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Journal Cover Journal of Cellular Physiology
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   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]   [SJR: 1.608]   [H-I: 118]
  • Characterization of Cardiac Anoctamin1 Ca2+-activated Chloride Channels
           and Functional Role in Ischemia-Induced Arrhythmias
    • Authors: Zhen Ye; Ming-Ming Wu, Chun-Yu Wang, Yan-Chao Li, Chang-Jiang Yu, Yuan-Feng Gong, Jun Zhang, Qiu-Shi Wang, Bin-Lin Song, Kuai Yu, H. Criss Hartzell, Dayue Darrel Duan, Dan Zha, Zhi-Ren Zhang
      Pages: n/a - n/a
      Abstract: Anoctamin1 (ANO1) encodes a Ca2+-activated chloride (Cl-) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1-encoded CaCCs in ischemia-induced arrhythmias in the heart. Quantitative real-time RT-PCR, immunofluorescence staining assays and immunohistochemisrty identified the molecular expression, location and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch-clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca2+-activated Cl- current (ICl.Ca) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of ICl.Ca, which was inhibited by a specific ANO1 inhibitor, T16Ainh-A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia-induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper “spike and dome” compared to control cardiomyocytes from non-ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia-induced early phase 1 repolarization acceleration and caused a much shallower “spike and dome”. We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia-induced increase in ANO1 expression may be responsible for the increased density of ICl.Ca in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T10:20:25.073373-05:
      DOI: 10.1002/jcp.24709
  • Function of Latent TGFβ Binding Protein 4 and Fibulin 5 in
           Elastogenesis and Lung Development
    • Authors: Branka Dabovic; Ian B. Robertson, Lior Zilberberg, Melinda Vassallo, Elaine C. Davis, Daniel B Rifkin
      Pages: n/a - n/a
      Abstract: Mice deficient in Latent TGFβ Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFβ2 levels. However, the elastic fiber assembly is not improved in Tgfb2-/-;Ltbp4S-/- compared to Ltbp4S-/- lungs. We found that decreased levels of TGFβ1 or TGFβ3 did not improve lung septation indicating that the TGFβ isoform elevated in Ltbp4S-/- lungs is TGFβ2. Expression of a form of Ltbp4 that could not bind latent TGFβ did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFβ complexes. Therefore, the change in TGFβ-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S-/- mutation with a fibulin-5 null mutant in Fbln5-/-;Ltbp4S-/- mice improves the lung septation compared to Ltbp4S-/- lungs. Large globular elastin aggregates characteristic for Ltbp4S-/- lungs do not form in Fbln5-/-;Ltbp4S-/- lungs and EM studies showed that elastic fibers in Fbln5-/-;Ltbp4S-/- lungs resemble those found in Fbln5-/- mice. These results are consistent with a role for TGFβ2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:59:02.003792-05:
      DOI: 10.1002/jcp.24704
  • M-CSF priming of osteoclast precursors can cause
           osteoclastogenesis-insensitivity, which can be prevented and overcome on
    • Authors: Teun J. de Vries; Ton Schoenmaker, David Aerts, Lilyanne C. Grevers, Pedro P.C. Souza, Kamran Nazmi, Mark van de Wiel, Bauke Ylstra, Peter L. van Lent, Pieter J.M. Leenen, Vincent Everts
      Pages: n/a - n/a
      Abstract: Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:58:43.763942-05:
      DOI: 10.1002/jcp.24702
  • The regulation of TGFβ1 induced fibronectin EDA exon alternative
           splicing in human renal proximal tubule epithelial cells
    • Authors: Mysore Keshavmurthy Phanish; Felicia Heidebrecht, Mohammad E Nabi, Nileshkumar Shah, Ioana Niculescu-Duvaz, Mark Edward Carl Dockrell
      Pages: n/a - n/a
      Abstract: The EDA+ splice variant of fibronectin (Fn) is an early and important component of the extracellular matrix in renal fibrosis. In this work, we investigate cellular mechanisms of EDA+ Fn production in human primary proximal tubule epithelial cells (PTECs). TGFβ1-induced EDA+Fn production was assessed by immunocytochemistry, PCR and Western blotting. SRp40 knockdown was achieved by siRNA. The role of the PI3 kinase-AKT signalling and splicing regulatory protein SRp40 in the production of EDA+ Fn was studied by using the chemical inhibitor LY294002 and siRNA targeted to SRp40 respectively. Interaction between PI3 kinase-AKT signalling and SRp40 were assessed by immunofluorescence and immunoprecipitation. To assess the specificity of SRp40 in regulating the splicing of EDA+ exon, we studied the effect of SRp40 knockdown on TGFβ1 induced splicing of FGF receptor 2. Primary human PTECs expressed EDA+ and EDA- Fn. TGFβ1 treatment resulted in increases in the production and deposition of EDA+ Fn as well as an increase in the ratio of EDA+/EDA- Fn mRNA. The TGFβ1 induced EDA+ production was dependent on PI3 kinase-AKT signalling and SRp40 expression. Immunoprecipitation experiments demonstrated direct binding between AKT and SRp40 with an increase in the amount of SRp40 bound to AKT upon TGFβ1 treatment. TGFβ1 treatment resulted in reduction in the FGF receptor2 IIIb splice variant which was unaffected by SRp40 knockdown. In this work, we have presented the first evidence for the regulation of Fn pre-mRNA splicing by PI3 kinase-AKT signalling and SRp40 in human PTECs. Targeting the splicing of Fn pre-mRNA to skip the EDA exon is an attractive option to combat fibrosis. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:58:29.371336-05:
      DOI: 10.1002/jcp.24703
  • Contribution of Sp1 to Telomerase Expression and Activity in Skin
           Keratinocytes Cultured with a Feeder Layer
    • Authors: Francis Bisson; Claudie Paquet, Jean-Michel Bourget, Karine Zaniolo, Patrick J. Rochette, Solange Landreville, Odile Damour, François Boudreau, François A. Auger, Sylvain L. Guérin, Lucie Germain
      Pages: n/a - n/a
      Abstract: The growth of primary keratinocytes is improved by culturing them with a feeder layer. The aim of this study was to assess whether the feeder layer increases the lifespan of cultured epithelial cells by maintaining or improving telomerase activity and expression. The addition of an irradiated fibroblast feeder layer of either human or mouse origin (i3T3) helped maintain telomerase activity as well as expression of the transcription factor Sp1 in cultured keratinocytes. In contrast, senescence occurred earlier, together with a reduction of Sp1 expression and telomerase activity, in keratinocytes cultured without a feeder layer. Telomerase activity was consistently higher in keratinocytes grown on the three different feeder layers tested relative to cells grown without them. Suppression of Sp1 expression by RNA inhibition (RNAi) reduced both telomerase expression and activity in keratinocytes and also abolished their long-term growth capacity suggesting that Sp1 is a key regulator of both telomerase gene expression and cell cycle progression of primary cultured human skin keratinocytes. The results of the present study therefore suggest that the beneficial influence of the feeder layer relies on its ability to preserve telomerase activity in cultured human keratinocytes through the maintenance of stable levels of Sp1 expression. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:58:15.623077-05:
      DOI: 10.1002/jcp.24706
  • TGF-beta1 Suppresses Plasmin and MMP Activity in Flexor Tendon Cells via
           PAI-1: Implications for Scarless Flexor Tendon Repair
    • Authors: Youssef M. Farhat; Alaa A. Al-Maliki, Anas Easa, Regis J. O'Keefe, Edward M. Schwarz, Hani A. Awad
      Pages: n/a - n/a
      Abstract: Flexor tendon injuries caused by deep lacerations to the hands are a challenging problem as they often result in debilitating adhesions that prevent the movement of the afflicted fingers. Evidence exists that tendon adhesions as well as scarring throughout the body are largely precipitated by the pleiotropic growth factor, TGF-β1, but the effects of TGF-β1 are poorly understood in tendon healing. Using an in vitro model of tendon healing, we previously found that TGF-β1 causes gene expression changes in tenocytes that are consistent with scar tissue and adhesion formation, including upregulation of the anti-fibrinolytic protein, PAI-1. Therefore, we hypothesized that TGF-β1 contributes to scarring and adhesions by reducing the activity of proteases responsible for ECM degradation and remodeling, such as plasmin and MMPs, via upregulation of PAI-1. To test our hypothesis, we examined the effects of TGF-β1 on the protease activity of tendon cells. We found that flexor tendon tenocytes treated with TGF-β1 had significantly reduced levels of active MMP-2 and plasmin. Interestingly, the effects of TGF-β1 on protease activity were completely abolished in tendon cells from homozygous PAI-1 KO mice, which are unable to express PAI-1. Our findings support the hypothesis that TGF-β1 induces PAI-1, which suppresses plasmin and plasmin-mediated MMP activity, and provide evidence that PAI-1 may be a novel therapeutic target for preventing adhesions and promoting a scarless, regenerative repair of flexor tendon injuries. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:57:12.284745-05:
      DOI: 10.1002/jcp.24707
  • Glycated collagen induces α11 integrin expression through TGF-β2
           and Smad3
    • Authors: Ilana Talior-Volodarsky; Pamma D. Arora, Yongqiang Wang, Cédric Zeltz, Kim A. Connelly, Donald Gullberg, Christopher A. McCulloch
      Pages: n/a - n/a
      Abstract: The adhesion of cardiac fibroblasts to the glycated collagen interstitium in diabetics is associated with de novo expression of the α11 integrin, myofibroblast formation and cardiac fibrosis. We examined how methylglyoxal-glycated collagen regulates α11 integrin expression. In cardiac fibroblasts plated on glycated collagen but not glycated fibronectin, there was markedly increased α11 integrin and α-smooth muscle actin expression. Compared with native collagen, binding of purified α11β1 integrin to glycated collagen was reduced by>4-fold, which was consistent with reduced fibroblast attachment to glycated collagen. Glycated collagen strongly enhanced the expression of TGF-β2 but not TGF-β1 or TGF-β3. The increased expression of TGF-β2 was inhibited by triple helical collagen peptides that mimic the α11β1 integrin binding site on type I collagen. In cardiac fibroblasts transfected with α;11 integrin luciferase promoter constructs, glycated collagen activated the α11 integrin promoter. Analysis of α11 integrin promoter truncation mutants showed a novel Smad2/3 binding site located between -809 and -1300 nt that was required for promoter activation. We conclude that glycated collagen in the cardiac interstitium triggers an autocrine TGF-β2 signalling pathway that stimulates α11 integrin expression through Smad2/3 binding elements in the α11 integrin promoter, which is important for myofibroblast formation and fibrosis. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:56:39.920407-05:
      DOI: 10.1002/jcp.24708
  • Intrinsic Sex-Linked Variations in Osteogenic and Adipogenic
           Differentiation Potential of Bone Marrow Multipotent Stromal Cells
    • Authors: Beth Bragdon; Robert Burns, Amelia H. Baker, Anna C. Belkina, Elise F. Morgan, Gerald V. Denis, Louis C. Gerstenfeld, Jennifer J. Schlezinger
      Pages: n/a - n/a
      Abstract: Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSC) in bone is lacking. This study assessed sex-linked differences in MSC differentiation in 3-, 6-, and 9-month-old C57BL/6J mice. Analysis of tibiae showed that female mice had lower bone volume fraction and higher adipocyte content in the bone marrow compared to age-matched males. While both males and females lost bone mass in early aging, the rate of loss was higher in males. Similar expression of bone- and adipocyte-related genes was seen in males and females at 3 and 9 months, while at 6 months, females exhibited a two-fold greater expression of these genes. Under osteogenic culture conditions, bone marrow MSCs from female 3- and 6-month-old mice expressed similar levels of bone-related genes, but significantly greater levels of adipocyterelated genes, than male MSCs. Female MSCs also responded to rosiglitazone-induced suppression of osteogenesis at a 5-fold lower (10 nM) concentration than male MSCs. Female MSCs grown in estrogen-stripped medium showed similar responses to rosiglitazone as MSCs grown in serum containing estrogen. MSCs from female mice that had undergone ovariectomy before sexual maturity also were sensitive to rosiglitazone-induced effects on osteogenesis. These results suggest that female MSCs are more sensitive to modulation of differentiation by PPARγ and that these differences are intrinsic to the sex of the animal from which the MSCs came. These results also may explain the sensitivity of women to the deleterious effects of rosiglitazone on bone. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:56:21.032911-05:
      DOI: 10.1002/jcp.24705
  • Ghrelin augments the expressions and secretions of proinflammatory
           adipokines, VEGF120 and MCP-1, in differentiated 3T3-L1 adipocytes.
    • Authors: Atsuko Kitahara; Kazuto Takahashi, Rie Moriya, Hirohisa Onuma, Keiko Handa, Yoshikazu Sumitani, Toshiaki Tanaka, Hidenori Katsuta, Susumu Nishida, Takuya Sakurai, Kouichi Inukai, Hideki Ohno, Hitoshi Ishida
      Pages: n/a - n/a
      Abstract: Ghrelin is a physiological-active peptide with growth hormone-releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin-direct-effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3-L1 adipocytes stimulated with ghrelin in vitro. The stimulation with 50 nmol/L ghrelin for 24 h resulted in the significant 1.9-fold increase on vascular endothelial growth factor-120 (VEGF120) releases (p 
      PubDate: 2014-06-23T07:12:03.363548-05:
      DOI: 10.1002/jcp.24699
  • Loss of GLUT4 induces metabolic reprogramming and impairs viability of
           breast cancer cells
    • Authors: Pablo Garrido; Fernando G. Osorio, Javier Morán, Estefanía Cabello, Ana Alonso, José M.P. Freije, Celestino González
      Pages: n/a - n/a
      Abstract: Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and constitute promising targets for cancer treatment. Here we demonstrate that the glucose transporter 4 (GLUT4) has a prominent role in basal glucose uptake in MCF7 and MDA-MB-231 breast cancer cells. We show that shRNA-mediated down-regulation of GLUT4 diminishes glucose uptake and induces metabolic reprogramming by reallocating metabolic flux to oxidative phosphorylation. This reallocation is reflected on an increased activity of the mitochondrial oxidation of pyruvate and lower lactate release. Altogether, GLUT4 inhibition compromises cell proliferation and critically affects cell viability under hypoxic conditions, providing proof-of-principle for the feasibility of using pharmacological approaches to inhibit GLUT4 in order to induce metabolic reprogramming in vivo in breast cancer models. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-13T05:54:04.090888-05:
      DOI: 10.1002/jcp.24698
  • Enhancement of Runx2 expression is potentially linked to β-catenin
           accumulation in canine intervertebral disc degeneration
    • Authors: Munetaka Iwata; Takeshi Aikawa, Takaharu Hakozaki, Kiyotaka Arai, Hiroki Ochi, Hirotaka Haro, Masahiro Tagawa, Yoshinori Asou, Yasushi Hara
      Pages: n/a - n/a
      Abstract: Intervertebral disc degeneration (IVDD) greatly affects the quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP because the cells disappear with age and are replaced by fibrochondrocyte-like cells. Because IVDD develops as early as within the first year of life, we used canines as a model to investigate the in vitro mechanisms underlying IVDD. The mechanism underlying age-related IVDD, however, is poorly understood. Several research groups have suggested that Wnt/β-catenin signaling plays an important role in IVDD. However, the role of Wnt/β-catenin signals in IVD cells is not yet well understood. Here, we demonstrate that Wnt/β-catenin signaling could enhance Runx2 expression in IVDD and lead to IVD calcification. NP tissue was obtained from Beagle dogs after evaluation of the degeneration based on magnetic resonance imaging (MRI). Histological analysis showed that lack of Safranin-O staining, calcified area, and matrix metalloproteinase13-positive cells increased with progression of the degeneration. Furthermore, the levels of β-catenin- and Runx2-positive cells also increased. Real-time reverse-transcription polymerase chain reaction analysis showed that the MRI signal intensity and mRNA expression levels of β-catenin and Runx2 are correlated in NP tissues. Moreover, supplementation of LiCl induced β-catenin accumulation and Runx2 expression. In contrast, FH535 inhibited LiCl-induced upregulation. These results suggest that Runx2 transcript and protein expression, potentially in combination with β-catenin accumulation, are enhanced in degenerated and calcified intervertebral discs of CDBs. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-11T04:03:49.549056-05:
      DOI: 10.1002/jcp.24697
  • Nestin is a Marker of Lung Remodeling Secondary to Myocardial Infarction
           and Type I Diabetes in the Rat
    • Authors: Andréanne Chabot; Marc-Andre Meus, Patrice Naud, Vanessa Hertig, Jocelyn Dupuis, Louis Villeneuve, Nabil Elkhoury, Celine Fiset, Stanley Nattel, Jean-Francois Jasmin, Angelino Calderone
      Pages: n/a - n/a
      Abstract: Upregulation of the intermediate filament protein nestin was identified in a subpopulation of fibroblasts during reactive and reparative fibrosis and directly contributed to the enhanced proliferative phenotype. The present study tested the hypothesis that nestin was expressed in lung fibroblasts and the pattern of expression represented a distinct marker of pulmonary remodeling secondary to myocardial infarction and type I diabetes. Nestin(+) fibroblasts were detected in rat lungs and a subpopulation exhibited a myofibroblast phenotype delineated by the co-expression of smooth muscle α-actin. In the lungs of myocardial infarcted rats, interstitial collagen content and nestin mRNA/protein levels were significantly increased despite the absence of secondary pulmonary hypertension, whereas smooth muscle α-actin protein expression was unchanged. Exposure of rat pulmonary fibroblasts to pro-fibrotic stimuli angiotensin II and transforming growth factor-β significantly increased nestin protein levels. In the lungs of type I diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of nestin mRNA/protein expression. Nestin was reported a target of miR-125b, albeit miR-125b levels were unchanged in pulmonary fibroblasts treated with pro-fibrotic stimuli. Nestin(+) cells lacking smooth muscle α-actin/collagen staining were also identified in rodent lungs and a transgenic approach revealed that expression of the intermediate filament protein was driven by intron 2 of the nestin gene. The disparate regulation of nestin characterized a distinct pattern of pulmonary remodeling secondary to myocardial infarction and type I diabetes and upregulation of the intermediate filament protein in lung fibroblasts may have facilitated in part the reactive fibrotic response. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-11T03:55:59.250545-05:
      DOI: 10.1002/jcp.24696
  • Loss of p53 Enhances NF‐κB‐Dependent Lamellipodia
  • Glucosamine‐Induced Sp1 O‐GlcNAcylation Ameliorates
           Hypoxia‐Induced SGLT Dysfunction in Primary Cultured Renal Proximal
           Tubule Cells
    • Abstract: The aim of this study is to determine whether GlcN could recover the endoplasmic reticulum (ER) stress‐induced dysfunction of Na+/glucose cotransporter (SGLT) in renal proximal tubule cells (PTCs) under hypoxia. With the rabbit model, the renal ischemia induced tubulointerstitial abnormalities and decreased SGLTs expression in tubular brush‐border, which were recovered by GlcN. Thus, the protective mechanism of GlcN against renal ischemia was being examined by using PTCs. Hypoxia decreased the level of protein O‐GlcNAc and the expression of O‐GlcNAc transferase (OGT) while increased O‐GlcNAcase (OGA) and these were reversed by GlcN. Hypoxia also decreased the expression of SGLTs (SGLT1 and 2) and [14C]‐α‐methyl‐D‐glucopyranoside (α‐MG) uptake which were recovered by GlcN and PUGNAc (OGA inhibitor). Hypoxia enhanced reactive oxygen species (ROS) and then ER stress proteins, glucose‐regulated protein 78 (GRP78), and C/EBP‐homologous protein (CHOP). However, the expression of GRP78 increased till 6 h and then decreased whereas CHOP increased gradually. Moreover, decreased GRP78 and increased CHOP were reversed by NAC (antioxidant) and GlcN. GlcN ameliorated hypoxia‐induced decrease of O‐GlcNAc modification of Sp1 but OGT or Sp1 siRNAs blocked the recovery effect of GlcN on SGLT expression and α‐MG uptake. In addition, hypoxia‐decreased GRP78 and HIF‐1α expression was reversed by GlcN but OGT siRNA or Sp1 siRNA ameliorated the effect of GlcN. When PTCs were transfected with GRP78 siRNA or HIF‐1α siRNA, SGLT expression and α‐MG uptake was decreased. Taken together, these data suggest that GlcN‐induced O‐GlcNAc modified Sp1 with stimulating GRP78 and HIF‐1α activity ameliorate hypoxia‐induced SGLT dysfunction in renal PTCs. J. Cell. Physiol. 229: 1557–1568, 2014. © 2014 Wiley Periodicals, Inc.
