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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
           bone
    • 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
       
  • Review: CXCR4/CXCR7 molecular involvement in neuronal and neural
           progenitor migration: focus in CNS repair
    • Authors: José Joaquín Merino; Victor Bellver-Landete, María Jesús Oset-Gasque, Beatriz Cubelos
      Pages: n/a - n/a
      Abstract: In the adult brain, Neural Progenitor Cells (NPCs) reside in the subventricular zone (SVZ) of the lateral ventricles, the dentate gyrus and the olfactory bulb. Following CNS insult, NPCs from the SVZ can migrate along the rostral migratory stream (RMS), a migration of NPCs that is directed by proinflammatory cytokines. Indeed, cells expressing CXCR4 follow a homing signal that ultimately leads to neuronal integration and CNS repair, although such molecules can also promote NPC quiescence. The ligand, SDF1 alpha (or CXCL12) is one of the chemokines secreted at sites of injury that it is known to attract NSC-derived neuroblasts, cells that express CXCR4. In function of its concentration, CXCL12 can induce different responses, promoting NPC migration at low concentrations while favoring cell adhesion via EGF and the alpha 6 integrin at high CXCL12 concentrations. However, the preclinical effectiveness of chemokines and their relationship with NPC mobilization requires further study, particularly with respect to CNS repair. Indeed, NPC migration may also be affected by the release of cytokines or chemokines induced by local inflammation, through autocrine or paracrine mechanisms, as well as through erythropoietin (EPO) or nitric oxide (NO) release. CXCL12 activity requires G-coupled proteins and the availability of its ligand may be modulated by its binding to CXCR7, for which it shows a stronger affinity than for CXCR4. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-10T04:35:01.992366-05:
      DOI: 10.1002/jcp.24695
       
  • A FUNCTIONAL TRANSIENT RECEPTOR POTENTIAL VANILLOID 4 (TRPV4) CHANNEL IS
           EPXRESSED IN HUMAN ENDOTHELIAL PROGENITOR CELLS
    • Authors: Silvia Dragoni; Germano Guerra, Alessandra Fiorio Pla, Giuseppe Bertoni, Alessandra Rappa, Valentina Poletto, Cinzia Bottino, Adele Aronica, Francesco Lodola, Maria Pia Cinelli, Umberto Laforenza, Vittorio Rosti, Franco Tanzi, Luca Munaron, Francesco Moccia
      Pages: n/a - n/a
      Abstract: Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial and recapitulate the vascular network of injured tissues. Intracellular Ca2+ signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca2+-permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store-operated Ca2+ entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays a master signalling role in mature endothelium, by controlling both vascular remodelling and arterial pressure. We found that EPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca2+ signals uniquely in presence of extracellular Ca2+. GSK- and PMA-induced Ca2+ elevations were inhibited by RN-1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor-enriched culture medium. Conversely, SOCE inhibition with BTP-2, La3+ and Gd3+ dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-09T06:28:35.242331-05:
      DOI: 10.1002/jcp.24686
       
  • EGF regulates claudin-2 and -4 expression through STAT3 and Src in MDCK
           cells
    • Authors: Vicky García-Hernández; Catalina Flores-Maldonado, Ruth Rincon-Heredia, Odette Verdejo-Torres, José Bonilla-Delgado, Ivan Meneses-Morales, Patricio Gariglio, Rubén G. Contreras
      Pages: n/a - n/a
      Abstract: Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-09T06:16:47.503243-05:
      DOI: 10.1002/jcp.24687
       
  • CCN1 secreted by tonsil-derived mesenchymal stem cells promotes
           endothelial cell angiogenesis via integrin αvβ3 and AMPK
    • Authors: Yoon Shin Park; Soojin Hwang, Yoon Mi Jin, Yeonsil Yu, Sung-Ae Jung, Sung-Chul Jung, Kyung-Ha Ryu, Han Su Kim, Inho Jo
      Pages: n/a - n/a
      Abstract: CCN1 is highly expressed in cancer cells and has been identified in the secretome of bone marrow-derived mesenchymal stem cells (BM-MSC). Although secreted CCN1 is known to promote angiogenesis, its underlying mechanism remains unclear. Here, we examined whether our recently-established tonsil-derived MSC (T-MSC) secrete CCN1 and, if any, how CCN1 promotes the angiogenesis of human umbilical vein endothelial cells (HUVEC). Compared with untreated control T-MSC, a higher level of CCN1 was secreted by T-MSC treated with activin A and sonic hedgehog, drugs known to induce endodermal differentiation. Expectedly, conditioned medium collected from differentiated T-MSC (DCM) significantly increased HUVEC migration and tube formation compared with that from control T-MSC (CCM), and these stimulatory effects were reversed by neutralization with anti-CCN1 antibody. Treatment with recombinant human CCN1 (rh-CCN1) alone also mimicked the stimulatory effects of DCM. Furthermore, treatment with either DCM or rh-CCN1 increased the phosphorylation of AMP kinase (AMPK), and ectopic expression of siRNA of the AMPK gene inhibited all observed effects of both DCM and rh-CCN1. However, no alteration of intracellular ATP levels or phosphorylation of LKB1, a well-known upstream factor of AMPK activation, was observed under our conditions. Finally, the neutralization of integrin αvβ3 with anti-integrin αvβ3 antibody almost completely reversed the effects of CCN1 on AMPK phosphorylation, and EC migration and tube formation. Taken together, we demonstrated that T-MSC increase the secretion of CCN1 in response to endodermal differentiation and that integrin αvβ3 and AMPK mediate CCN1-induced EC migration and tube formation independent of intracellular ATP levels alteration. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-09T06:11:32.787331-05:
      DOI: 10.1002/jcp.24690
       
