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Journal Cover   Journal of Cellular Physiology
  [SJR: 1.608]   [H-I: 118]   [4 followers]  Follow
    
   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  [1606 journals]
  • DJ‐1 protects breast cancer cells against
           2'‐benzoyloxycinnamaldehyde‐induced oxidative stress
           independent of Nrf2
    • Authors: Ismail Ahmed Ismail; Abo bakr Abdel shakor, Su‐Hyung Hong
      Pages: n/a - n/a
      Abstract: 2'‐benzoyloxycinnamaldehyde (BCA) is a promising antitumor agent. BCA effectively inhibited proliferation of MDA‐MB‐435 more than in MCF‐7 breast cancer cells. Our recent findings showed that DJ‐1 protects MCF7 cells from BCA‐induced oxidative stress via its mitochondrial translocation and inhibition of the mitochondrial perturbation (Ismail et al., 2012). In this study, we addressed the question of whether Nrf2 works downstream to DJ‐1 in mediating differential antiproliferation effects in MCF‐7 and MDAMB‐435 breast cancer cells induced by BCA treatment. BCA upregulated the expression and induced nuclear translocalization of DJ‐1 and Nrf2 in only MCF‐7 cells. However, in MDA‐MB‐435, BCA increased only Nrf2 expression without inducing DJ‐1 and/or Nrf2 protein translocalization to the nucleus. Furthermore, DJ‐1 knockdown decreased DJ‐1 expression in both cells without affecting Nrf2 and its downstream target γ‐GCS; suggesting that DJ‐1‐induced cell protection and works independent of Nrf2 signaling pathway. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-09T17:20:46.477227-05:
      DOI: 10.1002/jcp.24957
       
  • Targeting osteopontin, the silent partner of NA+/H+ exchanger isoform 1 in
           cardiac remodeling
    • Authors: Iman A. Mohamed; Fatima Mraiche
      Pages: n/a - n/a
      Abstract: Cardiac hypertrophy (CH), characterized by the enlargement of cardiomyocytes, fibrosis and apoptosis, contributes to cardiac remodeling, which if left unresolved results in heart failure. Understanding the signaling pathways underlying CH is necessary to identify potential therapeutic targets. The Na+/H+‐exchanger isoform I (NHE1), a ubiquitously expressed glycoprotein and cardiac specific isoform, regulates intracellular pH. Recent studies have demonstrated that enhanced expression/activity of NHE1 contributes to cardiac remodeling and CH. Inhibition of NHE1 in both in vitro and in vivo models have suggested that inhibition of NHE1 protects against hypertrophy. However, clinical trials using NHE1 inhibitors have proven to be unsuccessful, suggesting that additional factors maybe contributing to cardiac remodeling. Recent studies have indicated that the upregulation of NHE1 is associated with enhanced levels of osteopontin (OPN) in the setting of CH. OPN has been demonstrated to be upregulated in left ventricular hypertrophy, dilated cardiomyopathy and in diabetic cardiomyopathy. The cellular interplay between OPN and NHE1 in the setting of CH remains unknown. This review focuses on the role of NHE1 and OPN in cardiac remodeling and emphasizes the signaling pathways implicating OPN in the NHE1‐induced hypertrophic response. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-09T17:20:20.178571-05:
      DOI: 10.1002/jcp.24958
       
