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Journal Cover Journal of Cellular Physiology     [SJR: 1.608]   [H-I: 118]
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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  [1605 journals]
  • 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
       
  • 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
       
  • Prostaglandin Dependent Control of an Endogenous Estrogen Receptor Agonist
           by Osteoblasts
    • 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. 230: 1104–1114, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Alpha‐Synuclein as a Pathological Link Between Chronic Traumatic
           Brain Injury and Parkinson's Disease
    • 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, α‐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 α‐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 α‐synuclein as a pathological link between chronic effects of TBI and PD symptoms as evidenced by significant overexpression and abnormal accumulation of α‐synuclein in inflammation‐infiltrated SN of rats exposed to chronic TBI. J. Cell. Physiol. 230: 1024–1032, 2015. © 2014 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
       
  • microRNA‐151–3p Regulates Slow Muscle Gene Expression by
           Targeting ATP2a2 in Skeletal Muscle Cells
    • Abstract: MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate the stability or translation of cognate mRNAs at the post‐transcriptional level. Accumulating evidence indicates that miRNAs play important roles in many aspects of muscle function, including muscle growth and development, regeneration, contractility, and muscle fiber type plasticity. In the current study, we examined the function of miR‐151–3p in myoblast proliferation and differentiation. Results show that overexpression of miR‐151–3p not only upregulates myoblast proliferation, but also decreases slow muscle gene expression (such as MHC‐β/slow and slow muscle troponin I) in both C2C12 myotubes and in primary cultures. Alternatively, inhibition of miR‐151–3p by antisense RNA was found to upregulate MHC‐β/slow expression, indicating that miR‐151–3p plays a role in muscle fiber type determination. Further investigation into the underlying mechanisms revealed for the first time that miR‐151–3p directly targets ATP2a2, a gene encoding for a slow skeletal and cardiac muscle specific Ca2+ ATPase, SERCA2 thus downregulating slow muscle gene expression. Mechanisms by which the alteration in SERCA2 expression induces changes in other slow muscle gene expression levels needs to be defined in future research. J. Cell. Physiol. 230: 1003–1012, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • FoxK2 is Required for Cellular Proliferation and Survival
    • 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 proapoptotic 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. 230: 1013–1023, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • DNA‐PKcs Deficiency Inhibits Glioblastoma Cell‐Derived
           Angiogenesis After Ionizing Radiation
    • 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 FAKTry925 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. 230: 1094–1103, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Effect of rMnSOD on Survival Signaling in Pediatric High Risk T‐Cell
           Acute Lymphoblastic Leukaemia
    • 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 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. 230: 1086–1093, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • The Role of Epigenetic Mechanisms in Notch Signaling During Development
    • 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. 230: 969–981, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Role of Methionine Adenosyltransferase α2 and β Phosphorylation
           and Stabilization in Human Hepatic Stellate Cell
           Trans‐Differentiation
    • 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. 230: 1075–1085, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in
           Dividing Human Cells
    • 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. 230: 1064–1074, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • UCP2‐Related Mitochondrial Pathway Participates in Oroxylin
           A‐Induced Apoptosis in Human Colon Cancer Cells
    • 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 demonstrated that UCP2 plays a key role in mitochondrial apoptotic pathway; UCP2s inhibition by oroxylin A triggers the MPTP opening, and promotes the apoptosis in CaCo‐2 cells. J. Cell. Physiol. 230: 1054–1063, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • LIGHT Is a Crucial Mediator of Airway Remodeling
    • 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. 230: 1042–1053, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Lkb1 Deletion Promotes Ectopic Lipid Accumulation in Muscle Progenitor
           Cells and Mature Muscles
    • 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. 230: 1033–1041, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Table of Contents: Volume 230, Number 5
    •  
  • Editor's Choice
    •  
  • Highlights: Volume 230, Number 5
    •  
  • Insights Into RNA Transcriptome Profiling of Cardiac Tissue in Obesity and
           Hypertension Conditions
    • Abstract: Several epidemiologic 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 physiologic mechanisms, pathways or genes involved in many biologic 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 physiologic and biochemical mechanisms involved in gene expression changes produced by these events, emphasizing the use of new technologies for gene expression analyses. J. Cell. Physiol. 230: 959–968, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Journal of Cellular Physiology: Volume 230, Number 5, May 2015
    • Abstract: Cover: Protein binding partners of human Notch1 are visualized using the BioGRID website. Only physical interactions are shown and interspecies interactions are excluded. Canonical Notch binding partners are labeled in green, chromatin factors in red, and some examples of crosstalk to other signaling pathway mentioned in the text are labeled yellow. See reviews on Nuclear Receptors and Epigenomics in this issue: Schwanbeck, Wang et al., and Stachowiak et al. on pages 969–1002.
       
  • Epoxyeicosatrienoic Acids Regulate Macrophage Polarization and Prevent
           LPS‐Induced Cardiac Dysfunction
    • Abstract: Macrophages, owning tremendous phenotypic plasticity and diverse functions, were becoming the target cells in various inflammatory, metabolic and immune diseases. Cytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acid to form epoxyeicosatrienoic acids (EETs), which possess various beneficial effects on cardiovascular system. In the present study, we evaluated the effects of EETs treatment on macrophage polarization and recombinant adeno‐associated virus (rAAV)‐mediated CYP2J2 expression on LPS‐induced cardiac dysfunction, and sought to investigate the underlying mechanisms. In vitro studies showed that EETs (1µmol/l) significantly inhibited LPS‐induced M1 macrophage polarization and diminished the proinflammatory cytokines at transcriptional and post‐transcriptional level; meanwhile it preserved M2 macrophage related molecules expression and upregulated anti‐inflammatory cytokine IL‐10. Furthermore, EETs down‐regulated NF‐κB activation and up‐regulated PPARα/γ and HO‐1 expression, which play important roles in regulating M1 and M2 polarization. In addition, LPS treatment in mice induced cardiac dysfunction, heart tissue damage and infiltration of M1 macrophages, as well as the increase of inflammatory cytokines in serum and heart tissue, but rAAV‐mediated CYP2J2 expression increased EETs generation in heart and significantly attenuated the LPS‐induced harmful effects, which mechanisms were similar as the in vitro study. Taken together, the results indicate that CYP2J2/EETs regulates macrophage polarization by attenuating NF‐κB signaling pathway via PPARα/γ and HO‐1 activation and its potential use in treatment of inflammatory disease. This article is protected by copyright. All rights reserved
       
  • Identification of genes selectively regulated in human hepatoma cells by
           treatment with dyslipidemic sera and PUFAs
    • Abstract: Serum composition is linked to metabolic diseases not only to understand their pathogenesis but also for diagnostic purposes. Quality and quantity of nutritional intake can affect disease risk and serum composition. It is then possible that diet derived serum components directly affect pathogenetic mechanisms. To identify involved factors, we evaluated the effect on gene expression of direct addition of dyslipidemic human serum samples to cultured human hepatoma cells (HepG2). Sera were selected on the basis of cholesterol level, considering this parameter as mostly linked to dietary intake. Cells were treated with 32 sera from hypercholesterolemic and normocholesterolemic subjects to identify differentially regulated mRNAs using DNA microarray analysis. We identified several mRNAs with the highest modulations in cells treated with dyslipidemic sera versus cells treated with normal sera. Since the two serum groups had variable polyunsaturated fatty acids (PUFAs) contents, selected mRNAs were further assessed for their regulation by docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AA). Four genes resulted both affected by serum composition and PUFAs: 3‐hydroxy‐3‐methylglutaryl‐CoenzymeA synthase 2 (HMGCS2), glutathione S‐transferase alpha 1 (GSTA1), liver expressed antimicrobial peptide 2 (LEAP2) and apolipoprotein M (ApoM). HMGCS2 expression appears the most relevant and was also found modulated via transcription factors peroxysome proliferator activated receptor α (PPARα) and forkhead box O1 (FoxO1). Our data indicate that expression levels of the selected mRNAs, primarily of HMGCS2, could represent a reference of nutritional intake, PUFAs effects and dyslipidemic diseases pathogenesis. This article is protected by copyright. All rights reserved
       
  • Molecular Mechanisms of Helicobacter Pylori Pathogenesis
    • Abstract: Helicobacter pylori infects 50% of mankind. The vast majority of H. pylori infection occurs in the developing countries where up to 80% of the middle‐aged adults may be infected. Bacterial infection causes an inflammatory response that proceeds through a series of intermediated stages of precancerous lesions (gastritis, atrophy, intestinal metaplasia and dysplasia). Among infected individuals, approximately 10% develops severe gastric lesions such as peptic ulcer disease, 1‐3% progresses to gastric cancer (GC) with a low 5 year survival rate, and 0.1% develops mucosa‐associated lymphoid tissue (MALT). GC is one of the most common cancer and the third leading cause of cancer‐related deaths worldwide. In this review we have summarized the most recent papers about molecular mechanisms of Helicobacter pylori pathogenesis. The main important steps of H. pylori infection such as adhesion, entry in epithelial gastric cells, activation of intracellular pathways until epigenetic modifications have been described. This article is protected by copyright. All rights reserved
       
