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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  [1603 journals]   [SJR: 1.608]   [H-I: 118]
  • Phospholipase C epsilon‐induced TRPC6 activation: A common but
           redundant mechanism in primary podocytes
    • Authors: Hermann Kalwa; Ursula Storch, Jana Demleitner, Susanne Fiedler, Tim Mayer, Martina Kannler, Meike Fahlbusch, Holger Barth, Alan Smrcka, Friedhelm Hildebrandt, Thomas Gudermann, Alexander Dietrich
      Pages: n/a - n/a
      Abstract: In eukaryotic cells, activation of phospholipase C (PLC)‐coupled membrane receptors by hormones leads to an increase in the intracellular Ca2+ concentration [Ca2+]i. Catalytic activity of PLCs results in the hydrolysis of phosphatidylinositol 4,5‐bisphosphate to generate inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG) which opens DAG‐sensitive classical transient receptor channels 3, 6 and 7 (TRPC3/6/7), initiating Ca2+ influx from the extracellular space. Patients with focal segmental glomerulosclerosis (FSGS) express gain‐of‐function mutants of TRPC6, while others carry loss‐of‐function mutants of PLCε, raising the intriguing possibility that both proteins interact and might work in the same signalling pathway. While TRPC6 activation by PLCβ and PLCγ isozymes was extensively studied, the role of PLCε in TRPC6 activation remains elusive. TRPC6 was co‐immunoprecipitated with PLCε in a heterologous overexpression system in HEK293 cells as well as in freshly isolated murine podocytes. Receptor‐operated TRPC6 currents in HEK293 cells expressing TRPC6 were reduced by a specific PLCε siRNA and by a PLCε loss‐of‐function mutant isolated from a patient with FSGS. PLCε‐induced TRPC6 activation was also identified in murine embryonic fibroblasts (MEFs) lacking Gαq/11 proteins. Further analysis of the signal transduction pathway revealed a Gα12/13 Rho‐GEF activation which induced Rho‐mediated PLCε stimulation. Therefore, we identified a new pathway for TRPC6 activation by PLCε. PLCε‐/‐ podocytes however, were undistinguishable from WT podocytes in their angiotensin II‐induced formation of actin stress fibers and their GTPγS‐induced TRPC6 activation, pointing to a redundant role of PLCε‐mediated TRPC6 activation at least in podocytes. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-18T00:13:09.618887-05:
      DOI: 10.1002/jcp.24883
       
  • Clinical predictive circulating peptides in rectal cancer patients treated
           with neoadjuvant chemoradiotherapy
    • Authors: Sara Crotti; Maria Vittoria Enzo, Chiara Bedin, Salvatore Pucciarelli, Isacco Maretto, Paola Del Bianco, Pietro Traldi, Ennio Tasciotti, Mauro Ferrari, Flavio Rizzolio, Giuseppe Toffoli, Antonio Giordano, Donato Nitti, Marco Agostini
      Pages: n/a - n/a
      Abstract: Preoperative chemoradiotherapy is worldwide accepted as a standard treatment for locally advanced rectal cancer. Current standard of treatment includes administration of ionizing radiation for 45‐50.4 Gy in 25‐28 fractions associated with 5‐fluorouracil administration during radiation therapy. Unfortunately, forty percent of patients have a poor or absent response and novel predictive biomarkers are demanding. For the first time, we apply a novel peptidomic methodology and analysis in rectal cancer patients treated with preoperative chemoradiotherapy. Circulating peptides (Molecular Weight
      PubDate: 2014-12-17T05:24:51.215198-05:
      DOI: 10.1002/jcp.24894
       
  • Mixl1 and Flk1 are Key Players of Wnt/TGF‐β Signaling During
           DMSO‐Induced Mesodermal Specification in P19 cells
    • Authors: Seung‐Cheol Choi; Ji‐Hyun Choi, Long‐Hui Cui, Ha‐Rim Seo, Jong‐Ho Kim, Chi‐Yeon Park, Hyung‐Joon Joo, Jae‐Hyoung Park, Soon‐Jun Hong, Cheol‐Woong Yu, Do‐Sun Lim
      Pages: n/a - n/a
      Abstract: Dimethyl sulfoxide (DMSO) is widely used to induce multilineage differentiation of embryonic and adult progenitor cells. To date, little is known about the mechanisms underlying DMSO‐induced mesodermal specification. In this study, we investigated the signaling pathways and lineage‐determining genes involved in DMSO‐induced mesodermal specification in P19 cells. Wnt/β‐catenin and TGF‐β superfamily signaling pathways such as BMP, TGF‐β and GDF1 signaling were significantly activated during DMSO‐induced mesodermal specification. In contrast, Nodal/Cripto signaling pathway molecules, required for endoderm specification, were severely downregulated. DMSO significantly upregulated the expression of cardiac mesoderm markers but inhibited the expression of endodermal and hematopoietic lineage markers. Among the DMSO‐activated cell lineage markers, the expression of Mixl1 and Flk1 was dramatically upregulated at both the transcript and protein levels, and the populations of Mixl1+, Flk1+ and Mixl1+/Flk1+ cells also increased significantly. DMSO modulated cell cycle molecules and induced cell apoptosis, resulting in significant cell death during EB formation of P19 cells. An inhibitor of Flk1, SU5416 significantly blocked expressions of TGF‐β superfamily members, mesodermal cell lineage markers and cell cycle molecules but it did not affect Wnt molecules. These results demonstrate that Mixl1 and Flk1 play roles as key downstream or interacting effectors of Wnt/TGF‐β signaling pathway during DMSO‐induced mesodermal specification in P19 cells. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-17T05:24:40.308746-05:
      DOI: 10.1002/jcp.24892
       
  • Matrix rigidity mediates TGFβ1‐induced
           epithelial‐myofibroblast transition by controlling cytoskeletal
           organization and MRTF‐A localization
    • Authors: Joseph W. O'Connor; Patrick N. Riley, Sandeep M. Nalluri, Parth K. Ashar, Esther W. Gomez
      Pages: n/a - n/a
      Abstract: Myofibroblasts mediate normal wound healing and upon chronic activation can contribute to the development of pathological conditions including organ fibrosis and cancer. Myofibroblasts can develop from epithelial cells through an epithelial‐mesenchymal transition (EMT) during which epithelial cells exhibit drastic morphological changes and upregulate cytoskeletal associated proteins that enable exertion of large contractile forces and remodeling of the surrounding microenvironment. Increased matrix rigidity is a hallmark of fibrosis and tumor progression and mechanical tension has been identified as a regulator of EMT; however, the mechanisms governing the mechanical regulation of EMT are not completely understood. Here, we find that matrix rigidity regulates transforming growth factor (TGF)‐β1‐induced EMT, with rigid substrata enabling increased myofibroblast marker expression, cell morphology changes, and cytoskeletal reorganization while soft matrices block these changes. Furthermore, we find that matrix rigidity controls the subcellular localization of myocardin related transcription factor (MRTF)‐A, a regulator of cytoskeletal protein expression that contributes to the acquisition of myogenic features during EMT. Results from these studies provide insight into how biophysical cues contribute to myofibroblast development from epithelial cells and may suggest ways to enhance wound healing or to engineer therapeutic solutions for fibrosis and cancer. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-17T05:24:24.580027-05:
      DOI: 10.1002/jcp.24895
       
  • 15‐Lipoxygenase Promotes Chronic Hypoxia‐Induced Phenotype
           Changes of PASMCs via Positive Feedback‐Loop of BMP4
    • Authors: Xiufeng Yu; Liuping Wei, Ping Lu, Tingting Shen, Xia Liu, Tingting Li, Bo Zhang, Hao Yu, Daling Zhu
      Pages: n/a - n/a
      Abstract: Our laboratory has previously demonstrated that 15‐lipoxygenase (15‐LO)/15‐hydroxyeicosatetr‐aenoic acid (15‐HETE) is involved in hypoxic pulmonary arterial hypertension (PAH). Phenotypical alterations of vascular smooth muscle cells are considered to be an important stage in the development of PAH, whereas the underlying mechanisms and signaling systems are still unclear. Here, we determined the contribution of 15‐LO/15‐HETE signaling in the hypoxia–induced phenotype changes of pulmonary arterial smooth muscle cells (PASMCs). To accomplish this, cellular and molecular changes in pulmonary vascular remodeling were detected in PAH patients and rats exposed to hypoxia. We found that the hypoxia‐induced alterations in PASMCs phenotypes were reversed by the inhibition of 15‐LO/15‐HETE or inhibition of BMP4/BMPRI. Hypoxia‐induced 15‐LO1/2 expression in rat PASMCs was significantly abolished by small interfering RNA targeted at BMP4. Meanwhile, BMP4/BMPRI‐15‐LO/15‐HETE had a positive feedback mechanism. Furthermore, ERK and p38MAPK act as the downstream of the 15‐LO/15‐HETE‐BMP4/BMPRI signaling. Our results suggest that chronic hypoxia promotes phenotypical alterations of PASMCs due to the interaction between 15‐LO/15‐HETE and BMP4/BMPRI. Our study reveals a novel mechanism of hypoxia‐induced pulmonary vascular remodeling and suggested new therapeutic strategies for the targeting of 15‐LO/15‐HETE and BMP4/BMPRI in PAH treatment. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-17T05:23:26.346096-05:
      DOI: 10.1002/jcp.24893
       
