley Periodicals, Inc.
  • Glycation of Wild‐Type Apomyoglobin Induces Formation of Highly
           Cytotoxic Oligomeric Species
    • Abstract: Protein glycation is a non‐enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild‐type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild‐type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild‐type apomyoglobin have been also investigated. J. Cell. Physiol. 9999: 2807–2820, 2015. © 2015 Wiley Periodicals, Inc.
  • Sustained Inhibition of Proliferative Response After Transient FGF
           Stimulation is Mediated by Interleukin 1 Signaling
    • Abstract: Transient FGF stimulation of various cell types results in FGF memory – a sustained blockage of efficient proliferative response to FGF and other growth factors. FGF memory establishment requires HDAC activity, indicating its epigenetic character. FGF treatment stimulates proinflammatory NFκB signaling, which is also critical for FGF memory formation. The search for FGF‐induced mediators of FGF memory revealed that FGF stimulates HDAC‐dependent expression of the inflammatory cytokine IL1α. Similarly to FGF, transient cell treatment with recombinant IL1α inhibits the proliferative response to further FGF and EGF stimulation, but does not prevent FGF receptor‐mediated signaling. Interestingly, like cells pretreated with FGF1, cells pretreated with IL1α exhibit enhanced restructuring of actin cytoskeleton and increased migration in response to FGF stimulation. IRAP, a specific inhibitor of IL1 receptor, and a neutralizing anti‐IL1α antibody prevent the formation of FGF memory and rescue an efficient proliferative response to FGF restimulation. A similar effect results following treatment with the anti‐inflammatory agents aspirin and dexamethasone. Thus, FGF memory is mediated by proinflammatory IL1 signaling. It may play a role in the limitation of proliferative response to tissue damage and prevention of wound‐induced hyperplasia. This article is protected by copyright. All rights reserved
  • Failure to Target RANKL Signaling Through p38‐MAPK Results in
           Defective Osteoclastogenesis in the Microphthalmia Cloudy‐eyed
    • Abstract: The microphthalmia‐associated transcription factor (MITF) is a basic helix‐loop‐helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of receptor activator of NFκB ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C‐terminal end. Here we show that the Mitfce allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitfce/ce mice. The loss of the leucine zipper domain in Mitfce/ce mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitfce. This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site‐directed mutations in the docking site interfered with the interaction between MITF and its co‐factors FUS and BRG1. MITF‐ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/ p38 MAPK interactions in osteoclast differentiation. This article is protected by copyright. All rights reserved
  • Leptin Effect on Acetylation and Phosphorylation of PGC1α in Muscle
           Cells Associated With AMPK and Akt Activation in High‐Glucose Medium
    • Abstract: Leptin is crucial in energy metabolism, including muscle regulation. Peroxisome proliferator activated receptor gamma co‐activator 1α (PGC1α) orchestrates energy metabolism and is tightly controlled by post‐translational covalent modifications such as phosphorylation and acetylation. We aimed to further the knowledge of PGC1α control by leptin (at physiological levels) in muscle cells by time‐sequentially analysing the activation of AMP activated protein kinase (AMPK), p38 mitogen‐activated protein kinase (p38 MAPK) and Akt (Protein kinase B) – all known to phosphorylate PGC1α and to be involved in the regulation of its acetylation status – in C2C12 myotubes placed in a high‐glucose serum‐free medium. We also studied the protein levels of PGC1α, Sirtuin 1, adiponectin, COX IV, mitofusin 2 (Mfn2) and pyruvate dehydrogenase kinase 4 (PDK4). Our main findings suggest an important role of leptin regulating AMPK and Akt phosphorylation, mitofusin 2 induction and PGC1α acetylation status, with the novelty that the latter in transitorily increased in response to leptin, an effect dependent, at least in part, on AMPK regulation. These post‐translational reversible changes in PGC1α in response to leptin, especially the increase in acetylation status, may be related to the physiological role of the hormone in modulating muscle cell response to the physiological/nutritional status. This article is protected by copyright. All rights reserved