  • Dynamically regulated CFTR expression and its functional role in cutaneous
           wound healing
    • Abstract: The physiological role of cystic fibrosis transmembrane conductance regulator (CFTR) in keratinocytes and skin wound healing is completely unknown. The present study shows that CFTR is expressed in the multiple layers of keratinocytes in mouse epidermis and exhibits a dynamic expression pattern in a dorsal skin wound healing model, with diminishing levels observed from Day 3 to Day 5 and re‐appearing from Day 7 to Day 10 after wounding. Knockdown of CFTR in cultured human keratinocytes promotes cell migration but inhibits differentiation, while overexpression of CFTR suppresses migration but enhances differentiation, indicating an important role of CFTR in regulating keratinocyte behavior. In addition, we have demonstrated a direct association of CFTR with epithelial junction formation as knockdown of CFTR downregulates the expression of adhesion molecules, such as E‐cadherin, ZO‐1 and β‐catenin, and disrupts the formation of cell junction, while overexpression of CFTR enhances cell junction formation. More importantly, we have shown that ΔF508cftr‐/‐ mice with defective CFTR exhibit delayed wound healing as compared to wild type mice, indicating that normal function of CFTR is critical for wound repair. Taken together, the present study has revealed a previously undefined role of CFTR in regulating skin wound healing processes, which may have implications in injury repair of other epithelial tissues. This article is protected by copyright. All rights reserved
       
  • SIRT1 protects against oxidative stress‐induced endothelial
           progenitor cells apoptosis by inhibiting FOXO3a via FOXO3a ubiquitination
           and degradation
    • Abstract: Cell loss due to apoptosis induced by oxidative stress is a major hurdle for endothelial progenitor cells (EPCs)‐based therapy. Sirtuin 1 (SIRT1) plays important roles in many pathophysiological processes by deacetylating various substrates, including forkhead transcription factor (FOXO). However, after deacetylation, the fate of FOXO protein remains to be explored. In the present study, we investigated whether SIRT1 exerted a protective effect on hydrogen peroxide (H2O2)‐induced EPCs apoptosis and, if so, what the underlying mechanism might be. EPCs were isolated and obtained from human umbilical cord blood by density gradient centrifugation and identified by morphology, tube formation ability, cell surface markers and the ability to take up acetylated low density lipoprotein (Dil‐Ac‐LDL) and bind ulex europaeus agglutinin 1 (FITC‐UEA‐1). Immunofluorescence showed that SIRT1 localized in the nucleus of EPCs in the presence or absence of H2O2. SIRT1 protein level in EPCs was increased by the treatment with H2O2 for 24 h. Incubation of EPCs with H2O2 dose dependently induced EPCs apoptosis. SIRT1 overexpression reduced the rate of EPCs apoptosis induced by H2O2, while SIRT1 downregulation and EX527, a specific SIRT1 inhibitor, exerted the opposite effect. SIRT1 overexpression decreased the total FOXO3a protein expression, whereas SIRT1 downregulation and EX527 increased the amount of FOXO3a protein. SIRT1 reduced FOXO3a transcriptional activity according to Bim expression. Co‐immunoprecipitation (Co‐IP) assay showed that SIRT1 could bind to FOXO3a, reduce its acetylation level and increase its ubiquitination level. To sum up, our work demonstrated that SIRT1 had a pivotally protective role in the regulation of EPCs apoptosis induced by H2O2 and that SIRT1 protected against apoptosis by inhibiting FOXO3a via FOXO3a ubiquitination and subsequent degradation. This article is protected by copyright. All rights reserved
       
  • MechanosensitiveStore‐Operated Calcium Entry Regulatesthe Formation
           of Cell Polarity
    • Abstract: Ca2+‐mediated formation of cell polarity is essential for directional migration, which plays an important role inphysiological and pathological processes in organisms. To examine the critical role of store‐operated Ca2+ entry, which is the major form of extracellular Ca2+ influx in non‐excitable cells, in the formation of cell polarity, we employed human bone osteosarcoma U2OS cells, which exhibit distinct morphological polarity during directional migration. Our analyses showed that Ca2+ was concentrated at the rear end of cells and that extracellular Ca2+ influx was important for cell polarization. Inhibition of store‐operated Ca2+ entry using specific inhibitors disrupted the formation of cell polarity in a dose‐dependent manner. Moreover, the channelosomal components caveolin‐1, TRPC1and Orai1 were concentrated at the rear end of polarized cells. Knockdown of TRPC1 or a TRPC inhibitor, but not knockdown of Orai1,reduced cell polarization. Furthermore, disruption of lipid rafts or overexpression of caveolin‐1 contributed to the downregulation of cell polarity.On the other hand, we also found that cell polarity, store‐operated Ca2+ entry activity and cell stiffness were markedly decreased by low substrate rigidity, which may be caused by the disorganization of actin filaments and microtubules that occurs while regulating the activity of the mechanosensitive TRPC1 channel. This article is protected by copyright. All rights reserved
       
  • Y‐Box binding protein‐1 is part of a complex molecular network
           linking ΔNp63α to the PI3K/AKT pathway in cutaneous squamous
           cell carcinoma
    • Abstract: Cutaneous squamous cell carcinomas (SCCs) typically lack somatic oncogene‐activating mutations and most of them contain p53 mutations. However, the presence of p53 mutations in skin premalignant lesions suggests that these represent early events during tumor progression and additional alterations may be required for SCC development. SCC cells frequently express high levels of ΔNp63α and Y‐box binding 1 (YB‐1 or YBX1) oncoproteins. Here, we show that knockdown of YB‐1 in spontaneously immortalized HaCaT and non‐metastatic SCC011 cells led to a dramatic decrease of ΔNp63α, cell detachment and death. In highly metastatic SCC022 cells, instead, YB‐1 silencing induces PI3K/AKT signaling hyperactivation which counteracts the effect of YB‐1 depletion and promotes cell survival. In summary, our results unveil a functional cross‐talk between YB‐1, ΔNp63α and the PI3K/AKT pathway critically governing survival of squamous carcinoma cells. This article is protected by copyright. All rights reserved
       
  • Hairless up‐regulates Tgf‐β2 expression via
           down‐regulation of miR‐31 in the skin of
           ‘Hairpoor’ (HrHp) mice
    • Abstract: Hairless (HR) has been shown to regulate hair follicle (HF) morphogenesis and hair cycling. The Hr mutant hair loss mouse referred to as ‘hairpoor’ (HrHp) displays overexpression of the HR protein through translational derepression. In this study, we found that 64 miRNAs were differentially expressed between the skin of HrHp/HrHp and wild type mice at P7 using miRNA‐microarray analysis and miR‐31 displayed the most reduced expression in HrHp/HrHp skin. In vivo observation and investigation using an in vitro reporter expression system revealed that miR‐31 and pri‐miR‐31 were consistently down‐regulated in the HR over‐expressed condition. In addition, we found that the transforming growth factor β 2 (Tgf‐β2), a known catagen inducer, is the putative target of miR‐31. Furthermore, Tgf‐β2 level was also increased in HR over‐expressed keratinocyte and HrHp/HrHp mice. These study results suggest that HR controls Tgf‐β2 expression via regulation of miR‐31, thus causing abnormal hair cycle in HrHp/HrHp mice. This article is protected by copyright. All rights reserved
       
  • Chondroptosis in Alkaptonuric Cartilage
    • 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. 230: 1148–1157, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • IGF‐1 Induces IP3‐dependent Calcium Signal Involved in the
           Regulation of Myostatin Gene Expression Mediated by NFAT During Myoblast
           Differentiation
    •  
  • 7‐Ketocholesterol and 5,6‐Secosterol Modulate Differently the
           Stress‐Activated Mitogen‐Activated Protein Kinases (MAPKs) in
           Liver Cells
    •  
  • Short Time Tripterine Treatment Enhances Endothelial Progenitor Cell
           Function via Heat Shock Protein 32
    • Abstract: 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. 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. 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. 230: 1139–1147, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Permanent Culture of Macrophages at Physiological Oxygen Attenuates the
           Antioxidant and Immunomodulatory Properties of Dimethyl Fumarate
    • 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. 230: 1128–1138, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • PDE5 Inhibitors Enhance Celecoxib Killing in Multiple Tumor Types
    • 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. 230: 1115–1127, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • β2‐adrenoreceptor–mediated proliferation inhibition of
           embryonic pluripotent stem cells
    • Abstract: Adrenoreceptors (ARs) are widely expressed and play essential roles throughout the body. Different subtype adrenoceptors elicit distinct effects on cell proliferation, but knowledge remains scarce about the subtype‐specific effects of β2‐ARs on the proliferation of embryonic pluripotent stem (PS) cells that represent different characteristics of proliferation and cell cycle regulation with the somatic cells. Herein, we identified a β2‐AR/AC/cAMP/PKA signaling pathway in embryonic PS cells and found that the pathway stimulation inhibited proliferation and cell cycle progression involving modulating the stem cell growth and cycle regulatory machinery. Embryonic stem (ES) cells and embryonal carcinoma stem (ECS) cells expressed functional β‐ARs coupled to AC/cAMP/PKA signaling. Agonistic activation of β‐ARs led to embryonic PS cell cycle arrest and proliferation inhibition. Pharmacological and genetic analyses using receptor subtype blocking and RNA interference approaches revealed that this effect selectively depended on β2‐AR signaling involving the regulation of AKT, ERK, Rb, and Cyclin E molecules. Better understanding of the effects of β2‐ARs on embryonic PS cell proliferation and cycle progression may provide new insights to stem cell biology and afford the opportunity for exploiting more selective ligands targeting the receptor subtype for the modulation of stem cells. This article is protected by copyright. All rights reserved
       