  • A High Fat Diet Increases Bone Marrow Adipose Tissue (MAT) But Does Not
           Alter Trabecular or Cortical Bone Mass in C57BL/6J Mice
    • Authors: Casey R. Doucette; Mark C. Horowitz, Ryan Berry, Ormond A. MacDougald, Rea Anunciado‐Koza, Robert A. Koza, Clifford J. Rosen
      Pages: n/a - n/a
      Abstract: Obesity has been associated with high bone mineral density (BMD) but a greater propensity to fracture. Some obese individuals have increased marrow adipose tissue (MAT), but the impact of MAT on bone turnover remains controversial, as do changes in BMD associated with a high fat diet (HFD). In this study we hypothesized that MAT volume would increase in response to HFD but would be independent of changes in BMD. Hence, we fed C57BL/6J (B6) male mice at 3 weeks of age either a high fat diet (60 kcal %) or regular diet (10 kcal %) for 12 weeks (n = 10/group). We measured MAT volume by osmium staining and micro‐CT (µCT) as well as bone parameters by µCT, histomorphometry, and dual‐energy X‐ray absorptiometry. We also performed a short‐term pilot study using 13‐week‐old B6 males and females fed a HFD (58 kcal %) for 2 weeks (n = 3/sex). Both long‐ and short‐term HFD feedings were associated with high MAT volume, however, femoral trabecular bone volume fraction (BV/TV), bone formation rate and cortical bone mass were not altered in the long‐term study. In the short‐term pilot study, areal BMD was unchanged after two weeks of HFD. We conclude that, for B6 mice fed a HFD starting at wean or 13 weeks of age, MAT increases whereas bone mass is not altered. More studies are needed to define the mechanism responsible for the rapid storage of energy in the marrow and its distinction from other adipose depots. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-07T03:44:02.295724-05:
      DOI: 10.1002/jcp.24954
       
  • LB‐1 Exerts Antitumor Activity in Pancreatic Cancer by Inhibiting
           HIF‐1α and Stat3 Signaling
    • Authors: Fei Niu; Yan Li, Fang‐Fang Lai, Lin Ni, Ming Ji, Jing Jin, Han‐Ze Yang, Chao Wang, Dong‐Ming Zhang, Xiao‐Guang Chen
      Abstract: Hypoxia is widely present in pancreatic cancer and subsequently causes the overexpression of hypoxia‐inducible factor‐1α (HIF‐1α) and signal transducer and activator of transcription‐3 (Stat3). HIF‐1α and Stat3 function cooperatively to regulate a number of downstream genes that are implicated in tumorigenesis. Thus, inhibition of HIF‐1α and Stat3 is a potential therapeutic strategy for pancreatic cancer. In this study, we explored how LB‐1, a novel triptolide (LA) derivative, exerted its antitumor effect through blockade of HIF‐1α and Stat3 signaling. Our data showed that LB‐1 was able to inhibit the proliferation and colony formation of Mia‐PaCa2 and SW1990 cells. LB‐1 suppressed HIF‐1α protein accumulation by promoting its proteasome degradation and reducing transactivation. Moreover, the silence of HIF‐1α by shRNA partially prevented the proliferation inhibition triggered by LB‐1. As expected, LB‐1 also decreased Stat3 protein accumulation and blocked the physical interactions between HIF‐1α/p300/phosphor‐Stat3 (p‐Stat3) at the pharmacological concentration to reduce VEGF expression, thereby hypoxia‐induced angiogenesis. In the Mia‐PaCa2 nude xenograft model, therapeutic treatment with LB‐1 significantly inhibited tumor growth and had minimal systemic toxicity compared to the mother drug LA. Furthermore, in accordance with in vitro results, HIF‐1α activation and Stat3 expression in tumors were blocked by LB‐1 through mTOR‐dependent pathway. Taken together, these results illustrate that, as a potent inhibitor of HIF‐1α and Stat3 signaling, LB‐1 exhibits antitumor effect and could be potentially used to treat pancreatic cancer. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:37:27.355969-05:
      DOI: 10.1002/jcp.24949
       
  • TSH/TSHR Signaling Suppresses Fatty Acid Synthase (FASN) Expression in
           Adipocytes
    • Authors: Jicui Chen; Jianmin Ren, Qingping Jing, Sumei Lu, Yuchao Zhang, Yuantao Liu, Cong Yu, Peng Gao, Chen Zong, Xia Li, Xiangdong Wang
      Abstract: TSH/TSHR signaling plays a role in the regulation of lipid metabolism in adipocytes. However, the precise mechanisms are not known. In the present study, we determined the effect of TSH on fatty acid synthase (FASN) expression, and explored the underlying mechanisms. In vitro, TSH reduced FASN expression in both mRNA and protein levels in mature adipocytes and was accompanied by protein kinase A (PKA) activation, cAMP‐response element binding protein (CREB) phosphorylation, as well as extracellular signal‐regulated kinase 1/2 (ERK1/2) and c‐Jun NH2‐terminal kinase (JNK) activation. TSH‐induced down‐regulation of FASN was partially abolished by inhibition of PKA and ERK, but not JNK. TSHR and FASN expression in visceral tissue was significantly increased in C57BL/6 mice with diet‐induced obesity compared with control animals, whereas thyroid TSHR expression was normal. These findings suggest that activation of TSHR directly inhibits FASN expression in mature adipocytes, possibly mediated by PKA and ERK. In obese animals, this function of TSHR seems to be counteracted. The precise mechanisms need further investigation. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:37:11.744617-05:
      DOI: 10.1002/jcp.24952
       