  • EGFL7 is expressed in bone microenvironment and promotes angiogenesis via
           ERK, STAT3, and integrin signaling cascades
    • Authors: Shek Man Chim; Vincent Kuek, Siu To Chow, Bay Sie Lim, Jennifer Tickner, Jinmin Zhao, Rosa Chung, Yu-Wen Su, Ge Zhang, Wendy Erber, Cory J Xian, Vicki Rosen, Jiake Xu
      Pages: n/a - n/a
      Abstract: Angiogenesis plays a pivotal role in bone formation, remodeling, and fracture healing. The regulation of angiogenesis in the bone microenvironment is highly complex and orchestrated by intercellular communication between bone cells and endothelial cells. Here, we report that EGF-like domain 7 (EGFL7), a member of the epidermal growth factor (EGF) repeat protein superfamily is expressed in both the osteoclast and osteoblast lineages, and promotes endothelial cell activities. Addition of exogenous recombinant EGFL7 potentiates SVEC (simian virus 40-transformed mouse microvascular endothelial cell line) cell migration and tube-like structure formation in vitro. Moreover, recombinant EGFL7 promotes angiogenesis featuring web-like structures in ex vivo fetal mouse metatarsal angiogenesis assay. We show that recombinant EGFL7 induces phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), and focal adhesion kinase (FAK) in SVEC cells. Inhibition of ERK1/2 and STAT3 signaling impairs EGFL7-induced endothelial cell migration, and angiogenesis in fetal mouse metatarsal explants. Bioinformatic analyses indicate that EGFL7 contains a conserved RGD/QGD motif and EGFL7-induced endothelial cell migration is significantly reduced in the presence of RGD peptides. Moreover, EGFL7 gene expression is significantly upregulated during growth plate injury repair. Together, these results demonstrate that EGFL7 expressed by bone cells regulates endothelial cell activities through integrin-mediated signaling. This study highlights the important role that EGFL7 expressed in bone microenvironment plays in the regulation of angiogenesis in bone. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-09T06:11:19.231353-05:
      DOI: 10.1002/jcp.24684
       
  • Human Olfactory Bulb Neural Stem Cells expressing hNGF Restore Cognitive
           Deficit in Alzheimer's Disease Rat Model
    • Authors: Hany E. S. Marei; Amany Farag, Asma Althani, Nahla Afifi, Abd-Elmaksoud A , Samah Lashen, Shaymaa Rezk, Roberto Pallini, Patrizia Casalbore, Carlo Cenciarelli
      Pages: n/a - n/a
      Abstract: In this study, we aim at demonstrating the fate of allogenic adult human olfactory bulb neural stem/progenitor cells (OBNSC/NPCs) transplanted into the rat hippocampus treated with ibotenic acid (IBO), a neurotoxicant specific to hippocampal cholinergic neurons that are lost in Alzheimer's disease. We assessed their possible ability to survive, integrate, proliferate, and differentiate into different neuronal and glial elements; and tried to evaluate their possible therapeutic potential, and the mechanism(s) relevant to neuroprotection following their engraftment into the CNS milieu. OBNSC/NPCs were isolated from adult human olfactory bulb patients, genetically engineered to express GFP and human nerve growth factor (hNGF) by lentivirus-mediated infection, and stereotaxically transplanted into the hippocampus of IBO-treated animals and controls. Stereological analysis of engrafted OBNSCs eight weeks post transplantation revealed a 1.89 fold increase with respect to the initial cell population, indicating a marked ability for survival and proliferation. In addition, 54.71 ± 11.38%, 30.18 ± 6.00%, and 15.09 ± 5.38% of engrafted OBNSCs were identified by morphological criteria suggestive of mature neurons, oligodendrocytes and astrocytes respectively. Taken together, this work demonstrated that human OBNSCs expressing NGF ameliorate the cognitive deficiencies associated with IBO-induced lesions in AD model rats, and the improvement can probably be attributed primarily to neuronal and glial cell replacement as well as the trophic influence exerted by the secreted NGF. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-09T06:09:11.317525-05:
      DOI: 10.1002/jcp.24688
       
  • MicroRNA-based Biotechnology for Plant Improvement
    • Authors: Baohong Zhang; Qinglian Wang
      Pages: n/a - n/a
      Abstract: MicroRNAs (miRNAs) are an extensive class of newly discovered endogenous small RNAs, which negatively regulate gene expression at the post-transcription levels. As the application of next-generation deep sequencing and advanced bioinformatics, the miRNA-related study has been expended to non-model plant species and the number of identified miRNAs has dramatically increased in the past years. miRNAs play a critical role in almost all biological and metabolic processes, which provides an unique strategy for plant improvement. Here, we first review the discover history and biogenesis of miRNAs in brief, but focus on more importantly on the application of miRNAs on plant breeding and the future directions. Increasing plant biomass through controlling plant development and phase change is one achievement for miRNA-based biotechnology; plant tolerance to abiotic and biotic stress was also significantly enhanced by regulating the expression of an individual miRNA. Both endogenous and artificial miRNAs may serve as an important tool for plant improvement. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-09T06:09:09.022283-05:
      DOI: 10.1002/jcp.24685
       