  • The Na+/HCO3‐ Co‐Transporter SLC4A4 Plays a Role in Growth and
           Migration of Colon and Breast Cancer Cells
    • Abstract: The hypoxic and acidic tumor environment necessitates intracellular pH (pHi) regulation for tumor progression. Carbonic anhydrase IX (CA IX; hypoxia‐induced) is known to facilitate CO2 export and generate HCO3‐ in the extracellular tumor space. It has been proposed that HCO3‐ is re‐captured by the cell to maintain an alkaline pHi. A diverse range of HCO3‐ transporters, coupled with a lack of a clear over‐expression in cancers have limited molecular identification of this cellular process. Here we report that hypoxia induces the Na+/HCO3‐ co‐transporter (NBCe1) SLC4A4 mRNA expression exclusively in the LS174 colon adenocarcinoma cell line in a HIF1α dependent manner. HCO3‐ dependent pHi recovery observations revealed the predominant use of an NBC mechanism suggesting that reversal of a Cl‐/HCO3‐ exchanger is not utilized for tumor cell pHi regulation. Knockdown of SLC4A4 via shRNA reduced cell proliferation and increased mortality during external acidosis and spheroid growth. pHi recovery from acidosis was partially reduced with knockdown of SLC4A4. In MDA‐MB‐231 breast cancer cells expressing high levels of SLC4A4 compared to LS174 cells, SLC4A4 knockdown had a strong impact on cell proliferation, migration, and invasion. SLC4A4 knockdown also altered expression of other proteins including CA IX. Furthermore the Na+/HCO3‐ dependent pHi recovery from acidosis was reduced with SLC4A4 knockdown in MDA‐MB‐231 cells. Combined our results indicate that SLC4A4 contributes to the HCO3‐ transport and tumor cell phenotype. This study complements the on‐going molecular characterization of the HCO3‐ re‐uptake mechanism in other tumor cells for future strategies targeting these potentially important drug targets. This article is protected by copyright. All rights reserved
       
  • Quantifying Heterogeneity and Dynamics of Clonal Fitness in Response to
           Perturbation
    • Abstract: The heterogeneous dynamics of clonal lineages within a cell population, in aggregate, shape both normal and pathological biological processes. Studies of clonality typically relate the fitness of clones to their relative abundance, thus requiring long‐term experiments and limiting conclusions about the heterogeneity of clonal fitness in response to perturbation. We present, for the first time, a method that enables a dynamic, global picture of clonal fitness within a mammalian cell population. This novel assay allows facile comparison of the structure of clonal fitness in a cell population across many perturbations. By utilizing high‐throughput imaging, our methodology provides ample statistical power to define clonal fitness dynamically and to visualize the structure of perturbation‐induced clonal fitness within a cell population. We envision that this technique will be a powerful tool to investigate heterogeneity in biological processes involving cell proliferation, including development and drug response. This article is protected by copyright. All rights reserved
       
  • Biphasic Role of Calcium in Mouse Sperm Capacitation Signaling Pathways
    • Abstract: Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up‐regulation of a cAMP‐dependent pathway, changes in intracellular pH, intracellular Ca2+ and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca2+ ions have a biphasic role in the regulation of cAMP‐dependent signaling. Media without added Ca2+ salts (nominal zero Ca2+) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca2+. However, chelation of the extracellular Ca2+ traces by EGTA induced both cAMP‐dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca2+ media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca2+ ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ media. Therefore, sperm lacking Catsper Ca2+ channels behave as wild‐type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca2+ involved in the regulation of cAMP‐dependent and tyrosine phosphorylation pathways required for sperm capacitation. This article is protected by copyright. All rights reserved
       
  • Bisphosphonate treatment of type I diabetic mice prevents early bone loss
           but accentuates suppression of bone formation
    • Abstract: Type I (T1) diabetes is an autoimmune and metabolic disease associated with bone loss. Previous studies demonstrate that T1‐diabetes decreases osteoblast activity and viability. Bisphosphonate therapy, commonly used to treat osteoporosis, is demonstrated to inhibit osteoclast activity as well as osteoblast apoptosis. Therefore, we examined the effect of weekly alendronate treatments on T1‐diabetes induced osteoblast apoptosis and bone loss. Bone TUNEL assays identified that alendronate therapy prevents the diabetes‐induced osteoblast death observed during early stages of diabetes development. Consistent with this, alendronate treatment for 40 days was able to prevent diabetes‐induced trabecular bone loss. Alendronate was also able to reduce marrow adiposity in both control diabetic mice compared to untreated mice. Mechanical testing indicated that 40 days of alendronate treatment increased bone stiffness but decreased the work required for fracture in T1‐diabetic and alendronate treated mice. Of concern at this later time point, bone formation rate and osteoblast markers, which were already decreased in diabetic mice, were further suppressed in alendronate treated diabetic mice. Taken together, our results suggest that short term alendronate treatment can prevent T1‐diabetes‐induced bone loss in mice, possibly in part by inhibiting diabetes onset associated osteoblast death, while longer treatment enhanced bone density but at the cost of further suppressing bone formation in diabetic mice. This article is protected by copyright. All rights reserved
       
  • Acetate supplementation as a means of inducing glioblastoma
           stem‐like cell growth arrest
    • Abstract: Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem‐like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non‐GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N‐acetyl‐L‐aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM‐derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose‐dependent reduction of GSC growth, it also reduced cell viability. GTA‐mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA‐mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth. This article is protected by copyright. All rights reserved
       
  • Activation of Nfatc2 in Osteoblasts Causes Osteopenia
    • Abstract: Nuclear factor of activated T‐cells (Nfat)c1 to c4 are transcription factors that play an undisputable role in osteoclastogenesis. However, Nfat function in osteoblastic cells is controversial. Constitutive activation of Nfatc1 and c2 in osteoblasts suppresses cell function, although the study of Nfat in vivo has yielded conflicting results. To establish the consequences of Nfatc2 activation in osteoblasts, we generated transgenic mice where a 3.6 kilobase fragment of the collagen type I α1 promoter directs expression of a constitutively active Nfatc2 mutant (Col3.6‐Nfatc2). The skeletal phenotype of Col3.6‐Nfatc2 mice of both sexes and of sex‐matched littermate controls was investigated by microcomputed tomography and histomorphometry. Col3.6‐Nfatc2 mice were born at the expected Mendelian ratio and appeared normal. Nfatc2 expression was confirmed in parietal bones from 1 and 3 month old transgenic mice. One month old Col3.6‐Nfatc2 female mice exhibited cancellous bone compartment osteopenia secondary to a 30% reduction in bone formation. In contrast, cancellous femoral bone volume and bone formation were not altered in male transgenics, whereas osteoblast number was higher, suggesting incomplete osteoblast maturation. Indices of bone resorption were not affected in either sex. At 3 months of age, the skeletal phenotype evolved; and Col3.6‐Nfatc2 male mice exhibited vertebral osteopenia, whereas femoral cancellous bone was not affected in either sex. Nfatc2 activation in osteoblasts had no impact on cortical bone structure. Nfatc2 activation inhibited alkaline phosphatase activity and mineralized nodule formation in bone marrow stromal cell cultures. In conclusion, Nfatc2 activation in osteoblasts inhibits bone formation and causes cancellous bone osteopenia. This article is protected by copyright. All rights reserved
       
  • FADD phosphorylation impaired islet morphology and function
    • Abstract: Previous studies have indicated that Fas‐FasL pathway and its downstream caspase‐8 can regulate islet mass and insulin secretion. As a classical adaptor in Fas‐FasL signaling, Fas‐associated death domain‐containing protein (FADD) takes part in many non‐apoptosis processes regulated by its phosphorylation. However, its role in islets has not been evaluated to date. Here, through comparative proteomics and bioinformatic analysis on FADD phosphorylated (FADD‐D) and wild‐type (WT) MEFs, we found three proteins involved in islet differentiation and function were dysregulated due to FADD phosphorylation. The mouse model of FADD‐D, which mimics constitutive phosphorylated FADD expression in mice, was further analyzed to address this issue. We confirmed the proteomic results by immunohistological analyses on pancreatic islets. In addition, we found that FADD‐D mice displayed decreased islet area, and the glucose stimulated insulin secretion (GSIS) of FADD‐D islets was impaired. These data suggest a novel role of FADD in islet development and insulin secretion. This article is protected by copyright. All rights reserved Abbreviations FADD, Fas‐associated death domain‐containing protein; MEFs, mouse embryonic fibroblasts; GSIS, glucose stimulated insulin secretion; Glut2, glucose transporter 2; Pdx1, Pancreas duodenum homeobox 1; Gck, glucokinase.
       
