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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  [1604 journals]   [SJR: 1.608]   [H-I: 118]
  • Vitamin D Receptor and RXR in the Post‐Genomic Era
    • Authors: Mark D. Long; Lara E. Sucheston‐Campbell, Moray J. Campbell
      Pages: n/a - n/a
      Abstract: Following the elucidation of the human genome and components of the epigenome, it is timely to revisit what is known of vitamin D receptor (VDR) function. Early transcriptomic studies using microarray approaches focused on the protein coding mRNA that were regulated by the VDR, usually following treatment with ligand. These studies quickly established the approximate size and surprising diversity of the VDR transcriptome, revealing it to be highly heterogenous and cell type and time dependent. Investigators also considered VDR regulation of non‐protein coding RNA and again, cell and time dependency was observed. Attempts to integrate mRNA and miRNA regulation patterns are beginning to reveal patterns of co‐regulation and interaction that allow for greater control of mRNA expression, and the capacity to govern more complex cellular events. Alternative splicing in the trasncriptome has emerged as a critical process in transcriptional control and there is evidence of the VDR interacting with components of the splicesome. ChIP‐Seq approaches have proved to be pivotal to reveal the diversity of the VDR binding choices across cell types and following treatment, and have revealed that the majority of these are non‐canonical in nature. The underlying causes driving the diversity of VDR binding choices remain enigmatic. Finally, genetic variation has emerged as important to impact the transcription factor affinity towards genomic binding sites, and recently the impact of this on VDR function has begun to be considered. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:34:03.202427-05:
      DOI: 10.1002/jcp.24847
       
  • Regulation of Ribosomal Gene Expression in Cancer
    • Authors: Le Xuan Truong Nguyen; Aparna Raval, Jacqueline S. Garcia, Beverly S. Mitchell
      Pages: n/a - n/a
      Abstract: The ability of a cell to undergo malignant transformation is both associated with and dependent on a concomitant increase in protein synthesis due to increased cell division rates and biosynthetic activities. Protein synthesis, in turn, depends upon the synthesis of ribosomes and thus ultimately on the transcription of ribosomal RNA by RNA polymerase I that occurs in the nucleolus. Enlargement of nucleoli has long been considered a hallmark of the malignant cell, but it is only recently that the rate of synthesis of rRNA in the nucleolus has been recognized as both a critical regulator of cellular proliferation and a potential target for therapeutic intervention. As might be expected, the factors regulating rRNA synthesis are both numerous and complex. It is the objective of this review to highlight recent advances in understanding how rRNA synthesis is perturbed in transformed mammalian cells and to consider the impact of these findings on the development of new approaches to the treatment of malignancies. In‐depth analysis of the process of rRNA transcription itself may be found in several recently published reviews (Drygin et al., 2010, Annu Rev Pharmacol Toxicol 50:131–156; Bywater et al., 2013,Cancer Cell 22: 51–65; Hein et al., 2013,Trends Mol Med 19:643–654). J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:04:46.345249-05:
      DOI: 10.1002/jcp.24854
       
  • Short time tripterine treatment enhances endothelial progenitor cell
           function via heat shock protein 32
    • Authors: Chenhui Lu; Xiaoping Zhang, Denghai Zhang, Erli Pei, Jichong Xu, Tao Tang, Meng Ye, Georges Uzan, Kangkang Zhi, Maoquan Li
      Pages: n/a - n/a
      Abstract: Aims The dysfunction of endothelial progenitor cells (EPCs) limits their potential for the treatment of ischemia and atherosclerosis. Therefore, we investigated the effect of tripterine on EPC function and examined the underlying mechanisms. Methods and Results The effect of tripterine, an active component of Tripterygium wilfordii Hook, on the enhancement of EPC function and the efficiency of EPC transplantation was investigated in vitro and in vivo. Treatment of EPCs with tripterine at 2.5 µM for 4 h inhibited oxidized low‐density lipoprotein (ox‐LDL) induced ROS production, cell apoptosis, and cell senescence and improved the migration and tube formation capacities of EPCs treated with ox‐LDL (200 µg/ml). In vivo studies showed that tripterine conditioning of EPCs administered to ischemic foci improved blood perfusion and microvascular density in a mouse hindlimb ischemia model. Examination of the underlying mechanisms indicated that the effect of tripterine is mediated by the induction of heat shock protein 32 expression and the inhibition of JNK activation. Conclusion(s) The present results are of clinical significance because they suggest the potential of tripterine as a therapeutic agent to improve the efficacy of EPC transplantation for the treatment of ischemic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:04:43.882389-05:
      DOI: 10.1002/jcp.24849
       
  • The Role of Epigenetic Mechanisms in Notch Signaling During Development
    • Authors: Ralf Schwanbeck
      Pages: n/a - n/a
      Abstract: The Notch pathway is a highly conserved cell–cell communication pathway in metazoan involved in numerous processes during embryogenesis, development, and adult organisms. Ligand‐receptor interaction of Notch components on adjacent cells facilitates controlled sequential proteolytic cleavage resulting in the nuclear translocation of the intracellular domain of Notch (NICD). There it binds to the Notch effector protein RBP‐J, displaces a corepressor complex and enables the induction of target genes by recruitment of coactivators in a cell‐context dependent manner. Both, the gene‐specific repression and the context dependent activation require an intense communication with the underlying chromatin of the regulatory regions. Since the epigenetic landscape determines the function of the genome, processes like cell fate decision, differentiation, and self‐renewal depend on chromatin structure and its remodeling during development. In this review, structural features enabling the Notch pathway to read these epigenetic marks by proteins interacting with RBP‐J/Notch will be discussed. Furthermore, mechanisms of the Notch pathway to write and erase chromatin marks like histone acetylation and methylation are depicted as well as ATP‐dependent chromatin remodeling during the activation of target genes. An additional fine‐tuning of transcriptional regulation upon Notch activation seems to be controlled by the commitment of miRNAs. Since cells within an organism have to react to environmental changes, and developmental and differentiation cues in a proper manner, different signaling pathways have to crosstalk to each other. The chromatin status may represent one major platform to integrate these different pathways including the canonical Notch signaling. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:04:41.22321-05:0
      DOI: 10.1002/jcp.24851
       
  • Keeping Wnt Signalosome in Check by Vesicular Traffic
    • Authors: Qiang Feng; Nan Gao
      Pages: n/a - n/a
      Abstract: Wg/Wnts are paracrine and autocrine ligands that activate distinct signaling pathways while being internalized through surface receptors. In the field, contrasting views exist on whether, where, and how endocytosis may modulate Wnt signaling. We gather considerable amount of evidences to elaborate the point that signal‐receiving cells utilize distinct, flexible, and sophisticated vesicular trafficking mechanisms to keep Wnt signaling activity in check. Same molecules in a highly context‐dependent fashion serve as regulatory hub for various signaling purposes: amplification, maintenance, inhibition, and termination. Updates are provided for the regulatory mechanisms related to the three relevant cell surface complexes, Wnt‐Fzd‐LRP6, Dkk1‐Kremen‐LRP6, and R‐spondin‐LGR5‐RNF43, which potently influence Wnt signaling. We pay particular attentions to how cells achieve sustained and delicate control of Wnt signaling strength by employing comprehensive aspects of vesicular trafficking. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:03:37.979712-05:
      DOI: 10.1002/jcp.24853
       
  • Contractile and Metabolic Properties of Engineered Skeletal Muscle Derived
           From Slow and Fast Phenotype Mouse Muscle
    • Authors: Alastair Khodabukus; Keith Baar
      Pages: n/a - n/a
      Abstract: Satellite cells derived from fast and slow muscles have been shown to adopt contractile and metabolic properties of their parent muscle. Mouse muscle shows less distinctive fiber‐type profiles than rat or rabbit muscle. Therefore, in this study we sought to determine whether three‐dimensional muscle constructs engineered from slow soleus (SOL) and fast tibialis anterior (TA) from mice would adopt the contractile and metabolic properties of their parent muscle. Time‐to‐peak tension (TPT) and half‐relaxation time (1/2RT) was significantly slower in SOL constructs. In agreement with TPT, TA constructs contained significantly higher levels of fast myosin heavy chain (MHC) and fast troponin C, I, and T isoforms. Fast SERCA protein, both slow and fast calsequestrin isoforms and parvalbumin were found at higher levels in TA constructs. SOL constructs were more fatigue resistant and contained higher levels of the mitochondrial proteins SDH and ATP synthase and the fatty acid transporter CPT‐1. SOL constructs contained lower levels of the glycolytic enzyme phosphofructokinase but higher levels of the β‐oxidation enzymes LCAD and VLCAD suggesting greater fat oxidation. Despite no changes in PGC‐1α protein, SOL constructs contained higher levels of SIRT1 and PRC. TA constructs contained higher levels of the slow‐fiber program repressor SOX6 and the six transcriptional complex (STC) proteins Eya1and Six4 which may underlie the higher in fast‐fiber and lower slow‐fiber program proteins. Overall, we have found that muscles engineered from predominantly slow and fast mouse muscle retain contractile and metabolic properties of their native muscle. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T13:52:34.672339-05:
      DOI: 10.1002/jcp.24848
       
  • Chondroptosis in Alkaptonuric Cartilage
    • Authors: Lia Millucci; Giovanna Giorgetti, Cecilia Viti, Lorenzo Ghezzi, Silvia Gambassi, Daniela Braconi, Barbara Marzocchi, Alessandro Paffetti, Pietro Lupetti, Giulia Bernardini, Maurizio Orlandini, Annalisa Santucci
      Pages: n/a - n/a
      Abstract: Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above‐mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T13:52:27.162952-05:
      DOI: 10.1002/jcp.24850
       
  • Enhanced Phenotypic Alterations of Alveolar Type II Cells in Response to
           Aflatoxin G1‐Induced Lung Inflammation
    • Authors: Haitao Shen; Chunping Liu, Peilu Shao, Li Yi, Yuan Wang, Emily Mills Ko, Ziqiang Tian, Xin Zhao, Juan Wang, Lingxiao Xing, Xianghong Zhang
      Pages: n/a - n/a
      Abstract: Recently, we discovered that Aflatoxin G1 (AFG1) induces chronic lung inflammatory responses, which may contribute to lung tumorigenesis in Balb/C mice. The cancer cells originate from alveolar type II cells (AT‐II cells). The activated AT‐II cells express high levels of MHC‐II and COX‐2, may exhibit altered phenotypes, and likely inhibit antitumor immunity by triggering regulatory T cells (Tregs). However, the mechanism underlying phenotypic alterations of AT‐II cells caused by AFG1‐induced inflammation remains unknown. In this study, increased MHC‐II expression in alveolar epithelium was observed and associated with enhanced Treg infiltration in mouse lung tissues with AFG1‐induced inflammation. This provides a link between phenotypically altered AT‐II cells and Treg activity in the AFG1‐induced inflammatory microenvironment. AFG1‐activated AT‐II cells underwent phenotypic maturation since AFG1 upregulated MHC‐II expression on A549 cells and primary human AT‐II cells in vitro. However, mature AT‐II cells may exhibit insufficient antigen presentation, which is necessary to activate effector T cells, due to the absence of CD80 and CD86. Furthermore, we treated A549 cells with AFG1 and TNF‐α together to mimic an AFG1‐induced inflammatory response in vitro, and we found that TNF‐α and AFG1 coordinately enhanced MHC‐II, CD54, COX‐2, IL‐10, and TGF‐β expression levels in A549 cells compared to AFG1 alone. The phenotypic alterations of A549 cells in response to the combination of TNF‐α and AFG1 were mainly regulated by TNF‐α‐mediated induction of the NF‐κB pathway. Thus, enhanced phenotypic alterations of AT‐II cells were induced in response to AFG1‐induced inflammation. Thus, AT‐II cells are likely to suppress anti‐tumor immunity by triggering Treg activity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T13:52:14.909451-05:
      DOI: 10.1002/jcp.24852
       
  • PDE5 inhibitors enhance Celecoxib killing in multiple tumor types
    • Authors: Laurence Booth; Jane L. Roberts, Nichola Cruickshanks, Seyedmehrad Tavallai, Timothy Webb, Peter Samuel, Adam Conley, Brittany Binion, Harold F. Young, Andrew Poklepovic, Sarah Spiegel, Paul Dent
      Pages: n/a - n/a
      Abstract: The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L‐NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over‐expression of c‐FLIP‐s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-09T12:42:59.57235-05:0
      DOI: 10.1002/jcp.24843
       
  • Permanent culture of macrophages at physiological oxygen attenuates the
           antioxidant and immunomodulatory properties of dimethyl fumarate
    • Authors: Benjamin Haas; Sandra Chrusciel, Sarah Fayad‐Kobeissi, Jean‐Luc Dubois‐Randé, Francisco Azuaje, Jorge Boczkowski, Roberto Motterlini, Roberta Foresti
      Pages: n/a - n/a
      Abstract: We hypothesized that O2 tension influences the redox state and the immunomodulatory responses of inflammatory cells to dimethyl fumarate (DMF), an activator of the nuclear factor Nrf2 that controls antioxidant genes expression. This concept was investigated in macrophages permanently cultured at either physiological (5% O2) or atmospheric (20% O2) oxygen levels and then treated with DMF or challenged with lipopolysaccharide (LPS) to induce inflammation. RAW 264.7 macrophages cultured at 20% O2 exhibited a pro‐oxidant phenotype, reflected by a lower content of reduced glutathione, higher oxidized glutathione and increased production of reactive oxygen species when compared to macrophages continuously grown at 5% O2. At 20% O2, DMF induced a stronger antioxidant response compared to 5% O2 as evidenced by a higher expression of heme oxygenase‐1, NAD(P)H:quinone oxydoreductase‐1 and superoxide dismutase‐2. After challenge of macrophages with LPS, several pro‐inflammatory (iNOS, TNF‐α, MMP‐2, MMP‐9), anti‐inflammatory (arginase‐1, IL‐10) and pro‐angiogenic (VEGF‐A) mediators were evaluated in the presence or absence of DMF. All markers, with few interesting exceptions, were significantly reduced at 5% O2. This study brings new insights on the effects of O2 in the cellular adaptation to oxidative and inflammatory stimuli and highlights the importance of characterizing the effects of chemicals and drugs at physiologically relevant O2 tension. Our results demonstrate that the common practice of culturing cells at atmospheric O2 drives the endogenous cellular environment towards an oxidative stress phenotype, affecting inflammation and the expression of antioxidant pathways by exogenous modulators. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-09T12:42:44.639233-05:
      DOI: 10.1002/jcp.24844
       