  • FLRT2 Interacts With Fibronectin in the ATDC5 Chondroprogenitor Cells
    • Abstract: Expression studies have implicated FLRT2 in cranial neural crest cell migration and prechondrogenic cell condensation during craniofacial skeletogenesis. We aimed to determine whether FLRT2 was involved in mediating cell‐matrix interactions in the ATDC5 chondroprogenitor cell line. Immunolocalization experiments of ATDC5 cells revealed that FLRT2 was present on the cell membrane as well as extracellularly, where it colocalized with Fibronectin (Fn). After cell extraction of the matrix, FLRT2 was identified in the ATDC5‐derived extracellular matrix (ECM) and was further found to be associated with Fn‐coated beads in cell cultures. Blockage of Fn fibril formation via a blocking peptide resulted in a concomitant decrease in extracellular FLRT2 accumulation. Over a 7‐day period following the replenishment of the Fn blocking peptide to the cultures, there was a partial rebound in Fn fibril formation that was accompanied by a concomitant reappearance of FLRT2 co‐expression. Co‐immunoprecipitation confirmed that FLRT2 and Fn interacted, either directly or indirectly. Immunoprecipitation and Western blot analyses with antibodies recognizing epitopes located on the extra‐ and intracellular domains of FLRT2 further revealed the presence of different sized bands, suggesting that FLRT2 may exist in both membrane‐bound and shed forms. Our data therefore provide evidence that FLRT2 and/or its cleavage products may be cooperating with Fn and other ECM proteins to regulate critical cellular events. Further studies will be necessary in delineate more precisely the roles of FLRT2 in mediating cell‐ and cell‐matrix interactions during normal development. J. Cell. Physiol. 229: 1538–1547, 2014. © 2014 Wiley Periodicals, Inc.
  • Efficacy of Different Sequences of Radio‐ and Chemotherapy in
           Experimental Models of Human Melanoma
    • Abstract: Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation‐induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real‐time RT‐PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to >100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet‐shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation → cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair. J. Cell. Physiol. 229: 1548–1556, 2014. © 2014 Wiley Periodicals, Inc.
  • ERK3 Promotes Endothelial Cell Functions by Upregulating
           SRC‐3/SP1‐Mediated VEGFR2 Expression
    • Abstract: Despite a regain of interest recently in ERK3 kinase signaling, the molecular regulations of both ERK3 gene expression and protein kinase activity are still largely unknown. While it is shown that disruption of ERK3 gene causes neonatal lethality, cell type‐specific functions of ERK3 signaling remain to be explored. In this study, we report that ERK3 gene expression is upregulated by cytokines through c‐Jun in endothelial cells; c‐Jun binds to the ERK3 gene and regulates its transcription. We further reveal a new role for ERK3 in regulating endothelial cell migration, proliferation and tube formation by upregulating SRC‐3/SP‐1‐mediated VEGFR2 expression. The underlying molecular mechanism involves ERK3‐stimulated formation of a transcriptional complex involving coactivator SRC‐3, transcription factor SP‐1 and the secondary coactivator CBP. Taken together, our study identified a molecular regulatory mechanism of ERK3 gene expression and revealed a previously unknown role of ERK3 in regulating endothelial cell functions. J. Cell. Physiol. 229: 1529–1537, 2014. © 2014 Wiley Periodicals, Inc.
  • A Functional N‐terminal Domain in C/EBPβ‐LAP* is Required
           for Interacting with SWI/SNF and to Repress Ric‐8B Gene
           Transcription in Osteoblasts
    • Abstract: The chromatin remodeling complex SWI/SNF and the transcription factor C/EBPβ play critical roles in osteoblastic cells as they jointly control transcription of a number of bone‐related target genes. The largest C/EBPβ isoform, LAP*, possesses a short additional N‐terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N‐terminus in C/EBPβ‐LAP* for binding SWI/SNF and for recruiting this complex to the Ric‐8B gene to mediate transcriptional repression. We find that both C/EBPβ‐LAP* and SWI/SNF simultaneously bind to the Ric‐8B promoter in differentiating osteoblasts that repress Ric‐8B expression. This decreased expression of Ric‐8B is not accompanied by significant changes in histone acetylation at the Ric‐8B gene promoter sequence. A single aminoacid change at the C/EBPβ‐LAP* N‐terminus (R3L) that inhibits C/EBPβ‐LAP*‐SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric‐8B promoter as well as C/EBPβ‐LAP*‐dependent repression of the Ric‐8B gene. Inducible expression of the C/EBPβ‐LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPβ‐LAP*‐target promoters and competes with the endogenous C/EBPβ factor. Together our results indicate that a functional N‐terminus in C/EBPβ‐LAP* is required for interacting with SWI/SNF and for Ric‐8B gene repression in osteoblasts. J. Cell. Physiol. 229: 1521–1528, 2014. © 2014 Wiley Periodicals, Inc.
  • HIF‐1 Alpha‐Induced Up‐Regulation of miR‐9
           Contributes to Phenotypic Modulation in Pulmonary Artery Smooth Muscle
           Cells During Hypoxia
    • Abstract: Pulmonary artery smooth muscle cells (PASMCs) are associated with the development of hypoxic pulmonary hypertension (HPH). Recent studies have implicated a critical role for microRNAs (miRNAs) in HPH; however, their expression and regulation in hypoxia‐mediated phenotypic modulation of PASMCs remains largely unclear. Here, we report that miR‐9 was induced in hypoxia and involved in a hypoxia‐induced phenotypic switch in rat primary PASMCs. Knockdown of miR‐9 followed by hypoxia exposure attenuated PASMCs proliferation and enhanced the expression of contractile genes in vascular smooth muscle cells (VSMCs), while overexpression of miR‐9 in normoxia promoted a proliferative phenotype in PASMCs. The primary transcripts of miR‐9‐1 and miR‐9‐3, but not miR‐9‐2, increased dramatically after hypoxia, whereas silencing of the hypoxia‐associated transcription factor HIF‐1α following hypoxia exposure abolished the enhancement of both primary transcripts in PASMCs. Using in silico analysis, we found three putative HIF‐1α binding motifs on miR‐9‐1 and one motif on miR‐9‐3 located within the 5‐kb region upstream of the transcriptional start sites. Chromatin immunoprecipitation assay revealed that hypoxia enhanced the direct interaction between HIF‐1α and the regulatory elements of miR‐9‐1 and miR‐9‐3. Reporter assays showed that the regulatory regions of miR‐9‐1 and miR‐9‐3 behaved as enhancers in a HIF‐1α‐dependent manner during hypoxia. Taken together, our data uncover a regulatory mechanism involving HIF‐1α‐mediated up‐regulation of miR‐9, which plays a role in the hypoxia‐induced phenotypic switch of PASMCs. J. Cell. Physiol. 229: 1511–1520, 2014. © 2014 Wiley Periodicals, Inc.