  • Brain natriuretic peptide and C-type natriuretic peptide maintain porcine
           oocyte meiotic arrest
    • Authors: Wenqiang Zhang; Ye Yang, Wei Liu, Qian Chen, Huarong Wang, Xiao Wang, Yanhao Zhang, Meijia Zhang, Guoliang Xia
      Pages: n/a - n/a
      Abstract: Recent studies have shown that C-type natriuretic peptide (CNP) serves as a key control system during mouse oocyte maturation. We used pig models (in vitro and in vivo) to explore the role played by the natriuretic peptide family in porcine oocyte maturation. We reported the expression and location of natriuretic peptide system in different stages of porcine antral follicles. Atrial natriuretic peptide (ANP) and CNP were expressed primarily in granulosa cells, whereas brain natriuretic peptide (BNP) and natriuretic peptide receptor-B (NPRB) receptor were expressed in granulosa cells (both cumulus and mural granulosa cells) and thecal internal cells, and the natriuretic peptide receptor-A (NPRA) receptor predominantly in thecal cells. Upon in vitro culture, BNP and CNP maintained meiotic arrest of oocytes associated with cumulus cells. The expression levels of BNP, CNP, and the NPRB receptor increased upon treatment of prepubertal gilts with pregnant mare's serum gonadotropin and decreased upon subsequent human chorionic gonadotropin injection. Such dynamic changes in the expression of natriuretic peptides and their receptor paralleled the proportions of oocytes exhibiting nuclear maturation in vivo. These data indicated that BNP and CNP co-contributed to maintaining porcine meiotic arrest under physiological condition and lutenizing hormone (LH) relieved this inhibitory effect by decreasing the expression levels of BNP and CNP in vivo. Our present work, combined with previous data, improved the understanding of the oocyte meiotic arrest mechanisms and further revealed that natriuretic peptides serve as oocyte maturation inhibitor (OMI) to inhibit oocyte maturation in mammals. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-09T06:08:03.925794-05:
      DOI: 10.1002/jcp.24682
       
  • Journal of Cellular Physiology: Volume 229, Number 9, September 2014
    • Abstract: Cover: Micrograph of osteoprogenitor cells. See article by Vimalraj et al. on pages (1236–1244).
      PubDate: 2014-05-23T12:05:18.212085-05:
      DOI: 10.1002/jcp.24673
       
  • Editor's Choice
    • PubDate: 2014-05-23T12:05:15.574933-05:
      DOI: 10.1002/jcp.24675
       
  • Table of Contents: Volume 229, Number 9
    • PubDate: 2014-05-23T12:05:14.713865-05:
      DOI: 10.1002/jcp.24674
       
  • Highlights: Volume 229, Number 9
    • PubDate: 2014-05-23T12:05:13.182589-05:
      DOI: 10.1002/jcp.24676
       
  • Monitoring Collagen Synthesis in Fibroblasts Using Fluorescently Labeled
           tRNA Pairs
    • Authors: Jiaqi Liu; Macarena Pampillo, Fen Guo, Shangxi Liu, Barry S. Cooperman, Ian Farrell, Dvir Dahary, Bing S. Gan, David B. O'Gorman, Zeev Smilansky, Andy V. Babwah, Andrew Leask
      First page: 1121
      Abstract: There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET‐based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti‐fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells. J. Cell. Physiol. 229: 1121–1129, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:16.801072-05:
      DOI: 10.1002/jcp.24630
       
  • Nerve Growth Factor Modulation of Retinal Ganglion Cell Physiology
    • Authors: Gloria Roberti; Flavio Mantelli, Ilaria Macchi, Mina Massaro-Giordano, Marco Centofanti
      First page: 1130
      Abstract: Nerve growth factor (NGF) is an endogenous neurotrophin involved in the development, maintenance and regeneration of mammalian sympathetic and sensory neurons. Additionally, NGF is known to have trophic and differentiating activity on several populations of cholinergic neurons of the central nervous system (CNS), and to act as a differentiation factor in the development of the visual cortex. The paramount functions of NGF in the visual system are also highlighted by the presence of this neurotrophin and both its receptors TrkA and p75 in most intra‐ocular tissues, including lens, vitreous, choroid, iris, and trabecular meshwork. In the retina, NGF is produced and utilized specifically by retinal ganglion cells (RGC), bipolar neurons and glial cells, and is thought to have crucial protective effects in several disease states. Studies on the role of NGF on RGCs survival following optic nerve transection, ischemic injury, ocular hypertension and glaucoma are discussed in this review. J. Cell. Physiol. 229: 1130–1133, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:13.526805-05:
      DOI: 10.1002/jcp.24573
       
  • Assessing the Functional Mechanical Properties of Bioengineered Organs
           With Emphasis on the Lung
    • Authors: Béla Suki
      First page: 1134
      Abstract: Recently, an exciting new approach has emerged in regenerative medicine pushing the forefront of tissue engineering to create bioartificial organs. The basic idea is to create biological scaffolds made of extracellular matrix (ECM) that preserves the three‐dimensional architecture of an entire organ. These scaffolds are then used as templates for functional tissue and organ reconstruction after re‐seeding the structure with stem cells or appropriately differentiated cells. In order to make sure that these bioartificial organs will be able to function in the mechanical environment of the native tissue, it is imperative to fully characterize their mechanical properties and match them with those of the normal native organs. This mini‐review briefly summarizes modern measurement techniques of mechanical function characterized mostly by the material or volumetric stiffness. Micro‐scale and macro‐scale techniques such as atomic force microscopy and the tissue strip stress–strain approach are discussed with emphasis on those that combine mechanical measurements with structural visualization. Proper micro‐scale stiffness helps attachment and differentiation of cells in the bioartificial organ whereas macro‐scale functionality is provided by the overall mechanical properties of the construct. Several approaches including failure mechanics are also described, which specifically probe the contributions of the main ECM components including collagen, elastin, and proteoglycans to organ level ECM function. Advantages, drawbacks, and possible pitfalls as well as interpretation of the data are given throughout. Finally, specific techniques to assess the functionality of the ECM of bioartificial lungs are separately discussed. J. Cell. Physiol. 229: 1134–1140, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:18.737273-05:
      DOI: 10.1002/jcp.24600
       