  • Cyclooxygenase‐2 Regulates NLRP3 Inflammasome‐Derived
           IL‐1β Production
    • Authors: Kuo‐Feng Hua; Ju‐Ching Chou, Shuk‐Man Ka, Yu‐Ling Tasi, Ann Chen, Shih‐Hsiung Wu, Hsiao‐Wen Chiu, Wei‐Ting Wong, Yih‐Fuh Wang, Change‐Ling Tsai, Chen‐Lung Ho, Cheng‐Hsiu Lin
      Pages: n/a - n/a
      Abstract: The NLR family, pyrin domain‐containing 3 (NLRP3) inflammasome is a reactive oxygen species‐sensitive multiprotein complex that regulates IL‐1β maturation via caspase‐1. It also plays an important role in the pathogenesis of inflammation‐related disease. Cyclooxygenase‐2 (COX‐2) is induced by inflammatory stimuli and contributes to the pathogenesis of inflammation‐related diseases. However, there is currently little known about the relationship between COX‐2 and the NLRP3 inflammasome. Here, we describe a novel role for COX‐2 in regulating the activation of the NLRP3 inflammasome. NLRP3 inflammasome‐derived IL‐1β secretion and pyroptosis in macrophages were reduced by pharmaceutical inhibition or genetic knockdown of COX‐2. COX‐2 catalyzes the synthesis of prostaglandin E2 and increases IL‐1β secretion. Conversely, pharmaceutical inhibition or genetic knockdown of prostaglandin E2 receptor 3 reduced IL‐1β secretion. The underlying mechanisms for the COX‐2‐mediated increase in NLRP3 inflammasome activation were determined to be the following: (1) enhancement of lipopolysaccharide‐induced proIL‐1β and NLRP3 expression by increasing NF‐κB activation and (2) enhancement of the caspase‐1 activation by increasing damaged mitochondria, mitochondrial reactive oxygen species production and release of mitochondrial DNA into cytosol. Furthermore, inhibition of COX‐2 in mice in vivo with celecoxib reduced serum levels of IL‐1β and caspase‐1 activity in the spleen and liver in response to lipopolysaccharide (LPS) challenge. These findings provide new insights into how COX‐2 regulates the activation of the NLRP3 inflammasome and suggest that it may be a new potential therapeutic target in NLRP3 inflammasome‐related diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T14:19:29.251235-05:
      DOI: 10.1002/jcp.24815
       
  • Effect of rMnSOD on survival signaling in pediatric high risk T‐cell
           acute lymphoblastic leukaemia
    • Authors: A Pica; A Di Santi, V D'angelo, A Iannotta, M Ramaglia, M Di Martino, Ml Pollio, A Schiattarella, A Borrelli, A Mancini, P Indolfi, F. Casale
      Pages: n/a - n/a
      Abstract: Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that defends against oxidative damage due to reactive oxygen species (ROS). A new isoform of MnSOD with cytotoxic activity was recently discovered in liposarcoma cells. Here, we tested the effectiveness of a recombinant form of this isoform (rMnSOD) on leukemic T cells, Jurkat cells, and normal lymphocytes. Our results confirm that leukemic T cells can internalize rMnSOD and that rMnSOD causes apoptosis of 99% of leukemic cells without showing toxic effects on healthy cells. Using light and electron microscopy, we determined that an rMnSOD concentration of 0.067 μM most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 μM rMnSOD resulted in high expression of the pro‐apoptotic protein Bax and low expression of the anti‐apoptotic protein Bcl‐2 in leukemia cells. Concerning signal transduction pathway no influence was observed after treatment except for Jurkat cells showing a slightly decreased expression of ERK phosphorylation. These results suggest that rMnSOD may be an effective and non‐toxic treatment option for T‐cell leukemia. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T11:36:06.767904-05:
      DOI: 10.1002/jcp.24840
       
  • Prostaglandin Dependent Control of an Endogenous Estrogen Receptor Agonist
           by Osteoblasts
    • Authors: Thomas L. McCarthy; Michael Centrella
      Pages: n/a - n/a
      Abstract: Estrogen receptor (ER) activation has complex effects on bone cells, and loss of circulating estradiol adversely affects skeletal status in women. Hormone replacement therapy effectively circumvents bone loss after menopause, but enhances disease risk in other tissues. Here we show that prostaglandin E2 (PGE2) augments the activity of an osteoblast‐derived selective ER modulator, ObSERM. The stimulatory effect of PGE2 is replicated in part by either the PG receptor EP3 agonist 17‐phenyl trinor PGE2 or by the PG receptor FP agonist PGF2α. Whereas activation of the various PG receptors induces multiple downstream signals, the response to PGE2 was mimicked by activators of protein kinase C, and suppressed by inhibition of protein kinase C but not by inhibition of protein kinase A. Moreover, inhibition of nitric oxide synthesis and activation of the PTH and Wnt pathways increases ObSERM activity. Our studies therefore reveal that ObSERM activity is controlled in distinct ways and revise our understanding of ER activation within bone by agents or events associated with PG expression. They also predict ways to sustain or improve bone formation, fracture repair, and surgical healing without adding the risk of disease in other tissues where ER activation also has important biological functions. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T11:35:38.523439-05:
      DOI: 10.1002/jcp.24842
       
  • DNA‐PKcs deficiency inhibits glioblastoma cell‐derived
           angiogenesis after ionizing radiation
    • Authors: Yang Liu; Luwei Zhang, Yuanyuan Liu, Chao sun, Hong Zhang, Guoying Miao, Cui Xiao Di, Xin Zhou, Rong Zhou, Zhenhua Wang
      Pages: n/a - n/a
      Abstract: DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) plays a critical role in non‐homologous end‐joining repair of DNA double‐strand breaks (DSB) induced by ionizing radiation (IR). Little is known, however, regarding the relationship between DNA‐PKcs and IR‐induced angiogenesis; thus, in this study we aimed to further elucidate this relationship. Our findings revealed that lack of DNA‐PKcs expression or activity sensitized glioma cells to radiation due to the defective DNA DSB repairs and inhibition of phosphorylated AktSer473. Moreover, DNA‐PKcs deficiency apparently mitigated IR‐induced migration, invasion and tube formation of human microvascular endothelial cell (HMEC‐1) in conditioned media derived from irradiated DNA‐PKcs mutant M059J glioma cells or M059K glioma cells that have inhibited DNA‐PKcs kinase activity due to the specific inhibitor NU7026 or siRNA knockdown. Moreover, IR‐elevated vascular endothelial growth factor (VEGF) secretion was abrogated by DNA‐PKcs suppression. Supplemental VEGF antibody to irradiated‐conditioned media was negated enhanced cell motility with a concomitant decrease in phosphorylation of the FAK Try925 and SrcTry416. Furthermore, DNA‐PKcs suppression was markedly abrogated in IR‐induced transcription factor hypoxia inducible factor‐1α (HIF‐1α) accumulation, which is related to activation of VEGF transcription. These findings, taken together, demonstrate that depletion of DNA‐PKcs in glioblastoma cells at least partly suppressed IR‐inflicted migration, invasion and tube formation of HMEC‐1 cells, which may be associated with the reduced HIF‐1α level and VEGF secretion. Inhibition of DNA‐PKcs may be a promising therapeutic approach to enhance radio‐therapeutic efficacy for glioblastoma by hindering its angiogenesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T11:34:26.23108-05:0
      DOI: 10.1002/jcp.24841
       
  • Role of Methionine Adenosyltransferase α2 and β Phosphorylation
           and Stabilization in Human Hepatic Stellate Cell
           Trans‐Differentiation
    • Authors: Komal Ramani; Shant Donoyan, Maria Lauda Tomasi, Sunhee Park
      Pages: n/a - n/a
      Abstract: Myofibroblastic trans‐differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S‐adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up‐regulation during trans‐differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post‐translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans‐differentiation. Culture‐activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen‐activated protein kinase (MAPK) family members [ERK1/2, V‐Raf Murine Sarcoma Viral Oncogene Homolog B1 (B‐Raf), MEK]. Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B‐Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho‐sites destabilized them and prevented HSC trans‐differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans‐differentiation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T09:36:32.892568-05:
      DOI: 10.1002/jcp.24839
       
  • Methods and Procedures in Adipose Stem Cells: State of the Art and
           Perspective For Translation Medicine
    • Authors: G.F. Nicoletti; F. De Francesco, F. D'Andrea, G.A. Ferraro
      Pages: n/a - n/a
      Abstract: Stem cells have potential in the retrieval and repair of injured tissue and renovation of organ function. To date, several studies have been carried out to elucidate how differentiation of stem cells can be used in regenerative medicine applications. Adipose tissue is an abundant and accessible source of stem cell, useful for regenerative therapeutic use. Adipose stem cells (ASCs) are favorable for future translational research and can be applied in many clinical settings. Adipose tissue repair has been recently adopted in clinical trials to prove that ASCs can be successfully used in patients. Variability in cell culture procedures (isolation, characterization and differentiation) may have an influence on the experimental outcome. In this report, we consider the selection mechanisms of ASCs using flow cytometry, cell culture, freezing/thawing, cell cycle evaluation, histochemistry/immunofluorescence and differentiation of ASCs. Both researchers and regulatory institutions should consider a new policy for GMP procedures and protocols, paying special attention to stem cell bio‐physiology, to facilitate more clinically oriented studies. ASCs show angiogenic properties, with prospects of repairing tissue damaged by radiotherapy, as well as possessing the ability to heal chronic wounds. They can also be useful in surgical practice. We focus on the potential clinical application of ASCs that are currently available regarding translational medicine and the methods and procedures for their isolation, differentiation and characterization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T09:34:51.035164-05:
      DOI: 10.1002/jcp.24837
       
  • Pregnane X Receptor as the “Sensor and Effector” in Regulating
           Epigenome
    • Authors: Xi Ma; Jingshu Chen, Yanan Tian
      Pages: n/a - n/a
      Abstract: The pregnane X receptor (PXR, NR1I2) is a ligand‐activated nuclear receptor which plays an essential role in organism's metabolic detoxification system by sensing the presence of xenobiotics and triggering detoxification responses. In addition to its role in xenobiotic metabolism, PXR has pleiotropic functions in regulating immune/inflammatory responses, cell proliferation, bile acid/cholesterol metabolism, glucose and lipid metabolism, steroid/endocrine homeostasis and bone metabolism. Recent research suggests that the PXR is required for maintaining healthy commensalism between microbiota and gut. Interestingly, the metabolites such as indole derivatives from commensal microbes serve as the ligands for the PXR in intestinal epithelium forming an intricate mutualistic interaction between host and microbiota. PXR‐regulated gene responses are controlled at epigenetic level by chromatin modifications, DNA methylation and noncoding RNA. Developmental alterations of the epigenome by exposure to the xenobiotics or diseases may produce persistent changes in PXR‐regulated physiological responses. These new areas of research promise to vastly increase our understanding of PXR‐regulated responses. In this review we highlight recent results on the epigenetic mechanisms for the PXR‐regulated gene expression and discuss the physiological significance of these findings. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T09:33:13.495063-05:
      DOI: 10.1002/jcp.24838
       
  • Gastroprotective Effects of L‐Lysine Salification of Ketoprofen in
           Ethanol‐Injured Gastric Mucosa
    • Authors: A. Cimini; L. Brandolini, R. Gentile, L. Cristiano, P. Menghini, A. Fidoamore, A. Antonosante, E. Benedetti, A. Giordano, M. Allegretti
      Pages: n/a - n/a
      Abstract: Ketoprofen L‐lysine salt (KLS), a NSAID, is widely used for its analgesic efficacy and tolerability. L‐lysine salification was reported to increase the solubility and the gastric absorption and tolerance of Ketoprofen. Since the management of NSAIDs gastrotoxicity still represents a major limitation in prolonged therapies, mainly when gastric lesions are present, this study investigated the gastro‐protective activity of L‐lysine by using a well‐established model of gastric mucosa injury, the ethanol‐gastric injury model. Several evidences show that the damaging action of ethanol could be attributed to the increase of ROS, which plays a key role in the increase of lipid peroxidation products, including malonyldialdehyde and 4‐hydroxy‐2‐nonenal. With the aim to unravel the mechanism of L‐lysine gastroprotection, cellular MDA levels and 4‐HNE protein adducts as markers of lipid peroxidation and a panel of key endogenous gastro‐protective proteins were assayed. The data obtained indicate a gastroprotective effect of L‐lysine on gastric mucosa integrity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-06T22:53:18.120733-05:
      DOI: 10.1002/jcp.24809
       
  • Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in
           Dividing Human Cells
    • Authors: Anna Sobol; Paola Galluzzo, Shuang Liang, Brittany Rambo, Sylvia Skucha, Megan J. Weber, Sara Alani, Maurizio Bocchetta
      Pages: n/a - n/a
      Abstract: Hypoxic non‐small cell lung cancer (NSCLC) is dependent on Notch‐1 signaling for survival. Targeting Notch‐1 by means of γ‐secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post‐mortem analysis of GSI‐treated, NSCLC‐burdened mice suggested enhanced phosphorylation of 4E‐BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non‐canonical 4E‐BP1 phosphorylation pattern rearrangement‐a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF‐4F composition indicating increased recruitment of eIF‐4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF‐4A assembly into eIF‐4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap‐ and IRES‐dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin‐1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC‐1) inhibition affected 4E‐BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC‐1. Key phenomena described in this study were reversed by overexpression of the APP C‐terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC‐1 regulation of cap‐dependent protein synthesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-06T01:57:08.048627-05:
      DOI: 10.1002/jcp.24835
       
  • Emerging Concept in DNA Methylation: Role of Transcription Factors in
           Shaping DNA Methylation Patterns
    • Authors: Claire Marchal; Benoit Miotto
      Pages: n/a - n/a
      Abstract: DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site‐specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-06T01:56:48.790137-05:
      DOI: 10.1002/jcp.24836
       