  • Intrinsic resistance triggered under acid loading within normal esophageal
           epithelial cells: NHE1‐ and ROS‐mediated survival
    • Authors: Sun Young Park; Yeon Joo Lee, Eun Jeong Cho, Chang Yell Shin, Uy Dong Sohn
      Pages: n/a - n/a
      Abstract: The transition to a pathological phenotype such as Barrett's esophagus occurs via induction of resistance upon repeated contact with gastric refluxate in esophagus. This study examined the molecular changes within normal esophageal epithelial cells (EECs) under short‐term acid loading and the role of these changes in defensive resistance against acidic cytotoxicity. After primary cultured EECs were exposed to pH 4‐acidified medium (AM4), cell viability was determined by the MTT assay. Reactive oxygen species (ROS) and NAD(P)H oxidase (NOX) activity were measured. Activation of the mitogen‐activated protein kinases (MAPKs) MEK/ERK1/2, p38 and JNK; phosphoinositol‐3‐kinase (PI3K)/Akt, and nuclear factor‐kappa B (NF‐κB) were detected by Western blot analysis or immunofluorescence staining. AM4 incubation induced intracellular ROS generation accompanied by increase in NOX activity, which was further increased by Na+/H+ exchange‐1 (NHE1)‐dependent inhibition but was prevented by inhibition of NOX or mitochondria complex I. AM4 also induced phosphorylation of MEK/ERK1/2, p38 MAPK, PI3K/Akt, and nuclear translocation of NF‐κB, and all these effects, except for p38 MAPK phosphorylation, were abolished by inhibition of ROS. ROS‐dependent PI3K/Akt activation, which mediates NF‐κB nuclear translocation, was inhibited by protein tyrosine kinase (PTK) inhibitors and NHE1‐specific inhibitor. All inhibitors of NHE, ROS, PTK, PI3K or NF‐κB further decreased AM4‐induced cell viability. Acid loading in the presence of NHE1‐dependent protection induced ROS generation by activating NOX and mitochondria complex I, which stimulated PTK/PI3K/Akt/NF‐κB‐dependent survival in EEC. Our data indicate that normal EEC initially respond to acid loading through intrinsic survival activation. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-17T05:22:34.404688-05:
      DOI: 10.1002/jcp.24896
       
  • GABARAP is a Determinant of Apoptosis in Growth‐Arrested Chicken
           Embryo Fibroblasts
    • Authors: Scott Maynard; Romita Ghosh, Ying Wu, Shi Yan, Tetsuaki Miyake, Mark Gagliardi, Karen Rethoret, P‐A. Bédard
      Pages: n/a - n/a
      Abstract: Nutrient depletion triggers a series of adaptive processes as part of the unfolded protein response or UPR. These processes reduce stress to the endoplasmic reticulum by enhancing its protein folding capacity or ability to promote the degradation of dysfunctional proteins. Failure to restore ER homeostasis causes the activation of lethal pathways. The expression of a dominant negative mutant of C/EBPβ (Δ184‐C/EBPβ) alters this balance in chicken embryo fibroblasts (CEF). As a result, CEF display enhanced survival upon prolonged nutrient depletion. Starved Δ184‐C/EBPβ–expressing CEF display pronounced features of autophagy characterized by the appearance of large vesicles containing amorphous material, the formation of smaller double‐membrane vesicles (autophagosomes) and processing of LC3 and GABARAP. However, there were marked differences in the expression and processing of these proteins. In both normal and Δ184‐C/EBPβ expressing CEF, the lipidated form of LC3 (form II) accumulated during starvation but was detectable even when cells were actively dividing in complete medium. In contrast, GABARAP expression and lipidation were strongly stimulated in response to starvation. Inhibition of LC3 expression by RNA interference led to apoptosis in normal CEF even in the absence of starvation but stable and near complete repression of GABARAP was tolerated. Moreover, the inhibition of GABARAP enhanced CEF survival and abolished the expression of the pro‐apoptotic CHOP factor in conditions of starvation, suggesting a reduced level of ER stress. Therefore, GABARAP is a determinant of apoptosis in CEF subjected to prolonged nutrient depletion. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-16T15:26:08.44154-05:0
      DOI: 10.1002/jcp.24889
       
  • Anthropometric, metabolic and molecular determinants of human epidermal
           growth factor receptor 2 expression in luminal B breast cancer
    • Authors: Patrizia Vici; Anna Crispo, Antonio Giordano, Luigi Di Lauro, Francesca Sperati, Irene Terrenato, Laura Pizzuti, Domenico Sergi, Marcella Mottolese, Claudio Botti, Maria Grimaldi, Immacolata Capasso, Giuseppe D'Aiuto, Maurizio Di Bonito, Flaviano Di Paola, Marcello Maugeri‐Saccà, Maurizio Montella, Maddalena Barba
      Pages: n/a - n/a
      Abstract: Genomic and trascriptomic profiling has recently contributed details to the characterization of luminal B breast cancer. We explored the contribution of anthropometric, metabolic and molecular determinants to the multifaceted heterogeneity of this breast cancer subtype, with a specific focus on the association between body mass index (BMI), pre‐treatment fasting glucose, hormone receptors and expression of human epidermal growth factor receptor 2 (HER2). Extensively annotated specimens were obtained from 154 women with luminal B breast cancer diagnosed at two Italian comprehensive cancer centres. Participants' characteristics were descriptively analyzed overall and by HER2 status (positive vs negative). BMI (
      PubDate: 2014-12-16T03:55:43.480921-05:
      DOI: 10.1002/jcp.24891
       
  • Functional interaction between the ESCRT‐I component TSG101 and the
           HSV‐1 tegument ubiquitin specific protease
    • Authors: Calistri A; Munegato D, Toffoletto M, Celestino M, Franchin E, Comin A, Sartori E, Salata C, Parolin C, Palù G
      Pages: n/a - n/a
      Abstract: Similar to phosphorylation, transient conjugation of ubiquitin to target proteins (ubiquitination) mediated by the concerted action of ubiquitin ligases and de‐ubiquitinating enzymes (DUBs) can affect substrate function. As obligate intracellular parasites, viruses rely on different cellular pathways for their own replication and the well conserved ubiquitin conjugating/de‐conjugating system is not an exception. Viruses not only usurp the host proteins involved in the ubiquitination/de‐ubiquitination process, but they also encode their own ubiquitin ligases and DUBs. Here we report that an N‐terminal variant of the herpes simplex virus (HSV) type‐1 large tegument protein VP1/2 (VP1/21–767), encompassing an active DUB domain (herpesvirus tegument ubiquitin specific protease, htUSP), and TSG101, a component of the endosomal sorting complex required for transport (ESCRT)‐I, functionally interact. In particular, VP1/21–767 modulates TSG101 ubiquitination and influences its intracellular distribution. Given the role played by the ESCRT machinery in crucial steps of both cellular pathways and viral life cycle, the identification of TSG101 as a cellular target for the HSV‐1 specific de‐ubiquitinating enzyme contributes to the clarification of the still under debate function of viral encoded DUBs highly conserved throughout the Herpesviridae family. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-16T03:51:42.885361-05:
      DOI: 10.1002/jcp.24890
       
  • “Nuclear FGF Receptor‐1 and CREB Binding Protein ‐ an
           integrative signaling module”
    • Authors: M.K. Stachowiak; B. Birkaya, J. Aletta, S. Narla, C.A. Benson, B. Decker, E.K. Stachowiak
      Pages: n/a - n/a
      Abstract: In this review we summarize the current understanding of a novel integrative function of Fibroblast Growth Factor Receptor‐1 (FGFR1) and its partner CREB Binding Protein (CBP) acting as a nuclear regulatory complex. Nuclear FGFR1 and CBP interact with and regulate numerous genes on various chromosomes. FGFR1 dynamic oscillatory interactions with chromatin and with specific genes, underwrites gene regulation mediated by diverse developmental signals. Integrative Nuclear FGFR1 Signaling (INFS) effects the differentiation of stem cells and neural progenitor cells via the gene‐controlling Feed‐Forward‐And‐Gate mechanism. Nuclear accumulation of FGFR1 occurs in numerous cell types and disruption of INFS may play an important role in developmental disorders such as schizophrenia, and in metastatic diseases such as cancer. Enhancement of INFS may be used to coordinate the gene regulation needed to activate cell differentiation for regenerative purposes or to provide interruption of cancer stem cell proliferation. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-11T07:03:12.569542-05:
      DOI: 10.1002/jcp.24879
       
  • Absence of Bone Sialoprotein (BSP) alters profoundly haematopoiesis and
           upregulates Osteopontin
    • Authors: RN Granito; W Bouleftour, O Sabido, C Lescale, M Thomas, JE Aubin, M Goodhardt, L Vico, L Malaval
      Pages: n/a - n/a
      Abstract: Matrix proteins of the SIBLING family interact with bone cells, extracellular matrix and mineral, and are thus in a key position to regulate the microenvironment of the bone tissue, including its haematopoietic component. In this respect, osteopontin (OPN) has been implicated in the haematopoietic stem cell (HSC) niche as negative regulator of the HSC function. We investigated the impact on haematopoietic regulation of the absence of the cognate bone sialoprotein (BSP). BSP knockout (‐/‐) mice display increased bone marrow cellularity, and an altered commitment of haematopoietic precursors to myeloid lineages, leading in particular to an increased frequency of monocyte/macrophage cells. The B cell pool is increased in ‐/‐ bone marrow, and its composition is shifted towards more mature lymphocyte stages. BSP‐null mice display a decreased HSC fraction among LSK cells and a higher percentage of more committed progenitors as compared to +/+. The fraction of proliferating LSK progenitors is higher in ‐/‐ mice, and after PTH treatment the mutant HSC pool is lower than in +/+. Strikingly, circulating levels of OPN as well as its expression in the bone tissue are much higher in the ‐/‐. Thus, a BSP‐null bone microenvironment affects the haematopoietic sytem both quantitatively and qualitatively, in a manner in part opposite to the OPN knockout, suggesting that the effects might in part reflect the higher OPN expression in the absence of BSP. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-11T07:02:58.295958-05:
      DOI: 10.1002/jcp.24877
       