  • MicroRNA‐338‐3p Regulates Osteogenic Differentiation of Mouse
           Bone Marrow Stromal Stem Cells by Targeting Runx2 and Fgfr2
    • Abstract: The elucidation of the molecular mechanisms that govern the differentiation of bone marrow stromal cells (BMSCs) could provide new insight into the treatment of bone loss diseases. MicroRNAs (miRNAs) are small non‐coding RNAs that regulate gene expression during stem cell growth, proliferation, and differentiation. In the present study, we showed that miR‐338‐3p expression was significantly down‐regulated during the osteoblastic differentiation of BMSCs. Additionally, miR‐338‐3p was up‐regulated in ovariectomized (OVX) mice compared with sham mice. An in vitro analysis revealed that the over‐expression of miR‐338‐3p can inhibit the expression of osteoblast differentiation markers such as Osterix (Osx), thus reducing osteoblast differentiation. Bioinformatic analysis and dual luciferase assays confirmed that miR‐338‐3p can repress gene expression by targeting Runx2 and Fgfr2. In the BMSCs derived from OVX mice, the inhibition of miR‐338‐3p partially rescued mineralization and osteoblast differentiation. Taken together, our data show that miR‐338‐3p plays an important role during osteoblast differentiation of BMSCs and serves as a potential modulator of osteoporosis via its effect on osteoblasts. J. Cell. Physiol. 229: 1494–1502, 2014. © 2014 Wiley Periodicals, Inc.
  • Bone Morphogenetic Protein‐2 Antagonizes Bone Morphogenetic
           Protein‐4 Induced Cardiomyocyte Hypertrophy and Apoptosis
    • Abstract: Our previous work showed that the expression of bone morphogenetic protein‐4 (BMP4) was up‐regulated in pathological cardiac hypertrophy models and BMP4 induced cardiomyocyte hypertrophy and apoptosis. Bone morphogenetic protein‐2 (BMP2) and BMP4 share greater than 80% amino acid homology and there exists an interaction between BMP2 and BMP4, so the aim of the present study was to elucidate the changes of BMP2 in the cardiac hypertrophy models and the effects of BMP2 on BMP4‐induced cardiomyocyte hypertrophy and apoptosis. The in vivo cardiac hypertrophy models were induced by pressure‐overload and swimming exercise in mice. BMP2 mRNA and protein expressions increased in pressure‐overload and swimming‐exercise induced cardiac hypertrophy. BMP2 itself did not elicit cardiomyocyte hypertrophy and apoptosis, but antagonized BMP4‐induced cardiomyocyte hypertrophy and apoptosis. BMP2 stimulated Akt in cardiomyocytes and Akt inhibitor prevented the antagonism of BMP2 on BMP4‐induced cardiomyocyte apoptosis. Furthermore, BMP2 inhibited BMP4‐induced JNK activation in cardiomyocytes. In conclusion, BMP2 antagonizes BMP4‐induced cardiomyocyte hypertrophy and apoptosis. The anti‐apoptotic effects of BMP2 on BMP4‐induced cardiomyocyte apoptosis might be through activating Akt and inhibiting JNK activation. J. Cell. Physiol. 229: 1503–1510, 2014. © 2014 Wiley Periodicals, Inc.
  • Modulating Endothelial Barrier Function by Targeting Vimentin
    • Abstract: Vimentin is a major intermediate filament protein in vascular endothelial cells which might be involved in their function as a barrier tissue. It is proposed to dynamically maintain integrity of the endothelium as a tightly regulated permeability barrier that is subjected to a variety of shear and contractile forces. The results described in this report demonstrate that vimentin plays that role through mechanisms that are dependent on its phosphorylation state. Withaferin A (WFA), a vimentin targeting drug is shown to disrupt endothelial barrier function through its effects on vimentin filament distribution and physical properties. These effects are related to WFA's ability to increase vimentin phosphorylation. Through overexpressing a non‐phosphorylatable vimentin mutant we can block the effects of WFA on vimentin distribution and barrier permeability. The barrier augmentation effect appears to extend to endothelial cells that do not express detectable mutant vimentin which might suggest transmissible effects across cells. Blocking vimentin phosphorylation also protects the endothelial barrier against LPS endotoxin, implicating it as a target for drug development against pulmonary edema and acute respiratory distress syndrome (ARDS). J. Cell. Physiol. 229: 1484–1493, 2014. © 2014 Wiley Periodicals, Inc.
  • Core Binding Factor β (CBFβ) Is Retained in the Midbody During
    • Abstract: Core Binding Factor β (CBFβ) is complexed with the RUNX family of transcription factors in the nucleus to support activation or repression of genes related to bone (RUNX2), hematopoiesis (RUNX1) and gastrointestinal (RUNX3) development. Furthermore, RUNX proteins contribute to the onset and progression of different types of cancer. Although CBFβ localizes to cytoskeletal architecture, its biological role in the cytoplasmic compartment remains to be established. Additionally, the function and localization of CBFβ during the cell cycle are important questions relevant to its biological role. Here we show that CBFβ dynamically distributes in different stages of cell division and importantly is present during telophase at the midbody, a temporal structure important for successful cytokinesis. A functional role for CBFβ localization at the midbody is supported by striking defects in cytokinesis that include polyploidy and abscission failure following siRNA‐mediated downregulation of endogenous CBFβ or overexpression of the inv(16) fusion protein CBFβ‐SMMHC. Our results suggest that CBFβ retention in the midbody during cytokinesis reflects a novel function that contributes to epigenetic control. J. Cell. Physiol. 229: 1466–1474, 2014. © 2014 Wiley Periodicals, Inc.
  • P53 Dependent Mitochondrial Permeability Transition Pore Opening Is
           Required for Dexamethasone‐Induced Death of Osteoblasts
    • Abstract: Prolonged or overdose glucocorticoids (GCs) usage is the common cause of osteoporosis. In the present study, we studied the cellular mechanism of dexamethasone (Dex)‐induce osteoblast cell death by focusing on the role of mitochondrial permeability transition pore (mPTP). In cultured osteoblastic MC3T3‐E1 cells, Dex‐induced mPTP opening, which was demonstrated by mitochondrial membrane potential (MPP) decrease, cyclophilin‐D (CyPD)–adenine nucleotide translocator 1 (ANT‐1) mitochondrial complexation and cytochrome C (cyto‐C) release. The mPTP inhibitor sanglifehrin A (SfA) dramatically inhibited Dex‐induced MPP loss, cyto‐C release and MC3T3‐E1 cell death. Dex‐induced cell death requires mPTP composing protein CyPD, as CyPD inhibitor cyclosporin A (CsA) and CyPD siRNA knockdown inhibited Dex‐induced MC3T3‐E1 cell death, while CyPD overexpression aggravated Dex's cytotoxic effect. We found that Dex induced P53 phosphorylation and translocation to mitochondria, where it formed a complex with CyPD. Glucocorticoid receptor (GR) siRNA knockdown, or P53 inhibition (by its inhibitor pifithrin‐α or shRNA silencing) suppressed Dex‐induced CyPD‐P53 mitochondrial association and subsequent MC3T3‐E1 cell death. Finally, in primary cultured osteoblasts, Dex‐induced cell death was inhibited by CsA, SfA or pifithrin‐α. Together, our data suggest that Dex‐induced osteoblast cell death is associated with GR‐P53‐regulated mPTP opening. J. Cell. Physiol. 229: 1475–1483, 2014. © 2014 Wiley Periodicals, Inc.
  • Adiponectin as Novel Regulator of Cell Proliferation in Human Glioblastoma
    • Abstract: Adiponectin (Acrp30) is an adipocyte‐secreted hormone with pleiotropic metabolic effects, whose reduced levels were related to development and progression of several malignancies. We looked at the presence of Acrp30 receptors in human glioblastomas (GBM), hypothesizing a role for Acrp30 also in this untreatable cancer. Here we demonstrate that human GBM express Acrp30 receptors (AdipoR1 and AdipoR2), which are often co‐expressed in GBM samples (70% of the analyzed tumors). To investigate the effects of Acrp30 on GBM growth, we used human GBM cell lines U87‐MG and U251, expressing both AdipoR1 and AdipoR2 receptors. In these cells, Acrp30 treatment inhibits DNA synthesis and cell proliferation rate, inducing arrest in G1 phase of the cell cycle. These effects were correlated to a sustained activation of ERK1/2 and Akt kinases, upon Acrp30 treatment. Our results suggest that Acrp30 may represent a novel endogenous negative regulator of GBM cell proliferation, to be evaluated for the possible development of novel pharmacological approaches. J. Cell. Physiol. 229: 1444–1454, 2014. © 2014 Wiley Periodicals, Inc.