  • Could lncRNAs be the Missing Links in Control of Mesenchymal Stem Cell
           Differentiation'
    • Authors: Coralee E. Tye; Jonathan A.R. Gordon, Lori A. Martin‐Buley, Janet L. Stein, Jane B. Lian, Gary S. Stein
      Abstract: Long suspected, recently recognized, and increasingly studied, non protein‐coding RNAs (ncRNAs) are emerging as key drivers of biological control and pathology. Since their discovery in 1993, microRNAs (miRNAs) have been the subject of intense research focus and investigations have revealed striking findings, establishing that these molecules can exert a shocking level of biological control in numerous tissues. More recently, long ncRNAs (lncRNAs), the lesser‐studied siblings of miRNA, have been suggested to have a similar robust role in developmental and adult tissue regulation. Mesenchymal stem cells (MSCs) are an important source of multipotent cells for normal and therapeutic tissue repair. Much is known about the critical role of miRNAs in biogenesis and differentiation of MSCs however; recent studies have suggested lncRNAs may play an equally important role in the regulation of these cells. Here we highlight the role of lncRNAs in the regulation of mesenchymal stem cell lineages including adipocytes, chondrocytes, myoblasts, and osteoblasts. In addition, the potential for these noncoding RNAs to be used as biomarkers for disease or therapeutic targets is also discussed. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-25T00:21:51.876637-05:
      DOI: 10.1002/jcp.24834
       
  • LIGHT is a crucial mediator of airway remodeling
    • Authors: Jen‐Yu Hung; Shyh‐Ren Chiang, Ming‐Ju Tsai, Ying‐Ming Tsai, Inn‐Wen Chong, Jiunn‐Min Shieh, Ya‐Ling Hsu
      Abstract: Chronic inflammatory airway diseases like asthma and chronic obstructive pulmonary disease are major health problems globally. Airway epithelial cells play important role in airway remodeling, which is a critical process in the pathogenesis of diseases. This study aimed to demonstrate that LIGHT, an inflammatory factor secreted by T cells after allergen exposure, is responsible for promoting airway remodeling. LIGHT increased primary human bronchial epithelial cells (HBECs) undergoing epithelial‐mesenchymal transition (EMT) and expressing MMP‐9. The induction of EMT was associated with increased NF‐κB activation and p300/NF‐κB association. The interaction of NF‐κB with p300 facilitated NF‐κB acetylation, which in turn, was bound to the promoter of ZEB1, resulting in E‐cadherin downregulation. LIGHT also stimulated HBECs to produce numerous cytokines/chemokines that could worsen airway inflammation. Furthermore, LIGHT enhanced HBECs to secrete activin A, which increased bronchial smooth muscle cell (BSMC) migration. In contrast, depletion of activin A decreased such migration. The findings suggest a new molecular determinant of LIGHT‐mediated pathogenic changes in HBECs and that the LIGHT‐related vicious cycle involving HBECs and BSMCs may be a potential target for the treatment of chronic inflammation airway diseases with airway remodeling. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-24T00:46:50.134114-05:
      DOI: 10.1002/jcp.24832
       
  • Lkb1 deletion promotes ectopic lipid accumulation in muscle progenitor
           cells and mature muscles
    • Authors: Tizhong Shan; Pengpeng Zhang, Pengpeng Bi, Shihuan Kuang
      Abstract: Excessive intramyocellular triglycerides (muscle lipids) are associated with reduced contractile function, insulin resistance and Type 2 diabetes, but what governs lipid accumulation in muscle is unclear. Here we report a role of Lkb1 in regulating lipid metabolism in muscle stem cells and their descendent mature muscles. We used MyodCre and Lkb1flox/flox mice to specifically delete Lkb1 in myogenic cells including stem and differentiated cells, and examined the lipid accumulation and gene expression of myoblasts cultured from muscle stem cells (satellite cells). Genetic deletion of Lkb1 in myogenic progenitors led to elevated expression of lipogenic genes and ectopic lipid accumulation in proliferating myoblasts. Interestingly, the Lkb1‐deficient myoblasts differentiated into adipocyte‐like cells upon adipogenic induction. However, these adipocyte‐like cells maintained myogenic gene expression with reduced ability to form myotubes efficiently. Activation of AMPK by AICAR prevented ectopic lipid formation in the Lkb1 null myoblasts. Notably, Lkb1‐deficient muscles accumulated excessive lipids in vivo in response to high‐fat diet feeding. These results demonstrate that Lkb1 acts through AMPK to limit lipid deposition in muscle stem cells and their derivative mature muscles, and point to the possibility of controlling muscle lipid content using AMPK activating drugs. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-24T00:44:13.870582-05:
      DOI: 10.1002/jcp.24831
       
  • UCP2‐related mitochondrial pathway participates in oroxylin
           A‐induced apoptosis in human colon cancer cells
    • Authors: Chen Qiao; Libin Wei, Qinsheng Dai, Yuxin Zhou, Qian Yin, Zhiyu Li, Yuanming Xiao, Qinglong Guo, Na Lu
      Abstract: Oroxylin A is a flavonoid extracted from the root of Scutellaria baicalensis Georgi. Our previous research demonstrated that oroxylin A have various anti‐tumor effects including apoptosis, cell cycle arrest, drug‐resistant reversion and others. This paper explores the mechanism how oroxylin A induce apoptosis by regulating uncoupling protein 2 (UCP2) in human colon cancer cells. We found that the inhibition of UCP2 by UCP2 siRNA significantly increased the sensitivity of cells to drugs, reactive oxygen species (ROS) generation and the opening of mitochondrial permeability transition pore (MPTP) of CaCo‐2 cells. We also found that UCP2 inhibition could lead to ROS‐mediated MPTP activation. Furthermore, we demonstrated that oroxylin A triggered MPTP‐dependent pro‐apoptotic protein release from mitochondria to matrix and then induced apoptotic cascade by inhibiting UCP2. Intriguingly, the inhibition of UCP2 by oroxylin A was able to block Bcl‐2 translocation to the mitochondria, keeping MPTP at open‐state. In conclusion, we have demonstrate that UCP2 play a key role in mitochondrial apoptotic pathway; UCP2's inhibition by oroxylin A triggers the MPTP opening, and promotes the apoptosis in CaCo‐2 cells. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-24T00:44:07.232297-05:
      DOI: 10.1002/jcp.24833
       
  • Alpha‐synuclein as a Pathological Link between Chronic Traumatic
           Brain Injury and Parkinson's disease
    • Authors: Sandra A. Acosta; Naoki Tajiri, Ike de la Pena, Marina Bastawrous, Paul R. Sanberg, Yuji Kaneko, Cesar V. Borlongan
      Abstract: The long‐term consequences of traumatic brain injury (TBI) are closely associated with the development of histopathological deficits. Notably, TBI may predispose long‐term survivors to age‐related neurodegenerative diseases, such as Parkinson's disease (PD), which is characterized by a gradual degeneration of the nigrostriatal dopaminergic neurons. However, preclinical studies on the pathophysiological changes in substantia nigra (SN) after chronic TBI are lacking. In the present in vivo study, we examined the pathological link between PD‐associated dopaminergic neuronal loss and chronic TBI. Sixty days post TBI, rats were euthanized and brain tissues harvested. Immunostaining was performed using tyrosine hydroxylase (TH), an enzyme required for the synthesis of dopamine in neurons, á‐synuclein, a presynaptic protein that plays a role in synaptic vesicle recycling, and major histocompatibility complex II (MHCII), a protein found in antigen presenting cells such as inflammatory microglia cells, all key players in PD pathology. Unbiased stereology analyses revealed significant decrease of TH‐positive expression in the surviving dopaminergic neurons of the SN pars compacta (SNpc) relative to sham control. In parallel, increased á‐synuclein accumulation was detected in the ipsilateral SN compared to the contralateral SN in TBI animals or sham control. In addition, exacerbation of MHCII+ cells was recognized in the SN and cerebral peduncle ipsilateral to injury relative to contralateral side and sham control. These results suggest á‐synuclein as a pathological link between chronic effects of TBI and PD symptoms as evidenced by significant overexpression and abnormal accumulation of á‐synuclein in inflammation‐infiltrated SN of rats exposed to chronic TBI. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-24T00:43:55.605155-05:
      DOI: 10.1002/jcp.24830
       
  • Genome‐wide transcriptional and functional analysis of endoglin
           isoforms in the human promonocytic cell line U937
    • Authors: Francisco J Blanco; Luisa Ojeda‐Fernandez, Mikel Aristorena, Eunate Gallardo‐Vara, Alberto Benguria, Ana Dopazo, Carmen Langa, Luisa M Botella, Carmelo Bernabeu
      Abstract: Endoglin is an auxiliary cell surface receptor for TGF‐β family members. Two different alternatively spliced isoforms, long (L)‐endoglin and short (S)‐endoglin, have been reported. S‐endoglin and L‐endoglin proteins vary from each other in their cytoplasmic tails that contain 14 and 47 amino acids, respectively. A critical role for endoglin in vascular development has primarily been studied in endothelial cells. In addition, endoglin expression is upregulated during monocyte‐to‐macrophage differentiation, however, little is known about its role in this myeloid context. To investigate the function of endoglin in monocytes, stable transfectants expressing the two endoglin isoforms in the promonocytic human cell line U937 were generated. The differential gene expression fingerprinting of these endoglin transfectants using DNA microarrays and further bioinformatics analysis showed a clear alteration in essential biological functions, mainly those related to “Cellular Movement”, including cell adhesion and transmigration. Interestingly, these cellular functions are highly dependent on adhesion molecules, including integrins α1 (CD49a, ITGA1 gene), αL (CD11a, ITGAL gene), αM (CD11b, ITGAM gene) and β2 (CD18, ITGB2 gene) and the chemokine receptor CCR2 (CD192, CCR2 gene), which are downregulated in endoglin transfectants. Moreover, activin A (INHBA gene), a TGF‐β superfamily member involved in macrophage polarization, was distinctly affected in each endoglin transfectant, and may contribute to the regulated expression of integrins. These data were confirmed by quantitative PCR, flow cytometry and functional tests. Taken together, these results provide new insight into endoglin function in monocytes. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:48:01.205103-05:
      DOI: 10.1002/jcp.24827
       
  • Novel Hedgehog Agonists Promote Osteoblast Differentiation in Mesenchymal
           Stem Cells
    • Authors: Takashi Nakamura; Masahiro Naruse, Yuta Chiba, Toshihisa Komori, Keiichi Sasaki, Masahiro Iwamoto, Satoshi Fukumoto
      Abstract: Hedgehog (Hh) family members are involved in multiple cellular processes including proliferation, migration, differentiation, and cell fate determination. Recently, the novel Hh agonists Hh‐Ag 1.3 and 1.7 were identified in a high‐throughput screening of small molecule compounds that activate the expression of Gli1, a target of Hh signaling. This study demonstrates that Hh‐Ag 1.3 and 1.7 strongly activate the expression of endogenous Gli1 and promote osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Both compounds stimulated alkaline phosphatase activity in a dose‐dependent manner, and induced osteoblast marker gene expression in C3H10T1/2 cells, which indicated that they had acquired an osteoblast identity. Of the markers, the expression of osterix/Sp7, a downstream target of runt‐related transcription factor (Runx)2, was induced by Hh‐Ag 1.7, which also rescued the osteoblast differentiation defect ofRD‐127, a mesenchymal cell line from Runx2‐deficient mice. Hh‐Ags also activated canonical Wnt signaling and synergized with low doses of BMP‐2 to enhance osteoblastic potential. Thus, Hh‐Ag 1.7 could be useful for bone healing in individuals with abnormalities in osteogenesis, such as osteoporosis patients and the elderly, and can contribute to the development of novel therapeutics for the treatment of bone fractures and defects. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:47:21.000731-05:
      DOI: 10.1002/jcp.24823
       
  • MicroRNA‐302a Stimulates Osteoblastic Differentiation by Repressing
           COUP‐TFII Expression
    • Authors: In‐Hong Kang; Byung‐Chul Jeong, Sung‐Woong Hur, Hyuck Choi, Seung‐Ho Choi, Je‐Hwang Ryu, Yun‐Chan Hwang, Jeong‐Tae Koh
      Abstract: Chicken ovalbumin upstream promoter transcription factor II (COUP‐TFII) is a potent transcription factor that represses osteoblast differentiation and bone formation. Previously we observed that stimuli for osteoblast differentiation, such as bone morphogenetic protein 2 (BMP2), inhibits COUP‐TFII expression. This study was undertaken to identify BMP2‐regulated and COUP‐TFII‐targeting microRNAs (miRNAs), and to explore their regulatory roles in osteoblast differentiation. Based on in silico analysis, 12 miRNAs were selected and their expression in BMP2‐treated MC3T3‐E1 cells was examined. BMP2 induced miR‐302a expression in dose‐ and time‐dependent manners with the decrease in COUP‐TFII expression. Runx2, a BMP2‐downstream transcription factor, specifically regulated miR‐302a expression and its promoter activity. A computer‐based prediction algorithm led to the identification of two miR‐302a binding sites on the 3'‐untranslational region of COUP‐TFII mRNA (S1: 620‐626 bp, S2: 1016‐1022 bp), and a luciferase assay showed that miR‐302a directly targeted S1 and S2. Transfection of miR‐302a precursor significantly enhanced expression of osteogenic marker genes with decreasing COUP‐TFII mRNA and protein level, alkaline phosphatase activity and matrix mineralization. On the other hand, inhibition of miR‐302a significantly attenuated BMP2‐induced osteoblast specific gene expression, alkaline phosphatase activity, and matrix mineralization with increasing COUP‐TFII mRNA and protein level. These results indicate that miR‐302a is induced by osteogenic stimuli and promotes osteoblast differentiation by targeting COUP‐TFII. MiR‐302a could be a positive regulator for osteoblast differentiation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:45:31.80351-05:0
      DOI: 10.1002/jcp.24822
       
  • miR‐203 is a Direct Transcriptional Target of E2F1 and Causes G1
           Arrest in Esophageal Cancer Cells
    • Authors: Kun Zhang; Limeng Dai, Bo Zhang, Xueqing Xu, Jiazhong Shi, Liyuan Fu, Xuedan Chen, Juan Li, Yun Bai
      Abstract: miR‐203 act as tumor repressor by inhibiting cell proliferation and is repressed in a variety of human tumors, although the molecular mechanisms responsible have not been elucidated. Here, we reveal that miR‐203 is regulated by E2F1, an important transcription factor that can induce cell proliferation by controlling cell cycle progression. We found that miR‐203 expression was induced by cisplatin, which also induced E2F1 protein accumulation in esophageal squamous cell carcinoma (ESCC) cell lines. miR‐203 expression was elevated upon activation of ectopic E2F1, whereas this induction was abolished when the E2F1 gene was silenced. Moreover, with luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays, we demonstrated that E2F1 transactivates miR‐203 by directly binding to the miR‐203 gene promoter. In addition, we found that miR‐203 inhibited cell proliferation by inducing G1/S cell cycle arrest, but not apoptosis, in ESCC cell lines. Finally, we observed that miR‐203 negatively inhibited the expression of CDK6, subsequently decreasing E2F1 expression possibly through Rb phosphorylation. Taken together, our data show that cancer‐related miR‐203 is a novel transcriptional target of E2F1 and that it regulates cell cycle arrest by participating in a feedback loop with E2F1. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:43:18.257391-05:
      DOI: 10.1002/jcp.24821
       