  • Wnt5a Induces Collagen Production by Human Periodontal Ligament Cells
           through TGFβ1‐mediated Upregulation of Periostin Expression
    • Authors: Daigaku Hasegawa; Naohisa Wada, Hidefumi Maeda, Shinichiro Yoshida, Hiromi Mitarai, Atsushi Tomokiyo, Satoshi Monnouchi, Sayuri Hamano, Asuka Yuda, Akifumi Akamine
      Abstract: Wnt5a, a member of the noncanonicalWnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this study, we examined the localization and potential function of Wnt5a in PDL tissue. Immunohistochemical analysis revealed that Wnt5a was expressed predominantly in rat PDL tissue. Semi‐quantitative reverse‐transcription polymerase chain reaction and western blotting analysis demonstrated that human PDL cells (HPDLCs) expressed Wnt5a and its receptors (Ror2, Fzd2, Fzd4, and Fzd5). Removal of occlusal pressure by extraction of opposing teeth decreased Wnt5a expression in rat PDL tissue, and the expression of Wnt5a and its receptors in HPDLCs was upregulated by exposure to mechanical stress. Stimulation with Wnt5a significantly enhanced the proliferation and migration of HPDLCs. Furthermore, Wnt5a suppressed osteoblastic differentiation of HPDLCs cultivated in osteogenic induction medium, while it significantly enhanced the expression of PDL‐related genes, such as periostin, type‐I collagen, and fibrillin‐1 genes, and the production of collagen in HPDLCs cultivated in normal medium. Both knockdown of periostin gene expression by siRNA and inhibition of TGFβ1 function by neutralizing antibody suppressed the Wnt5a‐induced PDL‐related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFβ1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFβ1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFβ1‐mediated upregulation of periostin expression. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:36:55.648137-05:
      DOI: 10.1002/jcp.24950
       
  • Concise Review: The Role of Oxygen in Hematopoietic Stem Cells Physiology
    • Authors: Mojca Jež; Primož Rožman, Zoran Ivanović, Tuba Bas
      Abstract: Molecular dioxygen, O2, is an important element in cellular microenvironment in vivo, and often overlooked in standard in vitro and ex vivo cell culture systems. Molecular oxygen is the ultimate electron acceptor in oxidative cellular respiration, and also a signal that regulates cell fate through concentration gradients. Recent advances in physiology of oxygen and adult stem cell research have shown that apart from being important for oxidative phosphorylation, thus energy metabolism, oxygen is also important as a signaling molecule and an integral part of stem cell niche. This review article covers the influence of physiologically relevant oxygen levels on adult stem cells through highlighting the research on the effect of oxygen concentration on hematopoietic stem cell maintenance, proliferation and differentiation. This is important particularly to understand the embryonic and adult stem cell biology and physiology. The new discoveries in this field will help to further improve current tissue engineering and clinical applications. In addition, understanding the relationship between oxygen and stemness is invaluable for the advanced treatments of neoplastic diseases.
      Authors believe that in future, active and programmed dynamic of oxygen levels will be routinely used for the programmed in vitro and ex‐vivo expansion of different adult stem cell types and tissue regeneration purposes. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:36:36.448372-05:
      DOI: 10.1002/jcp.24953
       