  • New Advances of microRNAs in Glioma Stem Cells, With Special Emphasis on
           Aberrant Methylation of microRNAs
    • Authors: Bing Zhao; Er-Bao Bian, Jia Li, Jun Li
      First page: 1141
      Abstract: Malignant brain tumors are thought to be originate from a small population of cells that display stem cell properties, including the capacity of self‐renewal, multipotent differentiation, initiation of tumor tissues. Cancer stem cells (CSCs) have been identified in gliomas in which they are named as glioma stem cells (GSCs). GSCs, sharing some characteristics with normal neural stem cells (NSCs), contribute to the cellular origin for primary gliomas and the recurrence of malignant gliomas after current conventional therapy. Recently, increasing evidences have showed that miRNAs play a central role in GSCs. In this review we focus on the role of GSCs in gliomas and in the abnomal expression of miRNAs in GSCs. Furthermore, we also discuss epigenetic dysregulation of tumor‐suppressor miRNAs by promoter DNA methylation is involved in the regulation of GSCs biology. Recent advances in understanding dysregulated expression of miRNAs and methylation of tumor‐suppressor miRNAs in GSCs and their possible use as new therapeutic targets of gliomas. J. Cell. Physiol. 229: 1141–1147, 2014. © 2013 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:11.93198-05:0
      DOI: 10.1002/jcp.24540
       
  • Basic Models Modeling Resistance Training: An Update for Basic Scientists
           Interested in Study Skeletal Muscle Hypertrophy
    • Authors: Jason Cholewa; Lucas Guimarães-Ferreira, Tamiris da Silva Teixeira, Marshall Alan Naimo, Xia Zhi, Rafaele Bis Dal Ponte de Sá, Alice Lodetti, Mayara Quadros Cardozo, Nelo Eidy Zanchi
      First page: 1148
      Abstract: Human muscle hypertrophy brought about by voluntary exercise in laboratorial conditions is the most common way to study resistance exercise training, especially because of its reliability, stimulus control and easy application to resistance training exercise sessions at fitness centers. However, because of the complexity of blood factors and organs involved, invasive data is difficult to obtain in human exercise training studies due to the integration of several organs, including adipose tissue, liver, brain and skeletal muscle. In contrast, studying skeletal muscle remodeling in animal models are easier to perform as the organs can be easily obtained after euthanasia; however, not all models of resistance training in animals displays a robust capacity to hypertrophy the desired muscle. Moreover, some models of resistance training rely on voluntary effort, which complicates the results observed when animal models are employed since voluntary capacity is something theoretically impossible to measure in rodents. With this information in mind, we will review the modalities used to simulate resistance training in animals in order to present to investigators the benefits and risks of different animal models capable to provoke skeletal muscle hypertrophy. Our second objective is to help investigators analyze and select the experimental resistance training model that best promotes the research question and desired endpoints. J. Cell. Physiol. 229: 1148–1156, 2014. © 2013 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:11.516799-05:
      DOI: 10.1002/jcp.24542
       
  • Proteasome Modulator 9 Gene SNPs, Responsible for Anti‐Depressant
           Response, Are in Linkage With Generalized Anxiety Disorder
    • Authors: Claudia Gragnoli
      First page: 1157
      Abstract: Proteasome modulator 9 (PSMD9) gene single nucleotide polymorphism (SNP) rs1043307/rs2514259 (E197G) is associated with significant clinical response to the anti‐depressant desipramine. PSMD9 SNP rs74421874 [intervening sequence (IVS) 3 + nt460 G>A], rs3825172 (IVS3 + nt437 C>T) and rs1043307/rs2514259 (E197G A>G) are all linked to type 2 diabetes (T2D), maturity‐onset‐diabetes‐of the young 3 (MODY3), obesity and waist circumference, hypertension, hypercholesterolemia, T2D‐macrovascular and T2D‐microvascular disease, T2D‐neuropathy, T2D‐carpal tunnel syndrome, T2D‐nephropathy, T2D‐retinopathy, non‐diabetic retinopathy and depression. PSMD9 rs149556654 rare SNP (N166S A>G) and the variant S143G A>G also contribute to T2D. PSMD9 is located in the chromosome 12q24 locus, which per se is in linkage with depression, bipolar disorder and anxiety. In the present study, we wanted to determine whether PSMD9 is linked to general anxiety disorder in Italian T2D families. Two‐hundred Italian T2D families were phenotyped for generalized anxiety disorder, using the diagnostic criteria of DSM‐IV. When the diagnosis was unavailable or unclear, the trait was reported as unknown. The 200 Italians families were tested for the PSMD9 T2D risk SNPs rs74421874 (IVS3 + nt460 G>A), rs3825172 (IVS3 +nt437 T>C) and for the T2D risk and anti‐depressant response SNP rs1043307/rs2514259 (E197G A>G) for evidence of linkage with generalized anxiety disorder. Non‐parametric linkage analysis was executed via Merlin software. One‐thousand simulation tests were performed to exclude results due to random chance. In our study, the PSMD9 gene SNPs rs74421874, rs3825172, and rs1043307/rs2514259 result in linkage to generalized anxiety disorder. This is the first report describing PSMD9 gene SNPs in linkage to generalized anxiety disorder in T2D families. J. Cell. Physiol. 229: 1157–1159, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:14.750969-05:
      DOI: 10.1002/jcp.24581
       