  • FoxK2 is required for cellular proliferation and survival
    • Authors: Lars P. van der Heide; Patrick J. E. C. Wijchers, Lars von Oerthel, J. Peter H. Burbach, Marco F. M. Hoekman, Marten P. Smidt
      Abstract: FoxK2 is a forkhead transcription factor expressed ubiquitously in the developing murine central nervous system. Here we investigated the role of FoxK2 in vitro and focused on proliferation and cellular survival. Knockdown of FoxK2 results in a decrease in BrdU incorporation and H3 phosphorylation, suggesting attenuation of proliferation. In the absence of growth factors FoxK2 knockdown results in a dramatic increase in caspase 3 activity and propidium iodide positive cells, indicative of cell death. Additionally, knockdown of FoxK2 results in an increase in the mRNA of Gadd45α, Gadd45γ, as well as an increase in the phosphorylation of the mTOR dependent kinase p70S6K. Rapamycin treatment completely blocked the increase in p70S6K and synergistically potentiated the decrease in H3 phosphorylation upon FoxK2 knockdown. To gain more insight into the pro‐apoptotic effects upon FoxK2 knockdown we screened for changes in Bcl2 genes. Upon FoxK2 knockdown both Puma and Noxa were significantly upregulated. Both genes were not inhibited by rapamycin treatment, instead rapamycin increased Noxa mRNA. FoxK2 requirement in cellular survival is further emphasized by the fact that resistance to TGFβ‐induced cell death was greatly diminished after FoxK2 knockdown. Overall our data suggest FoxK2 is required for proliferation and survival, that mTOR is part of a feedback loop partly compensating for FoxK2 loss, possibly by upregulating Gadd45s, whereas cell death upon FoxK2 loss is induced in a Bcl2 dependent manner via Puma and Noxa. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:42:38.06738-05:0
      DOI: 10.1002/jcp.24828
       
  • Involvement of intracellular signalling in the IL‐1β inhibitory
           effect on fructose intestinal absorption
    • Authors: M Jesús Rodríguez‐Yoldi; Sonia Gascón, Cristina Barranquero, Alberto García‐Barrios, Jesús Osada
      Abstract: A variety of bacteria and their excreted/secreted products having direct effects on epithelial ion transport and permeability and the release of cytokines during bacterial infection may impact directly on epithelial function. Interleukin‐1β (IL‐1β) is a pleiotropic cytokine that affects the intestinal absorption of nutrients. The aim of this work was to study the intracellular signalling pathways involved in the inhibitory effect of IL‐1β on D‐fructose intestinal transport in rabbit jejunum and Caco‐2 cells. The results show that the cytokine inhibitory effect was completely reversed in presence of proteasome or PKC selective inhibitors in IL‐1β treated rabbits. In addition, the activation of PI3K abolished the IL‐1β effect. Likewise, these results were confirmed in Caco‐2 cells. In addition, p‐PI3K expression was increased by IL‐1β‐treatment whereas the expression of p‐PKCα was not significantly affected. In summary, the results suggest that IL‐1β could regulate the activation of pPKCα 73, pPI3K 55 and NF‐kB proteins. These events could exert an inhibitory effect on fructose intestinal absorption by a modification of GLUT5 insertion to brush‐border membrane and/or the functional transporter activity. This effect is independent of hormonal milieu and nervous stimuli. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:42:20.411333-05:
      DOI: 10.1002/jcp.24820
       
  • The dysfunction of the trabecular meshwork during glaucoma course
    • Authors: Sergio Claudio Saccà; Alessandra Pulliero, Alberto Izzotti
      Abstract: Primary open angle glaucoma is a multi‐tissue disease that targets, in an ascending order, the trabecular meshwork, the optic nerve head, the lateral geniculate nuclei and the visual cortex. Oxidative stress and vascular damage play major roles in triggering apoptotic cell loss in these tissues. Molecular alterations occurring in the ocular anterior chamber during the early course of glaucoma trigger this cell loss. These molecular events are mainly of endogenous origin and related to the long‐term accumulation of oxidative damages arising from mitochondrial failure and endothelial dysfunction. This situation results in decreased antioxidant defences in aqueous humour and apoptosis activation in trabecular meshwork cells as triggered by severe mitochondrial damage altering tissue function and integrity. The presence of neural proteins in glaucomatous aqueous humour indicate that a molecular interconnection exists between the anterior and the posterior chamber tissues. Trabecular meshwork and lamina cribrosa share a common neuro‐ectodermal embryological, which contribute to explain the interconnection between anterior and the posterior chamber during glaucoma pathogenesis. During glaucoma, proteins deriving from the damage occurring in endothelial trabecular meshwork cells are released into aqueous humour. Accordingly, aqueous humour composition is characterised in glaucomatous patients by the presence of proteins deriving from apoptosis activation, mitochondrial damage, loss of intercellular connections, antioxidant decrease. Many questions remain unanswered, but molecular events illuminate TM damage and indicate that trabecular cells protection plays a role in the treatment and prevention of glaucoma. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:41:42.077463-05:
      DOI: 10.1002/jcp.24826
       
  • Flt3 ligand induces monocyte proliferation and enhances the function of
           monocyte‐derived dendritic cells in vitro
    • Authors: Sung‐Whan Kim; Seong‐Mi Choi, Yee Shin Choo, Il‐Kwon Kim, Byeung‐Wook Song, Han‐Soo Kim
      Abstract: Flt3 ligand (FL), a potent hematopoietic cytokine, plays an important role in development and activation of dendritic cells (DCs) and natural killer cells (NK). Although some post‐receptor signaling events of FL have been characterized, the role of FL on Flt3 expressing human peripheral blood monocyte is unclear. In the current study, we examined the role of FL on cell survival and growth of peripheral blood monocytes and function of monocyte‐derived DCs. FL promoted monocyte proliferation in a dose‐dependent manner and prevented spontaneous apoptosis. FL induced ERK phosphorylation and a specific ERK inhibitor completely abrogated FL‐mediated cellular growth, while p38 MAPK, JNK and AKT were relatively unaffected. Addition of FL to GM‐CSF and IL‐4 during DCs generation from monocytes increased the yield of DCs through induction of cell proliferation. DCs generated in the presence of FL expressed more costimulatory molecules on their surfaces and stimulated allogeneic T cell proliferation in MLR to a higher magnitude. Furthermore, FL partially antagonized IL‐10‐mediated inhibition on DCs function. Further characterization of FL actions may provide new and important information for immunotherapeutic approaches utilizing DCs. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:41:13.443492-05:
      DOI: 10.1002/jcp.24824
       
  • Justicidin A‐Induced Autophagy Flux Enhances Apoptosis of Human
           Colorectal Cancer Cells via Class III PI3K and Atg5 Pathway
    • Authors: Shen‐Jeu Won; Cheng‐Hsin Yen, Hsiao‐Sheng Liu, Shan‐Ying Wu, Sheng‐Hui Lan, Ya‐Fen Jiang‐Shieh, Chun‐Nan Lin, Chun‐Li Su
      Abstract: Our previous reports showed that justicidin A (JA), a novel and pure arylnaphthalide lignan isolated from Justicia procumbens, induces apoptosis of human colorectal cancer cells and hepatocellular carcinoma cells, leading to the suppression of both tumor cell growth in NOD‐SCID mice. Here, we reveal that JA induces autophagy in human colorectal cancer HT‐29 cells by conversion of autophagic marker LC3‐I to LC3‐II. Furthermore, LC3 puncta and autophagic vesicle formation, and SQSTM1/p62 suppression were observed. Administration of autophagy inhibitor (bafilomycin A1 and chloroquine) and transfection of a tandem fluorescent‐tagged LC3 (mRFP‐GFP) reporter plasmid (ptfLC3) demonstrated that JA induces autophagy flux in HT‐29 cells. Expression of LC3, SQSTM1, Beclin 1, and nuclear DNA double‐strand breaks (representing apoptosis) were also detected in the tumor tissue of HT‐29 cells transplanted into NOD‐SCID mice orally administrated with JA. In addition, the expression of autophagy signaling pathway‐related molecules p‐PDK1, p‐mTOR, p‐p70S6k/p‐RPS6KB2 was decreased, whereas that of class III PI3K, Beclin 1, Atg5‐Atg12, and mitochondrial BNIP3 was increased in response to JA. Pre‐treatment of the cells with class III PI3K inhibitor 3‐methyladenine or Atg5 shRNA attenuated JA‐induced LC3‐II expression and LC3 puncta formation, indicating the involvement of class III PI3K and Atg5. A novel mechanism was demonstrated in the anticancer compound JA; pre‐treatment with 3‐methyladenine or Atg5 shRNA blocked JA‐induced suppression in cell growth and colony formation, respectively, via inhibition of apoptosis. In contrast, administration of apoptosis inhibitor Z‐VAD did not affect JA‐induced autophagy. Our data suggest the chemotherapeutic potential of JA for treatment of human colorectal cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:28:03.183994-05:
      DOI: 10.1002/jcp.24825
       
  • p27 and Leukemia: Cell cycle and beyond
    • Authors: Anita Roy; Subrata Banerjee
      Abstract: Cell division is the foundation to development and the regulation of cell cycle progression is therefore of paramount importance to the living organisms. Primary control of cell cycle is achieved by an array of cyclins and cyclin dependent kinases (CDKs). The functions of these cyclin‐CDK complexes are again regulated by a host of cyclin dependent kinase inhibitors (CDKI). Till date CDKIs are broadly classified into two groups – INK4 family (p15, p16, p18 and p19) and the cip/kip family (p21, p27 and p57). Collectively these CDKIs regulate the progression from G1 to S phase of cell cycle. This review summarizes the functions of p27 while highlighting its emerging roles in leukemia. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-10T00:28:21.380767-05:
      DOI: 10.1002/jcp.24819
       
  • Hesperetin induces apoptosis in breast carcinoma by triggering
           accumulation of ROS and activation of ASK1/JNK pathway
    • Authors: Shreyasi Palit; Susanta Kar, Gunjan Sharma, Pijush K. Das
      Abstract: Hesperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. In the present study hesperetin exhibited a significant cytotoxic effect in human breast carcinoma MCF‐7 cells in a concentration‐ and time‐dependent manner without affecting normal (HMEC) as well as immortalized normal mammary epithelial cells (MCF‐10A). The cytotoxic effect of hesperetin was due to the induction of apoptosis as evident from the phosphatidyl‐serine externalization, DNA fragmentation, caspase‐7 activation and PARP cleavage. Apoptosis was associated with caspase‐9 activation, mitochondrial membrane potential loss, release of cytochrome c and increase in Bax:Bcl‐2 ratio. Pre‐treatment with caspase‐9 specific inhibitor (Z‐LEHD‐fmk) markedly attenuated apoptosis suggesting an involvement of intrinsic mitochondrial apoptotic cascade. Further, DCFDA flow‐cytometric analysis revealed triggering of ROS in a time‐dependent manner. Pre‐treatment with ROS scavenger N‐acetylcysteine (NAC) and glutathione markedly abrogated hesperetin‐mediated apoptosis whereas carbonyl cyanide m‐chlorophenylhydrazone (CCCP) pretreatment along with DHR123‐based flow‐cytometry indicated the generation of cytosolic ROS. Profiling of MAPKs revealed activation of JNK upon hesperetin treatment which was abrogated upon NAC pretreatment. Additionally, inhibition of JNK by SP600125 significantly reversed hesperetinmediated apoptosis. The activation of JNK was associated with the activation of ASK1. Silencing of ASK1 resulted in significant attenuation of JNK activation as well as reversed the hesperetin‐mediated apoptosis suggesting that hesperetin‐mediated apoptosis of MCF‐7 cells involves accumulation of ROS and activation of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple negative breast cancer MDA‐MB‐231 cells via intrinsic pathway via activation of caspase ‐9 and ‐3 and increase in Bax:Bcl‐2 ratio. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-10T00:16:32.838662-05:
      DOI: 10.1002/jcp.24818
       
  • The GPER agonist G‐1 induces mitotic arrest and apoptosis in human
           vascular smooth muscle cells independent of GPER
    • Authors: Yu Gui; Zhan Shi, Zeng Yong Wang, Jing‐Jing Li, Can Xu, Rui Juan Tian, Xin Xing Song, Michael P. Walsh, Dong Li, Jie Gao, Xi‐Long Zheng
      Abstract: The G protein‐coupled estrogen receptor (GPER) has been implicated in the regulation of smooth muscle cell (SMC) proliferation. The GPER selective agonist G‐1 has been a useful tool for exploring the biological roles of GPER in a variety of experimental settings, including SMC proliferation. The present study, originally designed to investigate cellular and signaling mechanisms underlying the regulatory role of GPER in vascular SMC proliferation using G‐1, unexpectedly revealed off‐target effects of G‐1. G‐1 (1‐10 μM) inhibited bromodeoxyuridine (BrdU) incorporation of human SMCs and caused G2/M cell accumulation. G‐1 treatment also increased mitotic index concurrent with a decrease in phosphorylation of Cdk1 (Tyr 15) and an increase in phosphorylation of the mitotic checkpoint protein BuBR1. Furthermore, G‐1 caused microtubule disruption, mitotic spindle damage, and tubulin depolymerization. G‐1 induced cell apoptosis as indicated by the appearance of TUNEL‐positive and annexin V‐positive cells with enhanced cleavage of caspases 3 and 9. However, neither the GPER antagonist G‐15 nor the MAPK kinase inhibitor PD98059 prevented these G‐1 effects. Down‐regulation of GPER or p44/42 MAPK with siRNA transfection also did not affect the G‐1‐induced apoptosis. We conclude that G‐1 inhibits proliferation of SMCs through mechanisms involving mitotic arrest and apoptosis, independent of GPER and the MAPK pathway. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-10T00:12:53.242319-05:
      DOI: 10.1002/jcp.24817
       