  • Rac1 regulates myosin II phosphorylation through regulation of myosin
           light chain phosphatase
    • Authors: Keita Shibata; Hiroyasu Sakai, Qian Huang, Hirotoshi Kamata, Yoshihiko Chiba, Miwa Misawa, Reiko Ikebe, Mitsuo Ikebe
      Pages: n/a - n/a
      Abstract: Phosphorylation of regulatory light chain (MLC) activates myosin II, which enables it to promote contractile and motile activities of cells. We report here a signaling mechanism that activates MLC phosphorylation and smooth muscle contraction. Contractile agonists activated Rac1, and Rac1 inhibition diminished agonist‐induced MLC phosphorylation, thus inhibiting smooth muscle contraction. Rac1 inhibits the activity of MLC phosphatase (MLCP) but not that of MLC kinase, through a phosphatase that targets MYPT1 (a regulatory subunit of MLCP) and CPI‐17 (a MLCP specific inhibitor) rather than through the RhoA‐Rho dependent kinase (ROCK) pathway. Rac1 inhibition decreased the activity of protein kinase C (PKC), which also contributes to the change in CPI‐17 phosphorylation. We propose that activation of Rac1 increases the activity of PKC, which increases the phosphorylation of CPI‐17 and MYPT1 by inhibiting the phosphatase that targets these proteins, thereby decreasing the activity of MLCP and increasing phosphorylation of MLC. Our results suggest that Rac1 coordinates with RhoA to increase MLC phosphorylation by inactivation of CPI‐17/MYPT1 phosphatase, which decreases MLCP activity thus promoting MLC phosphorylation and cell contraction. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-11T07:02:43.56431-05:0
      DOI: 10.1002/jcp.24878
       
  • Regulation of Cigarette Smoke Induction of IL‐8 in Macrophages by
           AMP‐activated Protein Kinase Signaling
    • Authors: Hsin‐Kuo Ko; Hung‐Fu Lee, An‐Hsuan Lin, Meng‐Han Liu, Ching‐I Liu, Tzong‐Shyuan Lee, Yu Ru Kou
      Pages: n/a - n/a
      Abstract: Inhaled cigarette smoke (CS) causes persistent lung inflammation in smokers. Interleukin 8 (IL‐8) released from macrophages is a key chemokine during initiation and progression of CS‐induced lung inflammation, yet its regulation is largely unknown. AMP‐activated protein kinase (AMPK), a crucial energy homeostasis regulator, may modulate inflammation. Here we report that CS extract (CSE) increased the level of intracellular reactive oxygen species (ROS), activating AMPK, mitogen‐activated protein kinases (MAPKs), and NF‐κB, as well as inducing IL‐8, in human macrophages. N‐acetyl‐cysteine (ROS scavenger) or hexamethonium [nicotinic acetylcholine receptor (nAChR) antagonist] attenuated the CSE‐induced increase in intracellular ROS, activation of AMPK and NF‐κB, as well as IL‐8 induction, which suggests that nAChRs and ROS are important. Prevention of AMPK activation by compound C or AMPK siRNA reduced CSE‐induced IL‐8 production, confirming the role of AMPK. Compound C or AMPK siRNA also inhibited the activation of MAPKs and NF‐κB by CSE, which suggests that these molecules are downstream of AMPK. Additionally, exposure of human macrophages to nicotine activated AMPK and induced IL‐8 and that these effects were lessened by hexamethonium or compound C, implying that nicotine in CS may be causative. Furthermore, chronic CS exposure in mice promoted AMPK phosphorylation and expression of MIP‐2 (an IL‐8 homolog) in infiltrated macrophages and in lung tissues, as well as induced lung inflammation, all of which were reduced by compound C treatment. Thus, we identified a novel nAChRs‐dependent, ROS‐sensitive, AMPK/MAPKs/NF‐κB signaling pathway, which seems to be important to CS‐induced macrophage IL‐8 production and possibly to lung inflammation. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-11T07:02:29.09788-05:0
      DOI: 10.1002/jcp.24881
       
  • Depletion of Amyloid Precursor Protein (APP) causes G0 arrest in nonsmall
           cell lung cancer (NSCLC) cells
    • Authors: Anna Sobol; Paola Galluzzo, Megan J. Weber, Sara Alani, Maurizio Bocchetta
      Pages: n/a - n/a
      Abstract: We recently reported that Amyloid Precursor Protein (APP) regulates global protein synthesis in a variety of human dividing cells, including non‐small cell lung cancer (NSCLC) cells. More specifically, APP depletion causes an increase of both cap‐ and IRES‐dependent translation. Since growth and proliferation are tightly coupled processes, here we asked what effects artificial downregulation of APP could have elicited in NSCLC cells proliferation. APP depletion caused a G0/G1 arrest through destabilization of the cyclin‐C protein and reduced pRb phosphorylation at residues Ser802/811. siRNA to cyclin‐C mirrored the cell cycle distribution observed when silencing APP. Cells arrested in G0/G1 (and with augmented global protein synthesis) increased their size and underwent a necrotic cell death due to cell membrane permeabilization. These phenotypes were reversed by overexpression of the APP C‐terminal domain, indicating a novel role for APP in regulating early cell cycle entry decisions. It is seems that APP moderates the rate of protein synthesis before the cell clears growth factors‐and nutrients‐dependent checkpoint in mid G1. Our results raise questions on how such processes interact in the context of (at least) dividing NSCLC cells. The data presented here suggest that APP, although required for G0/G1 transitions, moderates the rate of protein synthesis before the cell fully commits to cell cycle progression following mechanisms, which seem additional to concurrent signals deriving from the PI3‐K/Akt/mTORC‐1 axis. APP appears to play a central role in regulating cell cycle entry with the rate of protein synthesis; and its loss‐of‐function causes cell size abnormalities and death. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-11T07:01:44.896143-05:
      DOI: 10.1002/jcp.24875
       
  • A miRNA signature in human cord blood stem and progenitor cells as
           potential biomarker of specific acute myeloid leukemia subtypes
    • Authors: M. Cattaneo; E. Pelosi, G. Castelli, A.M. Cerio, A. D'Angiò, L. Porretti, P. Rebulla, L. Pavesi, G. Russo, A. Giordano, J. Turri, L. Cicconi, F. Lo‐Coco, U. Testa, I. Biunno
      Pages: n/a - n/a
      Abstract: MicroRNAs (miRNAs) are important regulators of several cellular processes. During hematopoiesis, specific expression signatures have been reported in different blood cell lineages and stages of hematopoietic stem cell (HSC) differentiation. Here we explored the expression of miRNAs in umbilical cord blood stem (HSC) and progenitor cells (HPC) and compared it to unilineage granulocyte and granulo‐monocyte differentiation as well as to primary blasts from patients with acute myeloid leukemia (AML). CD34+CD38‐ ad CD34+CD38+ cells were profiled using a global array consisting of about 2000 miRNAs. An approach combining bioinformatic prediction of miRNA targets with mRNA expression profiling was used to search for putative biologically enriched functions and networks. At least fifteen miRNAs to be differentially expressed between HSC and HPC cell population, a cluster of 7 miRNAs are located in the q32 region of human chromosome 14 (miR‐377‐3p, ‐136‐5p, 376a‐3p, 495‐3p, 654‐3p, 376c‐3p and 381‐3p) whose expression decreased during the early stages of normal myelopoiesis but were markedly increased in a small set of AML. Interestingly, miR‐4739 and ‐4516, two novel microRNA whose function and targets are presently unknown, showed specific and peculiar expression profile during the hematopoietic stem cells differentiation into unilineages and resulted strongly upregulated in almost all AML subsets. miR‐181, ‐126‐5p, ‐29b‐3p and ‐22‐3p resulted dis‐regulated in specific leukemias phenotypes. This study provides the first evidence of a miRNA signature in human cord blood stem and progenitor cells with a potential role in hematopoietic stemness properties and possibly in leukemogenesis of specific AML subtypes. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-11T07:01:28.915805-05:
      DOI: 10.1002/jcp.24876
       
  • An alternative long‐term culture system for highly‐pure mouse
           spermatogonial stem cells
    • Authors: Bao‐Rong He; Fan Lu, Lingling Zhang, Ding‐Jun Hao, Hao Yang
      Pages: n/a - n/a
      Abstract: Increasing evidence suggests that spermatogonial stem cells (SSCs) have great clinical potential to give rise to a variety of cell types besides all spermatogenic lineage cells. The development of an efficient method for long‐term culture of highly‐pure SSCs is essential for further studies related to SSC biological events. Here, we describe an in vitro culture system obtaining mouse SSC cultures of high purity, viability and proliferation. For establishing long‐term cultures of SSCs, we mainly focused on isolation procedures and culture conditions. These included co‐coating of extracellular substrates, i.e. poly‐L‐lysine (PLL) and Laminin, as well as combinatiorial use of three milder enzymes and simultaneously less trypsin to minimize enzyme‐mediated degradation of SSCs. Furthermore, a unique purification procedure was performed to effectively eliminate contaminating non‐SSCs. Finally, a critical step is to ensure SSC maintenance and expansion by utilizing optimal culture medium. Obtained data suggest that applying our optimally modified method, SSCs can be cultured for over 90 days with high purity (around 93.5%). Moreover, SSCs isolated and expanded using our protocol fulfills all criteria of SSCs without losing their stemness‐characterized by SSC‐phenotypic gene expression and long‐term self‐renewal. This study describes for the first time a protocol allowing isolation and expansion of SSCs suitable for numerous studies related to SSC‐based clinical therapies of various diseases. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-11T07:01:15.263505-05:
      DOI: 10.1002/jcp.24880
       