  • RhoA‐Mediated Inhibition of Vascular Endothelial Cell Mobility:
           Positive Feedback Through Reduced Cytosolic p21 and p27
    • Abstract: We previously identified that activation of the aryl hydrocarbon receptor (AhR) by 3‐methylcholanthrene (3MC) exerts antiproliferative and antimigratory effects on human umbilical vein endothelial cells (HUVECs) through the upregulation of p21/p27 transcription and RhoA activation. In this study, we investigated the mechanisms of 3MC‐mediated downregulation of cytosolic p21/ p27, and the effects of 3MC on RhoA activation and cell migration, in mouse cerebral vascular endothelial cells (MCVECs). Our results indicated that 3MC reduced the phosphorylation of p21/p27 through AhR/RhoA/PTEN‐mediated PI3K/Akt inactivation, which reduced cytosolic p21/p27 retention, causing RhoA activation through positive feedback. Downregulation of p21/p27 by siRNA, and cytosolic p21/p27 by the nuclear export blocker leptomycin B, further reduced cell migration in the 3MC‐treated cells. Reduced cytosolic p21/p27 expression led to reduced interaction between RhoA and the RhoA inhibitor p190RhoGAP, causing RhoA activation. Treatment with YS‐49 activated PI3K/Akt, a downstream target of RhoA, to reduce RhoA/PTEN activation in the 3MC‐treated cells, whereas treatment with wortmannin, a PI3K inhibitor, activated RhoA/PTEN. Gain‐ and loss‐of‐function analyses revealed that constitutively active (CA) Akt1, but not CA Akt2, inactivated RhoA and stimulated migratory activity. Considering the essential role of RhoA activation in cell migration, we evaluated the potential use of simvastatin, a RhoA inhibitor, as a therapeutic intervention in vivo using matrigel plug formation assays. Our results provide a molecular basis for the therapeutic application of simvastatin to reduce RhoA/PTEN activation, restore cytosolic levels of phosphorylated p21/p27, and induce angiogenic processes. J. Cell. Physiol. 229: 1455–1465, 2014. © 2014 Wiley Periodicals, Inc.
  • Pancreatic Cancer‐Induced Cachexia Is Jak2‐Dependent in Mice
    • Abstract: Cancer cachexia syndrome is observed in 80% of patients with advanced‐stage cancer, and it is one of the most frequent causes of death. Severe wasting accounts for more than 80% in patients with advanced pancreatic cancer. Here we wanted to define, by using an microarray approach and the Pdx1‐cre;LSL‐KrasG12D;INK4a/arffl/fl mice model, the pathways involved in muscle, liver, and white adipose tissue wasting. These mice, which develop systematically pancreatic cancer, successfully reproduced many human symptoms afflicted with this disease, and particularly cachexia. Using the profiling analysis of pancreatic cancer‐dependent cachectic tissues we found that Jak2/Stat3 pathways, p53 and NFkB results activated. Thus, our interest was focused on the Jak2 pathways because it is pharmacologically targetable with low toxicity and FDA approved drugs are available. Therefore, Pdx1‐cre;LSL‐KrasG12D;INK4a/arffl/fl mice were treated with the Jak2 inhibitor AG490 compound daily starting at 7 weeks old and for a period of 3 weeks and animals were sacrificed at 10 weeks old. Body weight for control mice was 27.84 ± 2.14 g, for untreated Pdx1‐cre;LSL‐KrasG12D;INK4a/arffl/fl was 14.97 ± 1.99 g, whereas in animals treated with the AG490 compound the weight loss was significantly less to 24.53 ± 2.04 g. Treatment with AG490 compound was efficient since phosphorylation of Jak2 and circulating interleukin‐6 (IL6) levels were significantly reduced in cachectic tissues and in mice respectively. In conclusion, we found that Jak2/Stat3‐dependent intracellular pathway plays an essential role since its pharmacological inhibition strongly attenuates cachexia progression in a lethal transgenic pancreatic cancer model. J. Cell. Physiol. 229: 1437–1443, 2014. © 2014 Wiley Periodicals, Inc.
  • Link Between DNA Damage and Centriole Disengagement/Reduplication in
           Untransformed Human Cells
    • Abstract: The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ‐tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53‐dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. J. Cell. Physiol. 229: 1427–1436, 2014. © 2014 Wiley Periodicals, Inc.
  • GRP78 Mediates Cell Growth and Invasiveness in Endometrial Cancer
    • Abstract: Recent studies have indicated that endoplasmic reticulum stress, the unfolded protein response activation and altered GRP78 expression can play an important role in a variety of tumors development and progression. Very recently we reported for the first time that GRP78 is increased in endometrial tumors. However, whether GRP78 could play a role in the growth and/or invasiveness of endometrial cancer cells is still unknown. Here we report that the silencing of GRP78 expression affects both cell growth and invasiveness of Ishikawa and AN3CA cells, analyzed by the (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide) and transwell migration assay, respectively. At variance with Ishikawa cells, AN3CA cells showed, besides an endoplasmic reticulum, also a plasma membrane GRP78 localization, evidenced by both immunofluorescence and cell membrane biotinylation experiments. Intriguingly, flow cytometry experiments showed that the treatment with a specific antibody targeting GRP78 C‐terminal domain caused apoptosis in AN3CA but not in Ishikawa cells. Induction of apoptosis in AN3CA cells was not mediated by the p53 pathway activation but was rather associated to reduced AKT phosphorylation. Interestingly, immunofluorescence analysis evidenced that endometrioid adenocarcinoma tissues displayed, similarly to AN3CA cells, also a GRP78 plasma membrane localization. These data suggest that GRP78 and its plasma membrane localization, might play a role in endometrial cancer development and progression and might constitute a novel target for the treatment of endometrial cancer. J. Cell. Physiol. 229: 1417–1426, 2014. © 2014 Wiley Periodicals, Inc.
  • Altered Histone Mark Deposition and DNA Methylation at Homeobox Genes in
           Human Oral Squamous Cell Carcinoma
    • Abstract: We recently reported a role of polycomb repressive complex 2 (PRC2) and PRC2 trimethylation of histone 3 lysine 27 (H3K27me3) in the regulation of homeobox (HOX) (Marcinkiewicz and Gudas, 2013, Exp Cell Res) gene transcript levels in human oral keratinocytes (OKF6‐TERT1R) and tongue squamous cell carcinoma (SCC) cells. Here, we assessed both the levels of various histone modifications at a subset of homeobox genes and genome wide DNA methylation patterns in OKF6‐TERT1R and SCC‐9 cells by using ERRBS (enhanced reduced representation bisulfite sequencing). We detected the H3K9me3 mark at HOXB7, HOXC10, HOXC13, and HOXD8 at levels higher in OKF6‐TERT1R than in SCC‐9 cells; at IRX1 and SIX2 the H3K9me3 levels were conversely higher in SCC‐9 than in OKF6‐TERT1R. The H3K79me3 mark was detectable only at IRX1 in OKF6‐TERT1R and at IRX4 in SCC‐9 cells. The levels of H3K4me3 and H3K36me3 marks correlate with the transcript levels of the assessed homeobox genes in both OKF6‐TERT1R and SCC‐9. We detected generally lower CpG methylation levels on DNA in SCC‐9 cells at annotated genomic regions which were differentially methylated between OKF6‐TERT1R and SCC‐9 cells; however, some genomic regions, including the HOX gene clusters, showed DNA methylation at higher levels in SCC‐9 than OKF6‐TERT1R. Thus, both altered histone modification patterns and changes in DNA methylation are associated with dysregulation of homeobox gene expression in human oral cavity SCC cells, and this dysregulation potentially plays a role in the neoplastic phenotype of oral keratinocytes. J. Cell. Physiol. 229: 1405–1416, 2014. © 2014 Wiley Periodicals, Inc.
  • Reelin/DAB‐1 Signaling in the Embryonic Limb Regulates the
           Chondrogenic Differentiation of Digit Mesodermal Progenitors
    • Abstract: Reelin is a bioactive component of some extracellular matrices. Most studies on this signaling glycoprotein have been performed in the developing nervous system, where Reelin binds to the very‐low‐density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) of target cells. This induces phosphorylation of the intracellular adaptor protein Disabled‐1 (Dab‐1), which subsequently activates downstream effectors to regulate important aspects of neuroblast biology. Here, we show that the components of the Reelin signaling pathway exhibit a dynamic expression pattern during the development of the digits in chick and mouse embryonic limbs. Reelin and Dab‐1 are highly expressed in the differentiating digit cartilages and tendinous blastemas. Immunolabeling of phospho‐Dab‐1 indicates that the pattern of gene expression correlates with zones of active signaling. Intense signaling is also present in the early stages of cartilage differentiation in micromass cultures of digit mesodermal progenitors. In this in vitro assay, disruption of the Reelin signaling pathway by gene silencing causes cystoskeletal and cell shape modifications accompanied by reduced chondrogenesis and down‐regulation of specific cartilage molecular markers. Of note, Scleraxis and Six2, which are master genes of tendinous blastemas, become up‐regulated in these experiments. We further show that the receptors ApoER2 and VLDLR are differentially expressed in cartilage and tendons and that these receptors show temporal expression differences in the micromass cultures. Sox9 and other chondrogenic markers were downregulated in micromass cultures after ApoER2 gene silencing, while gene silencing of VLDLR up‐regulates Scleraxis. In summary, our findings provide evidence of a role for Reelin signaling in skeletogenesis that promotes chondrogenesis through ApoER2 and inhibits tenogenic differentiation through VLDLR. J. Cell. Physiol. 229: 1397–1404, 2014. © 2014 Wiley Periodicals, Inc.