  • IL‐1β Promotes Malignant Transformation and Tumor
           Aggressiveness in Oral Cancer
    • Authors: Chia‐Huei Lee; Jeffrey Shu‐Ming Chang, Shih‐Han Syu, Thian‐Sze Wong, Jimmy Yu‐Wai Chan, Ya‐Chu Tang, Zhi‐Ping Yang, Wen‐Chan Yang, Chiung‐Tong Chen, Shao‐Chun Lu, Pei‐Hua Tang, Tzu‐Ching Yang, Pei‐Yi Chu, Jenn‐Ren Hsiao, Ko‐Jiunn Liu
      Abstract: Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin‐1 beta (IL‐1β) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro‐IL‐1β expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4‐Nitroquinolin‐1‐oxide (4‐NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro‐IL‐1β was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine‐derived nitrosamine ketone (NNK) and arecoline stimulated IL‐1β secretion in an inflammasome‐dependent manner. IL‐1β treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL‐6, IL‐8, and growth‐regulated oncogene‐α following IL‐1β stimulation. The conditioned medium of IL‐1β‐treated OSCC cells exerted significant proangiogenic effects. Crucially, IL‐1β increased the invasiveness of OSCC cells through the epithelial‐mesenchymal transition (EMT), characterized by downregulation of E‐cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL‐1β□ can be induced by tobacco and betel quid‐related carcinogens, and participate in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-10T00:11:17.930538-05:
      DOI: 10.1002/jcp.24816
       
  • Novel Insights into TRPM7 Function in Fibrotic Diseases: A Potential
           Therapeutic Target
    • Authors: Tao Xu; Bao‐ming Wu, Xiao‐ming Meng, Cheng Huang, Ming‐ming Ni, Jun Li
      Abstract: Transient receptor potential (TRP) channels are cellular sensors for a wide spectrum of physical and chemical stimuli. Activation of TRP channels changes the membrane potential, translocates important signaling ions crossing the cell membrane, alters enzymatic activity, and initiates endocytosis/exocytosis. Fibrosis is the leading cause of organ dysfunction in diseases, which is characterized by an imbalance in the turnover of extracellular matrix components. Accumulating evidence has demonstrated that TRPM7, a member of TRP channels superfamily, participates in the development and pathogenesis of fibrotic diseases, such as hepatic, pulmonary and cardiac fibrosis. In this review, we discuss the comprehensive role of TRPM7 in modulating profibrotic response and its potential as therapeutic target for fibrotic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T19:18:08.739893-05:
      DOI: 10.1002/jcp.24801
       
  • Epithelial‐to‐Mesenchymal Transition Induced by
           TGF‐β1 Is Mediated by AP1‐Dependent EpCAM Expression in
           MCF‐7 Cells
    • Authors: Jiujiao Gao; Qiu Yan, Jiao Wang, Shuai Liu, Xuesong Yang
      Abstract: The epithelial‐to‐mesenchymal transition (EMT), a process involving the breakdown of cell‐cell junctions and loss of epithelial polarity, is closely related to cancer metastasis and invasion. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane protein expressed in the majority of normal epithelial tissues and overexpressed in the majority of human epithelial cancers including breast cancer. EpCAM plays an important role in cancer progression. We showed that EpCAM participated in TGF‐β1‐induced EMT. TGF‐β1 treatment of MCF‐7 breast cancer cells was shown to induce EpCAM expression, which promoted the EMT and cell migration. EpCAM overexpression further enhanced TGF‐β1‐induced EMT, and EpCAM knockdown inhibited TGF‐β1‐induced EMT. We further demonstrated that TGF‐β1 treatment induced the phosphorylation of JNK that was in turn responsible for the increased expression of Jun and Fos. This result suggests an important role of the JNK to AP‐1 signaling to EpCAM downstream of TGF‐β1 for the induction of EMT in the breast cancer cells. Collectively, our study highlights a novel function for EpCAM in TGF‐β1‐induced EMT process and suggests that targeting of EpCAM may be an attractive strategy to treat breast cancer. This study implicates the potential value of EpCAM as a molecular marker for breast cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T19:17:54.701705-05:
      DOI: 10.1002/jcp.24802
       
  • WW Domain of BAG3 is Required for the Induction of Autophagy in Glioma
           Cells
    • Authors: Nana Merabova; Ilker Kudret Sariyer, A Sami Saribas, Tijana Knezevic, Jennifer Gordon, M. Caterina Turco, Alessandra Rosati, Michael Weaver, Jacques Landry, Kamel Khalili
      Abstract: Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co‐chaperone family of proteins that associates with Hsp70 through a conserved BAG domain positioned near the C‐terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin‐1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin‐1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:57:46.553115-05:
      DOI: 10.1002/jcp.24811
       
  • A Group of Novel HIF‐1α Inhibitors, Glyceollins, Blocks
           HIF‐1α Synthesis and Decreases Its Stability via Inhibition of
           the PI3K/AKT/mTOR Pathway and Hsp90 Binding
    • Authors: Sun Hee Lee; Jun‐Goo Jee, Kwanghyun Ryu, You Mie Lee
      Abstract: Glyceollins, a group of phytoalexins isolated from soybean, are known to exhibit anticancer, antiestrogenic, and antiangiogenic activities. However, whether glyceollins regulate tumor growth through regulation of hypoxia‐inducible factor (HIF)‐1α has not been investigated. We determined whether and how glyceollins regulate the synthesis and stability of HIF‐1α. Quantitative real‐time PCR revealed that glyceollins inhibited the expression of HIF‐1‐induced genes such as vascular endothelial growth factor (VEGF) in cancer cells. Enzyme‐linked immunosorbent assay and reporter luciferase assay showed that glyceollins decreased VEGF secretion and its promoter activity, respectively. Treatment of various cancer cells with 0.5‐100 µM glyceollins under hypoxic conditions reduced the expression of HIF‐1α. Glyceollins blocked translation of HIF‐1α by inhibiting the PI3K/AKT/mTOR pathway under hypoxic conditions. Glyceollins decreased the stability of HIF‐1α after treatment with cycloheximide, a protein synthesis inhibitor, and increased the ubiquitination of HIF‐1α after treatment with MG132, a proteasome inhibitor. Glyceollins blocked the interaction of Hsp90 with HIF‐1α, as shown by immunoprecipitation assay. Chemical binding of Hsp90 with glyceollins, as confirmed by computational docking analysis, was stronger than that with geldanamycin at the HSP90 ATP‐binding pocket. We found that glyceollins decreased microvessel density, as well as expression of phosphorylated AKT/mTOR and the Hsp90 client protein CDK4, in solid tumor tissues. Glyceollins potently inhibited HIF‐1α synthesis and decreased its stability by blocking the PI3K/AKT/mTOR pathway and HSP90 binding activity, respectively. These results may provide new perspectives into potential therapeutic application of glyceollins for the prevention and treatment of hypervascularized diseases and into the mechanism of their anticancer activity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:51:50.62946-05:0
      DOI: 10.1002/jcp.24813
       
  • Gene Expression Changes in Progression of Cervical Neoplasia Revealed by
           Microarray Analysis of Cervical Neoplastic Keratinocytes
    • Authors: John Charles Rotondo; Silvia Bosi, Cristian Bassi, Manuela Ferracin, Giovanni Lanza, Roberta Gafà, Eros Magri, Rita Selvatici, Stefania Torresani, Roberto Marci, Paola Garutti, Massimo Negrini, Mauro Tognon, Fernanda Martini
      Abstract: To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large‐scale gene expression profile was carried out on HPV16‐CIN2, HPV16‐CIN3 and normal cervical keratinocytes derived from two HPV16‐CIN2, two HPV‐CIN3 lesions and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16‐CIN2 keratinocytes and in HPV16‐CIN2 keratinocytes compared with HPV16‐CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription‐polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:48:46.34043-05:0
      DOI: 10.1002/jcp.24808
       
  • The Coiled‐Coil Domain Containing 80 (ccdc80) Gene Regulates
           gadd45β2 Expression in the Developing Somites of Zebrafish as a New
           Player of the Hedgehog Pathway
    • Authors: Isabella Della Noce; Silvia Carra, Chiara Brusegan, Rosina Critelli, Andrea Frassine, Carlo De Lorenzo, Antonio Giordano, Gianfranco Bellipanni, Erica Villa, Franco Cotelli, Anna Pistocchi, Filippo Schepis
      Abstract: The Coiled‐Coil Domain Containing 80 (CCDC80) gene has been identified as strongly induced in rat thyroid PC CL3 cells immortalized by the adenoviral E1A gene. In human, CCDC80 is a potential oncosoppressor due to its down‐regulation in several tumor cell lines and tissues and it is expressed in almost all tissues. CCDC80 has homologous in mouse, chicken and zebrafish. We cloned the zebrafish ccdc80 and analyzed its expression and function during embryonic development. The in silico translated zebrafish protein shares high similarity with its mammalian homologous, with nuclear localization signals and a signal peptide. Gene expression analysis demonstrates that zebrafish ccdc80 is maternally and zygotically expressed throughout the development. In particular, ccdc80 is strongly expressed in the notochord and it is under the regulation of the Hedgehog pathway. In this work we investigated the functional effects of ccdc80‐loss‐of‐function during embryonic development and verified its interaction with gadd45β2 in somitogenesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:47:10.174514-05:
      DOI: 10.1002/jcp.24810
       
  • Vitamin D Attenuates Cytokine‐Induced Remodeling in Human Fetal
           Airway Smooth Muscle Cells
    • Authors: Rodney D. Britt; Arij Faksh, Elizabeth R. Vogel, Michael A. Thompson, Vivian Chu, Hitesh C. Pandya, Yassine Amrani, Richard J. Martin, Christina M. Pabelick, Y.S. Prakash
      Abstract: Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25‐dihydroxyvitamin D3; 1,25(OH)2D3), modulates responses to inflammation; however this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro‐inflammatory cytokines. Cells were pre‐treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP‐9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ‐induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:41:49.922249-05:
      DOI: 10.1002/jcp.24814
       
  • Isoform‐Specific SCFFbw7 Ubiquitination Mediates Differential
           Regulation of PGC‐1α
    • Authors: Julie S. Trausch‐Azar; Mona Abed, Amir Orian, Alan L. Schwartz
      Abstract: The E3 ubiquitin ligase and tumor suppressor SCFFbw7 exists as three isoforms that govern the degradation of a host of critical cell regulators, including c‐Myc, cyclin E, and PGC‐1α. Peroxisome proliferator activated receptor‐gamma coactivator 1α (PGC‐1α) is a transcriptional coactivator with broad effects on cellular energy metabolism. Cellular PGC‐1α levels are tightly controlled in a dynamic state by the balance of synthesis and rapid degradation via the ubiquitin‐proteasome system. Yet, isoform‐specific functions of SCFFbw7 are yet to be determined. Here, we show that the E3 ubiquitin ligase, SCFFbw7, regulates cellular PGC‐1α levels via two independent, isoform specific, mechanisms. The cytoplasmic isoform (SCFFbw7β) reduces cellular PGC‐1α levels via accelerated ubiquitin‐proteasome degradation. In contrast, the nuclear isoform (SCFFbw7α) increases cellular PGC‐1α levels and protein stability via inhibition of ubiquitin‐proteasomal degradation. When nuclear Fbw7α proteins are redirected to the cytoplasm, cellular PGC‐1α protein levels are reduced through accelerated ubiquitin‐proteasomal degradation. We find that SCFFbw7β catalyzes high molecular weight PGC‐1α‐ubiquitin conjugation, whereas SCFFbw7α produces low molecular weight PGC‐1α‐ubiquitin conjugates that are not effective degradation signals. Thus, selective ubiquitination by specific Fbw7 isoforms represents a novel mechanism that tightly regulates cellular PGC‐1α levels. Fbw7 isoforms mediate degradation of a host of regulatory proteins. The E3 ubiquitin ligase, Fbw7, mediates PGC‐1α levels via selective isoform‐specific ubiquitination. Fbw7β reduces cellular PGC‐1α via ubiquitin‐mediated degradation, whereas Fbw7α increases cellular PGC‐1α via ubiquitin‐mediated stabilization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:40:19.811917-05:
      DOI: 10.1002/jcp.24812
       
  • pRb2/p130 is Delocalized in Cytoplasm in Diffuse Gastric Cancer: A
           Preliminary Study
    • Authors: Letizia Cito; Paola Indovina, Iris Maria Forte, Domenico Di Marzo, Pasquale Somma, Daniela Barone, Antonella Penon, Danila Penon, Elisa Ceccherini, Pietro Micheli, Luca Saragoni, Marina Di Domenico, Antonia Feola, Franco Roviello, Eliseo Mattioli, Giovan Giacomo Giordano, Antonio Giordano
      Abstract: pRb2/p130 phosphorylation occurs at G0/G1 transition, when cells exit from quiescence. This phenomenon leads to E2F4 and E2F5 transcription factors releasing, allowing them to activate their target genes. Hence, pRb2/p130 represents one of the most important cell cycle controller because of transcription factors binding/releasing capability. It was previously shown that the loss of expression, or function, of pRb2/p130 is involved in different neoplasms development, due to related loss of control of E2F4 and E2F5 transcription activity. Here we analyzed gastric cancer tissue samples of diffuse histotype, comparing them with the normal counterpart. Yet, we did not find any change of expression levels. Rather, we found a cytoplasmic localization of pRb2/p130 in cancer tissue samples whereas, in normal counterparts, we found the expected nuclear localization. This occurrence lead to hypothesize an almost completely uncontrolled transcriptional activity of E2F4 and E2F5 in this kind of neoplasm, actually contributing to maintenance of transformed status. Although in limited number of cases, our data support the necessity of further investigations to verify the possibility of using pRb2/p130 as diagnostic marker of diffuse gastric cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:40:00.89997-05:0
      DOI: 10.1002/jcp.24805
       