  • Transcription Factor MEF2C Suppresses Endothelial Cell Inflammation via
           Regulation of NF‐κB and KLF2
    • Authors: Zhenhua Xu ; Takeshi Yoshida, Lijuan Wu, Debasish Maiti, Liudmila Cebotaru, Elia J. Duh
      Abstract: Endothelial cells play a major role in the initiation and perpetuation of the inflammatory process in health and disease, including their pivotal role in leukocyte recruitment. The role of pro‐inflammatory transcription factors in this process has been well‐described, including NF‐κB. However, much less is known regarding transcription factors that play an anti‐inflammatory role in endothelial cells. Myocyte enhancer factor 2 C (MEF2C) is a transcription factor known to regulate angiogenesis in endothelial cells. Here, we report that MEF2C plays a critical function as an inhibitor of endothelial cell inflammation. Tumor necrosis factor (TNF)‐α inhibited MEF2C expression in endothelial cells. Knockdown of MEF2C in endothelial cells resulted in the upregulation of pro‐inflammatory molecules and stimulated leukocyte adhesion to endothelial cells. MEF2C knockdown also resulted in NF‐κB activation in endothelial cells. Conversely, MEF2C overexpression by adenovirus significantly repressed TNF‐α induction of pro‐inflammatory molecules, activation of NF‐κB, and leukocyte adhesion to endothelial cells. This inhibition of leukocyte adhesion by MEF2C was partially mediated by induction of KLF2. In mice, lipopolysaccharide (LPS)‐induced leukocyte adhesion to the retinal vasculature was significantly increased by endothelial cell‐specific ablation of MEF2C. Taken together, these results demonstrate that MEF2C is a novel negative regulator of inflammation in endothelial cells and may represent a therapeutic target for vascular inflammation. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-05T06:27:32.898913-05:
      DOI: 10.1002/jcp.24870
       
  • HDAC4 degradation mediates HDAC inhibition‐induced protective
           effects against hypoxia/reoxygenation injury
    • Authors: Jianfeng Du; Ling Zhang, Shougang Zhuang, Gang J Qin, Ting Zhao
      Abstract: Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression through remodeling of chromatin structures. However, the molecular mechanisms involved in this event remain unknown. In this study, we sought to examine whether HDAC inhibition‐mediated protective effects involved HDAC4 sumoylation, degradation, and the proteasome pathway. Isolated neonatal mouse ventricular myocytes (NMVM) and H9c2 cardiomyoblasts were subjected to 48 hours of hypoxia (H) (1% O2) and 2 hours of reoxygenation (R). Treatment of cardiomyocytes with trichostatin A (TSA) attenuated H/R‐elicited injury, as indicated by a reduction of lactate dehydrogenase (LDH) leakage, an increase in cell viability, and decrease in apoptotic positive cardiomyocytes. MG132, a potent proteasome pathway inhibitor, abrogated TSA‐induced protective effects, which was associated with the accumulation of ubiquitinated HDAC4. NMVM transduced with adenoviral HDAC4 led to an exaggeration of H/R‐induced injury. TSA treatment resulted in a decrease in HDAC4 in cardiomyocytes infected with adenoviral HDAC4, and HDAC4‐induced injury was attenuated by TSA. HDAC inhibition resulted in a significant reduction in reactive oxygen species (ROS) in cardiomyoblasts exposed to H/R, which was attenuated by blockade of the proteasome pathway. Cardiomyoblasts carrying wild type and sumoylation mutation (K559R) were established to examine effects of HDAC4 sumoylation and ubiquitination on H/R injury. Disruption of HDAC4 sumoylation brought about HDAC4 accumulation and impairment of HDAC4 ubiquitination in association with enhanced susceptibility of cardiomyoblasts to H/R. Taken together, these results demonstrated that HDAC inhibition stimulates proteasome dependent degradation of HDAC4, which is associated with HDAC4 sumoylation to induce these protective effects. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-05T06:27:19.785689-05:
      DOI: 10.1002/jcp.24871
       
  • Galectin‐1 triggers epithelial‐mesenchymal transition in human
           hepatocellular carcinoma cells
    • Authors: María L Bacigalupo; Malena Manzi, María V Espelt, Lucas D Gentilini, Daniel Compagno, Diego J Laderach, Carlota Wolfenstein‐Todel, Gabriel A Rabinovich, María F Troncoso
      Abstract: Galectin‐1 (Gal1), a β‐galactoside‐binding protein abundantly expressed in tumor microenvironments, is associated with the development of metastasis in hepatocellular carcinomas (HCC). However, the precise roles of Gal1 in HCC cell invasiveness and dissemination are uncertain. Here, we investigated whether Gal1 mediate epithelial‐mesenchymal transition (EMT) in HCC cells, a key process during cancer progression. We used the well‐differentiated and low invasive HepG2 cells and performed ‘gain‐of‐function’ and ‘loss‐function’ experiments by transfecting cells with Gal1 cDNA constructs or by siRNA strategies, respectively. Epithelial and mesenchymal markers expression, changes in apico‐basal polarity, independent‐anchorage growth and activation of specific signaling pathways were studied using Western blot, fluorescence microscopy, soft‐agar assays and FOP/TOP flash reporter system. Gal1 up‐regulation in HepG2 cells induced down‐regulation of the adherens junction protein E‐cadherin and increased expression of the transcription factor Snail, one of the main inducers of EMT in HCC. Enhanced Gal1 expression facilitated the transition from epithelial cell morphology towards a fibroblastoid phenotype and favored up‐regulation of the mesenchymal marker vimentin in HCC cells. Cells overexpressing Gal1 showed enhanced anchorage‐independent growth and loss of apico‐basal polarity. Remarkably, Gal1 promoted Akt activation, β‐catenin nuclear translocation, TCF4/LEF1 transcriptional activity and increased cyclin D1 and c‐Myc expression, suggesting activation of the Wnt pathway. Furthermore, Gal1 overexpression induced E‐cadherin downregulation through a PI3K/Akt‐dependent mechanism. Our results provide the first evidence of a role of Gal1 as an inducer of EMT in HCC cells, with critical implications in HCC metastasis. This article is protected by copyright. All rights reserved
      PubDate: 2014-12-03T06:06:48.347576-05:
      DOI: 10.1002/jcp.24865
       
  • The Role of Notch Receptors in Transcriptional Regulation
    • Authors: Hongfang Wang; Chongzhi Zang, X. Shirley Liu, Jon C. Aster
      Abstract: Notch signaling has pleiotropic context‐specific functions that have essential roles in many processes, including embryonic development and maintenance and homeostasis of adult tissues. Aberrant Notch signaling (both hyper‐ and hypoactive) is implicated in a number of human developmental disorders and many cancers. Notch receptor signaling is mediated by tightly regulated proteolytic cleavages that lead to the assembly of a nuclear Notch transcription complex, which drives the expression of downstream target genes and thereby executes Notch's functions. Thus, understanding regulation of gene expression by Notch is central to deciphering how Notch carries out its many activities. Here, we summarize the recent findings pertaining to the complex interplay between the Notch transcriptional complex and interacting factors involved in transcriptional regulation, including co‐activators, cooperating transcription factors, and chromatin regulators, and discuss emerging data pertaining to the role of Notch‐regulated noncoding RNAs in transcription. This article is protected by copyright. All rights reserved
      PubDate: 2014-11-22T16:29:29.313594-05:
      DOI: 10.1002/jcp.24872
       
  • Editor's Choice
    • PubDate: 2014-11-21T13:45:18.329913-05:
      DOI: 10.1002/jcp.24855
       
  • Journal of Cellular Physiology: Volume 230, Number 3, March 2015
    • Abstract: Cover: Mechanisms of lncRNA function. See article by Tye et al. on pages 526–534.
      PubDate: 2014-11-21T13:45:13.746834-05:
      DOI: 10.1002/jcp.24744
       
  • Highlights: Volume 230, Number 3
    • PubDate: 2014-11-21T13:45:13.29471-05:0
      DOI: 10.1002/jcp.24746
       
  • Table of Contents: Volume 230, Number 3
    • PubDate: 2014-11-21T13:45:06.603566-05:
      DOI: 10.1002/jcp.24745
       