  • CD40 Signaling Drives B Lymphocytes Into an Intermediate Memory‐Like
           State, Poised Between Naïve and Plasma Cells
    • Abstract: Immunological memory comprising of antigen‐specific B and T cells contributes to the acquisition of long‐term resistance to pathogens. Interactions between CD40 on B cells and CD40L on T cells are responsible for several aspects of acquired immune responses including generation of memory B cells. In order to gain insights into events leading to memory B cell formation, we analyzed the genome‐wide expression profile of murine naive B cells stimulated in the presence of anti‐CD40. We have identified over 8,000 genes whose expression is altered minimally 1.5‐fold at least at one time point over a 3‐day time course. The array analysis indicates that changes in expression level of maximum number of these genes occur within 24 h of anti‐CD40 treatment. In parallel, we have studied the events following CD40 ligation by examining the expression of known regulators of naive B cell to plasma cell transition, including Pax5 and BLIMP1. The expression profile of these regulatory genes indicates firstly, that CD40 signaling activates naïve B cells to a phenotype that is intermediate between the naive and plasma cell stages of the B cell differentiation. Secondly, the major known regulator of plasma cell differentiation, BLIMP1, gets irreversibly downregulated upon anti‐CD40 treatment. Additionally, our data reveal that CD40 signaling mediated BLIMP1 downregulation occurs by non‐Pax5/non‐Bcl6 dependent mechanisms, indicating novel mechanisms at work that add to the complexity of understanding of B cell master regulatory molecules like BLIMP1 and Pax5. J. Cell. Physiol. 229: 1387–1396, 2014. © 2014 Wiley Periodicals, Inc.
  • Calcineurin Regulates Progressive Motility Activation of Rhinella (Bufo)
           arenarum Sperm Through Dephosphorylation of PKC Substrates
    • Abstract: Animals with external fertilization, as amphibians, store their sperm in a quiescent state in the testis. When spermatozoa are released into natural fertilization media, the hypotonic shock triggers activation of sperm motility. Rhinella (Bufo) arenarum sperm are immotile in artificial seminal plasma (ASP, resembling testicular plasma tonicity) but acquire in situ flagellar beating upon dilution. However, if components from the egg shelly coat are added to this medium, motility shifts to a progressive pattern. Recently, we have shown that the signal transduction pathway required for in situ motility activation involves a rise in intracellular cAMP through a transmembrane adenylyl cyclase and activation of PKA, mostly in the midpiece and in the sperm head. In this report, we demonstrate that activation of calcineurin (aka PP2B and PPP3) is required for the shift from in situ to progressive sperm motility. The effect of calcineurin is manifested by dephosphorylation of PKC substrates, and can be promoted by intracellular calcium rise by Ca2+ ionophore. Both phosphorylated PKC substrates and calcineurin localized to the flagella, indicating a clear differentiation between compartmentalization of PKA and calcineurin pathways. Moreover, no crosstalk is observed between these signaling events, even though both pathways are required for progressive motility acquisition as discussed. J. Cell. Physiol. 229: 1378–1386, 2014. © 2014 Wiley Periodicals, Inc.
  • Midbrain Cues Dictate Differentiation of Human Dental Pulp Stem Cells
           Towards Functional Dopaminergic Neurons
    • Abstract: Dental pulp originating from the neural crest is considered a better source of postnatal stem cells for cell‐based therapies in neurodegenerative diseases. Dental Pulp Stem Cells (DPSCs) have been shown to differentiate into cell‐types of cranial neural crest ontology; however, their ability to differentiate to functional neurons of the central nervous system remains to be studied. We hypothesized that midbrain cues might commit DPSCs to differentiate to functional dopaminergic cell‐type. As expected, DPSCs in their naïve state spontaneously expressed early and mature neuronal markers like nestin, musashi12, β tubulin III, and Map2ab. On exposure to midbrain cues (sonic hedgehog, fibroblast growth factor 8 and basic fibroblast growth factor), DPSCs showed upregulation of dopaminergic neuron‐specific transcription factors Nuclear Receptor related protein 1 (Nurr1), Engrailed 1 (En1) and paired‐like homeodomain transcription factor 3 (Pitx3) as revealed by real‐time RT‐PCR. Immunofluorescence and flow cytometry analysis showed enhanced expression of mature neuronal marker Map2ab and dopaminergic‐neuronal markers [tyrosine hydroxylase (TH), En1, Nurr1, and Pitx3], with nearly 77% of the induced DPSCs positive for TH. Functional studies indicated that the induced DPSCs could secrete dopamine constitutively and upon stimulation with potassium chloride (KCl) and adenosine triphosphate (ATP), as measured by dopamine ELISA. Additionally, the induced DPSCs showed intracellular Ca2+ influx in the presence of KCl, unlike control DPSCs. ATP‐stimulated Ca2+ influx was observed in control and induced DPSCs, but only the induced cells secreted dopamine. Our data clearly demonstrate for the first time that DPSCs in the presence of embryonic midbrain cues show efficient propensity towards functional dopaminergic cell‐type. J. Cell. Physiol. 229: 1369–1377, 2014. © 2014 Wiley Periodicals, Inc.
  • Phosphorylation‐Regulated Degradation of the Tumor‐Suppressor
           Form of PED by Chaperone‐Mediated Autophagy in Lung Cancer Cells
    • Abstract: PED/PEA‐15 is a death effector domain (DED) family member with a variety of effects on cell growth and metabolism. To get further insight into the role of PED in cancer, we aimed to find new PED interactors. Using tandem affinity purification, we identified HSC70 (Heat Shock Cognate Protein of 70 kDa)—which, among other processes, is involved in chaperone‐mediated autophagy (CMA)—as a PED‐interacting protein. We found that PED has two CMA‐like motifs (i.e., KFERQ), one of which is located within a phosphorylation site, and demonstrate that PED is a bona fide CMA substrate and the first example in which phosphorylation modifies the ability of HSC70 to access KFERQ‐like motifs and target the protein for lysosomal degradation. Phosphorylation of PED switches its function from tumor suppression to tumor promotion, and we show that HSC70 preferentially targets the unphosphorylated form of PED to CMA. Therefore, we propose that the up‐regulated CMA activity characteristic of most types of cancer cell enhances oncogenesis by shifting the balance of PED function toward tumor promotion. This mechanism is consistent with the notion of a therapeutic potential for targeting CMA in cancer, as inhibition of this autophagic pathway may help restore a physiological ratio of PED forms. J. Cell. Physiol. 229: 1359–1368, 2014. © 2014 Wiley Periodicals, Inc.
  • Migration Linked to FUCCI‐Indicated Cell Cycle Is Controlled by PTH
           and Mechanical Stress
    • Abstract: Bone metabolism is maintained via balanced repetition of bone resorption by osteoclasts and bone formation by osteoblasts. Osteoblastic cells are capable of conducting self‐renewal and differentiation that are basically associated with cell‐cycle transition to enable cell specification and bone formation. Osteoblasts are also migrating to fill the resorption cavity curved by osteoclasts during bone remodeling to maintain homeostasis of bone mass whose imbalance leads to osteoporosis. However, technical difficulties have hampered the research on the dynamic relationship between cell cycle and migration in osteoblasts. In this report, we overcome these problems by introducing fluorescent ubiquitination‐based cell cycle indicator (FUCCI) reporter system in calvarial osteoblastic cells and reveal that the cells in G1 as well as S/G2/M phase are migrating. Furthermore, the osteoblastic cells in S/G2/M phase migrate faster than those in G1 phase. Interestingly, parathyroid hormone (PTH) as an anabolic agent enhances migration velocity of the cells. Mechanical stress, another anabolic signal, also enhances migration velocity. In contrast, in the presence of both PTH and mechanical stress, the migration velocity returns to the base line levels revealing the interaction between the two anabolic stimuli in the regulation of cell migration. Importantly, PTH and mechanical stress also interact when they regulate the transition of cell cycle. These data demonstrate that osteoblastic migration is linked to cell cycle and it is under the control of mechanical and chemical stimuli that coordinate to regulate bone mass. J. Cell. Physiol. 229: 1353–1358, 2014. © 2014 Wiley Periodicals, Inc.
  • Non‐Cell Autonomous or Secretory Tumor Suppression
    • Abstract: Many malignancies result from deletions or loss‐of‐function mutations in one or more tumor suppressor genes, the products of which curb unrestrained growth or induce cell death in those with dysregulated proliferative capacities. Most tumor suppressors act in a cell autonomous manner, and only very few proteins are shown to exert a non‐cell autonomous tumor suppressor function on other cells. Examples of these include members of the secreted frizzled‐related protein (SFRP) family and the secreted protein acidic and rich in cysteine (SPARC)‐related proteins. Very recent findings have, however, considerably expanded our appreciation of non‐cell autonomous tumor suppressor functions. Broadly, this may occur in two ways. Intracellular tumor suppressor proteins within cells could in principle inhibit aberrant growth of neighboring cells by conditioning an antitumor microenvironment through secreted factors. This is demonstrated by an apparent non‐cell autonomous tumor suppressing property of p53. On the other hand, a tumor suppressor produced by a cell may be secreted extracellularly, and taken up by another cell with its activity intact. Intriguingly, this has been recently shown to occur for the phosphatase and tensin homolog (PTEN) by both conventional and unconventional modes of secretion. These recent findings would aid the development of therapeutic strategies that seek to reinstate tumor suppression activity in therapeutically recalcitrant tumor cells, which have lost it in the first place. J. Cell. Physiol. 229: 1346–1352, 2014. © 2014 Wiley Periodicals, Inc.