  • Generation of tumor‐specific cytotoxic T‐lymphocytes from the
           peripheral blood of colorectal cancer patients for adoptive T‐cell
           transfer
    • Abstract: This study designs a strategy for an ACT protocol based on the ex‐vivo selection of autologous peripheral blood‐derived CD8‐enriched T‐cells, stimulated with dendritic cells (DCs) that had been pulsed with apoptotic tumor cells to generate cytotoxic T lymphocytes (CTLs) with anti‐tumor activity. Seventy‐eight CRC patients were enrolled in this study. Tumor tissues and peripheral blood (PB) were obtained at surgery. Tissues were mechanically dissociated and cultured to obtain a primary tumor cell line from each patient. DCs were derived from peripheral blood mononuclear cells (PBMCs) using magnetic positive selection of CD14+ monocytes. Anti‐tumor CTLs were elicited in co‐/micro‐cultures using DCs as antigen‐presenting cells, autologous apoptotic tumor cells as a source of antigens, and CD8+ T lymphocytes as effectors. Interferon‐γ (IFN‐γ) secretion was assessed by ELISpot assays to evaluate the activation of the CTLs against the autologous tumor cells. Primary tumor cell lines were obtained from 20 of 78 patients (25.6%). DCs were generated from 26 patients, and of them, corresponding tumor cell lines were derived from 6 patients. ELISpot results showed that significant IFN‐γ secretion was detected after different numbers of stimulations for two patients, whereas weak secretion was observed for three patients. Despite difficulties due to contamination of several primary tumor cell lines with gut intestinal flora, the results suggest that the generation of tumor‐specific CTLs is feasible from patients with colorectal cancer, and could be useful for supporting an ACT approach in CRC. This article is protected by copyright. All rights reserved
       
  • Exploration of molecular pathways mediating electric field‐directed
           Schwann cell migration by RNA‐Seq
    • Abstract: In peripheral nervous systems, Schwann cells wrap around axons of motor and sensory neurons to form the myelin sheath. Following spinal cord injury, Schwann cells regenerate and migrate to the lesion and are involved in the spinal cord regeneration process. Transplantation of Schwann cells into injured neural tissue results in enhanced spinal axonal regeneration. Effective directional migration of Schwann cells is critical in the neural regeneration process. In this study, we report that Schwann cells migrate anodally in an applied electric field (EF). The directedness and displacement of anodal migration increased significantly when the strength of the EF increased from 50 mV/mm to 200 mV/mm. The EF did not significantly affect the cell migration speed. To explore the genes and signaling pathways that regulate cell migration in EFs, we performed a comparative analysis of differential gene expression between cells stimulated with an EF (100 mV/mm) and those without using next‐generation RNA sequencing, verified by RT‐qPCR. Based on the cut‐off criteria (FC > 1.2, q < 0.05), we identified 1,045 up‐regulated and 1,636 down‐regulated genes in control cells versus EF‐stimulated cells. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that compared to the control group, 21 pathways are down‐regulated, while 10 pathways are up‐regulated. Differentially expressed genes participate in multiple cellular signaling pathways involved in the regulation of cell migration, including pathways of regulation of actin cytoskeleton, focal adhesion, and PI3K‐Akt. This article is protected by copyright. All rights reserved.
       
  • Biosafety evidence for human dedifferentiated adipocytes
    • Abstract: Mature adipocytes have shown dynamic plasticity to be converted into fibroblast‐like and lipid‐free cells. After the dedifferentiation process, these cells re‐entered the cell cycle and acquired a high proliferation potential, becoming a valid source of stem cells. However, many aspects of the cellular biosafety about dedifferentiated fat cells remained unclear. This study aimed to elucidate their potential susceptibility to malignant transformation and to ascertain the safety of these cells for clinical use. To evaluate the genomic stability of dedifferentiated adipocytes, telomere length, hTERT gene transcription, the capacity of these cells to grow in an anchorage‐independent manner and the presence of DNA damage by single cell gel electrophoresis assay were studied. Spontaneous chromosomal alterations were excluded by cytogenetic analysis and the expression level of c‐myc and p53, tumor associated genes, were assessed, evaluating also p53 loss of function mutations. Despite the high proliferation capacity of dedifferentiated adipocytes, these cells showed stable telomere length compared with mature adipocytes, no hTERT transcriptions and consequently no telomerase activity, suggesting that both transformation and senescence were avoided. A constant expression level of c‐myc and p53, the inability of dedifferentiated adipocytes to grow in an anchorage‐independent manner, the absence of DNA damage suggested the safety of these cells. Moreover, a normal karyotype was preserved throughout the dedifferentiation process. Data in vivo showed that dedifferentiated adipocytes analyzed for tumorigenicity did not develop tumors. In conclusion, our data indicated that dedifferentiated adipocytes could be a relatively easily accessible resource for cell therapy and regenerative medicine. This article is protected by copyright. All rights reserved
       
  • TGF‐β1 up‐regulates connexin43 expression: a potential
           mechanism for human trophoblast cell differentiation
    • Abstract: Connexin43 (Cx43)‐mediated gap junctional intercellular communication (GJIC) are required for human trophoblast differentiation. To date, whether Cx43 mediates TGF‐β1‐induced trophoblast differentiation has not been determined. We showed that treatment with TGF‐β1 increased Cx43 expression and GJIC in HTR‐8/SVneo human trophoblast cells. In addition, Smad and ERK1/2 signaling pathways were involved in TGF‐β1‐induced up‐regulation of Cx43. Moreover, TGF‐β1 increased the expression of the syncytiotrophoblast marker, β‐hCG. Importantly, knockdown of Cx43 abolished the TGF‐β1‐induced up‐regulation of β‐hCG. Furthermore, overexpression of Cx43 up‐regulated β‐hCG expression. These results provide evidence that Cx43 and GJIC activity are up‐regulated by TGF‐β1 in human trophoblast cells, which subsequently contributes to TGF‐β1‐induced trophoblast differentiation. This article is protected by copyright. All rights reserved
       
  • Impaired expression of HIF ‐2α induces compensatory expression
           of HIF‐1α for the recovery from anemia
    • Abstract: Erythropoiesis is strongly influenced by the interactions between stromal cells and erythroid progenitors, as well as by a key regulatory factor, erythropoietin (EPO). We previously generated mice with a knock‐down mutation of Hif‐2α (referred to as kd/kd) and found that these kd/kd mice exhibited normocytic anemia, even though the EPO expression was not severely affected. However, the VCAM‐1 expression in spleen endothelial cells (EC), which is regulated by HIF‐2α was impaired, resulting in defective erythroid maturation. A deficiency of HIF‐2α clearly led to pancytopenia. However, the critical level of HIF‐2α required for erythropoiesis has not yet been elucidated. In this study, we generated HIF‐2α knockdown/knockout heterozygous mice (kd/null). Strikingly, anemia was observed in the kd/null mice, but the red blood cell indices were significantly improved compared to those of kd/kd mice. In the spleens of kd/null mice, higher HIF‐1α activity and expansion of the red pulp area were observed compared to those of kd/kd mice. Importantly, EC isolated from kd/null spleens showed high expression of VEGF receptors, FLK‐1 and FLT‐1, which are regulated by HIF‐1α instead of HIF‐2α under hypoxic conditions. We also found higher expression of phosphorylated ERK and higher proliferative activity in the EC isolated from kd/null mice compared to those from kd/kd mice. While the HIF‐2α expression was diminished, HIF‐1α bound to the HRE region in the promoters of genes that are normally regulated by HIF‐2α. These results suggest that there is a compensatory pathway involving HIF‐1α that regulates the expression of some HIF‐2α target genes. This article is protected by copyright. All rights reserved
       
  • Endochondral ossification model system: Designed cell fate of human
           epiphyseal chondrocytes during long‐term implantation
    • Abstract: The aim of this study was to establish a recapitulation system of human endochondral ossification as a paradigm of developmental engineering. Chondrocytes were isolated from the epiphyseal cartilage of the supernumerary digits of infants with polydactyly. In vivo studies showed that implanted chondrocytes exhibited cartilaginous regeneration over a short period of time and subsequent endochondral ossification with a marrow cavity. Tracing studies revealed that cells of donor origin at the periphery of the cartilage migrated into the center of the cartilage and transformed into osteoblasts, adipocytes and endothelial cells. Bone marrow was formed through anastomosis with the recipient endothelial system at 13 weeks, and from the migration of recipient hematopoietic cells at 50 weeks. This study provides a human endochondral ossification model system with transdifferentiation of the donor cells at the periphery of the cartilage. This article is protected by copyright. All rights reserved
       