  • Anti‐inflammatory/tissue repair macrophages enhance the
           cartilage‐forming capacity of human bone marrow‐derived
           mesenchymal stromal cells
    • Authors: Sergio B. Sesia; Ralph Duhr, Carolina Medeiros da Cunha, Atanas Todorov, Stefan Schaeren, Elisabetta Padovan, Giulio Spagnoli, Ivan Martin, Andrea Barbero
      Abstract: Macrophages are key players in healing processes. However little is known on their capacity to modulate the differentiation potential of mesenchymal stem/stromal cells (MSC). Here we investigated whether macrophages (Mf) with, respectively, pro‐inflammatory and tissue‐remodeling traits differentially modulate chondrogenesis of bone marrow derived‐MSC (BM‐MSC). We demonstrated that coculture in collagen scaffolds of BM‐MSC with Mf derived from monocytes polarized with M‐CSF (M‐Mf), but not with GM‐CSF (GM‐Mf) resulted in significantly higher glycosaminoglycan (GAG) content than what would be expected from an equal number of BM‐MSC alone (defined as chondro‐induction). Moreover, type II collagen was expressed at significantly higher levels in BM‐MSC/M‐Mf as compared to BM‐MSC/GM‐Mf constructs, while type X collagen expression was unaffected. In order to understand the possible cellular mechanism accounting for chondro‐induction, developing monoculture and coculture tissues were digested and the properties of the isolated BM‐MSC analysed. We observed that as compared to monocultures, in coculture with M‐Mf, BM‐MSC decreased less markedly in number and exhibited higher clonogenic and chondrogenic capacity. Despite their chondro‐inductive effect in vitro, M‐Mf did not modulate the cartilage tissue maturation in subcutaneous pockets of nude mice, as evidenced by similar accumulation of type X collagen and calcified tissue. Our results demonstrate that coculture of BM‐MSC with M‐Mf results in synergistic cartilage tissue formation in vitro. Such effect seems to result from the survival of BM‐MSC with high chondrogenic capacity. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of cartilage repair. This article is protected by copyright. All rights reserved
      PubDate: 2014-11-21T03:18:14.664235-05:
      DOI: 10.1002/jcp.24861
       
  • In type 2 diabetes mellitus glycated albumin alters macrophage gene
           expression impairing ABCA1‐mediated cholesterol efflux
    • Authors: Adriana Machado‐Lima; Rodrigo T Iborra, Raphael S Pinto, Gabriela Castilho, Camila H Sartori, Erika R Oliveira, Edna R Nakandakare, Daniel Giannella‐Neto, Maria Lucia C Corrêa‐Giannella, Pietro Traldi, Simona Porcu, Marco Roverso, Annunziata Lapolla, Marisa Passarelli
      Abstract: Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE‐albumin induces oxidative stress, which is linked to a reduction in ABCA‐1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2‐HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C‐ or DM2‐HSA to measure the 14C‐cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA‐1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real‐time RT‐PCR. An increased mean mass was observed in DM2‐HSA compared with C‐HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3, and HDL2 was impaired in DM2‐HSA‐treated cells, which was related to greater intracellular lipid accumulation. DM2‐HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA‐1 protein content decreased. Compared with C‐HAS‐treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2‐HSA treatment. Stearoyl‐Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2‐HSA. The level of glycation that occurs in vivo in DM2‐HSA‐treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2. This article is protected by copyright. All rights reserved
      PubDate: 2014-11-21T03:17:20.807212-05:
      DOI: 10.1002/jcp.24860
       
  • High levels of GPR30 protein in human testicular carcinoma in situ and
           seminomas correlate with low levels of estrogen receptor‐beta and
           indicate a switch in estrogen responsiveness
    • Authors: Francesca Boscia; Carmela Passaro, Vincenzo Gigantino, Sisto Perdonà, Renato Franco, Giuseppe Portella, Sergio Chieffi, Paolo Chieffi
      Abstract: The G protein‐coupled estrogen receptor (GPR30) is suggested to be involved in non‐nuclear estrogen signalling and is expressed in a variety of hormone dependent cancer entities. It is well established that oestrogens are involved in pathological germ cell proliferation including testicular germ cell tumours. This study was performed to further elucidate the role of this receptor and the possible correlation with the estrogen receptor β in human testicular carcinoma in situ (CIS), seminomas and in GC1 and TCam‐2 germ cell lines; in addition, a Tissue Micro‐Array was built using the most representative areas from 25 cases of human testicular seminomas and 20 cases of CIS. The expression of ERβ and GPR30 were observed by using Western blot analysis in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that down regulation of ERβ associates with GPR30 over‐expression both in human testicular CIS and seminomas. In addition, we show that 17β‐oestradiol induces the ERK1/2 activation and increases c‐Fos expression through GPR30 associated with ERβ down‐regulation in TCam‐2 cell line. The present results suggest that exposure to oestrogens or oestrogen‐mimics, in some as of yet undefined manner, diminishes the ERβ‐mediated growth restraint in CIS and in human testicular seminoma, probably due to ERβ down‐regulation associated to GPR30 increased expression indicating that GPR30 could be a potential therapeutic target to design specific inhibitors. This article is protected by copyright. All rights reserved
      PubDate: 2014-11-21T03:14:29.54656-05:0
      DOI: 10.1002/jcp.24864
       
  • Insights Into RNA Transcriptome Profiling of Cardiac Tissue in Obesity and
           Hypertension Conditions
    • Authors: Alzenira Costa; Octavio Luiz Franco
      Pages: n/a - n/a
      Abstract: Several epidemiological studies suggest that obesity and hypertension are associated with cardiac transcriptome modifications that could be further associated with inflammatory processes and cardiac hypertrophy. In this field, transcriptome studies have demonstrated their importance to elucidate physiological mechanisms, pathways or genes involved in many biological processes. Over the past decade, RNA microarray and RNA‐seq analysis has become an essential component to examine metabolic pathways in terms of mRNA expression in cardiology. In this review, cardiac muscle gene expression in response to effects of obesity and hypertension will be focused, providing a broad view on cardiac transcriptome and physiological and biochemical mechanisms involved in gene expression changes produced by these events, emphasizing the use of new technologies for gene expression analyses. This article is protected by copyright. All rights reserved
      PubDate: 2014-11-12T18:27:22.005069-05:
      DOI: 10.1002/jcp.24807
       
  • The synergistic effect of SAHA and Parthenolide in MDA‐MB231 breast
           cancer cells
    • Authors: Daniela Carlisi; Marianna Lauricella, Antonella D'Anneo, Giuseppina Buttitta, Sonia Emanuele, Riccardo di Fiore, Roberta Martinez, Christian Rolfo, Renza Vento, Giovanni Tesoriere
      Abstract: The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA‐MB231 cells, a triple‐negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA‐MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down‐regulation of DNMT1 expression induced by PN. Inhibition of the DNA‐binding activity of NF‐kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-11-05T05:37:12.554035-05:
      DOI: 10.1002/jcp.24863
       
  • A tissue specific magnetic resonance contrast agent, Gd‐AMH, for
           diagnosis of stromal endometriosis lesions: A phase I study
    • Authors: Pietro G. Signorile; Alfonso Baldi
      Abstract: The anti‐mullerian hormone (AMH) is a homodimeric glycoprotein member of the transforming growth factor ƒÀ (TGF‐ƒÀ) superfamily, and is secreted by Sertoli cells in the embryonic testes and is responsible of the regression of the mullerian duct. The physiological functions of this protein remain largely unknown, and its expression in human tissues has yet to be completely determined. [objective]. Firstly, we analyzed AMH expression in human tissues by immunohistochemistry. It was found that AMH was distributed in many organs, although with different tissue and cell localization and various expression levels; we also demonstrated strong AMH expression in endometriosis tissues. Secondly, we demonstrated the ability of an anti‐AMH antibody, labelled with gadiolinium, to be directly detected by magnetic resonance in small endometriosis lesions (5 mm in diameter) in vivo in a mouse model. In conclusion, our data suggest that based on its expression pattern, AMH may serve to maintain physiological cellular homeostasis in different human tissues and organs. Moreover, it is strongly expressed in endometriosis lesions as a selective tissue specific contrast agent for in vivo detection of stromal endometriosis lesions. The potential significance of these findings could be further validated in a clinical setting. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-11-05T05:34:54.340738-05:
      DOI: 10.1002/jcp.24862
       
  • Pigment epithelium derived factor suppresses expression of Sost/sclerostin
           by osteocytes: Implication for its role in bone matrix mineralization
    • Authors: Feng Li; Na Song, Joyce Tombran‐Tink, Christopher Niyibizi
      Abstract: Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. Mechanisms by which PEDF regulates matrix mineralization remain unknown. We examined effect of exogenous PEDF on expression of osteoblastic and osteocytic related genes and proteins in mineralizing osteoblast culture. Mineralizing human osteoblasts supplemented with exogenous PEDF for 14 days deposited 47% more mineral than cells cultured without PEDF. Analysis of selected gene expression by cells in mineralizing cultures supplemented with exogenous PEDF showed reduction in expression of Sclerostin (Sost) by 70%, matrix extracellular phosphoglycoprotein (MEPE) by 75% and dentin matrix protein (DMP‐1) by 20% at day 14 of culture. Phosphate‐regulating gene with homologies to endopeptidases on the X chromosome (PHEX) expression was not affected. Western blotting and immunoprecipitation showed that sclerostin and MEPE synthesis by osteocytes were reduced by 50% and 60% respectively in mineralizing osteoblasts containing exogenous PEDF. Primary osteocytes exposed to PEDF also reduced synthesis of Sost/sclerostin by 50% within 24h. For osteoblastic genes, Bone sialoprotein (BSP) was expressed at 75% higher by day 7 in cultures containing exogenous PEDF while Col1A1 expression remained high at all‐time points. Total beta‐catenin was increased in mineralizing osteoblastic cells suggesting increased Wnt activity. Taken together, the data indicate that PEDF suppressed expression of factors that inhibit mineralization while enhancing those that promote mineralization. The findings also suggest that PEDF may regulate Sost expression by osteocytes leading to enhanced osteoblastic differentiation and increased matrix mineralization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-11-03T00:41:21.765712-05:
      DOI: 10.1002/jcp.24859
       