  • SKP2 Overexpression Is Associated With Increased Serine 10 Phosphorylation
           of p27 (pSer10p27) in Triple‐Negative Breast Cancer
    • Authors: Katerina D. Fagan-Solis; Brian T. Pentecost, Joseph M. Gozgit, Brooke A. Bentley, Sharon M. Marconi, Christopher N. Otis, Douglas L. Anderton, Sallie Smith Schneider, Kathleen F. Arcaro
      First page: 1160
      Abstract: S‐phase kinase‐associated protein 2 (SKP2) is an important cell cycle regulator, targeting the cyclin‐dependent kinase (CDK) inhibitor p27 for degradation, and is frequently overexpressed in breast cancer. p27 regulates G1/S transition by abrogating the activity of cyclin/CDK complexes. p27 can undergo phosphorylation at serine 10 (pSer10p27). This phosphorylation event is associated with increased cell proliferation and poor prognosis in patients with glioma. The relationship between SKP2 and pSer10p27 in breast cancer has not been previously investigated. Immunohistochemistry (IHC) of SKP2, p27, pSer10p27, and other genes involved in this pathway, was analyzed in 188 breast tumors and 50 benign reduction mammoplasty samples. IHC showed SKP2 to be more highly expressed in estrogen receptor α (ERα)‐negative breast cancers and demonstrated that triple‐negative tumors were more likely to have high expression of SKP2 than were non‐triple negative, ERα‐negative tumors. A significant positive relationship was discovered for SKP2 and pSer10p27. High levels of SKP2 and pSer10p27 were observed significantly more often in ERα‐negative and triple‐negative than in ERα‐positive breast cancers. Use of the triple‐negative TMX2‐28 breast cancer cell line to address the role of SKP2 in cell cycle progression confirmed that SKP2 contributes to a more rapid cell cycle progression and may regulates pSer10p27 levels. Together, the results indicate that presence of high SKP2 plus high pSer10p27 levels in triple‐negative breast cancers is associated with aggressive growth, and highlight the validity of using SKP2 inhibitors as a therapeutic approach for treating this subset of breast cancers. J. Cell. Physiol. 229: 1160–1169, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:15.095217-05:
      DOI: 10.1002/jcp.24545
       
  • Dependence of Castration‐Resistant Prostate Cancer (CRPC) Stem Cells
           on CRPC‐Associated Fibroblasts
    • Authors: Helty Adisetiyo; Mengmeng Liang, Chun-Peng Liao, Joseph H. Jeong, Michael B. Cohen, Pradip Roy-Burman, Baruch Frenkel
      First page: 1170
      Abstract: We previously established a role for cancer‐associated fibroblasts (CAF) in enhancing the self‐renewal and differentiation potentials of putative prostate cancer stem cells (CSC). Our published work focused on androgen‐dependent prostate cancer (ADPC) using the conditional Pten deletion mouse model. Employing the same model, we now describe the interaction of CAF and CSC in castration‐resistant prostate cancer (CRPC). CAF isolated from ADPC (ADPCAF) and from CRPC (CRPCAF) were compared in terms of their ability to support organoid formation and tumor initiation by CSC from CRPC (CRPCSC) in vitro and in vivo. CRPCSC formed spheroids in vitro and well‐differentiated glandular structures under the renal capsules of recipient mice in vivo more effectively in the presence of CRPCAF compared to ADPCAF. Furthermore, whereas CSC with CAF from ADPC formed mostly well‐differentiated tumors in our previous study, we now show that CRPCSC, when combined with CRPCAF (but not ADPCAF), can form aggressive, poorly‐differentiated tumors. The potential of CRPCAF to support organoid/tumor formation by CRPCSC remained greater even when compared to 10‐fold more ADPCAF, suggesting that paracrine factors produced specifically by CRPCAF preferentially potentiate the stemness and tumorigenic properties of the corresponding CSC. This apparently unique property of CRPCAF was notable when the CAF and CSC were grafted in either intact or castrated recipient mice. In both environments, CRPCAF induced in the epithelial compartment higher proliferative activity compared to ADPCAF, indicated by a higher Ki67 index. Factors released by CRPCAF to regulate CRPCSC may be targeted to develop novel therapeutic approaches to manage advanced prostate cancer. J. Cell. Physiol. 229: 1170–1176, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:11.007251-05:
      DOI: 10.1002/jcp.24546
       
  • Definition of Novel Electrochemotherapy Parameters and Validation of their
           in Vitro and in Vivo Effectiveness
    • Authors: Enrico P. Spugnini; Alessandro Melillo, Lucio Quagliuolo, Mariarosaria Boccellino, Bruno Vincenzi, Paola Pasquali, Alfonso Baldi
      First page: 1177
      Abstract: Electrochemotherapy (ECT) is a cancer therapy that conjugates the administration of a chemotherapy agent to the delivery of permeabilizing pulses released singularly or as bursts. This approach results in higher number of anticancer molecules delivered to their biological targets, but is also associated to undesirable side effects such as pain and muscular contractions. A new electroporator delivering train of eight biphasic pulses at the voltage of 1,300 V/cm lasting 50 + 50 µsec each, with a frequency of 1 Hz, and with 10‐µsec interpulse intervals (total treatment time: 870 µsec/cm2 of treated area) was tested in vitro on the human lung cancer cell line A549 and in vivo, both in mice xenografts and privately owned rabbits with spontaneous tumors. The tumor cell line was treated with electroporation using the new parameters, that showed improved drug efficacy in causing cell death. Mice with chemoresistant xenografts were treated as well with either the new parameters and with a previous protocol, confirming the higher tolerability and efficacy of the novel parameters. Finally, a cohort of six pet rabbits with advanced skin neoplasms were enrolled in a compassionate trial using the new parameters in adjuvant fashion. In terms of efficacy, none of the rabbits experienced tumor recurrence, showing minimal discomfort during the ECT sessions. The data described, demonstrate that the new permeabilizing protocol adopting biphasic electric pulses displays a significant higher efficacy compared to previous ECT treatments and substantial reduction of the associated morbidity. J. Cell. Physiol. 229: 1177–1181, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:13.258554-05:
      DOI: 10.1002/jcp.24548
       