  • Correction of the FSHD Myoblast Differentiation Defect by Fusion With
           Healthy Myoblasts
    • Abstract: Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context. This article is protected by copyright. All rights reserved
  • Androgen Receptor Coactivators in Regulation of Growth and Differentiation
           in Prostate Cancer
    • Abstract: Androgen receptor (AR) is a key factor in regulation of growth and differentiation in normal and malignant prostate. Endocrine therapies for prostate cancer include inhibition of androgen production either by analogues of luteinizing hormone releasing hormone or abiraterone acetate and/or use of anti‐androgens such as hydroxyflutamide, bicalutamide, and enzalutamide. Castration therapy‐resistant cancer develops inevitably in patients who undergo treatment. AR coactivators are proteins which interact with one or more regions of the AR thus enhancing its function. Although several functions of AR coactivators may be redundant, specific functions have been identified and analyzed. The p160 group of coactivators, SRC‐1, ‐2, and ‐3 not only potentiate the activation of the AR, but are also implicated in potentiation of function of insulin‐like growth factor‐I and activation of the Akt pathway. Transcriptional integrators p300 and CBP are up‐regulated by androgen ablation and may influence antagonist/agonist balance of non‐steroidal anti‐androgens. A therapy approach designed to target p300 in prostate cancer revealed its role in regulation of proliferation of migration of androgen‐sensitive and –insensitive prostate cancer cells. Coactivators p300 and SRC‐1 are required for AR activation by interleukin‐6 (IL‐6), a cytokine that is overexpressed in castration‐therapy resistant prostate cancer. Some coactivators, such as Vav3 are involved in regulation of transcriptional activity of truncated AR, which emerge during endocrine thrapy. Stimulation of cellular migration and invasion by AR coactivators has also been described. Translational studies with aim to introduce anti‐AR coactivator therapy have not been successfuly implemented in the clinic so far. This article is protected by copyright. All rights reserved
  • Endoplasmic Reticulum Stress Interacts with Inflammation in Human Diseases
    • Abstract: The endoplasmic reticulum (ER) is a critical organelle for normal cell function and homeostasis. Disturbance in the protein folding process in the ER, termed ER stress, leads to the activation of unfolded protein response (UPR) that encompasses a complex network of intracellular signaling pathways. The UPR can either restore ER homeostasis or activate pro‐apoptotic pathways depending on the type of insults, intensity and duration of the stress, and cell types. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune, infectious, neurodegenerative, and metabolic disorders. In the cell, ER stress and inflammatory signaling share extensive regulators and effectors in a broad spectrum of biological processes. In spite of different etiologies, the two signaling pathways have been shown to form a vicious cycle in exacerbating cellular dysfunction and causing apoptosis in many cells and tissues. However, the interaction between ER stress and inflammation in many of these diseases remains poorly understood. Further understanding of the biochemistry, cell biology, and physiology may enable the development of novel therapies that spontaneously target these pathogenic pathways. This article is protected by copyright. All rights reserved
  • Selective Life‐Long Skeletal Myofiber – Targeted VEGF Gene
           Ablation Impairs Exercise Capacity in Adult Mice
    • Abstract: Exercise is dependent on adequate oxygen supply for mitochondrial respiration in both cardiac and locomotor muscle. To determine whether skeletal myofiber VEGF is critical for regulating exercise capacity, independent of VEGF function in the heart, ablation of the VEGF gene was targeted to skeletal myofibers (skmVEGF‐/‐) during embryogenesis (∼ E9.5), leaving intact VEGF expression by all other cells in muscle. In adult mice, VEGF levels were decreased in the soleus (by 65%), plantaris (94%), gastrocnemius (74%), EDL (99%) and diaphragm (64%) (p 