  • Protein Kinase C Inhibitor, GF109203X Attenuates Osteoclastogenesis, Bone
           Resorption and RANKL‐induced NF‐κB and NFAT activity
    • Authors: Jun Yao; Jia Li, Lin Zhou, Jianwen Cheng, Shek Man Chim, Ge Zhang, Julian M.W. Quinn, Jennifer Tickner, Jinmin Zhao, Jiake Xu
      Abstract: Osteolytic bone diseases are characterized by excessive osteoclast formation and activation. Protein kinase C (PKC)‐dependent pathways regulate cell growth, differentiation and apoptosis in many cellular systems, and have been implicated in cancer development and osteoclast formation. A number of PKC inhibitors with anti‐cancer properties have been developed, but whether they might also influence osteolysis (a common complication of bone invading cancers) is unclear. We studied the effects of the PKC inhibitor compound, GF109203X on osteoclast formation and activity, processes driven by receptor activator of NFκB ligand (RANKL). We found that GF109203X strongly and dose dependently suppresses osteoclastogenesis and osteoclast activity in RANKL‐treated primary mouse bone marrow cells. Consistent with this GF109203X reduced expression of key osteoclastic genes, including cathepsin K, calcitonin receptor, tartrate resistant acid phosphatase (TRAP) and the proton pump subunit V‐ATPase‐d2 in RANKL‐treated primary mouse bone marrow cells. Expression of these proteins is dependent upon RANKL‐induced NF‐κB and NFAT transcription factor actions; both were reduced in osteoclast progenitor populations by GF109203X treatment, notably NFATc1 levels. Furthermore, we showed that GF109203X inhibits RANKL‐induced calcium oscillation. Together, this study shows GF109203X may block osteoclast functions, suggesting that pharmacological blockade of PKC‐dependent pathways has therapeutic potential in osteolytic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-11-03T00:41:06.579636-05:
      DOI: 10.1002/jcp.24858
       
  • Glucose concentration and streptomycin alter in vitro muscle function and
           metabolism
    • Authors: Alastair Khodabukus; Keith Baar
      Abstract: Cell culture conditions can vary between laboratories and have been optimised for 2D cell culture. In this study, engineered muscle was cultured in 5.5 mM low glucose (LG) or 25 mM high glucose (HG) and in the absence or presence (+S) of streptomycin and the effect on C2C12 tissue‐engineered muscle function and metabolism was determined. Following 2 weeks differentiation, streptomycin (3‐fold) and LG (0.5‐fold) significantly decreased force generation. LG and/or streptomycin resulted in upward and leftward shifts in the force‐frequency curve and slowed time‐to‐peak tension and half‐relaxation time. Despite changes in contractile dynamics, no change in myosin isoform was detected. Instead, changes in troponin isoform, calcium sequestering proteins (CSQ and parvalbumin) and the calcium uptake protein SERCA predicted the changes in contractile dynamics. Culturing in LG and/or streptomycin resulted in increased fatigue resistance despite no change in the mitochondrial enzymes SDH, ATPsynthase and cytochrome C. However, LG resulted in increases in the β‐oxidation enzymes LCAD and VLCAD and the fatty acid transporter CPT‐1, indicative of a greater capacity for fat oxidation. In contrast, HG resulted in increased GLUT4 content and the glycolytic enzyme PFK, indicative of a more glycolytic phenotype. These data suggest that streptomycin has negative effects on force generation and that glucose can be used to shift engineered muscle phenotype via changes in calcium‐handling and metabolic proteins. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-30T15:18:36.324637-05:
      DOI: 10.1002/jcp.24857
       
  • TGFβ3 Regulates Periderm Removal through ΔNp63 in the Developing
           Palate
    • Authors: Lihua Hu; Jingpeng Liu, Zhi Li, Ferhat Ozturk, Channabasavaiah Gurumurthy, Rose‐Anne Romano, Satrajit Sinha, Ali Nawshad
      Abstract: The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor β3 (TGFβ3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (‐/‐) TGFβ3, interferon regulatory factor 6 (IRF6) (‐/‐), and truncated p63 (ΔNp63) (‐/‐) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFβ3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFβ3 (‐/‐), ΔNp63 (‐/‐), and wild‐type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFβ3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFβ3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFβ3 (‐/‐) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (‐/‐) palates because of premature fusion. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-30T15:14:43.751759-05:
      DOI: 10.1002/jcp.24856
       
  • 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
       
  • 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
       
  • 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
       
  • LncRNAs: New Players in Gliomas, With Special Emphasis on the Interaction
           of lncRNAs With EZH2
    • Authors: Er‐Bao Bian; Jia Li, Yong‐Sheng Xie, Gang Zong, Jun Li, Bing Zhao
      First page: 496
      Abstract: Gliomas are the most common primary malignancy in the brain, accounting for 50–60%. Despite all the efforts of cytoreductive surgery in combination with intense chemoradiotherapy, glioma remains an incurable disease. Recent studies have shown that long noncoding RNAs (lncRNAs) are involved in the pathology of gliomas. LncRNAs are involved in many cellular processes, such as angiogenesis, invasion, cell proliferation, and apoptosis. In this review we focus on the dysregulation of lncRNAs in gliomas. We also address that epigenetic modification such as DNA methylation and microRNAs interact with lncRNAs in gliomas. In addition, the interaction of lncRNAs with signaling pathways in gliomas is discussed systematically, with particular emphasis on the interaction of lncRNAs with EZH2. Such approaches provide valuable insights into the potential future applications of lncRNAs in the treatment of gliomas. J. Cell. Physiol. 230: 496–503, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:15.210125-05:
      DOI: 10.1002/jcp.24549
       
  • p27 and Leukemia: Cell cycle and beyond
    • Authors: Anita Roy; Subrata Banerjee
      First page: 504
      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
       
  • The dysfunction of the trabecular meshwork during glaucoma course
    • Authors: Sergio Claudio Saccà; Alessandra Pulliero, Alberto Izzotti
      First page: 510
      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
       
  • 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
      First page: 526
      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
       
  • MicroRNA‐195 Chemosensitizes Colon Cancer Cells to the
           Chemotherapeutic Drug Doxorubicin by Targeting the First Binding Site of
           BCL2L2 mRNA
    • Authors: Juan Qu; Liang Zhao, Pengzhi Zhang, Juan Wang, Ning Xu, Wenjuan Mi, Xingwang Jiang, Changming Zhang, Juan Qu
      First page: 535
      Abstract: The mechanisms underlying doxorubicin (Dox) resistance in colon cancer cells are not fully understood. MicroRNA (miRNA) play important roles in tumorigenesis and drug resistance. However, the relationship between miRNA and Dox resistance in colon cancer cells has not been previously explored. In this study, we utilized microRNA array and real‐time PCR to verify that miR‐127, miR‐195, miR‐22, miR‐137 were significantly down‐regulated, while miR‐21, miR‐592 were up‐regulated in both HT29/DOX and LOVO/DOX cell lines. In vitro cell viability assay showed that knockdown of miR‐195 in HT29 and LOVO cells caused a marked inhibition of Dox‐induced cytotoxicity. Moreover, we explored that miR‐195 is involved in repression of BCL2L2 expression through targeting its 3′‐untranslated region, especially the first binding site within its mRNA. Furthermore, down‐regulation of miR‐195 conferred DOX resistance in parental cells and reduced cell apoptosis activity, while over‐expression of miR‐195 sensitized resistant cells to DOX and enhanced cell apoptosis activity, all of which can be partly rescued by BCL2L2 siRNA and cDNA expression. These results may have implications for therapeutic strategies aiming to overcome colon cancer cell resistance to Dox. J. Cell. Physiol. 230: 535–545, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:08.670001-05:
      DOI: 10.1002/jcp.24366
       
  • Fibronectin Peptides as Potential Regulators of Hepatic Fibrosis Through
           Apoptosis of Hepatic Stellate Cells
    • Authors: Teresa Mòdol; Natalia Brice, Marina Ruiz de Galarreta, Antonia García Garzón, María J. Iraburu, Juan J. Martínez‐Irujo, María J. López‐Zabalza
      First page: 546
      Abstract: The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX‐2 based on oligonucleosomal DNA fragmentation, caspase‐3 and ‐9 activation, Bcl‐2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease‐9 (MMP‐9) expression in human monocytes. J. Cell. Physiol. 230: 546–553, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:15.889854-05:
      DOI: 10.1002/jcp.24714
       
  • LPS Promote the Odontoblastic Differentiation of Human Dental Pulp Stem
           Cells via MAPK Signaling Pathway
    • Authors: Wenxi He; Zhihua Wang, Zhirong Luo, Qing Yu, Yong Jiang, Yaqing Zhang, Zeyuan Zhou, Anthony J. Smith, Paul R. Cooper
      First page: 554
      Abstract: Human dental pulp stem cells (hDPSCs) show significant potential for exploitation in novel regeneration strategies, although lack of understanding of their responses to bacterial challenge constrains their application. The present study aimed to investigate whether lipopolysaccharide (LPS), the major pathogenic factor of Gram‐negative bacteria, regulates the differentiation of hDPSCs and which intracellular signaling pathways may be involved. LPS treatment significantly promoted the differentiation of hDPSCs demonstrable by increased mineralized nodule formation and mRNA expression of several odontoblastic markers in a dose‐dependent manner. While inhibition of TLR4, p38, and ERK signaling markedly antagonized LPS‐mediated differentiation of hDPSCs. The inhibition of JNK and NF‐κB signaling had no detectable effect on LPS activation of hDPSCs. LPS stimulation resulted in phosphorylation of NF‐κB p65, IκB‐α, extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (MAPK) in DPSCs in a time‐dependent manner, which was markedly suppressed by their specific inhibitors, respectively. Data demonstrated that LPS promoted odontoblastic differentiation of hDPSCs via TLR4, ERK, and P38 MAPK signaling pathways, but not NF‐κB signaling. J. Cell. Physiol. 230: 554–561, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:11.7046-05:00
      DOI: 10.1002/jcp.24732
       