  • Protective Effect of the Y220C Mutant p53 Against Steatosis: Good
           News'
    • Authors: Manuele Gori; Barbara Barbaro, Mario Arciello, Roberta Maggio, Carmela Viscomi, Alessia Longo, Clara Balsano
      First page: 1182
      Abstract: Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis to steatohepatitis, which may progress to fibrosis, and cirrhosis, leading eventually to hepatocarcinoma development. Recently, cases of hepatocarcinoma have been diagnosed in steatotic patients without nonalcoholic steatohepatitis (NASH) and cirrhosis. The p53 protein, besides its function as tumor suppressor, is emerging as an important regulator of cellular metabolism, but its role in steatosis remains unclear. We induced steatosis in HepG2 (wt‐p53) and Huh7.5.1 (Y220C‐mutant p53) cells using free fatty acids. We observed a different modulation of p53, different intracellular lipid content, and similar down‐regulation of the de novo lipid synthesis genes but opposite modulation of the fatty acid β‐oxidation pathway between HepG2 and Huh7.5.1. Accordingly, we found a diverse amount of apoptosis and reactive oxygen species between the two cell lines. Transfection of the wt‐p53 in Huh7.5.1 cells reverted the different lipid metabolism behavior observed in these cells. In conclusion, unlike the wt‐p53, the Y220C mutant provides a specific protection against steatosis and potentially against its progression. Our findings highlight for the first time an unknown role of a p53 mutant in the setting of steatosis. Being this mutation very frequent in human cancers, this study could be a breakthrough in explaining the occurrence of hepatocarcinoma in steatotic patients without NASH and cirrhosis. J. Cell. Physiol. 229: 1182–1192, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:16.396848-05:
      DOI: 10.1002/jcp.24550
       
  • Tanshinone II A Inhibits Tat‐Induced HIV‐1 Transactivation
           Through Redox‐Regulated AMPK/Nampt Pathway
    • Authors: Hong-Sheng Zhang; Xin-Yu Chen, Tong-Chao Wu, Feng-Juan Zhang
      First page: 1193
      Abstract: Tat transactivating activity regulated by NAD+‐dependent histone deacetylase sirtuin1 (SIRT1) connects HIV transcription with the metabolic state of the cell. Nicotinamide phosphoribosyltransferase (Nampt) is a rate‐limiting enzyme in the mammalian NAD+ biosynthesis. Nampt, SIRT1, and AMPK were involved in inhibiting HIV‐1 transactivation through redox‐regulated pathway. Tanshinone II A is a main lipid‐soluble monomer derivative from the root of Salvia miltiorrhiza (Danshen) and tanshinone II A possess a variety of biological activities through redox signaling pathway. Here we investigated the effect of tanshinone II A on Tat‐induced HIV‐1 transactivation and the redox signaling pathway involved in it. As the results were shown, tanshinone II A reversed Tat‐induced reactive oxygen species (ROS) production and down‐regulation of glutathione (GSH) levels in TZM‐bl cells through up‐regulation of Nrf2 expression. Tanshinone II A reversed Tat‐induced inhibition of SIRT1 activity but not SIRT1 protein expression. Tanshinone II A reversed Tat‐induced inhibition of Nampt protein expression and depletion of NAD+ levels in TZM‐bl cells in a dose‐dependent manner. Tanshinone II A‐evoked Nampt expression was mediated by AMPK signaling pathway. Tanshinone II A inhibited Tat‐induced HIV‐1 LTR transactivation dependent on AMPK‐Nampt pathway. Collectively, our data provide new insights into understanding of the molecular mechanisms of tanshinone II A inhibited Tat‐regulated transcription, suggesting that targeting AMPK/Nampt/SIRT1 pathway could serve as new anti‐HIV‐1 agents. J. Cell. Physiol. 229: 1193–1201, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:17.835059-05:
      DOI: 10.1002/jcp.24552
       
  • Exosomal ATF3 RNA Attenuates Pro‐Inflammatory Gene MCP‐1
           Transcription in Renal Ischemia‐Reperfusion
    • Authors: Hsi-Hsien Chen; Pei-Fang Lai, Yi-Fan Lan, Ching-Feng Cheng, Wen-Bing Zhong, Yuh-Feng Lin, Tzen-Wen Chen, Heng Lin
      First page: 1202
      Abstract: Transcriptional repressor activating transcription factor 3 (ATF3) is induced by various stress stimuli, including inflammation‐induced renal injury. In addition, ATF3 also down‐regulates adhesion molecules like intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), and monocyte chemotactic protein‐1 (MCP‐1). However, the relation between up‐regulated ATF3 after renal ischemia/reperfusion (I/R) injury and MCP‐1 is not completely understood. In this study, we demonstrated that, in renal I/R induced inflammation, induction of adhesion molecules (interleukin‐6, P‐selectin, E‐selectin, ICAM, VCAM, and MCP‐1) was higher in ATF3‐knockout mice than in wild‐type animals. Molecular and biochemical analyses revealed that ATF3 binds to the ATF/CRE sites in the MCP‐1 promoter and inhibits the secretion of MCP‐1 from renal epithelial cells after I/R injury. Urinary exosome containing ATF3 RNA was 60‐fold higher in patients with acute kidney injury than in normal controls, but no difference in total urinary ATF3 RNA levels was found. In addition, in vitro study showed that exosome containing ATF3 RNA derived from epithelial cells also inhibits MCP‐1 expression in the epithelial cells and macrophage migration. Furthermore, direct administration of the epithelium‐derived exosomal ATF3 RNA attenuates I/R induced kidney injury. Together, our studies reveal a novel regulatory mechanism of MCP‐1 expression mediated by the exosomal ATF3 RNA under renal I/R insult and suggest a potential targeted therapy for I/R induced acute kidney injury. J. Cell. Physiol. 229: 1202–1211, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:14.315745-05:
      DOI: 10.1002/jcp.24554
       