  • The SRE Motif in the Human PNPLA3 Promoter (–97 to –88bp)
           Mediates Transactivational Effects of SREBP‐1c
    • Authors: Hua Liang; Jing Xu, Fen Xu, Hongxia Liu, Ding Yuan, Shuhua Yuan, Mengyin Cai, Jinhua Yan, Jianping Weng
      Abstract: Patatin‐like phospholipase domain containing 3(PNPLA3) is a non‐secreted protein primarily expressed in liver and adipose tissue. Recently, numerous genetic studies have shown that PNPLA3 is a major susceptibility gene for nonalcoholic fatty liver disease (NAFLD). However, the mechanism involved in transcriptional regulation of the PNPLA3 gene remains unknown. We performed a detailed analysis of the human PNPLA3 gene promoter and identified two novel cis‐acting elements (SRE and NFY binding motifs) located at ‐97/‐88 and ‐26/‐22 bp, respectively. Overexpression of SREBP‐1c in HepG2 cells significantly increased PNPLA3 promoter activity. Mutation of either of the putative SRE or NFY binding motifs blocked the transactivation effects of SREBP‐1c on the promoter. Overexpression of SREBP‐1c and NFY together increased PNPLA3 promoter activity twice as much as that of SREBP‐1c or NFY expression alone. This result suggests that SREBP‐1c and NFY synergistically transactivate the human PNPLA3 gene. The ability of SREBP‐1c and NFY to bind these cis‐elements was confirmed using gel shift analysis. Putative SRE and NFY motifs also mediated synergistic insulin‐induced transactivation of the PNPLA3 promoter in HepG2 cells. Additionally, the ability of SREBP‐1c to bind to the PNPLA3 promoter was increased by insulin in a dose‐dependent manner. Moreover, the treatment of HepG2 cells with the PI3K inhibitor LY294002 led to reduced insulin promoter‐activating ability accompanied by a decrease in PNPLA3 and SREBP‐1c protein expression. These results demonstrate that SREBP‐1c is a direct activator of the human PNPLA3 gene and insulin transactivates the PNPLA3 gene via the PI3K‐SREBP‐1c/NFY pathway in HepG2 cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:36:20.953847-05:
      DOI: 10.1002/jcp.24951
       
  • Extracellular Ca2+ Promotes Odontoblastic Differentiation of Dental Pulp
           Stem Cells Via BMP2‐Mediated Smad1/5/8 and Erk1/2 Pathways
    • Authors: Shiting Li; Jing Hu, Gang Zhang, Wei Qi, Ping Zhang, Pengfei Li, Yong Zeng, Wenfeng Zhao, Yinghui Tan
      Abstract: Ca2+ is the main element of many pulp capping materials that are used to promote the regeneration of tertiary dentin, but the underlying molecular mechanism is not clear. In this study, we found that Ca2+ increased the expression of the odontoblastic differentiation marker gene DSPP and promoted odontoblastic differentiation and mineralization of DPSCs, but inhibited ALP activity. Ca2+ increases the expression of endogenous BMP2, which activates the Smad1/5/8 pathway and promotes the Smad1‐Runx2 and Runx2‐DSPP interaction in DPSCs. Inhibition of Smad1/5/8 with dorsomorphin partially blocked Runx2 activity; however, inhibition of the BMP2 receptor with Noggin nearly fully suppressed Runx2 activity. These results indicate that Ca2+ promotes cell differentiation mainly via BMP2‐mediated Smad‐dependent and Smad‐independent pathways. We then determined that the phosphorylation level of Erk1/2, but not JNK or p38, was significantly increased as a result of Ca2+ stimulation. Blockage of Erk1/2 was found to inhibit Runx2 activity, indicating that Ca2+ triggers the Erk1/2 pathway, which subsequently regulates Runx2 activity. In addition, inhibition of Erk1/2 differentially attenuated the phosphorylation levels of Smad1/5/8 and Smad2/3. Collectively, this study demonstrates that Ca2+ activates the BMP2‐mediated Smad1/5/8 and Erk1/2 pathways in DPSCs and that Smad1/5/8 and Erk1/2 signaling converge at Runx2 to control the odontoblastic differentiation of DPSCs. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:27:30.530524-05:
      DOI: 10.1002/jcp.24945
       