  • Detection of Phosphorylated Insulin Receptor in Colorectal Adenoma and
           Adenocarcinoma: Implications for Prognosis and Clinical Outcome
    • Authors: Claudia Abbruzzese; Maria Grazia Diodoro, Isabella Sperduti, Anna Maria Mileo, Giada Pattaro, Laura De Salvo, Maurizio Cosimelli, Nicola Perrotti, Marco G. Paggi
      First page: 562
      Abstract: Colorectal carcinoma remains among the most frequent causes of cancer death. Besides the well‐known genetic predisposition, a key role in colorectal adenoma and adenocarcinoma etio‐pathogenesis, mainly in sporadic cases, is played by definite risk factors, such as obesity, type 2 diabetes, insulin resistance, hyper‐insulinemia, and insulin therapy. These epidemiological data motivated us to determine, by means of immunohistochemistry, the amount of activated (phosphorylated) insulin receptor in archival samples from 22 colorectal adenoma and 117 adenocarcinoma patients, with the objective to estimate the role of this factor in colorectal epithelium transformation and cancer progression. Statistical analysis of the results clearly showed that positive staining for phosphorylated insulin receptor was significantly more frequent in adenomas than adenocarcinomas (P 
      PubDate: 2014-11-21T13:45:13.866926-05:
      DOI: 10.1002/jcp.24733
       
  • Blocking the Expression of Both Bone Sialoprotein (BSP) and Osteopontin
           (OPN) Impairs the Anabolic Action of PTH in Mouse Calvaria Bone
    • Authors: Wafa Bouleftour; Guenaelle Bouet, Renata Neves Granito, Mireille Thomas, Marie‐Thérèse Linossier, Arnaud Vanden‐Bossche, Jane E. Aubin, Marie‐hélène Lafage‐Proust, Laurence Vico, Luc Malaval
      First page: 568
      Abstract: Osteopontin (OPN) and bone sialoprotein (BSP) are coexpressed in osteoblasts and osteoclasts, and display overlapping properties. We used daily injection of parathyroid hormone 1–84 (iPTH) over the calvaria of BSP knockout (−/−) mice to investigate further their functional specificity and redundancy. iPTH stimulated bone formation in both +/+ and −/− mice, increasing to the same degree periosteum, osteoid and total bone thickness. Expression of OPN, osterix, osteocalcin (OCN) and DMP1 was also increased by iPTH in both genotypes. In contrast to +/+, calvaria cell cultures from −/− mice revealed few osteoblast colonies, no mineralization and little expression of OCN, MEPE or DMP1. In contrast, OPN levels were 5× higher in −/− versus +/+ cultures. iPTH increased alkaline phosphatase (ALP) activity in cell cultures of both genotypes, with higher OCN and the induction of mineralization in −/− cultures. siRNA blocking of OPN expression did not alter the anabolic action of the hormone in BSP +/+ calvaria, while it blunted iPTH effects in −/− mice, reduced to a modest increase in periosteum thickness. In −/− (not +/+) cell cultures, siOPN blocked the stimulation by iPTH of ALP activity and OCN expression, as well as the induction of mineralization. Thus, full expression of either OPN or BSP is necessary for the anabolic effect of PTH at least in the ectopic calvaria injection model. This suggests that OPN may compensate for the lack of BSP in the response to this hormonal challenge, and provides evidence of functional overlap between these cognate proteins. J. Cell. Physiol. 230: 568–577, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:12.41704-05:0
      DOI: 10.1002/jcp.24772
       
  • Signaling Pathways Involved in Megakaryocyte‐Mediated Proliferation
           of Osteoblast Lineage Cells
    • Authors: Ying‐Hua Cheng; Drew A. Streicher, David L. Waning, Brahmananda R. Chitteti, Rita Gerard‐O'Riley, Mark C. Horowitz, Joseph P. Bidwell, Fredrick M. Pavalko, Edward F. Srour, Lindsey D. Mayo, Melissa A. Kacena
      First page: 578
      Abstract: Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Lennert et al., 1975; Thiele et al., 1999; Chagraoui et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell–cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb‐mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK‐to‐OB signaling ultimately results in significant increases in the expression of c‐fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin‐mediated signaling mechanism, activating a novel response axis that de‐represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone‐related diseases. J. Cell. Physiol. 230: 578–586, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:09.098784-05:
      DOI: 10.1002/jcp.24774
       
  • PLCβ1a and PLCβ1b Selective Regulation and Cyclin D3 Modulation
           Reduced by Kinamycin F During K562 Cell Differentiation
    • Authors: Alberto Bavelloni; Gary I. Dmitrienko, Valerie J. Goodfellow, Ahmad Ghavami, Manuela Piazzi, William Blalock, Francesca Chiarini, Lucio Cocco, Irene Faenza
      First page: 587
      Abstract: Here we report that both PLCβ1a and PLCβ1b are relevant regulators of erythropoiesis in that kinamycin F, a potent inducer of γ‐globin production in K562 cells, caused a selectively reduction of both PLCβ1 isozymes even though the results point out that the effect of the drug is mainly directed toward the expression of the PLCβ1a isoform. We have identified a different role for the two isozymes as regulators of K562 differentiation process induced by kinamycin F. The overexpression of PLCβ1b induced an increase in γ‐globin expression even in the absence of kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCβ1 signaling pathway. Namely the amplification of the expression of the PLCβ1a, but not of PLCβ1b, is able to maintain high levels of expression of cyclin D3 even after treatment with kinamycin F. This could be due to their different distribution in the cell compartments since the amount of PLCβ1b is mainly present in the nucleus in respect to PLCβ1a. Our data indicate that the amplification of PLCβ1a expression, following treatment with kinamycin F, confers a real advantage to K562 cells viability and protects cells themselves from apoptosis. J. Cell. Physiol. 230: 587–594, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:09.558987-05:
      DOI: 10.1002/jcp.24776
       
  • Modulation of Mast Cell Proliferative and Inflammatory Responses by
           Leukotriene D4 and Stem Cell Factor Signaling Interactions
    • Authors: Nosayba Al‐Azzam; Vinay Kondeti, Ernest Duah, Farai Gombedza, Charles K. Thodeti, Sailaja Paruchuri
      First page: 595
      Abstract: Mast cells (MCs) are important effector cells in asthma and pulmonary inflammation, and their proliferation and maturation is maintained by stem cell factor (SCF) via its receptor, c‐Kit. Cysteinyl leukotrienes (cys‐LTs) are potent inflammatory mediators that signal through CysLT1R and CysLT2R located on the MC surface, and they enhance MC inflammatory responses. However, it is not known if SCF and cys‐LTs cross‐talk and influence MC hyperplasia and activation in inflammation. Here, we report the concerted effort of the growth factor SCF and the inflammatory mediator LTD4 in MC activation. Stimulation of MCs by LTD4 in the presence of SCF enhances c‐Kit‐mediated proliferative responses. Similarly, SCF synergistically enhances LTD4‐induced calcium, c‐fos expression and phosphorylation, as well as MIP1β generation in MCs. These findings suggest that integration of SCF and LTD4 signals may contribute to MC hyperplasia and hyper‐reactivity during airway hyper‐response and inflammation. J. Cell. Physiol. 230: 595–602, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:09.934342-05:
      DOI: 10.1002/jcp.24777
       
  • Inhibition of Metalloproteinase Activity in FANCA Is Linked to Altered
           Oxygen Metabolism
    • Authors: Silvia Ravera; Cristina Capanni, Danika Tognotti, Roberta Bottega, Marta Columbaro, Carlo Dufour, Enrico Cappelli, Paolo Degan
      First page: 603
      Abstract: Bone marrow (BM) failure, increased risk of myelodysplastic syndrome, acute leukaemia and solid tumors, endocrinopathies and congenital abnormalities are the major clinical problems in Fanconi anemia patients (FA). Chromosome instability and DNA repair defects are the cellular characteristics used for the clinical diagnosis. However, these biological defects are not sufficient to explain all the clinical phenotype of FA patients. The known defects are structural alteration in cell cytoskeleton, altered structural organization for intermediate filaments, nuclear lamina, and mitochondria. These are associated with different expression and/or maturation of the structural proteins vimentin, mitofilin, and lamin A/C suggesting the involvement of metalloproteinases (MPs). Matrix metalloproteinases (MMP) are involved in normal physiological processes such as human skeletal tissue development, maturation, and hematopoietic reconstitution after bone marrow suppression. Current observations upon the eventual role of MPs in FA cells are largely inconclusive. We evaluated the overall MPs activity in FA complementation group A (FANCA) cells by exposing them to the antioxidants N‐acetyl cysteine (NAC) and resveratrol (RV). This work supports the hypothesis that treatment of Fanconi patients with antioxidants may be important in FA therapy. J. Cell. Physiol. 230: 603–609, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:17.94399-05:0
      DOI: 10.1002/jcp.24778
       