  • GATA‐1 Deficiency Rescues Trabecular but not Cortical Bone in OPG
           Deficient Mice
    • Authors: Tomas E. Meijome; R. Adam Hooker, Ying‐Hua Cheng, Whitney Walker, Mark C. Horowitz, Robyn K. Fuchs, Melissa A. Kacena
      Abstract: GATA‐1low/low mice have an increase in megakaryocytes (MKs) and trabecular bone. The latter is thought to result from MKs directly stimulating osteoblastic bone formation while simultaneously inhibiting osteoclastogenesis. Osteoprotegerin (OPG) is known to inhibit osteoclastogenesis and OPG‐/‐ mice have reduced trabecular and cortical bone due to increased osteoclastogenesis. Interestingly, GATA‐1low/low mice have increased OPG levels. Here we sought to determine whether GATA‐1 knockdown in OPG‐/‐ mice could rescue the observed osteoporotic bone phenotype. GATA‐1low/low mice were bred with OPG‐/‐ mice and bone phenotype assessed. GATA‐1low/low X OPG‐/‐ mice have increased cortical bone porosity, similar to OPG‐/‐ mice. Both OPG‐/‐ and GATA‐1low/low X OPG‐/‐ mice, were found to have increased osteoclasts localized to cortical bone, possibly producing the observed elevated porosity. Biomechanical assessment indicates that OPG‐/‐ and GATA‐1low/low X OPG‐/‐ femurs are weaker and less stiff than C57BL/6 or GATA‐1low/low femurs. Notably, GATA‐1low/low X OPG‐/‐ mice had trabecular bone parameters that were not different from C57BL/6 values, suggesting that GATA‐1 deficiency can partially rescue the trabecular bone loss observed with OPG deficiency. The fact that GATA‐1 deficiency appears to be able to partially rescue the trabecular, but not the cortical bone phenotype suggests that MKs can locally enhance trabecular bone volume, but that MK secreted factors cannot access cortical bone sufficiently to inhibit osteoclastogenesis or that OPG itself is required to inhibit osteoclastogenesis in cortical bone. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:37:45.37493-05:0
      DOI: 10.1002/jcp.24803
       
  • Review: Cell Dynamics in Malignant Pleural Effusions
    • Authors: Enrico Giarnieri; Gianfranco Bellipanni, Marcella Macaluso, Rita Mancini, Adam Carl Holstein, Carla Milanese, Maria Rosaria Giovagnoli, Antonio Giordano, Giuseppe Russo
      Abstract: Malignant pleural effusions (MPEs) are a common manifestation found in patients with lung cancer. After cytological and histological confirmation of malignancy, talc pleurodesis still remains the treatment of choice in patients with MPEs resistant to chemotherapy. Despite this, primary challenges include reduced quality of life and life expectancy in general. Therefore, a better understanding of the cell biology of MPEs, along with improvements in treatment is greatly needed. It has recently been demonstrated that malignant pleural effusions (MPEs) may represent an excellent source for identification of molecular mechanisms within the tumor and its environment. The present review summarizes the current understanding of MPEs cells and tumor microenvironment, and particularly focuses on dissecting the cross‐talk between MPEs and epithelial to mesenchymal transition (EMT), inflammation and cancer stem cells. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:31:30.748438-05:
      DOI: 10.1002/jcp.24806
       
  • Role of Acinus in Regulating Retinoic Acid‐responsive Gene
           Pre‐mRNA Splicing
    • Authors: Fang Wang; Kenneth J. Soprano, Dianne Robert Soprano
      Abstract: Acinus‐S' is a co‐repressor for retinoic acid receptor (RAR)‐dependent gene transcription and has been suggested to be involved in RNA processing. In this study the role of Acinus isoforms in regulating pre‐mRNA splicing was explored using in vivo splicing assays. Both Acinus‐L and Acinus‐S', with the activity of Acinus‐L higher than that of Acinus‐S', increase the splicing of a retinoic acid (RA)‐responsive minigene containing a weak 5' splice site but not a RA‐responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing of the weak 5' splice site by Acinus in a dose‐ and time‐dependent manner, suggesting a RA‐dependent activity in addition to a RA‐independent activity of Acinus. The RA‐independent effect of Acinus occurs to varying degrees using minigene constructs containing several different promoters while the RA‐dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by a RA response element (RARE)‐containing promoter. This suggests that the ligand‐dependent splicing activity of Acinus is related to the RA‐activated RAR bound to the RARE. The RRM domain is necessary for the RA‐dependent splicing activity of Acinus and the RA‐independent splicing activity of Acinus is repressed by RNPS1. Importantly, measurement of the splicing of endogenous human RARβ and Bcl‐x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA‐dependent manner for RARβ and a RA‐independent manner for Bcl‐x. Taken together, these studies demonstrate that Acinus functions in both RAR‐dependent splicing and RAR‐dependent transcription. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:31:18.818058-05:
      DOI: 10.1002/jcp.24804
       
  • Palmitate‐stimulated monocytes induce adhesion molecule expression
           in endothelial cells via IL‐1 signaling pathway
    • Authors: Yosuke Shikama; Nanako Aki, Akiko Hata, Miho Nishimura, Seiichi Oyadomari, Makoto Funaki
      Abstract: Increased intake of saturated fatty acids (SFAs), such as palmitate (Pal), is linked to a higher risk of type 2 diabetes and cardiovascular disease. Although recent studies have investigated the direct effects of SFAs on inflammatory responses in vascular endothelial cells, it remains unknown whether SFAs also induce these responses mediated by circulating cells. In this study, especially focused on adhesion molecules and monocytes, we investigated the indirect effects of Pal on expression and release of ICAM‐1 and E‐selectin in vascular endothelial cells. Phorbol 12‐myristate 13‐acetate (PMA)‐treated THP‐1 (pTHP‐1) cells and human monocytes were stimulated with various free fatty acids (FFAs). SFAs, but not unsaturated fatty acids (UFAs), increased interleukin (IL)‐1β secretion and decreased IL‐1 receptor antagonist (IL‐1Ra) secretion, resulting in an increase in the IL‐1β/IL‐1Ra secretion ratio. UFAs dose‐dependently inhibited the increase in IL‐1β secretion and decrease in IL‐1Ra secretion induced by Pal. Moreover, in human aortic and vein endothelial cells, expression and release of ICAM‐1 and E‐selectin were induced by treatment with conditioned medium collected from Pal‐stimulated pTHP‐1 cells and human monocytes, but not by Pal itself. The up‐regulated expression and release of adhesion molecules by the conditioned medium were mostly abolished by recombinant human IL‐1Ra supplementation. These results suggest that the Pal‐induced increase in the ratio of IL‐1β/IL‐1Ra secretion in monocytes up‐regulates endothelial adhesion molecules, which could enhance leukocyte adhesion to endothelium. This study provides further evidence that IL‐1β neutralization through receptor antagonism may be useful for preventing the onset and development of cardiovascular disease. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T05:08:38.88911-05:0
      DOI: 10.1002/jcp.24797
       
  • microRNA‐151‐3p Regulates Slow Muscle Gene Expression by
           Targeting ATP2a2 in Skeletal Muscle Cells
    • Authors: Huan Wei; Zhongwen Li, Xiaobin Wang, Jie Wang, Weijun Pang, Gongshe Yang, Qingwu W. Shen
      Abstract: MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate the stability or translation of cognate mRNAs at the posttranscriptional level. Accumulating evidence indicates that miRNAs play important roles in many aspects of muscle function, including muscle growth and development, regeneration, contractility, and muscle fiber type plasticity. In the current study, we examined the function of miR‐151‐3p in myoblast proliferation and differentiation. Results show that overexpression of miR‐151‐3p not only upregulates myoblast proliferation, but also decreases slow muscle gene expression (such as MHC‐β/slow and slow muscle troponin I) in both C2C12 myotubes and in primary cultures. Alternatively, inhibition of miR‐151‐3p by antisense RNA was found to upregulate MHC‐β/slow expression, indicating that miR‐151‐3p plays a role in muscle fiber type determination. Further investigation into the underlying mechanisms revealed for the first time that miR‐151‐3p directly targets ATP2a2, a gene encoding for a slow skeletal and cardiac muscle specific Ca2+ ATPase, SERCA2 thus downregulating slow muscle gene expression. Mechanisms by which the alteration in SERCA2 expression induces changes in other slow muscle gene expression levels needs to be defined in future research. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T05:07:57.752466-05:
      DOI: 10.1002/jcp.24793
       
  • miR‐34c regulates the permeability of blood‐tumor barrier via
           MAZ‐mediated expression changes of ZO‐1, occludin and
           claudin‐5
    • Authors: Lini Zhao; Ping Wang, Yunhui Liu, Jun Ma, Yixue Xue
      Abstract: The purposes of this study were to investigate the potential roles of miR‐34c in regulating blood‐tumor barrier (BTB) functions and its possible molecular mechanisms. The over‐expression of miR‐34c significantly impaired the integrity and increased the permeability of BTB, which were detected in an in vitro BTB model by transendothelial electric resistance and horseradish peroxidase flux assays respectively. Meanwhile, real‐time quantitative PCR (qRT‐PCR), Western blot and immunofluorescence assays successively demonstrated down‐regulation of ZO‐1, occludin and claudin‐5. And miR‐34c silencing uncovered the opposite results. Dual‐luciferase reporter assays results revealed myc‐associated zinc‐finger protein (MAZ) is a target gene of miR‐34c. Besides, mRNA and protein expressions of MAZ were reversely regulated by miR‐34c. The down‐expression of MAZ significantly impaired the integrity and increased the permeability of BTB as well as down‐regulated the expressions of ZO‐1, occludin and claudin‐5. And chromatin immunoprecipitation verified that MAZ interacted with “GGGCGGG”, “CCCTCCC” and “GGGAGGG” DNA sequence of ZO‐1, occludin and claudin‐5 promoter respectively. The over‐expression or silencing of either miR‐34c or MAZ was performed simultaneously to further explore their functional relations, and results elucidated that miR‐34c and MAZ displayed reverse regulatory effects on the integrity and permeability of BTB as well as the expressions of ZO‐1, occludin and claudin‐5. In conclusion, our present study indicated that miR‐34c regulated the permeability of BTB via MAZ‐mediated expression changes of ZO‐1, occludin and claudin‐5. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:51:55.133271-05:
      DOI: 10.1002/jcp.24799
       
  • Sphingosine‐1‐phosphate mediates COX‐2 expression and
           PGE2/IL‐6 secretion via c‐Src‐dependent AP‐1
           activation
    • Authors: Chih‐Kai Hsu; I‐Ta Lee, Chih‐Chung Lin, Li‐Der Hsiao, Chuen‐Mao Yang
      Abstract: Sphingosine‐1‐phosphate (S1P) has been shown to regulate cyclooxygenase‐2 (COX‐2)/prostaglandin E2 (PGE2) expression and IL‐6 secretion in various respiratory diseases. However, the mechanisms underlying S1P‐induced COX‐2 expression and PGE2 production in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here we demonstrated that S1P markedly induced COX‐2 expression. S1P also induced PGE2 and IL‐6 secretion which were reduced by the inhibitors of COX‐2 (NS‐398 and celecoxib). Pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c‐Src (PP1), PYK2 (PF431396), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP‐1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c‐Src, PYK2, p38, p42, JNK2, c‐Jun, or c‐Fos reduced S1P‐induced COX‐2 expression and PGE2/IL‐6 secretion. Moreover, S1P induced c‐Src, PYK2, p42/p44 MAPK, JNK1/2, p38 MAPK, and c‐Jun phosphorylation. We observed that S1P‐induced p42/p44 MAPK and JNK1/2, but not p38 MAPK activation was mediated via a c‐Src/PYK2‐dependent pathway. S1P also enhanced c‐Fos, but not c‐Jun mRNA and protein expression and the AP‐1 promoter activity. S1P‐induced c‐Fos mRNA and protein expression, c‐Jun phosphorylation, and AP‐1 promoter activity was reduced by W123, CAY10444, PP1, PF431396, U0126, SP600125, or SB202190. These results demonstrated that S1P‐induced COX‐2 expression and PGE2/IL‐6 generation was mediated through S1PR1/3/c‐Src/PYK2/p42/p44 MAPK‐ or JNK1/2‐ and S1PR1/3/c‐Src/p38 MAPK‐dependent AP‐1 activation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:50:38.124258-05:
      DOI: 10.1002/jcp.24795
       
  • CRM197 in combination with shRNA interference of VCAM‐1 displays
           enhanced inhibitory effects on human glioblastoma cells
    • Authors: Yi Hu; Xing Lin, Ping Wang, Yi‐Xue Xue, Zhen Li, Li‐Bo Liu, Bo Yu, Tian‐Da Feng, Yun‐Hui Liu
      Abstract: CRM197 is a naturally nontoxic diphtheria toxin mutant that binds and inhibits heparin‐binding epidermal growth factor‐like growth factor. CRM197 serves as carrier protein for vaccine and other therapeutic agents. CRM197 also inhibits the growth, migration, invasion and induces apoptosis in various tumors. Vascular cell adhesion molecule‐1 (VCAM‐1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we aimed to investigate the role and mechanism of CRM197 combined with shRNA interference of VCAM‐1 (shRNA‐VCAM‐1) on the migration, invasion and apoptosis of glioblastoma cells. U87 and U251 human glioblastoma cells were treated with CRM197 (10 μg/ml) and shRNA interfering technology was employed to silence VCAM‐1 expression. Cell viability, migration, invasiveness and apoptosis were assessed with CCK8, Transwell and Annexin V‐PE/7‐AAD staining. Activation of cleaved caspase‐3, 8 and 9, activity of matrix metalloproteinase‐2/9 (MMP‐2/9) and expression of phosphorylated Akt (p‐Akt) were also checked. Results showed that CRM197 and shRNA‐VCAM‐1 not only significantly inhibited the cell proliferation, migration, invasion, but also promoted the apoptosis of U87 and U251 cells. Combined treatment of both displayed enhanced inhibitory effects on the malignant biological behavior of glioma cells. The activation of cleaved caspase‐3, 8, 9 was promoted, activity of MMP‐2 and MMP‐9 and expression of p‐Akt were inhibited significantly by the treatment of CRM197 and shRNA‐VCAM‐1 alone or in combination, indicating that the combination of CRM197 with shRNA‐VCAM‐1 additively inhibited the malignant behavior of human glioblastoma cells via activating caspase‐3, 8, 9 as well as inhibiting MMP‐2, MMP‐9 and Akt pathway. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:47:53.443367-05:
      DOI: 10.1002/jcp.24798
       