  • Alpha‐Lipoic Acid Promotes Osteoblastic Formation in
           H2O2‐Treated MC3T3‐E1 Cells and Prevents Bone Loss in
           Ovariectomized Rats
    • Authors: Chao Fu; Dong Xu, Chang‐Yuan Wang, Yue Jin, Qi Liu, Qiang Meng, Ke‐Xin Liu, Hui‐Jun Sun, Mo‐Zhen Liu
      Abstract: Alpha‐lipoic acid (ALA), a naturally occurring compound and dietary supplement, has been established as a potent antioxidant that is a strong scavenger of free radicals. Recently, accumulating evidences has indicated the relationship between oxidative stress and osteoporosis. Some studies have investigated the possible beneficial effects of ALA on osteoporosis both in vivo and in vitro; however, the precise mechanism(s) underlying the bone‐protective action of ALA remains unclear. Considering this, we focused on the anti‐oxidative capacity of ALA to exert bone‐protective effects in vitro and in vivo. In the present study, the effects of ALA on osteoblastic formation in H2O2‐treated MC3T3‐E1 pre‐osteoblasts and ovariectomy (OVX)‐induced bone loss in rats were investigated. The results showed that ALA promoted osteoblast differentiation, mineralization and maturation and inhibited osteoblast apoptosis, thus increasing the OPG/RANKL ratio and leading to enhanced bone formation in vitro and inhibited bone loss in vivo. Further study revealed that ALA exerted its bone‐protective effects by inhibiting reactive oxygen species (ROS) generation by down‐regulating Nox4 gene expression and protein synthesis and attenuating the transcriptional activation of NF‐κB. In addition, ALA might exert its bone‐protective effects by activating the Wnt/Lrp5/β‐catenin signaling pathway. Taken together, the present study indicated that ALA promoted osteoblastic formation in H2O2‐treated MC3T3‐E1 cells and prevented OVX‐induced bone loss in rats by regulating Nox4/ROS/NF‐κB and Wnt/Lrp5/β‐catenin signaling pathways, which provided possible mechanisms of bone‐protective effects in regulating osteoblastic formation and preventing bone loss. Taken together, the results suggest that ALA may a candidate for clinical osteoporosis treatment. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:27:12.995181-05:
      DOI: 10.1002/jcp.24947
       
  • Myocyte Enhancer Factor 2A Regulates Hydrogen Peroxide‐Induced
           Senescence of Vascular Smooth Muscle Cells via microRNA‐143
    • Authors: Wang Zhao; Xi‐Long Zheng, Dao‐Quan Peng, Shui‐Ping Zhao
      Abstract: Background Myocyte enhancer factor 2A (MEF2A) is involved in vascular smooth muscle cell (VSMC) proliferation, migration, and senescence. MicroRNA‐143/145 (miR‐143/145), which may be regulated by MEF2A, is known to promote cellular senescence. We hypothesized that MEF2A may promote VSMC senescence via miR‐143/145. Methods VSMC senescence was induced by hydrogen peroxide (H2O2), followed by detection using a senescence‐associated β‐galactosidase staining kit. The MEF2A protein, mRNA, and miR‐143/145 levels in VSMCs were detected using western blot analysis and SYBR green real‐time quantitative PCR, respectively. We further manipulated the expression levels of MEF2A and miR‐143 through viral or transient transfection. VSMC proliferation and migration were determined by methylthiazolyldiphenyl‐tetrazolium bromide and Millicell chamber, respectively. Results Both MEF2A and miR‐143, but not miRNA‐145, were up‐regulated in senescent VSMCs. Overexpression of either MEF2A or miR‐143 significantly enhanced VSMC senescence, but reduced proliferation and migration. MEF2A knockdown or miR‐143 inhibitor suppressed cellular senescence and increased proliferation and migration. We further revealed AKT signaling as a potential miR‐143 target, and an induction of miR‐143 expression by MEF2A via KLF2. Additionally, overexpression of MEF2A and miR‐143 resulted in synergistic effects on promotion of senescence, and MEF2A knockdown and miR‐143 reduction by inhibitor had synergistic inhibitory effects. Finally, MEF2A barely promoted VSMC senescence when miR‐143 was inhibited, and miR‐143 overexpression antagonized the inhibitory effect of MEF2A knockdown on VSMC senescence. Conclusions Our results revealed a link and interaction between MEF2A and miR‐143 and suggested a potential mechanism for MEF2A to regulate H2O2‐induced VSMC senescence. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:26:54.822511-05:
      DOI: 10.1002/jcp.24948
       