  • Reciprocal Control of Osteogenic and Adipogenic Differentiation by ERK/MAP
           Kinase Phosphorylation of Runx2 and PPARγ Transcription Factors
    • Abstract: In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPARγ, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPARγ. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPARγ phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPARγ‐dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant‐negative suppression of activity had the opposite effect. In rescue experiments using Runx2‐/‐ mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation‐deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation‐resistant S112A mutant PPARγ strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg ‐/‐ MEFs, while S112E mutant PPARγ was less active. Competition between RUNX2 and PPARγ was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPARγ phosphorylation in the control of osteoblast and adipocyte lineages. This article is protected by copyright. All rights reserved
  • CyclinD1 Down Regulation and Increased Apoptosis Are Common Features of
    • Abstract: Genetic variants within components of the cohesin complex (NIPBL, SMC1A, SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a spectrum of human syndromes known as “cohesinopathies” that includes Cornelia de Lange Syndrome (CdLS). CdLS is a multiple malformation syndrome affecting almost any organ and causing severe developmental delay. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in cohesin components. However, it is still unclear how mutations in different components of the cohesin complex affect the output of gene regulation. In this study, zebrafish embryos and SMC1A‐mutated patient‐derived fibroblasts were used to analyze abnormalities induced by SMC1A loss of function. We show that the knockdown of smc1a in zebrafish impairs neural development, increases apoptosis and specifically down‐regulates Ccnd1 levels. The same down‐regulation of cohesin targets is observed in SMC1A‐mutated patient fibroblasts. Previously, we have demonstrated that haploinsufficiency of NIPBL produces similar effects in zebrafish and in patients fibroblasts indicating a possible common feature for neurological defects and mental retardation in cohesinopathies. Interestingly, expression analysis of Smc1a and Nipbl in developing mouse embryos reveals a specific pattern in the hindbrain, suggesting a role for cohesin in neural development in vertebrates. This article is protected by copyright. All rights reserved
  • Osteogenic Potential of Human Oral‐Periosteal Cells (PCs) Isolated
           from Different Oral Origin: An In Vitro Study
    • Abstract: The periosteum is a specialized connective tissue containing multipotent stem cells capable of bone formation. In this study, we aimed at demonstrating that human oral periosteal cells derived from three different oral sites (upper vestibule, lower vestibule and hard palate) represent an innovative cell source for maxillo‐facial tissue engineering applications in terms of accessibility and self‐commitment towards osteogenic lineage. Periosteal cells (PCs) were isolated from patients with different ages (20‐30 yy, 40‐50 yy, 50‐60 yy); we then analyzed the in vitro proliferation capacity and the bone self‐commitment of cell clones culturing them without any osteogenic supplement to support their differentiation. We found that oral PCs, independently of their origin and age of patients, are mesenchymal stem cells with stem cell characteristics (clonogenical and proliferative activity) and that, even in absence of any osteogenic induction, they undertake the osteoblast lineage after 45 days of culture. These results suggest that oral periosteal cells could replace mesenchymal cells from bone marrow in oral tissue‐engineering applications. This article is protected by copyright. All rights reserved
  • Cellular and Molecular Mechanisms of Phenotypic Switch in Gastrointestinal
           Smooth Muscle