  • Angiotensin receptor I stimulates osteoprogenitor proliferation through
           tgfβ ‐mediated signaling
    • Abstract: Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti‐hypertensive drugs that target angiotensin II (Ang II) activity may also improve loss of bone mineral density. Here we identified in a genetic screen the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20‐17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20‐17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20‐17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)‐Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R‐dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20‐17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors. This article is protected by copyright. All rights reserved
       
  • Baicalein decreases hydrogen peroxide‐induced damage to
           NG108‐15 cells via upregulation of Nrf2
    • Abstract: Baicalein is a flavonoid inhibitor of 12‐lipoxygenase. Here, we investigated its effect on hydrogen peroxide‐induced damage to NG108‐15 cells. Hydrogen peroxide activated the mitochondrial apoptotic pathway, decreased Nrf2 expression, increased reactive oxygen species (ROS) levels, reduced viability, and increased cell death after 2 – 24 h treatment of NG108‐15 cells. Co‐treatment with hydrogen peroxide and baicalein completely suppressed the activation of mitochondrial apoptotic pathway by upregulating Nrf2 expression and reducing ROS stress and partially inhibited the effects on cell viability and cell death. Silencing of 12‐lipoxygenase had a similar protective effect to baicalein on hydrogen peroxide‐induced damage by blocking the hydrogen peroxide ‐induced decrease in Nrf2 expression and increase in ROS levels. Neither protective effect was altered by addition of 12‐hydroxyeicosatetraenoic acid, the product of 12‐lipoxygenase, suggesting that hydrogen peroxide induced damage via 12‐lipoxygenase by another, as yet unknown, mechanism, rather than activating it. Co‐treatment of cells with hydrogen peroxide and N‐acetylcysteine or the Nrf2 inducer sulforaphane reduced hydrogen peroxide‐induced damage in a similar fashion to baicalein, while the Nrf2 inhibitor retinoic acid blocked the protective effect of baicalein. Silencing Nrf2 also inhibited the protective effects of baicalein, sulforaphane , and N‐acetylcysteine and resulted in high ROS levels, suggesting ROS elimination was mediated by Nrf2. Taken together our results suggest that baicalein protects cells from hydrogen peroxide‐induced activation of the mitochondrial apoptotic pathway by upregulating Nrf2 and inhibiting 12‐lipoxygenase to block the increase in ROS levels. Hydrogen peroxide also activates a second mitochondrial dysfunction independent death pathway which is resista nt to baicalein. This article is protected by copyright. All rights reserved
       
  • The anti‐spasticity drug baclofen alleviates collagen‐induced
           arthritis and regulates dendritic cells
    • Abstract: Baclofen is used clinically as a drug that treats spasticity, which is a syndrome characterized by excessive contraction of the muscles and hyperflexia in the central nervous system (CNS), by activating GABAB receptors (GABABRs). Baclofen was recently reported to desensitize chemokine receptors and to suppress inflammation through the activation of GABABRs. GABABRs are expressed in various immune cells, but the functions of these receptors in autoimmune diseases remain largely unknown. In this study, we investigated the effects of baclofen in murine collagen‐induced arthritis (CIA). Oral administration of baclofen alleviated the clinical development of CIA, with a reduced number of IL‐17‐producing T helper 17 (TH17) cells. In addition, baclofen treatment suppressed dendritic cell (DC)‐primed TH17 cell differentiation by reducing the production of IL‐6 by DCs in vitro. Furthermore, the pharmacological and genetic blockade of GABABRs in DCs weakened the effects of baclofen, indicating that GABABRs are the molecular targets of baclofen on DCs. Thus, our findings revealed a potential role for baclofen in the treatment of CIA, as well as a previously unknown signaling pathway that regulates DC function. This article is protected by copyright. All rights reserved
       
  • Chronic Oxidative Stress Leads to Malignant Transformation Along with
           Acquisition of Stem Cell Characteristics, and Epithelial to Mesenchymal
           Transition in Human Renal Epithelial Cells
    • Abstract: Oxidative injury to cellular macromolecules has been suggested as a common pathway shared by multiple etiological factors for kidney cancer. Whether the chronic oxidative stress alone is sufficient to induce malignant transformation in human kidney cells is not clear. Therefore, the objective of this study was to evaluate the effect of H2O2‐induced chronic oxidative stress on growth, and malignant transformation of HK‐2 normal kidney epithelial cells. This study revealed that chronic oxidative stress causes increased growth and neoplastic transformation in normal kidney epithelial cells at non‐cytotoxic dose and increased adaptation to cytotoxic level. This was confirmed by gene expression changes, cell cycle analysis, anchorage independent growth assay and in vivo tumorigenicity in nude mice. Stem cells characteristics as revealed by up‐regulation of stem cell marker genes, and morphological changes indicative of EMT with up regulation of mesenchymal markers were also observed in cells exposed to chronic oxidative stress. Antioxidant NAC did not reverse the chronic oxidative stress‐induced growth, and adaptation suggesting that perturbed biological function in these cells are permanent. Partial reversal of oxidative stress‐induced growth, and adaptation by silencing of Oct 4 and Snail1, respectively, suggest that these changes are mediated by acquisition of stem cell and EMT characteristics. In summary, this study for the first time suggests that chronic exposure to elevated levels of oxidative stress is sufficient to induce malignant transformation in kidney epithelial cells through acquisition of stem cell characteristics. Additionally, the EMT plays an important role in increased adaptive response of renal cells to oxidative stress. This article is protected by copyright. All rights reserved
       
  • Role of the Unfolded Protein Response, GRP78 and GRP94 in Organ
           Homeostasis
    • Abstract: The endoplasmic reticulum (ER) is a cellular organelle where secretory and membrane proteins, as well as lipids, are synthesized and modified. When cells are subjected to ER stress, an adaptive mechanism referred to as the Unfolded Protein Response (UPR) is triggered to allow the cells to restore homeostasis. Evidence has accumulated that the UPR pathways provide specialized and unique roles in diverse development and metabolic processes. The glucose regulated proteins (GRPs) are traditionally regarded as ER proteins with chaperone and calcium binding properties. The GRPs are constitutively expressed at basal levels in all organs, and as stress‐inducible ER chaperones, they are major players in protein folding, assembly and degradation. This conventional concept is augmented by recent discoveries that GRPs can be actively translocated to other cellular locations such as the cell surface, where they assume novel functions that regulate signaling, proliferation, apoptosis and immunity. Recent construction and characterization of mouse models where the gene encoding for the UPR components and the GRPs is genetically altered provide new insights on the physiological contribution of these proteins in vivo. This review highlights recent progress towards the understanding of the role of the UPR and two major GRPs (GRP78 and GRP94) in regulating homeostasis of organs arising from the endoderm, mesoderm and ectoderm. GRP78 and GRP94 exhibit shared and unique functions, and in specific organs their depletion elicits adaptive responses with physiological consequences. This article is protected by copyright. All rights reserved
       
  • Functional role of the KCa3.1 potassium channel in synovial fibroblasts
           from rheumatoid arthritis patients
    • Abstract: Rheumatoid arthritis synovial fibroblasts (RA‐SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. New druggable targets in RA‐SFs would therefore be of high therapeutic interest. The present study shows that the intermediate‐conductance, calcium‐activated potassium channel KCa3.1 (KCNN4) is expressed at the mRNA and protein level in RA‐SFs, is functionally active, and has a regulatory impact on cell proliferation and secretion of pro‐inflammatory and pro‐destructive mediators. Whole‐cell patch‐clamp recordings identified KCa3.1 as the dominant potassium channel in the physiologically relevant membrane voltage range below 0 mV. Stimulation with transforming growth factor β1 (TGF‐β1) significantly increased transcription, translation, and channel function of KCa3.1. Inhibition of KCa3.1 by the selective, pore‐blocking inhibitor TRAM‐34, (and, in part by siRNA) significantly reduced cell proliferation, as well as expression and secretion of pro‐inflammatory factors (IL‐6, IL‐8, and MCP1) and the tissue‐destructive protease MMP3. These effects were observed in non‐stimulated and/or TGF‐β1‐stimulated RA‐SFs. Since small molecule‐based interference with KCa3.1 is principally well tolerated in clinical settings, further evaluation of channel blockers in models of rheumatoid arthritis may be a promising approach to identify new pharmacological targets and develop new therapeutic strategies for this debilitating disease. This article is protected by copyright. All rights reserved
       