  • Environmental Disruption of Circadian Rhythm Predisposes Mice to
           Osteoarthritis‐Like Changes in Knee Joint
    • Authors: Ranjan Kc; Xin Li, Robin M Voigt, Michael B Ellman, Keith C Summa, Martha Hotz Vitaterna, Ali Keshavarizian, Fred W Turek, Qing‐Jun Meng, Gary S. Stein, Andre J. van Wijnen, Di Chen, Christopher B Forsyth, Hee‐Jeong Im
      Abstract: Circadian rhythm dysfunction is linked to many diseases, yet pathophysiological roles in articular cartilage homeostasis and degenerative joint disease including osteoarthritis (OA) remains to be investigated in vivo. Here, we tested whether environmental or genetic disruption of circadian homeostasis predisposes to OA‐like pathological changes. Male mice were examined for circadian locomotor activity upon changes in the light:dark (LD) cycle or genetic disruption of circadian rhythms. Wild‐type (WT) mice were maintained on a constant 12 hour:12 hour LD cycle (12:12 LD) or exposed to weekly 12 hour phase shifts. Alternatively, male circadian mutant mice (ClockΔ19 or Csnk1etau mutants) were compared with age‐matched WT littermates that were maintained on a constant 12:12 LD cycle. Disruption of circadian rhythms promoted osteoarthritic changes by suppressing proteoglycan accumulation, upregulating matrix‐degrading enzymes and downregulating anabolic mediators in the mouse knee joint. Mechanistically, these effects involved activation of the PKCδ‐ERK‐RUNX2/NFκB and β‐catenin signaling pathways, stimulation of MMP‐13 and ADAMTS‐5, as well as suppression of the anabolic mediators SOX9 and TIMP‐3 in articular chondrocytes of phase‐shifted mice. Genetic disruption of circadian homeostasis does not predispose to OA‐like pathological changes in joints. Our results, for the first time, provide compelling in vivo evidence that environmental disruption of circadian rhythms is a risk factor for the development of OA‐like pathological changes in the mouse knee joint. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:26:37.299354-05:
      DOI: 10.1002/jcp.24946
       
  • Shear Stress Modulates Resistin‐Induced CC Chemokine Ligand 19
           Expression in Human Aortic Endothelial Cells
    • Authors: Hong‐Ren Yu; Mao‐Ling Sung, Hsing‐Chun Kuo, Chi‐Hui Lin, Cheng‐Nan Chen
      Abstract: Resistin may be an important link between obesity and diabetes. Recent studies have suggested an association between resistin and atherogenic processes. In addition, CCL19 is highly expressed in human atherosclerotic lesions. The interplays among resistin, CCL19, and shear stress in regulating vascular endothelial function are not clearly understood. In the present study, resistin stimulation induced dose‐ and time‐dependent CCL19 expression in human aortic endothelial cells (HAECs). By using neutralizing antibody and small interfering (si)RNA, we demonstrated that toll‐like receptor 4 (TLR4) is critical for resistin‐induced CCL19 expression. Transcription factor ELISA and chromatin immunoprecipitation assays showed that resistin increased NF‐κB‐DNA binding activities in ECs. Inhibition of NF‐κB activation by specific siRNA blocked the resistin‐induced CCL19 promoter activity and expression. Preshearing of ECs for 12 h at 20 dyn/cm2 inhibited the resistin‐induced NF‐κB activation and CCL19 expression. Our findings serve to elucidate the molecular mechanisms underlying the resistin induction of CCL19 expression in ECs and the shear‐stress protection against this induction. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:24:40.679468-05:
      DOI: 10.1002/jcp.24940
       