    • Abstract: As a general rule, smooth muscle cells (SMC) are able to switch from a contractile phenotype to a less mature synthetic phenotype. This switch is accompanied by a loss of differentiation with decreased expression of contractile markers, increased proliferation as well as the synthesis and the release of several signaling molecules such as pro‐inflammatory cytokines, chemotaxis‐associated molecules and growth factors. This SMC phenotypic plasticity has extensively been investigated in vascular diseases but interest is also emerging in the field of gastroenterology. It has in fact been postulated that altered micro‐environmental conditions, including the composition of microbiota, could trigger the remodeling of the enteric SMC, with phenotype changes and consequent alterations of contraction and impairment of gut motility. Several molecular actors participate in this phenotype remodeling. These include extracellular molecules such as cytokines and extracellular matrix proteins, as well as intracellular proteins, e.g. transcription factors. Epigenetic control mechanisms and miRNA have also been suggested to participate. In this review key roles and actors of smooth muscle phenotypic switch, mainly in GI tissue, are described and discussed in the light of literature data available so far. This article is protected by copyright. All rights reserved
  • The PPARβ/δ Agonist GW0742 Induces Early Neuronal Maturation of
           Cortical Post‐Mitotic Neurons: Role of PPARβ/δ in Neuronal
    • Abstract: Increasing evidences support that signalling lipids participate in synaptic plasticity and cell survival, and that the lipid signalling is closely associated with neuronal differentiation, learning and memory and with pathologic events such as epilepsy and Alzheimer's disease. The Peroxisome Proliferator‐Activated Receptors (PPAR) are strongly involved in the fatty acid cell signalling, as many of the natural lypophylic compounds are PPAR ligands. We have previously shown that PPARβ/δ is the main isotype present in cortical neuron primary cultures and that during neuronal maturation, PPARβ/δ is gradually increased and activated. To get more insight into the molecular mechanism by which PPARβ/δ may be involved in neuronal maturation processes, in this work a specific PPARβ/δ agonist, GW0742 was used administered alone or in association with a specific PPARβ/δ antagonist, the GSK0660 and the parameters involved in neuronal differentiation and maturation were assayed. The data obtained demonstrated the strong involvement of PPARβ/δ in neuronal maturation, triggering the agonist an anticipation of neuronal differentiation and the antagonist abolishing the observed effects. These effects appear to be mediated by the activation of both BDNF canonical (BDNF7TrkB) pathway. This article is protected by copyright. All rights reserved
  • IL‐6 Activates PI3K and PKCζ Signaling and Determines Cardiac
           Differentiation in Rat Embryonic H9c2 Cells
    • Abstract: Introduction IL‐6 influences several biological processes, including cardiac stem cell and cardiomyocyte physiology. Although JAK‐STAT3 activation is the defining feature of IL‐6 signaling, signaling molecules such as PI3K, PKCs and ERK1/2 are also activated and elicit different responses. Moreover, most studies on the specific role of these signaling molecules focus on the adult heart, and few studies are available on the biological effects evoked by IL‐6 in embryonic cardiomyocytes. Aim The aim of this study was to clarify the biological response of embryonic heart derived cells to IL‐6 by analyzing the morphological modifications and the signaling cascades evoked by the cytokine in H9c2 cells. Results IL‐6 stimulation determined the terminal differentiation of H9c2 cells, as evidenced by the increased expression of cardiac transcription factors (NKX2.5 and GATA4), structural proteins (α‐myosin heavy chain and cardiac Troponin T) and the gap junction protein Connexin 43. This process was mediated by the rapid modulation of PI3K, Akt, PTEN and PKCζ phosphorylation levels. PI3K recruitment was an upstream event in the signaling cascade and when PI3K was inhibited, IL‐6 failed to modify PKCζ, PTEN and Akt phosphorylation. Blocking PKCζ activity affected only PTEN and Akt. Finally, the overexpression of a constitutively active form of PKCζ in H9c2 cells largely mimicked the morphological and molecular effects evoked by IL‐6. Conclusion This study demonstrated that IL‐6 induces the cardiac differentiation of H9c2 embryonic cells though a signaling cascade that involves PI3K, PTEN and PKCζ activities. This article is protected by copyright. All rights reserved
  • RhoA‐Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate
    • Abstract: Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho‐family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M) and nanotopography (N) to evaluate the effect of surface topography on RhoA‐mediated functions (cell spreading, adhesion, migration and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S< M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness‐dependent (S 
  • D‐Aspartate Induces Proliferative Pathways in Spermatogonial
           GC‐1 Cells
    • Abstract: D‐aspartate (D‐Asp) is an endogenous amino acid present in vertebrate tissues, with particularly high levels in the testis. In vivo studies indicate that D‐Asp indirectly stimulates spermatogenesis through the hypothalamic‐pituitary‐gonadal axis. Moreover, in vitro studies have demonstrated that D‐Asp up‐regulates testosterone production in Leydig cells by enhancing expression of the steroidogenic acute regulatory protein. In this study, a cell line derived from immortalized type‐B mousespermatogonia retaining markers of mitotic germ cells (GC‐1) was employed to explore more direct involvement of D‐Asp in spermatogenesis.Activity and protein expression of markers of cell proliferation were determined at intervals during incubation in D‐Asp‐containing medium. D‐Asp induced phosphorylation of ERK and Akt proteins, stimulated expression of PCNA and Aurora B, and enhanced mRNA synthesis and protein expression of P450 aromatase and protein expressionof Estrogen Receptor β (ERβ). These results are the first demonstration of a direct effect of D‐Asp on spermatogonial mitotic activity. Considering thatspermatogonia express the NR1 subunit of the N‐Methyl‐D‐Aspartic Acid receptor (NMDAR), we suggest that their response to D‐Asp depends on NMDAR‐mediated activation of the ERK and Akt pathways and is further enhanced by activation of the P450 aromatase/ERβ pathway. This article is protected by copyright. All rights reserved
  • Hydroxytyrosol Inhibits Cannabinoid CB1 Receptor Gene Expression in
           3T3‐L1 Preadipocyte Cell Line
    • Abstract: The 3T3‐L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3‐L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR‐γ receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity‐associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3‐L1 pre‐adipocytes the effects of n‐3‐PUFA, α‐Linolenic acid (OM‐3), n‐6‐PUFA, Linoleic acid (OM‐6) and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis‐related genes PPAR‐γ, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3‐L1 cell differentiation by down‐regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti‐adipogenic effects, whereas OM‐3 and OM‐6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM‐3 and OM‐6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription. This article is protected by copyright. All rights reserved
  • Beta Adrenergic Receptor Stimulation Suppresses Cell Migration in
           Association With Cell Cycle Transition in Osteoblasts – Live Imaging
           Analyses Based on FUCCI System
    • Abstract: Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed‐ridden conditions in patients suffering from aging‐associated diseases including cardiovascular, neurological and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed‐ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis is still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppresses bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, FUCCI (Fluorescent Ubiquitination‐based Cell Cycle Indicator), in osteoblast biology and examined isoproterenol regulation of cell cycle transition cell migration in osteoblasts. Isoproterenol treatment suppresses the first entry peak of quiescent osteoblastic cells into cell cycle phase from G1/G0 to S/G2/M and also suppresses second major peak population size that enters into S/G2/M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1/S nor in G2/M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system. This article is protected by copyright. All rights reserved
  • Nitric Oxide Up‐Regulates RUNX2 in LNCaP Prostate Tumours:
           Implications for Tumour Growth In Vitro and In Vivo