  • TNF‐related weak inducer of apoptosis (TWEAK) regulates junctional
           proteins in tubular epithelial cells via canonical NF‐κB
           pathway and ERK activation
    • Abstract: The tubular epithelium may be intrinsically involved in promoting kidney injury by junctional instability, epithelial‐mesenchymal transition (EMT) and extracellular matrix remodelling. In this work, we investigated whether the pleiotropic and proinflammatory cytokine tumor necrosis factor‐like weak inducer of apoptosis (TWEAK), could be able to disturb junctional protein expression and to induce EMT of tubular cells. In cultured murine proximal tubular cells TWEAK induced phenotypic changes that were accompanied by F‐actin redistribution, loss of epithelial adherent (E‐cadherin, Cadherin‐16, β‐catenin) and tight junction (ZO‐1) proteins, and re‐expression of the mesenchymal protein Vimentin. The transcriptional repressors Snail and HNF1β were also modulated by TWEAK. In a murine model of obstructive renal pathology, TWEAK expression correlated with the appearance of the mesenchymal marker αSMA in kidney tubular cells. Mechanistically, the epithelial changes induced by TWEAK, including loss of epithelial integrity and EMT, via Fn14 were TGF‐β1 independent, but mediated by several intracellular signaling systems, including the canonical NF‐κB, ERK activation and the vitamin D receptor modulation. These results highlight potential contributions of TWEAK‐induced inflammatory mechanisms that could unveil new pathogenic effects of TWEAK starting tubulointerstitial damage and fibrosis. This article is protected by copyright. All rights reserved
       
  • Effect of NAD on PARP‐mediated insulin sensitivity in oleic acid
           treated hepatocytes
    • Abstract: High serum free fatty acids levels are associated with the development of insulin resistance in type 2 diabetes; however, the precise mechanisms underlying this lipid toxicity are unclear. To investigate whether PARP1 activation and NAD depletion are involved in the impairment of insulin sensitivity associated with lipotoxicity, HepG2 cells were cultured with 500 μM oleic acid for 48 hours. Oleic acid‐treated cells exhibited increased ROS generation, lipid accumulation and PARP1 activation. Treatment with the PARP1 inhibitor PJ34 and transfection with PARP1 small interfering RNA both prevented the oleic acid‐induced impairment of the insulin signaling pathway. Furthermore, treatment with PJ34 reversed the oleic acid‐induced decrease in intracellular NAD concentration, while exogenous NAD protected cells against oleic acid‐induced insulin insensitivity. Combined NAD and PJ34 administration did not enhance the effects obtained by treatment with either NAD or PJ34 alone. Interestingly, when cells were treated with the SIRT1 inhibitor EX527, the protective effects of PJ34 and NAD treatment were diminished. Taken together, these data suggest that NAD depletion by PARP1 activation is essential for the modulation of insulin sensitivity in oleic acid‐induced lipotoxicity. This article is protected by copyright. All rights reserved
       
  • KMUP‐1 promotes osteoblast differentiation through cAMP and cGMP
           pathways and signaling of BMP‐2/Smad1/5/8 and
           Wnt/β‐catenin
    • Abstract: Phosphodiesterase (PDE) inhibitors have been suggested as a possible candidate for the treatment of osteopenia, including osteoporosis. KMUP‐1 is a novel xanthine derivative with inhibitory activities on the PDE 3, 4, and 5 iso‐enzymes to suppress the degradation of cAMP and cGMP. This study aimed to investigate the effect of KMUP‐1 on osteoblast differentiation and the underlying cellular and molecular mechanisms. Primary osteoblasts and osteoblastic MC3T3‐E1 cells were examined. KMUP‐1 enhanced alkaline phosphatase (ALP) activity and mineralization compared to untreated controls in primary osteoblasts and MC3T3‐E1 cells. KMUP‐1 also increased the mRNA expression of the osteoblastic differentiation markers, including collagen type Ia, ALP, osteocalcin, osteoprotegerin, BMP‐2, and Runx2, a key transcription regulator for osteoblastic differentiation. The osteogenic effect of KMUP‐1 was abolished by BMP signalling inhibitor, noggin. Furthermore, we found that KMUP‐1 upregulated Smad1/5/8 phosphorylations with subsequent BRE‐Luc activation confirmed by transient transfection assay. In addition, KMUP‐1 inactivated glycogen synthase kinase‐3β (GSK‐3β), with associated nuclear translocation of β‐catenin. Co‐treatment with H89 and KT5823, cAMP and cGMP pathway inhibitors, respectively, reversed the KMUP‐1‐induced activations of Smad1/5/8, β‐catenin, and Runx2. The findings demonstrate for the first time that KMUP‐1 can promote osteoblast maturation and differentiation in vitro via BMP‐2/Smad1/5/8 and Wnt/β‐catenin pathways. These effects are mediated, in part, by the cAMP and cGMP signaling. Thus, KMUP‐1 may be a novel osteoblast activator and a potential new therapy for osteoporosis. This article is protected by copyright. All rights reserved
       
  • Wild‐type and Specific Mutant Androgen Receptor Mediates
           Transcription via 17β‐Estradiol in Sex Hormone‐sensitive
           Cancer Cells
    • Abstract: We previously encountered regulatory processes wherein dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone‐related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR), in breast cancer MCF‐7 cells. Here, we investigated whether such aberrant ligand‐nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed large amounts of AR at negligible levels of ERα/β or progesterone receptor. Both suppression of PTHrP and activation of prostate‐specific antigen genes were observed after independent administration of 17β‐estradiol (E2), DHT, or R5020. Consistent with the notion that the LNCaP AR lost its ligand specificity due to a mutation (Thr‐Ala877), experiments with siRNA targeting the respective NR revealed that the AR monopolized the role of the mediator of shared hormone‐dependent regulation, which was invariably associated with nuclear translocation of this mutant AR. Microarray analysis of gene regulation by DHT, E2, or R5020 disclosed that more than half of the genes downstream of the AR (Thr‐Ala877) overlapped in the LNCaP cells. Of particular interest, we realized that the AR (wild‐type (wt)) and AR (Thr‐Ala877) were equally responsible for the E2‐AR interactions. Fluorescence microscopy experiments demonstrated that both EGFP‐AR (wt) and EGFP‐AR (Thr‐Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Furthermore, reporter assays revealed that some other cancer cells exhibited aberrant E2‐AR (wt) signaling similar to that in the LNCaP cells. We herein postulate the presence of entangled interactions between wt AR and E2 in certain hormone‐sensitive cancer cells. This article is protected by copyright. All rights reserved
       
  • Human Olfactory Bulb Neural Stem Cells Mitigate Movement Disorders in a
           Rat Model of Parkinson's disease
    • Abstract: Parkinson's disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. Neural stem cells (NSCs) are multipotent stem cells that are capable of differentiating into different neuronal and glial elements. The production of DA neurons from NSCs could potentially alleviate behavioral deficits in Parkinsonian patients; timely intervention with NSCs might provide a therapeutic strategy for PD. We have isolated and generated highly enriched cultures of neural stem/progenitor cells from the human olfactory bulb (OB). If NSCs can be obtained from OB, it would alleviate ethical concerns associated with the use of embryonic tissue, and provide an easily accessible cell source that would preclude the need for invasive brain surgery. Following isolation and culture, olfactory bulb neural stem cells (OBNSCs) were genetically engineered to express hNGF and GFP. The hNFGGFP‐OBNSCs were transplanted into the striatum of 6‐hydroxydopamin (6‐OHDA) Parkinsonian rats. The grafted cells survived in the lesion environment for more than eight weeks after implantation with no tumor formation. The grafted cells differentiated in vivo into oligodendrocyte‐like (25±2.88%), neuron‐like (52.63±4.16%), and astrocyte ‐like (22.36±1.56%) lineages, which we differentiated based on morphological and immunohistochemical criteria. Transplanted rats exhibited a significant partial correction in stepping and placing in nonpharmacological behavioral tests, pole and rotarod tests. Taken together, our data encourage further investigations of the possible use of OBNSCs as a promising cell‐based therapeutic strategy for Parkinson's disease. This article is protected by copyright. All rights reserved
       
  • A hyaluronic acid‐based compound inhibits fibroblast senescence
           induced by oxidative stress in vitro and prevents oral mucositis in vivo
    • Abstract: Virtually all patients receiving radio‐ and chemotherapy for cancer develop oral mucositis, a severe and highly debilitating condition. The onset of mucositis is thought to involve the production of reactive oxygen species (ROS) in the submucosa. Here we investigated a possible protective effect of a commercial formulation of hyaluronic acid (HA) enriched with amino acids (Mucosamin®) against the damage induced by oxidative stress both in vitro and in vivo. Transient exposure of normal human oral fibroblasts to hydrogen peroxide (H2O2) led to irreversible senescence, as demonstrated by sustained increase in the levels of p16INK4A and SA‐βGal. Conditioned media from senescent fibroblasts induced detrimental effects on keratinocytes, as shown by reduced metabolic activity and migration capability. Pre‐treatment with Mucosamin® prevented H2O2‐induced, but not TGF‐β‐induced, fibroblast senescence with a concomitant reduction of fibroblast‐induced loss of keratinocyte vitality and functional activity. Finally, data from a case‐series of patients undergoing radio/chemotherapy strongly suggested that prophylactic use of the hyaluronic acid‐based compound in the form of a spray may be effective in preventing the onset of oral mucositis. This article is protected by copyright. All rights reserved
       