  • Functional Role of Connexins and Pannexins in the Interaction Between
           Vascular and Nervous System
    • Abstract: The microvascular network of the microcirculation works in tight communication with surrounding tissues to control blood supply and exchange of solutes. In cerebral circulation, microvascular endothelial cells constitute a selective permeability barrier that controls the environment of parenchymal brain tissue, which is known as the blood–brain barrier (BBB). Connexin‐ and pannexin‐formed channels (gap junctions and hemichannels) play a central role in the coordination of endothelial and smooth muscle cell function and connexin‐mediated signaling in endothelial cells is essential in the regulation of BBB permeability. Likewise, gap junction communication between astrocyte end‐feet also contributes to maintain the BBB integrity, but the participation of hemichannels in this process cannot be discarded. Sympathetic and sensory perivascular nerves are also involved in the control and coordination of vascular function through the release of vasoconstrictor or vasodilator signals and by the regulation of gap junction communication in the vessel wall. Conversely, ATP release through pannexin‐1‐formed channels mediates the α1‐adrenergic signaling. Furthermore, here we show that capsaicin‐induced CGRP release from mesenteric perivascular sensory nerves induces pannexin‐1‐formed channel opening, which in turn leads to reduction of pannexin‐1 and endothelial nitric oxide synthase (eNOS) expression along the time. Interestingly, blockade of CGRP receptors with CGRP8–37 increased eNOS expression by ∼5‐fold, suggesting that capsaicin‐sensitive sensory nerves are involved in the control of key signaling proteins for vascular function. In this review, we discuss the importance of connexin‐based channels in the control of BBB integrity and the functional interaction of vascular connexins and pannexins with the peripheral nervous system. J. Cell. Physiol. 229: 1336–1345, 2014. © 2014 Wiley Periodicals, Inc.
  • Telomere Length Maintenance, Shortening, and Lengthening
    • Abstract: Telomeres maintain chromosome stability and cell replicative capacity. Telomere shortening occurs concomitant with aging. Short telomeres are associated with some diseases, such as dyskeratosis congenita, idiopathic pulmonary fibrosis, and aplastic anemia. Telomeres are longer in pluripotent stem cells than in somatic cells and lengthen significantly during preimplantation development. Furthermore, telomere elongation during somatic cell reprogramming is of great importance in the acquisition of authentic pluripotency. This review focuses primarily on regulatory mechanisms of telomere length maintenance in pluripotent cells, telomere length extension in early embryo development, and also telomere rejuvenation in somatic cell reprogramming. Telomere related diseases are also discussed in this review. J. Cell. Physiol. 229: 1323–1329, 2014. © 2014 Wiley Periodicals, Inc.
  • DC‐STAMP, the Key Fusion‐Mediating Molecule in
    • Abstract: As a member of the mononuclear phagocyte system, osteoclasts (OC) absorb the bone matrix and participate in bone modeling by keeping a balance with osteoblasts (OB) and stromal cells. Mature OC derive from the fusion of mononuclear osteoclasts (mOC) and the fusion is considered as the indispensable process for the osteoclastogenesis and absorbing activity of OC. DC‐STAMP (dendritic cell‐specific transmembrane protein) has been validated playing a key role in the fusion of mOC. DC‐STAMP is mainly expressed in OC, macrophages and dendritic cells (DC). While DC‐STAMP was discovered in DC, more attentions have been paid to DC‐STAMP in OC in this decade. This review will mainly focus on the function of DC‐STAMP in OC. Studies on DC‐STAMP in DC may also provide new sight for the study of DC‐STAMP in OC. Since the function of DC‐STAMP is still poorly understood and few studies have been implemented for illustration, many issues are still unknown and need to be revealed. We will also discuss these questions in this review. J. Cell. Physiol. 229: 1330–1335, 2014. © 2014 Wiley Periodicals, Inc.
  • Signals Controlling Un‐Differentiated States in Embryonic Stem and
           Cancer Cells: Role of the Phosphatidylinositol 3′ Kinase Pathway
    • Abstract: The capacity of embryonic stem (ES) cells to differentiate into cell lineages comprising the three germ layers makes them powerful tools for studying mammalian early embryonic development in vitro. The human body consists of approximately 210 different somatic cell types, the majority of which have limited proliferative capacity. However, both stem cells and cancer cells bypass this replicative barrier and undergo symmetric division indefinitely when cultured under defined conditions. Several signal transduction pathways play important roles in regulating stem cell development, and aberrant expression of components of these pathways is linked to cancer. Among signaling systems, the critical role of leukemia inhibitory factor (LIF) coupled to the Jak/STAT3 (signal transduction and activation of transcription‐3) pathway in maintaining stem cell self‐renewal has been extensively reviewed. This pathway additionally plays multiple roles in tumorigenesis. Likewise, the phosphatidylinositide 3‐kinase (PI3K)/protein kinase B (PKB/Akt) pathway has been determined to play an important role in both stem cell maintenance and tumor development. This pathway is often induced in cancer with frequent mutational activation of the catalytic subunit of PI3K or loss of a primary PI3K antagonist, phosphatase and tensin homolog deleted on chromosome ten (PTEN). This review focusses on roles of the PI3K signal transduction pathway components, with emphasis on functions in stem cell maintenance and cancer. Since the PI3K pathway impinges on and collaborates with other signaling pathways in regulating stem cell development and/or cancer, aspects of the canonical Wnt, Ras/mitogen‐activated protein kinase (MAPK), and TGF‐β signaling pathways are also discussed. J. Cell. Physiol. 229: 1312–1322, 2014. © 2014 The
      Authors . Published by Wiley Periodicals, Inc.
  • Table of Contents: Volume 229, Number 10
  • Editor's Choice
  • Highlights: Volume 229, Number 10
  • Quantifying Mitotic Chromosome Dynamics and Positioning
    • Abstract: The proper organization and segregation of chromosomes during cell division is essential to the preservation of genomic integrity. To understand the mechanisms that spatially control the arrangement and dynamics of mitotic chromosomes requires imaging assays to quantitatively resolve their positions and movements. Here, we will discuss analytical approaches to investigate the position‐dependent control of mitotic chromosomes in cultured cells. These methods can be used to dissect the specific contributions of mitotic proteins to the molecular control of chromosome dynamics. J. Cell. Physiol. 229: 1301–1305, 2014. © 2014 Wiley Periodicals, Inc.
  • Role of PUF‐8/PUF Protein in Stem Cell Control, Sperm‐Oocyte
           Decision and Cell Fate Reprogramming
    • Abstract: Pumilio and FBF (PUF) proteins are conserved stem cell regulators that maintain germline stem cells (GSCs) in worms and flies. Moreover, they are also present in vertebrate stem cells. The nematode Caenorhabditis elegans has multiple PUF proteins with specialized roles. Among them, PUF‐8 protein controls multiple cellular processes, including proliferation, differentiation, sperm‐oocyte decision, and cell fate reprogramming, depending on the genetic context in the C. elegans germline. In this review, we describe the possible mechanisms of how PUF‐8 protein systematically controls multiple cellular processes in the C. elegans germline. Since PUF proteins are evolutionarily conserved, we suggest that a similar mechanism may be involved in controlling stem cell regulation and differentiation in other organisms, including humans. J. Cell. Physiol. 229: 1306–1311, 2014. © 2014 Wiley Periodicals, Inc.
  • Fibronectin Peptides as Potential Regulators of Hepatic Fibrosis through
           apoptosis of hepatic stellate cells
    • Abstract: The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX‐2 based on oligonucleosomal DNA fragmentation, caspase‐3 and ‐9 activation, Bcl‐2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease‐9 (MMP‐9) expression in human monocytes. © 2014 Wiley Periodicals, Inc.
  • Journal of Cellular Physiology: Volume 229, Number 10, October 2014
    • Abstract: Cover: Immunofluorescent micrographs of mitotic human cells with normal (center image) or disrupted (peripheral images) chromosome alignment. See article by Bissonette and Stumpff on pages 1301–1305. Cover designed by Jason Stumpff.
  • Distinct effects of inorganic phosphate on cell cycle and apoptosis in
           human vascular smooth muscle cells
    • Abstract: Objective Inorganic phosphate (Pi) is an essential nutrient to all living organisms. Nevertheless, hyperphosphatemia is now recognized as a risk factor for cardiovascular events and mortality in chronic kidney disease (CKD) patients. To our knowledge, the mechanisms by which elevated Pi alters smooth muscle cells proliferation have been poorly addressed. Therefore, in this study, we investigated the effects of Pi on cell cycle regulation and apoptosis in human aortic smooth muscle cells (HAoSMC). Approach and Results HAoSMC were treated with physiologic (1 mM) or high (2 and 3 mM) Pi concentrations. We showed that Pi not only decreased significantly cell viability (p 
  • Membrane‐Type 1 Matrix Metalloproteinase Downregulates Fibroblast
           Growth Factor‐2 Binding to the Cell Surface and Intracellular
    • Abstract: Membrane‐type 1 matrix metalloproteinase (MT1‐MMP, MMP‐14), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades extracellular matrix components and controls diverse cell functions through proteolytic and non‐proteolytic interactions with extracellular, intracellular and transmembrane proteins. Here we show that in tumor cells MT1‐MMP downregulates fibroblast growth factor‐2 (FGF‐2) signaling by reducing the amount of FGF‐2 bound to the cell surface with high and low affinity. FGF‐2 induces weaker activation of ERK1/2 MAP kinase in MT1‐MMP expressing cells than in cells devoid of MT1‐MMP. This effect is abolished in cells that express proteolytically inactive MT1‐MMP but persists in cells expressing MT1‐MMP mutants devoid of hemopexin‐like or cytoplasmic domain, showing that FGF‐2 signaling is downregulated by MT1‐MMP proteolytic activity. MT1‐MMP expression results in downregulation of FGFR‐1 and ‐4, and in decreased amount of cell surface‐associated FGF‐2. In addition, MT1‐MMP strongly reduces the amount of FGF‐2 bound to the cell surface with low affinity. Because FGF‐2 association with low‐affinity binding sites is a prerequisite for binding to its high‐affinity receptors, downregulation of low‐affinity binding to the cell surface results in decreased FGF‐2 signaling. Consistent with this conclusion, FGF‐2 induction of tumor cell migration and invasion in vitro is stronger in cells devoid of MT1‐MMP than in MT1‐MMP expressing cells. Thus, MT1‐MMP controls FGF‐2 signaling by a proteolytic mechanism that decreases the cell's biological response to FGF‐2. © 2014 Wiley Periodicals, Inc.