    • Abstract: Background Aberrant expression of the transcription factor RUNX2 in prostate cancer has a number of important consequences including increased resistance to apoptosis, invasion and metastasis to bone. We previously demonstrated that hypoxia up‐regulated RUNX2 in tumour cells, which in turn up‐regulated the anti‐apoptotic factor Bcl‐2. Here, we investigate the impact of nitric oxide (NO) on RUNX2 and Bcl‐2 expression in prostate cancer and further, how RUNX2 over‐expression can impact tumour growth, angiogenesis and oxygenation in vivo. Methods The effect of NO levels on RUNX2 and thus Bcl‐2 expression was examined in prostate cancer cells in vitro using methods including gene and protein expression analyses, nitrite quantitation, protein‐DNA interaction assays (ChIP) and viability assays (XTT). The effect of RUNX2 over‐expression on tumour physiology (growth, oxygenation and angiogenesis) was also assessed in vivo using LNCaP xenografts. Results A low (but not high) concentration of NO (10µM) induced expression of RUNX2 and Bcl‐2, conferring resistance to docetaxel. These effects were induced via the ERK and PI3K pathways and were dependent on intact AP‐1 binding sites in the RUNX2 promoter. RUNX2 over‐expression in LNCaP tumours in vivo decreased the time to tumour presentation and increased tumour growth. Moreover, these tumours exhibited improved tumour angiogenesis and oxygenation. Conclusion Low levels of NO increase expression of RUNX2 and Bcl‐2 in LNCaP prostate tumour cells, and in vivo up‐regulation of RUNX2 created tumours with a more malignant phenotype. Collectively, our data reveals the importance of NO‐regulation of key factors in prostate cancer disease progression. This article is protected by copyright. All rights reserved
  • Selective β2‐AR Blockage Suppresses Colorectal Cancer Growth
           through Regulation of EGFR‐Akt/ERK1/2 Signaling, G1‐phase
           Arrest, and Apoptosis
    • Abstract: The stress‐upregulated catecholamines‐activated β1‐ and β2‐adrenergic receptors (β1/2‐ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of β1/2‐ARs signaling for the treatment of CRC and elucidated the significance of β2‐AR expression in CRC in vitro and in clinical samples. The impacts of β1/2‐AR antagonists in CRC in vitro and CRC‐xenograft in vivo were examined. We found that repression of β2‐AR but not β1‐AR signaling selectively suppressed cell viability, induced G1‐phase cell cycle arrest, caused both intrinsic and extrinsic pathways‐mediated apoptosis of specific CRC cells and inhibited CRC‐xenograft growth in vivo. Moreover, the expression of β2‐AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidences that the expression profiles, signaling, and blockage of β2‐AR have a unique pattern in CRC comparing to other cancers. β2‐AR antagonism selectively suppresses the growth of CRC accompanying active β2‐AR signaling, which potentially carries wild‐type KRAS, in vitro and in vivo via the inhibition of β2‐AR transactivated EFGR‐Akt/ERK1/2 signaling pathway. Thus, β2‐AR blockage might be a potential therapeutic strategy for combating the progressions of β2‐AR‐dependent CRC. This article is protected by copyright. All rights reserved
  • Intravenous Immunoglobulin (IVIG) Attenuates TNF‐Induced Pathologic
           Bone Resorption and Suppresses Osteoclastogenesis by Inducing A20
    • Abstract: Investigations on the therapeutic effects of intravenous immunoglobulin (IVIG) have focused on the suppression of autoantibody‐ and immune complex‐mediated inflammatory pathogenesis. Inflammatory diseases such as rheumatoid arthritis are often accompanied by excessive bone erosion but the effect of IVIG on osteoclasts, bone‐resorbing cells, has not been studied. Here, we investigate whether IVIG directly regulates osteoclast differentiation and has therapeutic potential for suppressing osteoclast‐mediated pathologic bone resorption. IVIG or cross‐linking of Fcγ receptors with plate‐bound IgG suppressed receptor activator of nuclear factor‐kappa B ligand (RANKL)‐induced osteoclastogenesis and expression of osteoclast‐related genes such as integrin β3 and cathepsin K in a dose‐dependent manner. Mechanistically, IVIG or plate‐bound IgG suppressed osteoclastogenesis by downregulating RANKL‐induced expression of NFATC1, the master regulator of osteoclastogenesis. IVIG suppressed NFATC1 expression by attenuating RANKL‐induced NF‐κB signaling, explained in part by induction of the inflammatory signaling inhibitor A20. IVIG administration attenuated in vivo osteoclastogenesis and suppressed bone resorption in the tumor necrosis factor (TNF)‐induced calvarial osteolysis model. Our findings show that, in addition to suppressing inflammation, IVIG directly inhibits osteoclastogenesis through a mechanism involving suppression of RANK signaling. Direct suppression of osteoclast differentiation may provide beneficial effects on preserving bone mass when IVIG is used to treat rheumatic disorders. This article is protected by copyright. All rights reserved