  • Resveratrol inhibits NLRP3 inflammasome activation by preserving
           mitochondrial integrity and augmenting autophagy
    • Abstract: The NLRP3 inflammasome is a caspase‐1‐containing multi‐protein complex that controls the release of IL‐1β and plays important roles in the development of inflammatory disease. Here, we report that resveratrol, a polyphenolic compound naturally produced by plants, inhibits NLRP3 inflammasome‐derived IL‐1β secretion and pyroptosis in macrophages. Resveratrol inhibits the activation step of the NLRP3 inflammasome by suppressing mitochondrial damage. Resveratrol also induces autophagy by activating p38, and macrophages treated with an autophagy inhibitor are resistant to the suppressive effects of resveratrol. In addition, resveratrol administration mitigates glomerular proliferation, glomerular sclerosis, and glomerular inflammation in a mouse model of progressive IgA nephropathy. These findings were associated with decreased renal mononuclear leukocyte infiltration, reduced renal superoxide anion levels, and inhibited renal NLRP3 inflammasome activation. Our data indicate that resveratrol suppresses NLRP3 inflammasome activation by preserving mitochondrial integrity and by augmenting autophagy. This article is protected by copyright. All rights reserved
       
  • Lipidomic approach towards deciphering anandamide effects in rat decidual
           cell
    • Abstract: Altered phospholipid (PL) metabolism has been associated with pregnancy disorders. Moreover, lipid molecules such as endocannabinoids (eCBs) and prostaglandins (PGs) are important mediators of reproductive events. In humans, abnormal decidualization has been linked with unexplained infertility, miscarriage and endometrial pathologies. Anandamide (AEA), the major eCB, induces apoptosis in rat decidual cells. In this study, the PL profile of rat decidual cells was characterized by a Mass spectrometry (MS) based lipidomic approach. Furthermore, we analyzed a possible correlation between changes in PL of rat decidual cells' membrane and AEA‐induced apoptosis. We found an increase in phosphatidylserine and a reduction of cardiolipin and phophatidylinositol relative contents. In addition, we observed an increase in the content of alkyl(alkenyl)acylPL, plasmalogens, and of long chain fatty acids especially with high degrees of unsaturation, as well as an increase in lipid hydroperoxides in treated cells. These findings provide novel insights on deregulation of lipid metabolism by anandamide, which may display further implications in decidualization process. This article is protected by copyright. All rights reserved
       
  • miR‐1470 mediates lapatinib induced p27 upregulation by targeting
           c‐jun
    • Abstract: Our previous study indicated that lapatinib induces p27‐dependent G1 arrest through both transcriptional and post‐translational mechanisms. Using miRNA microarray technology and quantitative RT‐PCR, we further investigated the potential miRNAs that involved in p27 upregulation and Her‐2 signaling pathway alteration with lapatinib treatment. A subset of 7 miRNAs was significantly affected in both 0.5μM and 2.0μM and 24 hours and 48 hours lapatinib treatment. Among them, only miR‐1470, miR‐126 and miR‐1208 were identified in the Her‐2 pathway after KEGG pathway analysis. However, luciferase reporter assay confirmed that miR‐1470 directly recognized the 3'‐untranslated region of c‐jun transcripts, which was consistent with TargetScan analysis. miR‐1470 significantly decreased c‐jun expression, thus miR‐1470 may repressc‐jun activation of cyclinD1 expression, and consequently promoted the upregulation of p27, a key molecule in the cell cycle arrest. Taken together, the present study provided the first evidences that miR‐1470 mediated lapatinib induced p27 upregulation by targeting c‐jun. This article is protected by copyright. All rights reserved
       
  • Chemokine (C‐C motif) ligand 5 is involved in tumor‐associated
           dendritic cell‐mediated colon cancer progression through
           non‐coding RNA MALAT‐1
    • Abstract: Tumor micro‐environment is a critical factor in the development of cancer. The aim of this study was to investigate the inflammatory cytokines secreted by tumor‐associated dendritic cells (TADCs) that contribute to enhanced migration, invasion, and epithelial‐to‐mesenchymal transition (EMT) in colon cancer. The administration of recombinant human chemokine (C‐C motif) ligand 5 (CCL5), which is largely expressed by colon cancer surrounding TADCs, mimicked the stimulation of TADC‐conditioned medium on migration, invasion, and EMT in colon cancer cells. Blocking CCL5 by neutralizing antibodies or siRNA transfection diminished the promotion of cancer progression by TADCs. Tumor‐infiltrating CD11c+ DCs in human colon cancer specimens were shown to produce CCL5. The stimulation of colon cancer progression by TADC‐derived CCL5 was associated with the up‐regulation of non‐coding RNA metastasis‐associated lung adenocarcinoma transcript 1 (MALAT‐1), which subsequently increased the expression of Snail. Blocking MALAT‐1 significantly decreased the TADC‐conditioned medium and CCL5‐mediated migration and invasion by decreasing the enhancement of Snail, suggesting that the MALAT‐1/Snail pathway plays a critical role in TADC‐mediated cancer progression. In conclusion, the inhibition of CCL5 or CCL5‐related signaling may be an attractive therapeutic target in colon cancer patients. This article is protected by copyright. All rights reserved
       
  • GRP78 / BiP / HSPA5 / Dna K is a universal therapeutic target for human
           disease
    • Abstract: The chaperone GRP78 / Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU‐03012 (AR‐12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70, HSP90, GRP94, GRP58, HSP27, HSP40 and HSP60. OSU‐03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola / Marburg / Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively. Pre‐treatment with OSU‐03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya, Mumps, Measles, Rubella, RSV, CMV and Influenza viruses. OSU‐03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi‐drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU‐03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU‐03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus Dna K and bacterial phosphodiesterases are novel antibiotic targets, and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. This article is protected by copyright. All rights reserved
       
  • Ubiquitin‐specific Protease 22 (USP22) Positively Regulates RCAN1
           Protein Levels through RCAN1 De‐ubiquitination
    • Abstract: Protein ubiquitination can be reversed by de‐ubiquitinating enzymes (DUBs), which are classified into two main classes, cysteine proteases and metalloproteases. Cysteine proteases include ubiquitin‐specific proteases (USPs) and ubiquitin C‐terminal hydrolases. USP22 is a USP family member and a component of the mammalian SAGA transcriptional co‐activating complex. Regulator of calcineurin 1 (RCAN1; also known as DSCR1 or MCIP1) functions as an endogenous inhibitor of calcineurin signaling. In the present study, we have identified a novel interaction between USP22 and RCAN1 (RCAN1‐1S) in the mammalian cells. In addition, the overexpression of USP22 caused the increase of RCAN1 protein stability. USP22 antagonized the actions of FBW7, NEDD4‐2, and β‐TrCP E3 ligase on RCAN1 and promoted RCAN1 de‐ubiquitination. Moreover, we found that RCAN1 was bound to USP22 in basal conditions, and interferon‐α (IFN‐α) treatment caused the dissociation of RCAN1 from USP22, which subsequently triggered RCAN1 ubiquitination and proteasome degradation. Taken together, these results suggest that USP22 positively regulates RCAN1 levels, which would consequently affect diverse RCAN1‐linked cellular processes such as the inflammatory process involving the release of IFN‐α. This article is protected by copyright. All rights reserved
       
  • Dual‐functions of miR‐373 and miR‐520c by differently
           regulating the activities of MMP2 and MMP9
    • Abstract: MicroRNA‐520c (miR‐520c) and microRNA‐373 (miR‐373) are originally characterized as both oncogenes and tumor suppressors in different types of human cancers. In this study, we found that translation of mRNA of MT1‐MMP, an oncogene related to tumor metastasis, was well inhibited by miR‐520c and miR‐373 in several types of human cancer cells. Our experimental data demonstrated that these two microRNAs inhibited the translation of mRNA of MT1‐MMP and down‐regulated its proteolytic enzyme activities via targeting 3'UTR of mRNA of MT1‐MMP, further decreased activating proMMP2 into active MMP2 in fibrosarcoma HT1080, benign prostatic hyperplasia epithelial cell BPH‐1 and glioblastoma U87GM. More interestingly, from the effects of microRNAs on cell functions, we found that cell growth were all blocked on fibronectin and type IV collagen coated plates and also in three‐dimension type I collagen lattice but enhanced only in HT1080 cells on type IV collagen coated plates and in three‐dimension type I collagen lattice; cell migration results showed the same effect as that of cell growth. The difference was due to up‐regulating the expression of MMP9 gene by miR‐520c and miR‐373 in HT1080 cells but not in BPH‐1 and U87GM cells. Our findings suggest that miR‐520c and miR‐373, which have different roles in different type of cancer via regulating the translation of mRNA of MT1‐MMP and the expression of MMP9 gene, might have an important clue on clinic when selecting the therapeutic regimen and finding new drugs for intervention in different kinds of cancer. This article is protected by copyright. All rights reserved
       