  • Human Placental Extract Mediated Inhibition of Proteinase K: Implications
           of Heparin and Glycoproteins in Wound Physiology
    • Authors: Kanika Sharma; Chaitali Mukherjee, Siddhartha Roy, Debashree De, Debasish Bhattacharyya
      First page: 1212
      Abstract: Efficient debridement of the wound bed following the removal of microbial load prevents its progression into a chronic wound. Bacterial infection and excessive proteolysis characterize impaired healing and therefore, their inhibition might restore the disturbed equilibrium in the healing process. Human placental extract exhibits reversible, non‐competitive inhibition towards Proteinase K, a microbial protease, by stabilizing it against auto‐digestion. Scattering and fluorescence studies followed by biochemical analysis indicated the involvement of a glycan moiety. Surface plasmon resonance demonstrated specific interaction of heparin with Proteinase K having Kd in μM range. Further, Proteinase K contains sequence motifs similar to other heparin‐binding proteins. Molecular docking revealed presence of clefts suitable for binding of heparin‐derived oligosaccharides. Comprehensive analysis of this inhibitory property of placental extract partly explains its efficacy in curing wounds with common bacterial infections. J. Cell. Physiol. 229: 1212–1223, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:13.772478-05:
      DOI: 10.1002/jcp.24555
       
  • SIRT1 Knockdown Promotes Neural Differentiation and Attenuates the Heat
           Shock Response
    • Authors: Diana J. Liu; David Hammer, Daniel Komlos, Kuang Yu Chen, Bonnie L. Firestein, Alice Y.-C. Liu
      First page: 1224
      Abstract: Neurons have a limited capacity for heat shock protein (HSP) induction and are vulnerable to the pathogenic consequence of protein misfolding and aggregation as seen in age‐related neurodegenerative diseases. Sirtuin 1 (SIRT1), an NAD+‐dependent lysine deacetylase with important biological functions, has been shown to sustain the DNA‐binding state of HSF1 for HSP induction. Here we show that differentiation and maturation of embryonic cortical neurons and N2a neuroprogenitor cells is associated with decreases in SIRT1 expression and heat shock‐dependent induction of HSP70 protein. Tests of a pharmacological activator and an inhibitor of SIRT1 affirm the regulatory role of SIRT1 in HSP70 induction. Protein cross‐linking studies show that nuclear SIRT1 and HSF1 form a co‐migrating high molecular weight complex upon stress. The use of retroviral vectors to manipulate SIRT1 expression in N2a cells show that shRNA‐mediated knock down of SIRT1 causes spontaneous neurite outgrowth coincident with reduced growth rate and decreased induction of hsp70‐reporter gene, whereas SIRT1 over‐expression blocks the induced neural differentiation of N2a cells. Our results suggest that decreased SIRT1 expression is conducive to neuronal differentiation and this decrease contributes to the attenuated induction of HSPs in neurons. J. Cell. Physiol. 229: 1224–1235, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:12.354991-05:
      DOI: 10.1002/jcp.24556
       
  • A Positive Role of MicroRNA‐15b on Regulation of Osteoblast
           Differentiation
    • Authors: S. Vimalraj; Nicola C. Partridge, N. Selvamurugan
      First page: 1236
      Abstract: Osteoblast differentiation is tightly regulated by several factors including microRNAs (miRNAs). In this paper, we report that pre‐mir‐15b is highly expressed in differentiated osteoblasts. The functional role of miR‐15b in osteoblast differentiation was determined using miR‐15b mimic/inhibitor and the expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP), type I collagen genes was decreased by miR‐15b inhibitor. Runx2, a bone specific transcription factor is generally required for expression of osteoblast differentiation marker genes and in response to miR‐15b inhibitor treatment, Runx2 mRNA expression was not changed; whereas its protein expression was decreased. Even though Smurf1 (SMAD specific E3 ubiquitin protein ligase 1), HDAC4 (histone deacetylase 4), Smad7, and Crim1 were found to be few of miR‐15b's putative target genes, there was increased expression of only Smurf1 gene at mRNA and protein levels by miR‐15b inhibitor. miR‐15b mimic treatment significantly increased and decreased expressions of Runx2 and Smurf1 proteins, respectively. We further identified that the Smurf1 3′UTR is directly targeted by miR‐15b using the luciferase reporter gene system. This is well documented that Smurf1 interacts with Runx2 and degrades it by proteasomal pathway. Hence, based on our results we suggest that miR‐15b promotes osteoblast differentiation by indirectly protecting Runx2 protein from Smurf1 mediated degradation. Thus, this study identified that miR‐15b can act as a positive regulator for osteoblast differentiation. J. Cell. Physiol. 229: 1236–1244, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:19.105526-05:
      DOI: 10.1002/jcp.24557
       
  • Enzymatic and Regulatory Attributes of Trehalose‐6‐Phosphate
           Phosphatase from Candida utilis and its Role During Thermal Stress
    • Authors: Sagar Lahiri; Shakri Banerjee, Trina Dutta, Shinjinee Sengupta, Sandip Dey, Rusha Roy, Devlina Sengupta, Krishnananda Chattopadhyay, Anil K. Ghosh
      First page: 1245
      Abstract: Trehalose‐6‐phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti‐stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43‐fold purification. The protein sequence, determined by N‐terminal sequencing and MALDI‐TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length gene sequence of TPP of C. utilis was identified using rapid amplification of cDNA ends‐PCR reaction (RACE‐PCR). The gene was cloned and expressed in Escherichia coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steady‐state fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. Km was determined to be ~0.8 mM. Optimum temperature and pH were found to be 30 °C and 8.5, respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylation‐mediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations toward understanding the physico‐chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalose synthesis and survivability of the organism under thermal stress was established. J. Cell. Physiol. 229: 1245–1255, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:17.35782-05:0
      DOI: 10.1002/jcp.24562
       
  • The Purα/Purβ Single‐Strand DNA‐Binding Proteins
           Attenuate Smooth‐Muscle Actin Gene Transactivation in Myofibroblasts
           