  • The Differential Palmitoylation States of N‐Ras and H‐Ras
           Determine Their Distinct Golgi Subcompartment Localizations
    • Authors: Stephen J. Lynch; Harriet Snitkin, Iwona Gumper, Mark R. Philips, David Sabatini, Angel Pellicer
      First page: 610
      Abstract: Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C‐terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H‐Ras and N‐Ras) localize on the Golgi apparatus whereas K‐Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub‐organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live‐cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H‐Ras is distributed throughout the Golgi stacks, the singly palmitoylated N‐Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants, we show that the different sub‐Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane, but also their distribution within the Golgi stacks. J. Cell. Physiol. 230: 610–619, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:10.700845-05:
      DOI: 10.1002/jcp.24779
       
  • Thymoquinone Restores Radiation‐Induced TGF‐β Expression
           and Abrogates EMT in Chemoradiotherapy of Breast Cancer Cells
    • Authors: Shashi Rajput; B. N. Prashanth Kumar, Payel Banik, Sheetal Parida, Mahitosh Mandal
      First page: 620
      Abstract: Radiotherapy remains a prime approach to adjuvant therapies in patients with early and advanced breast cancer. In spite of therapeutic success, metastatic progression in patients undergoing therapy, limits its application. However, effective therapeutic strategies to understand the cellular and molecular machinery in inhibiting radiation‐induced metastatic progression, which is poorly understood so far, need to be strengthened. Ionizing radiation was known to prompt cancer cell's metastatic ability by eliciting Transforming Growth Factor‐beta (TGF‐β), a key regulator in epithelial–mesenchymal transdifferentiation and radio‐resistance. In this viewpoint, we employed thymoquinone as a radiosensitizer to investigate its migration and invasion reversal abilities in irradiated breast cancer cell lines by assessing their respective attributes. The role of metastasis regulatory molecules like TGF‐β, E‐cadherin, and integrin αV and its downstream molecules were determined using RT‐PCR, western blotting, immunofluorescence, and extracellular TGF‐β levels affirmed through ELISA assays. These studies affirmed the TGF‐β restoring ability of thymoquinone in radiation‐driven migration and invasion. Also, results demonstrated that the epithelial markers E‐cadherin and cytokeratin 19 were downregulated whereas mesenchymal markers like integrin αV, MMP9, and MMP2 were upregulated by irradiation treatment; however thymoquinone pre‐sensitization has reverted the expression of these proteins back to control proteins expression. Here, paclitaxel was chosen as an apoptosis inducer in TGF‐β restored cells and confirmed its cytotoxic effects in radiation alone and thymoquinone sensitized irradiated cells. We conclude that this therapeutic modality is effective in preventing radiation‐induced epithelial–mesenchymal transdifferentiation and concomitant induction of apoptosis in breast cancer. J. Cell. Physiol. 230: 620–629, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:14.707242-05:
      DOI: 10.1002/jcp.24780
       
  • Palmitate Induces ER Stress and Autophagy in H9c2 Cells: Implications for
           Apoptosis and Adiponectin Resistance
    • Authors: Min Park; Anna Sabetski, Yee Kwan Chan, Subat Turdi, Gary Sweeney
      First page: 630
      Abstract: The association between obesity and heart failure is well documented and recent studies have indicated that understanding the physiological role of autophagy will be of great significance. Cardiomyocyte apoptosis is one component of cardiac remodeling which leads to heart failure and in this study we used palmitate‐treated H9c2 cells as an in vitro model of lipotoxicity to investigate the role of autophagy in cell death. Temporal analysis revealed that palmitate (100 μM) treatment induced a gradual increase of intracellular lipid accumulation as well as apoptotic cell death. Palmitate induced autophagic flux, determined via increased LC3‐II formation and p62 degradation as well as by detecting reduced colocalization of GFP with RFP in cells overexpressing tandem fluorescent GFP/RFP‐LC3. The increased level of autophagy indicated by these measures were confirmed using transmission electron microscopy (TEM). Upon inhibiting autophagy using bafilomycin we observed an increased level of palmitate‐induced cell death assessed by Annexin V/PI staining, detection of active caspase‐3 and MTT cell viability assay. Interestingly, using TEM and p‐PERK or p‐eIF2α detection we observed increased endoplasmic reticulum (ER) stress in response to palmitate. Autophagy was induced as an adaptive response against ER stress since it was sensitive to ER stress inhibition. Palmitate‐induced ER stress also induced adiponectin resistance, assessed via AMPK phosphorylation, via reducing APPL1 expression. This effect was independent of palmitate‐induced autophagy. In summary, our data indicate that palmitate induces autophagy subsequent to ER stress and that this confers a prosurvival effect against lipotoxicity‐induced cell death. Palmitate‐induced ER stress also led to adiponecin resistance. J. Cell. Physiol. 230: 630–639, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:11.154765-05:
      DOI: 10.1002/jcp.24781
       
  • Pin1 Plays a Critical Role as a Molecular Switch in Canonical BMP
           Signaling
    • Authors: Won‐Joon Yoon; Rabia Islam, Young‐Dan Cho, Kyung‐Min Ryu, Hye‐Rim Shin, Kyung‐Mi Woo, Jeong‐Hwa Baek, Hyun‐Mo Ryoo
      First page: 640
      Abstract: Pin1 is a peptidyl prolyl cis‐trans isomerase that specifically binds to the phosphoserine–proline or phosphothreonine–proline motifs of numerous proteins. Previously, we reported that Pin1 deficiency resulted in defects in osteoblast differentiation during early bone development. In this study, we found that adult Pin1‐deficient mice developed osteoporotic phenotypes compared to age‐matched controls. Since BMP2 stored in the bone matrix plays a critical role in adult bone maintenance, we suspected that BMP R‐Smads (Smad1 and Smad5) could be critical targets for Pin1 action. Pin1 specifically binds to the phosphorylated linker region of Smad1, which leads to structural modification and stabilization of the Smad1 protein. In this process, Pin1‐mediated conformational modification of Smad1 directly suppresses the Smurf1 interaction with Smad1, thereby promoting sustained activation of the Smad1 molecule. Our data demonstrate that post‐phosphorylational prolyl isomerization of Smad1 is a converging signal to stabilize the Smad1 molecule against the ubiquitination process mediated by Smurf1. Therefore, Pin1 is a critical molecular switch in the determination of Smad1 fate, opposing the death signal transmitted to the Smad1 linker region by phosphorylation cascades after its nuclear localization and transcriptional activation. Thus, Pin1 could be developed as a major therapeutic target in many skeletal diseases. J. Cell. Physiol. 230: 640–647, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:16.289564-05:
      DOI: 10.1002/jcp.24787
       
  • Methotrexate‐Induced Bone Marrow Adiposity Is Mitigated by Folinic
           Acid Supplementation Through the Regulation of Wnt/β‐Catenin
           Signalling
    • Authors: Kristen R. Georgiou; Rethi Raghu Nadhanan, Chia‐Ming Fan, Cory J. Xian
      First page: 648
      Abstract: Antimetabolite Methotrexate (MTX) is commonly used in childhood oncology. As a dihydrofolate reductase inhibitor it exerts its action through the reduction of cellular folate, thus its intensive use is associated with damage to soft tissues, bone marrow, and bone. In the clinic, MTX is administered with folinic acid (FA) supplementation to alleviate some of this soft tissue damage. However, whether and how FA alleviates damage to the bone and bone marrow requires further investigation. As the Wnt/β‐catenin signalling pathway is critical for commitment and differentiation of mesenchymal stem cells down the osteogenic or adipogenic lineage, its deregulation has been found associated with increased marrow adiposity following MTX treatment. In order to elucidate whether FA supplementation prevents MTX‐induced bone marrow adiposity by regulating Wnt/β‐catenin signalling, young rats were given saline or 0.75 mg/kg MTX once daily for 5 days, receiving saline or 0.75 mg/kg FA 6 h after MTX. FA rescue alleviated the MTX‐induced bone marrow adiposity, as well as inducing up‐regulation of Wnt10b mRNA and β‐catenin protein expression in the bone. Furthermore, FA blocked up‐regulation of the secreted Wnt antagonist sFRP‐1 mRNA expression. Moreover, secreted sFRP‐1 protein in the bone marrow and its expression by osteoblasts and adipocytes was found increased following MTX treatment. This potentially indicates that sFRP‐1 is a major regulator of defective Wnt/β‐catenin signalling following MTX treatment. This study provides evidence that folate depletion caused by MTX chemotherapy results in increased bone marrow adiposity, and that FA rescue alleviates these defects by up‐regulating Wnt/β‐catenin signalling in the bone. J. Cell. Physiol. 230: 648–656, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:16.763649-05:
      DOI: 10.1002/jcp.24788
       
  • Influence of Nuclear Blebs and Micronuclei Status on the Growth Kinetics
           of Human Mesenchymal Stem Cells
    • Authors: Shikha Sharma; Ramesh Bhonde
      First page: 657
      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. 230: 657–666, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:13.323309-05:
      DOI: 10.1002/jcp.24789
       
  • The Cooperation of CREB and NFAT Is Required for PTHrP‐Induced RANKL
           Expression in Mouse Osteoblastic Cells
    • Authors: Hyun‐Jung Park; Kyunghwa Baek, Jeong‐Hwa Baek, Hyung‐Ryong Kim
      First page: 667
      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. 230: 667–679, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:14.24524-05:0
      DOI: 10.1002/jcp.24790
       
  • 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
      First page: 680
      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. 230: 680–690, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:12.036864-05:
      DOI: 10.1002/jcp.24792
       
  • 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
      First page: 691
      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, treatments that 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 lipopolysaccharide (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. 230: 691–701, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2014-11-21T13:45:06.715919-05:
      DOI: 10.1002/jcp.24794
       
  • 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
      First page: 702
      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
       
  • 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
      First page: 716
      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
       
  • 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
      First page: 732
      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
       
 
 
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