  • MSCs modified with ACE2 restore endothelial function following LPS
           challenge by inhibiting the activation of RAS
    • Authors: Hong‐li He; Ling Liu, Qi‐hong Chen, Shi‐xia Cai, Ji‐bin Han, Shu‐ling Hu, Pan Chun, Yi Yang, Feng‐mei Guo, Ying‐zi Huang, Hai‐bo Qiu
      Abstract: Angiotensin (Ang) II plays an important role in the process of endothelial dysfunction in acute lung injury(ALI) and is degraded by angiotensin‐converting enzyme2 (ACE2). However, treatmentsthat target ACE2 to injured endothelium and promote endothelial repair of ALI are lacking. Mesenchymal stem cells (MSCs) are capable of homing to the injured site and delivering a protective gene. Our study aimed to evaluate the effects of genetically modified MSCs, which overexpress the ACE2 protein in a sustained manner via a lentiviral vector, on Ang II production in endothelium and in vitro repair of LPS‐induced endothelial injury.We found that the efficiency of lentiviral vector transduction of MSCs was as high as 97.8% and was well maintained over 30 passages. MSCs modified with ACE2 showed a sustained high expression of ACE2 mRNA and protein. The modified MSCs secreted soluble ACE2 protein into the culture medium, which reduced the concentration of Ang II and increased the production of Ang 1–7. MSCs modified with ACE2 were more effective at restoring endothelial function than were unmodified MSCs, as shown by the enhanced survival of endothelial cells; the downregulated production of inflammatory mediators, including ICAM‐1, VCAM‐1, TNF‐α, and IL‐6; reduced paracellular permeability; and increased expression of VE‐cadherin. These data demonstrate that MSCs modified to overexpress the ACE2 gene can produce biologically active ACE2 protein over a sustained period of time and have an enhanced ability to promote endothelial repair after LPS challenge.These results encourage further testing of the beneficial effects of ACE2‐modified MSCs in an ALI animal model. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:40:42.77059-05:0
      DOI: 10.1002/jcp.24794
       
  • Interleukin‐17 induces CC chemokine receptor 6 expression and cell
           migration in colorectal cancer cells
    • Authors: Chih‐Chien Chin; Cheng‐Nan Chen, Hsing‐Chun Kuo, Chung‐Sheng Shi, Meng Chiao Hsieh, Yi‐Hung Kuo, Shui‐Yi Tung, Kam‐Fai Lee, Wen‐Shih Huang
      Abstract: The CC chemokine receptor 6 (CCR6) and its ligand CCL20 are involved in human colorectal cancer (CRC) carcinogenesis and can promote the progression of CRC. In addition, interleukin‐17 (IL‐17), produced by a T cell subset named “Th17,” has been identified as an important player in inflammatory responses, and has emerged as a mediator in inflammation‐associated cancer. However, the relevance of IL‐17 in the development and progression of CRC still remains to be explored. This study aimed to investigate the effect of IL‐17 on the cell migration of CRC cells. Human CRC HCT‐116 cells were used to study the effect of IL‐17 on CCR6 expression and cell migration in CRC cells. IL‐17 treatment induced migration of HCT‐116 cells across the Boyden chamber membrane and increased the expression level of the CCR6. Inhibition of CCR6 by small interfering RNA (siRNA) and neutralizing antibody inhibited IL‐17‐induced cell migration. By using specific inhibitors and short hairpin RNA (shRNA), we demonstrated that the activation of ERK and p38 pathways are critical for IL‐17‐induced CCR6 expression and cell migration. Promoter activity and transcription factor ELISA assays showed that IL‐17 increased NF‐κB‐DNA binding activity in HCT‐116 cells. Inhibition of NF‐κB activation by specific inhibitors and siRNA blocked the IL‐17‐induced CCR6 expression. Our findings support the hypothesis that CCR6 up‐regulation stimulated by IL‐17 may play an active role in CRC cell migration. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:40:26.168474-05:
      DOI: 10.1002/jcp.24796
       
  • Acetylcholine Inhibits Tumor Necrosis Factor α Activated Endoplasmic
           Reticulum Apoptotic Pathway via EGFR‐PI3K Signaling in
           Cardiomyocytes
    • Authors: Yi Miao; Xue‐Yuan Bi, Mei Zhao, Hong‐Ke Jiang, Jin‐Jun Liu, Dong‐Ling Li, Xiao‐Jiang Yu, Yong‐Hua Yang, Ning Huang, Wei‐Jin Zang
      Abstract: Previous findings have shown that acetycholine (ACh) decreased hypoxia‐induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α‐induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase‐3 activation and terminal deoxynucleotidyl transferase mediated dUTP‐biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α‐induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol‐3‐kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh‐elicited anti‐apoptotic property. Together, these data indicate that EGFR‐PI3K/Akt signaling is involved in M2 AChR‐mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:35:37.86881-05:0
      DOI: 10.1002/jcp.24800
       
  • Roles of SATB2 in Site‐Specific Stemness, Autophagy and Senescence
           of Bone Marrow Mesenchymal Stem Cells
    • Authors: WEIJIE DONG; PING ZHANG, YU FU, JIE GE, JIE CHENG, HUA YUAN, HONGBING JIANG
      Abstract: Craniofacial bone marrow mesenchymal stem cells (BMSCs) display some site‐specific properties that differ from those of BMSCs derived from the trunk and appendicular skeleton, but the characteristics of craniofacial BMSCs and the mechanisms that underlie their properties are not completely understood. Previous studies indicated that special AT‐rich binding protein 2 (SATB2) may be a potential regulator of craniofacial skeletal patterning and site‐specific osteogenic capacity. Here, we investigated the stemness, autophagy, and anti‐aging capacity of mandible‐derived BMSCs (M‐BMSCs) and tibia‐derived BMSCs (T‐BMSCs) and explored the role of SATB2 in regulating these properties. M‐BMSCs not only possessed stronger expression of SATB2 and stemness markers (pluripotency genes, such as Nanog, OCT‐4, Sox2, and Nestin) but also exhibited stronger autophagy and anti‐aging capacities under normal or hypoxia/serum deprivation conditions compared to T‐BMSCs. Exogenous expression of SATB2 in T‐BMSCs significantly enhanced the expression of pluripotency genes as well as autophagy and anti‐aging capacity. Moreover, SATB2 markedly enhanced osteogenic differentiation of BMSCs in vitro, and promoted bone defect regeneration and the survival of BMSCs that were transplanted into mandibles with critical size defects. Mechanistically, SATB2 upregulates pluripotency genes and autophagy‐related genes, which in turn activate the mechanistic target of rapamycin signaling pathway. Collectively, our results provide novel evidence that site‐specific BMSCs have distinct biological properties and suggest that SATB2 plays a potential role in regulating the stemness, autophagy, and anti‐aging properties of craniofacial BMSCs. The application of SATB2 to manipulate stem cells for the reconstruction of bone defects might represent a new approach. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:34:09.725711-05:
      DOI: 10.1002/jcp.24792
       
  • Gene therapies for cancer: strategies, challenges and successes
    • Authors: Swadesh K Das; Mitchell E. Menezes, Shilpa Bhatia, Xiang‐Yang Wang, Luni Emdad, Devanand Sarkar, Paul B. Fisher
      Abstract: Gene therapy, which involves replacement of a defective gene with a functional, healthy copy of that gene, is a potentially beneficial cancer treatment approach particularly over chemotherapy, which often lacks selectivity and can cause non‐specific toxicity. Despite significant progress pre‐clinically with respect to both enhanced targeting and expression in a tumor‐selective manner several hurdles still prevent success in the clinic, including non‐specific expression, low‐efficiency delivery and biosafety. Various innovative genetic approaches are under development to reconstruct vectors/transgenes to make them safer and more effective. Utilizing cutting‐edge delivery technologies, gene expression can now be targeted in a tissue‐ and organ‐specific manner. With these advances, gene therapy is poised to become amenable for routine cancer therapy with potential to elevate this methodology as a first line therapy for neoplastic diseases. This review discusses recent advances in gene therapy and their impact on a pre‐clinical and clinical level. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-06T03:27:34.725787-05:
      DOI: 10.1002/jcp.24791
       
  • Perlecan Heparan Sulfate Is Required for the Inhibition of Smooth Muscle
           Cell Proliferation by All‐trans‐Retinoic Acid
    • Abstract: Smooth muscle cell (SMC) proliferation is a key process in stabilization of atherosclerotic plaques, and during restenosis after interventions. A clearer understanding of SMC growth regulation is therefore needed to design specific anti‐proliferative therapies. Retinoic acid has been shown to inhibit proliferation of SMCs both in vitro and in vivo and to affect the expression of extracellular matrix molecules. To explore the mechanisms behind the growth inhibitory activity of retinoic acid, we hypothesized that retinoids may induce the expression of perlecan, a large heparan sulfate proteoglycan with anti‐proliferative properties. Perlecan expression and accumulation was induced in murine SMC cultures by all‐trans‐retinoic acid (AtRA). Moreover, the growth inhibitory effect of AtRA on wild‐type cells was greatly diminished in SMCs from transgenic mice expressing heparan sulfate‐deficient perlecan, indicating that the inhibition is perlecan heparan sulfate‐dependent. In addition, AtRA influenced activation and phosphorylation of PTEN and Akt differently in wild‐type and mutant SMCs, consistent with previous studies of perlecan‐dependent SMC growth inhibition. We demonstrate that AtRA regulates perlecan expression in SMCs and that the inhibition of SMC proliferation by AtRA is, at least in part, secondary to an increased expression of perlecan and dependent upon its heparan sulfate‐chains. J. Cell. Physiol. 230: 482–487, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Inflammation‐Induced Downregulation 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 1 and 24 h. Cells were additionally stimulated with butyrate to investigate its anti‐inflammatory potential. Butyrate uptake and oxidation were measured using 14C‐labeled 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 24 h 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, 1 h incubation with TNF‐α induced a significant increased IL‐8 expression and decreased butyrate uptake. Incubation with TNF‐α and/or IFN‐γ for 1 h did not induce cell damage nor influence butyrate oxidation. The inflammation‐induced downregulation 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. J. Cell. Physiol. 230: 418–426, 2015. © 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. Physiol. 230: 406–417, 2015. © 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. Physiol. 230: 395–405, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Neoadjuvant Chemotherapy in Breast Cancer Patients Induces miR‐34a
           and miR‐122 Expression
    • Abstract: Circulating microRNAs (miRNAs) have been extensively studied in cancer as biomarkers but little is known regarding the influence of anti‐cancer drugs on their expression levels. In this article, we describe the modifications of circulating miRNAs profile after neoadjuvant chemotherapy (NAC) for breast cancer. The expression of 188 circulating miRNAs was assessed in the plasma of 25 patients before and after NAC by RT‐qPCR. Two miRNAs, miR‐34a and miR‐122, that were significantly increased after NAC, were measured in tumor tissue before and after chemotherapy in 7 patients with pathological partial response (pPR) to NAC. These two chemotherapy‐induced miRNAs were further studied in the plasma of 22 patients with adjuvant chemotherapy (AC) as well as in 12 patients who did not receive any chemotherapy. Twenty‐five plasma miRNAs were modified by NAC. Among these miRNAs, miR‐34a and miR‐122 were highly upregulated, notably in pPR patients with aggressive breast cancer. Furthermore, miR‐34a level was elevated in the remaining tumor tissue after NAC treatment. Studying the kinetics of circulating miR‐34a and miR‐122 expression during NAC revealed that their levels were especially increased after anthracycline‐based chemotherapy. Comparisons of the plasma miRNA profiles after NAC and AC suggested that chemotherapy‐induced miRNAs originated from both tumoral and non‐tumoral compartments. This study is the first to demonstrate that NAC specifically induces miRNA expression in plasma and tumor tissue, which might be involved in the anti‐tumor effects of chemotherapy in breast cancer patients. J. Cell. Physiol. 230: 473–481, 2015. © 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 shown that homocysteine (Hcy) caused oxidative stress and altered mitochondrial function. Hydrogen sulfide (H2S) has potent anti‐inflammatory, anti‐oxidative, and anti‐apoptotic effects. Therefore, in the present study we examined whether H2S ameliorates Hcy‐induced mitochondrial toxicity which led to endothelial dysfunction in part, by epigenetic alterations in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 100 μM Hcy treatment in the presence or absence of 30 μM NaHS (donor of H2S) for 24 h. 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 (30 μM) attenuated the Hcy‐induced increased expression of DNMT1, DNMT3a, Ca2+, and decreased expression of DNMT3b in bEND3 cells. Furthermore, NaHS treatment also mitigated mitochondrial oxidative stress (NOX4, ROS, and NO) and restored ATP that indicates its protective effects against mitochondrial toxicity. Additional, NaHS significantly alleviated Hcy‐induced 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. Physiol. 230: 378–394, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Membrane‐Type 1 Matrix Metalloproteinase Downregulates Fibroblast
           Growth Factor‐2 Binding to the Cell Surface and Intracellular
           Signaling
    • 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. J. Cell. Physiol. 230: 366–377, 2015. © 2014 Wiley Periodicals, Inc.
       
  • C/EBPα and the Vitamin D Receptor Cooperate in the Regulation of
           Cathelicidin in Lung Epithelial Cells
    • Abstract: 1,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) and the vitamin D receptor (VDR) have been reported to have an important role in the regulation of innate immunity. We earlier reported that the antimicrobial peptide cathelicidin is induced by 1,25(OH)2D3 in normal human bronchial epithelial cells with a resultant increase in antimicrobial activity against airway pathogens. In this study, we demonstrate that C/EBP alpha (C/EBPα) is a potent enhancer of human cathelicidin antimicrobial peptide (CAMP) gene transcription in human lung epithelial cells. In addition we found that C/EBPα functionally cooperates with VDR in the regulation of CAMP transcription. A C/EBP binding site was identified at −627/−619 within the CAMP promoter, adjacent to the vitamin D response element (VDRE; −615/−600). Mutation of this site markedly attenuated the transcriptional response to C/EBPα as well as to 1,25(OH)2D3, further indicating cooperation between these two factors in the regulation of CAMP. ChIP analysis using 1,25(OH)2D3 treated human lung epithelial cells showed C/EBPα and VDR binding to the CAMP promoter. C/EBPα has previously been reported to cooperate with Brahma (Brm), an ATPase that is component of the SWI/SNF chromatin remodeling complex. We found that dominant negative Brm significantly inhibited C/EBPα as well as 1,25(OH)2D3 mediated induction of CAMP transcription, suggesting the functional involvement of Brm. These findings define novel mechanisms involving C/EBPα, SWI/SNF, and 1,25(OH)2D3 in the regulation of CAMP in lung epithelial cells. These mechanisms of enhanced activation of the CAMP gene in lung epithelial cells suggest potential candidates for the development of modulators of innate immune responses for adjunct therapy in the treatment of airway infections. J. Cell. Physiol. 230: 464–472, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane
           Reveals Roles of Acrosomal Proteins in ZP3 Binding
    • Abstract: The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm‐ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non‐capacitated and capacitated sperm. The results revealed that ZP‐binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far‐western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non‐capacitated and capacitated sperm, but the complexes (∼750–1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome‐intact sperm during capacitation. Co‐immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti‐zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm‐ZP binding during capacitation. J. Cell. Physiol. 230: 449–463, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Beta‐Adrenoceptor Activation Reduces Both Dermal Microvascular
           Endothelial Cell Migration via a cAMP‐Dependent Mechanism and Wound
           Angiogenesis
    • 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 foetal 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 (GPCRs) 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 cyclic adenosine monophosphate (cAMP)‐dependent and protein kinase A (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. 230: 356–365, 2015. © 2014 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
       
  • Distinct Effects of Inorganic Phosphate on Cell Cycle and Apoptosis in
           Human Vascular Smooth Muscle Cells
    • Abstract: 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 cell 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). 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 
       
  • Characterization of Cardiac Anoctamin1 Ca2+‐Activated Chloride
           Channels and Functional Role in Ischemia‐Induced Arrhythmias
    • 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 immunohistochemistry 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. J. Cell. Physiol. 230: 337–346, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Glycated Collagen Induces α11 Integrin Expression Through
           TGF‐β2 and Smad3
    • 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 >fourfold, 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 α11 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 signaling pathway that stimulates α11 integrin expression through Smad2/3 binding elements in the α11 integrin promoter, which is important for myofibroblast formation and fibrosis. J. Cell. Physiol. 230: 327–336, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Contribution of Sp1 to Telomerase Expression and Activity in Skin
           Keratinocytes Cultured With a Feeder Layer
    • 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. J. Cell. Physiol. 230: 308–317, 2015. © 2014 Wiley Periodicals, Inc.
       