  • Distinct Roles for Intestinal Epithelial Cell‐Specific Hdac1 and
           Hdac2 in the Regulation of Murine Intestinal Homeostasis
    • Abstract: The intestinal epithelium responds to and transmits signals from the microbiota and the mucosal immune system to insure intestinal homeostasis. These interactions are in part conveyed by epigenetic modifications, which respond to environmental changes. Protein acetylation is an epigenetic signal regulated by histone deacetylases, including Hdac1 and Hdac2. We have previously shown that villin‐Cre‐inducible intestinal epithelial cell (IEC)‐specific Hdac1 and Hdac2 deletions disturb intestinal homeostasis. To determine the role of Hdac1 and Hdac2 in the regulation of IEC function and the establishment of the dual knockout phenotype, we have generated villin‐Cre murine models expressing one Hdac1 allele without Hdac2, or one Hdac2 allele without Hdac1. We have also investigated the effect of short‐term deletion of both genes in naphtoflavone‐inducible Ah‐Cre and tamoxifen‐inducible villin‐CreER mice. Mice with one Hdac1 allele displayed normal tissue architecture, but increased sensitivity to DSS‐induced colitis. In contrast, mice with one Hdac2 allele displayed intestinal architecture defects, increased proliferation, decreased goblet cell numbers as opposed to Paneth cells, increased immune cell infiltration associated with fibrosis, and increased sensitivity to DSS‐induced colitis. In comparison to dual knockout mice, intermediary activation of Notch, mTOR and Stat3 signaling pathways was observed. While villin‐CreER Hdac1 and Hdac2 deletions led to an impaired epithelium and differentiation defects, Ah‐Cre‐mediated deletion resulted in blunted proliferation associated with the induction of a DNA damage response. Our results suggest that IEC determination and intestinal homeostasis are highly dependent on Hdac1 and Hdac2 activity levels, and that changes in the IEC acetylome may alter the mucosal environment. This article is protected by copyright. All rights reserved
  • S‐Adenosylmethionine Affects ERK1/2 and STAT3 Pathways and Induces
           Apoptosis in Osteosarcoma Cells
    • Abstract: Osteosarcoma is a very aggressive bone tumor. Its clinical outcome remains discouraging despite intensive surgery, radiotherapy and chemotherapy. Thus, novel therapeutic approaches are demanded. S‐Adenosylmethionine (AdoMet) is a naturally occurring molecule that is synthesized in our body by methionine adenosyltransferase isoenzymes and is also available as a nutritional supplement. AdoMet is the principal methyl donor in numerous methylation reactions and is involved in many biological functions. Interestingly, AdoMet has been shown to exert antiproliferative action in various cancer cells. However, the underlying molecular mechanisms are just starting to be studied. Here, we investigated the effects of AdoMet on the proliferation of osteosarcoma U2OS cells and the underlying mechanisms. We carried out direct cell number counting, MTT and flow cytometry‐based assays, and immunoblotting experiments in response to AdoMet treatment. We found that AdoMet strongly inhibits proliferation of U2OS cells by slowing‐down cell cycle progression and by inducing apoptosis. We also report that AdoMet consistently causes an increase of p53 and p21 cell‐cycle inhibitor, a decrease of cyclin A and cyclin E protein levels, and a marked increase of pro‐apoptotic Bax/Bcl‐2 ratio, with caspase‐3 activation and PARP cleavage. Moreover, the AdoMet‐induced antiproliferative effects were dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels. Altogether, our data enforce the evidence of AdoMet acting as a biomolecule with antiproliferative action in osteosarcoma cells, capable of down‐regulating ERK1/2 and STAT3 pathways leading to cell cycle inhibition and apoptosis, and provide a rationale for the possible use of AdoMet in osteosarcoma therapy. This article is protected by copyright. All rights reserved
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