    • Authors: Seethalakshmi Hariharan; Robert J. Kelm, Arthur Roger Strauch
      First page: 1256
      Abstract: Expression of smooth muscle alpha‐actin (SMαA) is essential for myofibroblast‐mediated wound contraction following tissue injury. The Pur α/β and YB‐1 transcriptional repressors govern the DNA‐binding activity of serum response factor (SRF) and phosphorylated Smad3 (pSmad3) transcriptional activators during induction of SMαA gene expression in human pulmonary myofibroblasts. In quiescent fibroblasts, Pur α exhibited a novel function in enhancing stability of pre‐existing SRF complexes with SMαA core promoter DNA, whereas Pur β was more effective in disrupting SRF‐DNA interaction. Pur proteins were less efficient competitors of pre‐existing, core‐promoter complexes containing both SRF and pSmad3 in nuclear extracts from TGFβ1‐activated myofibroblasts. TGFβ1 signaling dissociated a SRF/Pur protein complex with concurrent formation of a transient pSmad3/MRTF‐A/Pur β complex during early phase myofibroblast differentiation. Pur β was replaced by Pur α in the pSmad3/MRTF‐A complex in mature myofibroblasts. Combining all three repressors potently inhibited SRF and pSmad3 binding to promoter DNA in quiescent fibroblasts and TGFβ1‐activated myofibroblasts, respectively. The results point to dynamic interplay between transcriptional activators and repressors in regulating SMαA gene output during myofibroblast differentiation. Therapeutic targeting of nucleoprotein complexes regulating the SMαA promoter may prevent excessive myofibroblast accumulation associated with chronic cardiopulmonary fibrosis and dysfunctional tissue remodeling. J. Cell. Physiol. 229: 1256–1271, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:18.247781-05:
      DOI: 10.1002/jcp.24564
       
  • Cardioprotective Role of P38 MAPK During Myocardial Infarction Via
           Parallel Activation of α‐Crystallin B and Nrf2
    • Authors: Arkadeep Mitra; Aramita Ray, Ritwik Datta, Shantanu Sengupta, Sagartirtha Sarkar
      First page: 1272
      Abstract: Myocardial infarction (MI) is defined as cardiac cell death due to prolonged ischemia. Although necrotic cell death was considered to be solely responsible for myocyte death during MI, it was recently revealed that apoptosis also plays its part in this death process. Our laboratory has recently shown that endoplasmic reticulum (ER) stress‐induced apoptosis is the predominant route for apoptosis during MI and the conventional mitochondrial pathway is bypassed by activation of a small heat shock protein α‐crystallin B (CRYAB). Since CRYAB is a direct target of P38 mitogen‐activated protein kinase (MAPK) cascade, we were prompted to check the role of P38 MAPK in 20‐week‐old male Wister rats immediately after infarct formation. Interestingly, parallel activation of mitochondrial apoptotic pathway with an increase in ER stress‐induced apoptotic load was observed along with decreased activation of CRYAB and Nrf2 (a pro‐survival protein activated in response to ER stress) in MI rats treated with SB203580, a specific inhibitor of P38α and P38β compared to the MI alone. As a cumulative effect, this inhibitor treatment also resulted in significant increase in the levels of caspase3 activity and TUNEL positivity, the end point apoptotic markers. Furthermore, SB203580‐treated hypoxic adult cardiomyocytes showed formation of desmin aggregates which were previously associated with impaired cardiac function. Thus, this study shows for the first time the precise mechanism by which P38 MAPK plays a pro‐survival role and confers protection of cardiomyocytes, during infarct formation. J. Cell. Physiol. 229: 1272–1282, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:15.606689-05:
      DOI: 10.1002/jcp.24565
       
  • Novel Adeno‐Associated Viral Vector Delivering the Utrophin Gene
           Regulator Jazz Counteracts Dystrophic Pathology in mdx Mice
    • Authors: Georgios Strimpakos; Nicoletta Corbi, Cinzia Pisani, Maria Grazia Di Certo, Annalisa Onori, Siro Luvisetto, Cinzia Severini, Francesca Gabanella, Lucia Monaco, Elisabetta Mattei, Claudio Passananti
      First page: 1283
      Abstract: Over‐expression of the dystrophin‐related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor “Jazz” that up‐regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz‐transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno‐associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle‐specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α‐actin promoter to the AAV vector (mAAV). Injection of mAAV8‐Jazz viral preparations into mdx mice resulted in muscle‐specific Jazz expression coupled with up‐regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8‐Jazz‐treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF‐ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. J. Cell. Physiol. 229: 1283–1291, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:12.803772-05:
      DOI: 10.1002/jcp.24567
       
  • Adenosine Regulates the Proinflammatory Signaling Function of Thrombin in
           Endothelial Cells
    • Authors: Seyed Mahdi Hassanian; Peyman Dinarvand, Alireza R. Rezaie
      First page: 1292
      Abstract: The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin‐mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier‐disruptive effect of thrombin in HUVECs by a concentration‐dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A2A and A2B are expressed at the highest level among the four receptor subtypes (A2B > A2A > A1 > A3) on HUVECs. The barrier‐protective effect of adenosine in response to thrombin was recapitulated by the A2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A2A receptor and by the A2A‐specific antagonists, ZM‐241385 and SCH‐58261. The thrombin‐induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP‐dependent manner, providing a molecular mechanism through which adenosine exerts a barrier‐protective function. Adenosine also inhibited thrombin‐mediated activation of NF‐κB and decreased adhesion of monocytic THP‐1 cells to stimulated HUVECs via down‐regulation of expression of cell surface adhesion molecules, VCAM‐1, ICAM‐1, and E‐selectin. Moreover, adenosine inhibited thrombin‐induced elevated expression of proinflammatory cytokines, IL‐6 and HMGB‐1; and chemokines, MCP‐1, CXCL‐1, and CXCL‐3. Taken together, these results suggest that adenosine may inhibit thrombin‐mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation. J. Cell. Physiol. 229: 1292–1300, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-05-23T12:05:16.075091-05:
      DOI: 10.1002/jcp.24568
       
 
 
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