  • TGF‐β1 Suppresses Plasmin and MMP Activity in Flexor Tendon
           Cells via PAI‐1: Implications for Scarless Flexor Tendon Repair
    • 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, Transforming Growth Factor Beta 1(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 plasminogen activator inhibitor 1 (PAI‐1) knockout (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. J. Cell. Physiol. 230: 318–326, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Intrinsic Sex‐Linked Variations in Osteogenic and Adipogenic
           Differentiation Potential of Bone Marrow Multipotent Stromal Cells
    • Abstract: Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSCs) 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 twofold 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 adipocyte‐related 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. J. Cell. Physiol. 230: 296–307, 2015. © 2014 Wiley Periodicals, Inc.
       
  • The Regulation of TGFβ1 Induced Fibronectin EDA Exon Alternative
           Splicing in Human Renal Proximal Tubule Epithelial Cells
    • 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. J. Cell. Physiol. 230: 286–295, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Derivation of Endodermal Progenitors From Pluripotent Stem Cells
    • Abstract: Stem and progenitor cells play important roles in organogenesis during development and in tissue homeostasis and response to injury postnatally. As the regenerative capacity of many human tissues is limited, cell replacement therapies hold great promise for human disease management. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are prime candidates for the derivation of unlimited quantities of clinically relevant cell types through development of directed differentiation protocols, that is, the recapitulation of developmental milestones in in vitro cell culture. Tissue‐specific progenitors, including progenitors of endodermal origin, are important intermediates in such protocols since they give rise to all mature parenchymal cells. In this review, we focus on the in vivo biology of embryonic endodermal progenitors in terms of key transcription factors and signaling pathways. We critically review the emerging literature aiming to apply this basic knowledge to achieve the efficient and reproducible in vitro derivation of endodermal progenitors such as pancreas, liver and lung precursor cells. J. Cell. Physiol. 230: 246–258, 2015. © 2014 Wiley Periodicals, Inc.
       
  • VEGF Receptor 2 (VEGFR2) Activation Is Essential for Osteocyte Survival
           Induced by Mechanotransduction
    • Abstract: Mechanical loading plays a key role in bone formation and maintenance. While unloading induces osteocyte apoptosis and bone loss in vivo, mechanical stimuli prevents osteocyte death through a mechanism involving β‐catenin accumulation and ERK nuclear translocation. Vascular endothelial growth factor (VEGF) has a crucial role in bone formation, but its interaction with osteocytes is not completely understood. Of interest, VEGF receptor 2 (VEGFR2) has recently been shown to mediate the mechanical response of endothelial cells. The present study aimed to evaluate the putative role of the VEGF system in osteocyte mechanosensing. We show that either short (10 min) mechanical stimulus by pulsatile fluid flow (FF) (10 dyn/cm2, 8 Hz) or exogenous VEGF165 (6 ng/ml) similarly stimulated cell viability, ERK phosphorylation, and β‐catenin membrane translocation. A VEGFR2 antagonist (SU5416) or transfection with specific VEGFR2 siRNAs (siVEGFR2) decreased these events. FF for 10 min increased VEGFR2 phosphorylation at both Tyr‐1059 and Tyr‐1175; an effect that was mimicked by VEGF165 but was unaffected by a VEGF neutralizing antibody. Subsequently (at 6 h), this mechanical stimulus induced VEGF gene overexpression, which was prevented by siVEGFR2 transfection. Depletion of the structural protein caveolin‐1 by using siRNA technology impaired FF‐induced VEGFR2 phosphorylation. In conclusion, these in vitro findings point to caveolin‐1‐dependent VEGFR2 activation as an important mechanism whereby mechanical stimuli promote osteocyte viability. J. Cell. Physiol. 230: 278–285, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Recovery and Cultivation of Keratinocytes From Shipped Mouse Skin
    • Abstract: Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 h at 0 °C. The cultured keratinocytes from the control, non‐shipped skin and the 2‐day shipped skin were 43.6 +/− 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta‐1. However, under the same shipping conditions, the 3‐day shipped tissue failed to establish colonies in the culture. Therefore, this 2‐day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long‐distance collaborations. J. Cell. Physiol. 230: 242–245, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Runx1 Activities in Superficial Zone Chondrocytes, Osteoarthritic
           Chondrocyte Clones and Response to Mechanical Loading
    • Abstract: Runx1, the hematopoietic lineage determining transcription factor, is present in perichondrium and chondrocytes. Here we addressed Runx1 functions, by examining expression in cartilage during mouse and human osteoarthritis (OA) progression and in response to mechanical loading. Spared and diseased compartments in knees of OA patients and in mice with surgical destabilization of the medial meniscus were examined for changes in expression of Runx1 mRNA (Q‐PCR) and protein (immunoblot, immunohistochemistry). Runx1 levels were quantified in response to static mechanical compression of bovine articular cartilage. Runx1 function was assessed by cell proliferation (Ki67, PCNA) and cell type phenotypic markers. Runx1 is enriched in superficial zone (SZ) chondrocytes of normal bovine, mouse, and human tissues. Increasing loading conditions in bovine cartilage revealed a positive correlation with a significant elevation of Runx1. Runx1 becomes highly expressed at the periphery of mouse OA lesions and in human OA chondrocyte ‘clones’ where Runx1 co‐localizes with Vcam1, the mesenchymal stem cell (MSC) marker and lubricin (Prg4), a cartilage chondroprotective protein. These OA induced cells represent a proliferative cell population, Runx1 depletion in MPCs decreases cell growth, supporting Runx1 contribution to cell expansion. The highest Runx1 levels in SZC of normal cartilage suggest a function that supports the unique phenotype of articular chondrocytes, reflected by upregulation under conditions of compression. We propose Runx1 co‐expression with Vcam1 and lubricin in murine cell clusters and human ‘clones’ of OA cartilage, participate in a cooperative mechanism for a compensatory anabolic function. J. Cell. Physiol. 230: 440–448, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Non‐Random Patterns in the Distribution of NOR‐Bearing
           Chromosome Territories in Human Fibroblasts: A Network Model of
           Interactions
    • Abstract: We present a 3‐D mapping in WI38 human diploid fibroblast cells of chromosome territories (CT) 13,14,15,21, and 22, which contain the nucleolar organizing regions (NOR) and participate in the formation of nucleoli. The nuclear radial positioning of NOR‐CT correlated with the size of chromosomes with smaller CT more interior. A high frequency of pairwise associations between NOR‐CT ranging from 52% (CT13–21) to 82% (CT15–21) was detected as well as a triplet arrangement of CT15–21‐22 (72%). The associations of homologous CT were significantly lower (24–36%). Moreover, singular contacts between CT13–14 or CT13–22 were found in the majority of cells, while CT13–15 or CT13–21 predominantly exhibited multiple interactions. In cells with multiple nucleoli, one of the nucleoli (termed “dominant”) always associated with a higher number of CT. Moreover, certain CT pairs more frequently contributed to the same nucleolus than to others. This nonrandom pattern suggests that a large number of the NOR‐chromosomes are poised in close proximity during the postmitotic nucleolar recovery and through their NORs may contribute to the formation of the same nucleolus. A global data mining program termed the chromatic median determined the most probable interchromosomal arrangement of the entire NOR‐CT population. This interactive network model was significantly above randomized simulation and was composed of 13 connections among the NOR‐CT. We conclude that the NOR‐CT form a global interactive network in the cell nucleus that may be a fundamental feature for the regulation of nucleolar and other genomic functions. J. Cell. Physiol. 230: 427–439, 2015. © 2014 Wiley Periodicals, Inc.
       
  • The Cooperation of CREB and NFAT is Required for PTHrP‐Induced RANKL
           Expression in Mouse Osteoblastic Cells
    • Abstract: Parathyroid hormone‐related protein (PTHrP) is known to induce the expression of receptor activator of NF‐κB ligand (RANKL) in stromal cells/osteoblasts. However, the signaling pathways involved remain controversial. In the present study, we investigated the role of cAMP/protein kinase A (PKA) and calcineurin/NFAT pathways in PTHrP‐induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. PTHrP‐mediated induction of RANKL expression was significantly inhibited by H89 and FK506, an inhibitor of PKA and calcineurin, respectively. PTHrP upregulated CREB phosphorylation and the transcriptional activity of NFAT. Knockdown of CREB or NFATc1 blocked PTHrP‐induced RANKL expression. PTHrP increased the activity of the RANKL promoter reporter that contains approximately 2 kb mouse RANKL promoter DNA sequences. Insertions of mutations in CRE‐like element or in NFAT‐binding element abrogated PTHrP‐induced RANKL promoter activity. Chromatin immunoprecipitation assays showed that PTHrP increased the binding of CREB and NFATc1/NFATc3 to their cognate binding elements in the RANKL promoter. Inhibition of cAMP/PKA and its downstream ERK activity suppressed PTHrP‐induced expression and transcriptional activity of NFATc1. CREB knockdown prevented PTHrP induction of NFATc1 expression. Furthermore, NFATc1 and CREB were co‐immunoprecipitated. Mutations in CRE‐like element completely blocked NFATc1‐induced transactivation of the RANKL promoter reporter; however, mutations in NFAT‐binding element partially suppressed CREB‐induced RANKL promoter activity. Overexpression of CREB increased NFATc1 binding to the RANKL promoter and vice versa. These results suggest that PTHrP‐induced RANKL expression depends on the activation of both cAMP/PKA and calcineurin/NFAT pathways, and subsequently, CREB and NFAT cooperate to transactivate the mouse RANKL gene. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
  • Journal of Cellular Physiology: Volume 230, Number 2, February 2015
    • Abstract: Cover: Diagram illustrating the chromatic median analysis. See article by Pliss et al. on pages 427–439.
       
  • Table of Contents: Volume 230, Number 2
    •  
  • Editor's Choice
    •  
  • Highlights: Volume 230, Number 2
    •  
  • Initial Characterization of Osteoblast Differentiation and Loss of RUNX2
           Stability in the Newly Established SK11 Human Embryonic Stem
           Cell‐Derived Cell Line
    • Abstract: We describe a novel model for investigation of genetically normal human osteoblasts in culture. SK11 is a clonal progenitor cell line derived from human embryonic stem cells. Initially selected based on the expression of chondrogenic markers when differentiated in micromass culture, SK11 cells display typical mRNA expression patterns of bone phenotypic genes under osteogenic conditions. These include osterix, α1(I) collagen, alkaline phosphatase, osteonectin, osteopontin, and osteocalcin. Similar to well‐characterized murine osteoblast cultures, the osteoblast master regulator RUNX2 was present during the first few days after plating, but the protein disappeared during the first week of culture. Loss of RUNX2 expression is considered an important regulatory feature for osteoblast maturation. Indeed, following ∼2 weeks of differentiation, SK11 cultures exhibited robust calcium deposition, evidenced by alizarin red staining. We also introduced a lentiviral vector encoding doxycycline (dox)‐inducible FLAG‐tagged RUNX2 into SK11 cells. Dox‐mediated enhancement of RUNX2 expression resulted in accelerated mineralization, which was further increased by co‐treatment with BMP‐2. Like the endogenous RUNX2, expression of the virally coded FLAG‐RUNX2 was lost during the first week of culture despite persistent dox treatment. By following RUNX2 decay after dox withdrawal from day‐5 versus day‐3 cultures, we demonstrated a developmentally regulated decrease in RUNX2 stability. Availability of culture models for molecular investigation of genetically normal human osteoblasts is important because differences between murine and human osteoblasts, demonstrated here by the regulation of matrix Gla Protein, may have significant biomedical implications. J. Cell. Physiol. 230: 237–241, 2015. © 2014 Wiley Periodicals, Inc.
       
  • Influence of Nuclear Blebs and Micronuclei Status on the Growth Kinetics
           of Human Mesenchymal Stem Cells
    • Abstract: Therapeutic potential of mesenchymal stem cells (MSCs) demands assurance of the quality, safety, and genetic stability. Nuclear blebs (NBs) and Micronuclei (MNs) are considered as biomarkers for cancer and an increase in their numbers is associated with malignancy and other pathologic disorders. However, the status of NBs and MNs in MSCs is not known. Hence we examined the frequency of NBs and MNs in MSCs from umbilical cord (UC‐MSCs) and placenta (PD‐MSCs) and found a difference in the number of NBs and MNs depending on the source of the MSCs. The number of NBs and MNs was always found to be less in UC‐MSCs as compared to PD‐MSCs obtained from the same patient. Furthermore, we observed that the number of NBs was inversely proportional to the proliferation rate of the cells. The cryopreservation of these MSCs over 6 months also led to increase in the number of NBs and MNs thus slowing down their rate of proliferation on revival. MSCs from both the sources exhibiting high NBs and MNs showed longer S phase and G2‐M arrest and increase in senescent cells without altering their CD‐marker profile and differentiation potential. This feature was consistent with the upregulation of cell cycle checkpoint genes (p53, p21, Gadd45, ATM, ATR, chek2, p27, p16, and p10). In conclusion, our data demonstrates for the first time the importance of checking the occurrence of NBs and MNs in MSCs before using them for cellular therapy as a quality control measure to check their genetic stability. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
 
 
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