  • Beta‐Adrenoceptor Activation Reduces Both Dermal Microvascular
           Endothelial Cell Migration Via a cAMP‐Dependent Mechanism and Wound
    • Abstract: Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar‐free fetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta‐adrenoceptors (β‐AR) are G protein‐coupled receptors expressed on all skin cell‐types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β‐AR‐mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β‐AR activation reduced HDMEC migration via cAMP‐dependent and PKA‐independent mechanisms as demonstrated through use of an EPAC agonist that auto‐inhibited the cAMP‐mediated β‐AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β‐AR activation reduced pro‐angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β‐AR‐mediated autocrine and paracrine anti‐angiogenic mechanisms. In more complex environments, β‐AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β‐AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β‐AR agonists could be promising anti‐angiogenic modulators in skin. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
  • S‐nitrosylation of Cofilin‐1 Serves as a Novel Pathway for
           VEGF‐stimulated Endothelial Cell Migration
    • Abstract: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) mediates vascular endothelial growth factor (VEGF)‐stimulated endothelial cytoskeleton remodeling and migration; however, the underlying mechanisms are elusive. Covalent adduction of a NO moiety (NO•) to cysteines called S‐nitrosylation (SNO) is a key NO signaling pathway. The small actin‐binding protein cofilin‐1 (CFL1) is essential for actin cytoskeleton remodeling. We investigated whether S‐nitrosylation regulates CFL1 function and endothelial cytoskeleton remodeling and migration upon VEGF stimulation. VEGF rapidly stimulated S‐nitrosylation of CFL1, which was blocked by NO Synthase inhibition and eNOS knockdown by specific eNOS‐siRNA. Cys80 and Cys139 were identified as the major SNO‐sites in CFL1 by LC‐MS/MS. The actin severing activity of recombinant SNO‐mimetic CFL1 (C80/139A DMA‐CFL1), but not SNO‐deficient CFL1 (C80/139S DMS‐CFL1), was significantly greater than that of wild‐type CFL1 (wt‐CFL1). When wt‐CFL1 and its mutants were overexpressed in endothelial cells, basal actin bound wt‐CFL1 was undetectable but significantly increased by VEGF; basal actin bound DMA‐CFL1 was readily high and basal actin bound DMS‐CFL1 was detectable but low, and both were unresponsive to VEGF. Treatment with VEGF significantly increased filamentous (F‐) actin and filopodium formation and cell migration in endothelial cells. Overexpression of wt‐CFL1 inhibited VEGF‐induced F‐actin formation. Overexpression of DMA but not DMS CFL1 decreased basal but not VEGF‐stimulated F‐actin formation. Overexpression of DMA but not DMS CFL1 suppressed VEGF‐stimulated filopodium formation and migration in endothelial cells. Thus, S‐nitrosylation of CFL1 provides a novel signaling pathway post‐NO biosynthesis via eNOS‐derived NO for endothelial cytoskeleton remodeling and migration upon VEGF stimulation. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
  • Regulation of Osteoclast Multinucleation by the Actin Cytoskeleton
           Signaling Network
    • Abstract: Although it is known that osteoclasts are multinucleated cells that are responsible for bone resorption, the mechanism by which their size is regulated is unclear. We previously reported that an actin‐rich superstructure, termed the zipper‐like structure, specifically appears during the fusion of large osteoclast‐like cells (OCLs). Actin cytoskeleton reorganization in osteoclasts is regulated by a signaling network that includes the macrophage colony‐stimulating factor (M‐CSF) receptor, a proto‐oncogene, Src, and small GTPases. Here, we examined the role of actin reorganization in the multinucleation of OCLs differentiated from RAW 264.7 cells using various pharmacological agents. Jasplakinolide, which stabilizes actin stress fibers, induced the development of small OCLs, and the Src inhibitor SU6656 and the dynamin inhibitor dynasore impaired the maintenance of the podosome belt and the zipper‐like structure. These inhibitors decreased the formation of large OCLs but increased the number of small OCLs. M‐CSF is known to stimulate osteoclast fusion. M‐CSF signaling via Src up‐regulated Rac1 activity but down‐regulated Rho activity. Rac1 and Rho localized to the center of the zipper‐like structure. Rho activator II promoted the formation of small OCLs, whereas the Rho inhibitor Y27632 promoted the generation of large OCLs. These results suggest that the status of the actin cytoskeleton signaling network determines the size of OCLs during cell fusion. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
  • Hydrogen Sulfide Epigenetically Attenuates Homocysteine‐Induced
           Mitochondrial Toxicity Mediated through NMDA Receptor in Mouse Brain
           Endothelial (bEnd3) Cells
    • Abstract: Previously we have showed that homocysteine (Hcy) caused oxidative stress and altered mitochondrial function. Hydrogen sulphide (H2S) has potent anti‐inflammatory, anti‐oxidative and anti‐apoptotic effects. Therefore, in the present study we examined whether H2S ameliorates Hcyinduced mitochondrial toxicity which led to endothelial dysfunction in part, by epigenetic alterations in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 100μM Hcy treatment in the presence or absence of 30μM NaHS (donor of H2S) for 24hrs. Hcy‐activate NMDA receptor and induced mitochondrial toxicity by increased levels of Ca2+,NADPH‐oxidase‐4 (NOX‐4) expression, mitochondrial dehydrogenase activity and decreased the level of nitrate, superoxide dismutase (SOD‐2) expression, mitochondria membrane potentials, ATP production. To confirm the role of epigenetic, 5'‐azacitidine (an epigenetic modulator) treatment was given to the cells. Pretreatment with NaHS (30μM) attenuated the Hcy‐induced increased expression of DNMT1, DNMT3a, Ca2+ and decreased expression of DNMT3b in bEND3 cells. Furthermore, NaHS treatment also enhanced mitochondrial oxidative stress (NOX4, ROS, and NO) and restored ATP that indicates its protective effects against mitochondrial toxicity. Additional, NaHS significantly alleviated Hcyinduced LC3‐I/II, CSE, Atg3/7 and low p62 expression which confirm its effect on mitophagy. Likewise, NaHS also restored level of eNOS, CD31, VE‐Cadherin and ET‐1 and maintains endothelial function in Hcy treated cells. Molecular inhibition of NMDA receptor by using small interfering RNA showed protective effect whereas inhibition of H2S production by propargylglycine (PG) (inhibitor of enzyme CSE) showed mitotoxic effect. Taken together, results demonstrate that, administration of H2S protected the cells from HHcy‐induced mitochondrial toxicity and endothelial dysfunction. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
  • Inflammation‐induced down‐regulation of butyrate uptake and
           oxidation is not caused by a reduced gene expression
    • Abstract: In ulcerative colitis (UC) the butyrate metabolism is impaired, leading to energy‐deficiency in the colonic cells. The effect of inflammation on the butyrate metabolism was investigated. HT‐29 cells were incubated with pro‐inflammatory cytokines (TNF‐α and/or IFN‐γ) for 1h and 24h. Cells were additionally stimulated with butyrate to investigate its anti‐inflammatory potential. Butyrate uptake and oxidation were measured using 14C‐labelled butyrate. Gene expression of the butyrate metabolism enzymes, interleukin 8 (IL‐8; inflammatory marker) and villin‐1 (VIL‐1; epithelial cell damage marker) was measured via quantitative RT‐PCR. Significantly increased IL‐8 expression and decreased VIL‐1 expression after 24h incubation with TNF‐α and/or IFN‐γ confirmed the presence of inflammation. These conditions induced a decrease of both butyrate uptake and oxidation, whereas the gene expression was not reduced. Simultaneous incubation with butyrate counteracted the reduced butyrate oxidation. In contrast, 1h incubation with TNF‐α induced a significant increased IL‐8 expression and decreased butyrate uptake. Incubation with TNF‐α and/or IFN‐γ for 1h did not induce cell damage nor influence butyrate oxidation. The inflammation‐induced down‐regulation of the butyrate metabolism was not caused by a reduced gene expression, but appeared consequential to a decreased butyrate uptake. Increasing the luminal butyrate levels might have therapeutic potential in UC. © 2014 Wiley Periodicals, Inc.
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