  • Hepatic Regenerative Potential of Mouse Bone Marrow Very Small
           Embryonic‐Like Stem Cells
    • Abstract: Very small embryonic‐like stem cells (VSELs) are a Sca‐1+Lin‐CD45‐ cell population that has been isolated from the bone marrow of mice. The similarities and differences between the mRNA profiles of VSELs and embryonic stem (ES) cells have not yet been defined. Here, we report the whole genome gene expression profile of VSELs and ES cells. We analyzed the global gene expression of VSELs and compared it with ES cells by microarray analysis. We observed that 9,521 genes are expressed in both VSELs and ES cells, 1,159 genes are expressed uniquely in VSELs, and 420 genes are expressed uniquely in ES cells. We found that although VSELs are similar to ES cells in their expression of genes associated with stem cell behavior and pluripotency, there are also differences in their mRNA expression. We further analyzed the expression of stem cell‐associated genes in VSELs and ES cells, and found that there were differences in these genes. For instance, the Pkd2 and Yap1 gene were reduced in their expression in VSELs when compared with ES cells. But we also found Zfp54 gene expression was higher in VSELs compared with ES cells. More interestingly, we demonstrated that VSELs express c‐kit, the stem cell factor (SCF) receptor. In vitro, SCF promoted VSEL differentiation into hepatic colonies in the presence of hepatocyte growth factor. In vivo, transplantation of VSELs directly into CCl4‐induced injured livers significantly reduced serum ALT and AST levels. Therefore, these data suggest that VSELs play a role in the repair of injured livers. This article is protected by copyright. All rights reserved
       
  • Dual Effect of Cyanidin on RANKL‐Induced Differentiation and Fusion
           of Osteoclasts
    • Abstract: Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts are multinucleated cells derived from hematopoietic stem cells (HSCs) or monocyte/macrophage progenitor cells and formed by osteoclasts precursors (OCPs) fusion. Cyanidin is an anthocyanin widely distributed in food diet with novel antioxidant activity. However, the effect of cyanidin on osteoclasts is still unknown. We investigated the effect of cyanidin on RANKL‐induced osteoclasts differentiation and cell fusion. The results showed that cyanidin had a dual effect on RANKL‐induced osteoclastogenesis. Lower dosage of cyanidin (< 1μg/ml) has a promoting effect on osteoclastogenesis while higher dosage of cyanidin (> 10μg/ml) has an inhibitory effect. Fusogenic genes like CD9, ATP6v0d2, DC‐STAMP, OC‐STAMP and osteoclasts related genes like NFATc1, mitf and c‐fos were all regulated by cyanidin consistent to its dual effect. Further exploration showed that low concentration of cyanidin could increase osteoclasts fusion whereas higher dosage of cyanidin lead to the increase of LXR‐β expression and activation which is suppressive to osteoclasts differentiaton. All these results showed that cyanidin exhibits therapeutic potential in prevention of osteoclasts related bone disorders. This article is protected by copyright. All rights reserved
       
  • Angiopoietin‐1 protects the endothelial cells against advanced
           glycation end product injury by strengthening cell junctions and
           inhibiting cell apoptosis
    • Abstract: Endothelial dysfunction is a major characteristic of diabetic vasculopathy. Protection of the vascular endothelium is an essential aspect of preventing and treating diabetic vascular complications. Although Angiopoietin‐1 (Ang‐1) is an important endothelial‐specific protective factor, whether Ang‐1 protects vascular cells undergoing advanced glycation end product (AGE) injury has not been investigated. The aim of the present study was to determine the potential effects of Ang‐1 on endothelial cells after exposure to AGE. We show here that Ang‐1 prevented AGE‐induced vascular leakage by enhancing the adherens junctions between endothelial cells, and this process was mediated by the phosphorylation and membrane localization of VE‐cadherin. Furthermore, Ang‐1 also protected endothelial cells from AGE‐induced death by regulating phosphatidylinositol 3‐kinase (PI3K)/Akt‐dependent Bad phosphorylation. Our findings suggest that the novel protective mechanisms of Ang‐1 on endothelium are achieved by strengthening endothelial cell junctions and reducing endothelial cell death after AGE injury. This article is protected by copyright. All rights reserved
       
  • Cannabinoid receptor 1 but not 2 mediates macrophage phagocytosis by
           G(α)i/o/RhoA/ROCK signaling pathway
    • Abstract: Phagocytosis is critical to macrophages linking innate and adaptive immune reaction. Cannabinoid receptor 1 (CB1) and 2 (CB2) mediate immune modulation. However, the role of cannabinoid receptors in macrophage phagocytosis is undefined. In this study, we found that two murine macrophage lines (J774A.1 and RAW264.7) and peripheral blood macrophages all expressed CB1 and CB2 by immunofluorescence‐staining, real time RT‐PCR and Western blot. Macrophage phagocytic activity was determined by quantifying fluorescent intensity of the engulfed BioParticles or fluorescence‐activated cell sorting. mAEA (CB1 agonist) enhanced phagocytosis of macrophages, but JWH133 (CB2 agonist) had no influence. Pharmacological or genetic ablation of CB1 inhibited mAEA‐enhanced phagocytosis, while CB2 had no such effects. Meanwhile, activation of CB1 increased GTP‐bounding active form of small GTPase RhoA, but not Rac1 or Cdc42. AM281 (CB1 antagonist) and pertussis toxin (PTX, G(α)i/o protein inhibitor) decreased GTP‐bound RhoA protein level with mAEA. In addition, PTX, C3 Transferase (RhoA inhibitor) or Y27632 (Rho‐associated kinase ROCK inhibitor) attenuated CB1‐mediated phagocytosis. These results confirm that activation of CB1 regulates macrophage phagocytosis through G(α)i/o/RhoA/ROCK signaling pathway. Moreover, activation of CB1 induced significant up‐regulation of CB1 expression by real time RT‐PCR and Western blot analysis, but not CB2. It indicated the existence of a positive feedback between CB1 activation and CB1 expression. The up‐regulation of CB1 was RhoA‐independent but it may contribute to maintaining high phagocytic activity of macrophages for a longer time. In conclusion, CB1 mediates macrophage phagocytosis by G(α)i/o/RhoA/ROCK signal axis. These data further underline the role of CB1 in macrophage phagocytic process. This article is protected by copyright. All rights reserved
       
  • Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating
           enamel gene expression
    • Abstract: Although Bmp2 is essential for tooth formation, the role of Bmp2 during enamel formation remains unknown in vivo. In this study, the role of Bmp2 in regulation of enamel formation was investigated by the Bmp2 conditional knock out (Bmp2 cKO) mice. Teeth of Bmp2 cKO mice displayed severe and profound phenotypes with asymmetric and misshaped incisors as well as abrasion of incisors and molars. Scanning electron microscopy analysis showed that the enamel layer was hypoplastic and enamel lacked a typical prismatic pattern. Teeth from null mice were much more brittle as tested by shear and compressive moduli. Expression of enamel matrix protein genes, amelogenin, enamelin, and enamel‐processing proteases, Mmp‐20 and Klk4 was reduced in the Bmp2 cKO teeth as reflected in a reduced enamel formation. Exogenous Bmp2 up‐regulated those gene expressions in mouse enamel organ epithelial cells. This result for the first time indicates Bmp2 signaling is essential for proper enamel development and mineralization in vivo. This article is protected by copyright. All rights reserved
       
  • Down‐regulated CFTR during aging contributes to benign prostatic
           hyperplasia
    • Abstract: Benign prostatic hyperplasia (BPH) is a hyper‐proliferative disease of the aging prostate; however, the exact mechanism underlying the development of BPH remains incompletely understood. The present study investigated the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR), which has been previously shown to negatively regulate nuclear factor‐κB (NF‐κB)/cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) pathway, in the pathogenesis of BPH. Our results showed decreasing CFTR and increasing COX2 expression in rat prostate tissues with aging. Furthermore, suppression of CFTR led to increased expression of COX2 and over‐production of PGE2 in a normal human prostate epithelial cell line (PNT1A) with elevated NF‐κB activity. PGE2 stimulated the proliferation of primary rat prostate stromal cells but not epithelial cells, with increased PCNA expression. In addition, the condition medium from PNT1A cells after inhibition or knockdown of CFTR promoted cell proliferation of prostate stromal cells which could be reversed by COX2 or NF‐κB inhibitor. More importantly, the involvement of CFTR in BPH was further demonstrated by the down‐regulation of CFTR and up‐regulation of COX2/NF‐κB in human BPH samples. The present results suggest that CFTR may be involved in regulating PGE2 production through its negative regulation on NF‐κB/COX2 pathway in prostate epithelial cells, which consequently stimulates cell growth of prostate stromal cells. The overstimulation of prostate stromal cell proliferation by downregulation of CFTR‐enhanced PGE2 production and release during aging may contribute to the development of BPH. This article is protected by copyright. All rights reserved
       
 
 
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