  • Epigenetic Regulation of miR‐29s Affects the Lactation Activity of
           Dairy Cow Mammary Epithelial Cells
    • Authors: Yanjie Bian; Yu Lei, Chunmei Wang, Jie Wang, Lina Wang, Lili Liu, Lixin Liu, Xuejun Gao, Qingzhang Li
      Abstract: Milk is important for human nutrition, and enhanced milk quality has become a major selection criterion for the genetic improvement of livestock. Epigenetic modifications have been shown to be involved in mammary gland development; but the mechanisms underlying their effects remain unknown. MicroRNAs are involved in the regulation of milk synthesis and in mammary gland development. Our study is the first to investigate the roles of miR‐29s and epigenetic regulation in dairy cow mammary epithelial cells(DCMECs). Our results show that miR‐29s regulate the DNA methylation level by inversely targeting both DNMT3A and DNMT3B in DCMECs. The inhibition of miR‐29s caused global DNA hypermethylation and increased the methylation levels of the promoters of important lactation‐related genes, including casein alpha s1 (CSN1S1), E74‐like factor 5 (ElF5), peroxisome proliferator‐activated receptor gamma (PPARγ), sterol regulatory element binding protein‐1(SREBP1), and glucose transporter 1 (GLUT1). The inhibition of miR‐29s reduced the secretion of lactoprotein, triglycerides (TG) and lactose by DCMECs. Moreover, the treatment of DCMECs with 5‐aza‐2′‐deoxycytidine (5‐Aza‐dC) decreased the methylation levels of the miR‐29b promoter and increased the expression of miR‐29b. The link between miR‐29s and DNMT3A/3B enhances our understanding of the roles of miRNAs in mammary gland function, and our data will inform more experimentally oriented studies to identify new mechanisms of regulating lactation. We present new insights regarding the epigenetic regulation of lactation performance.Improved understanding of the molecular basis of lactation will aid in the development of strategies for optimizing milk quality in dairy cows and modifying the lactation performance of offspring. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:24:24.396909-05:
      DOI: 10.1002/jcp.24944
       
  • A Novel Role for Thrombopoietin in Regulating Osteoclast Development in
           Humans and Mice
    • Authors: Monique Bethel; Calvin L. T. Barnes, Amanda F. Taylor, Ying‐Hua Cheng, Brahmananda R. Chitteti, Mark C. Horowitz, Angela Bruzzaniti, Edward F. Srour, Melissa A. Kacena
      Abstract: Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK‐related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell‐cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c‐mpl or CD110) is expressed on cells of the OC lineage, c‐mpl is required for TPO to enhance OC formation in vitro, and TPO activates the MAPK, JAK/STAT, and NFκB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14 + CD110+ human OC progenitors derived from peripheral blood mononuclear cells (PBMCs), and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c‐mpl signaling as well as in patients suffering from bone disorders. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:24:07.472493-05:
      DOI: 10.1002/jcp.24943
       
  • Mitochondrial DNA Oxidative Damage Contributes to Cardiomyocyte
           Ischemia/Reperfusion‐Injury in Rats: Cardioprotective Role of
           Lycopene
    • Authors: Rongchuan Yue; Xuewei Xia, Jiahui Jiang, Dezhong Yang, Yu Han, Xiongwen Chen, Yue Cai, Liangpeng Li, Wei Eric Wang, Chunyu Zeng
      Abstract: Mitochondrial (mt) dysfunction and oxidative stress are involved in the pathogenesis of ischemia/reperfusion (I/R)‐injury. Lycopene, a lipophilic antioxidant found mainly in tomatoes and in other vegetables and fruits, can protect mtDNA against oxidative damage. However, the role of mtDNA in myocardial I/R‐injury is unclear. In the present study, we aimed to determine if and how lycopene protects cardiomyocytes from I/R‐injury. In both in vitro and in vivo studies, I/R‐injury increased mt 8‐hydroxyguanine (8‐OHdG) content, decreased mtDNA content and mtDNA transcription levels, and caused mitochondrial dysfunction in cardiomyocytes. These effects of I/R injury on cardiomycoytes were blocked by pre‐treatment with lycopene. MtDNA depletion alone was sufficient to induce cardiomyocyte death. I/R‐injury decreased the protein level of a key activator of mt transcription, mitochondrial transcription factor A (Tfam), which was blocked by lycopene. The protective effect of lycopene on mtDNA was associated with a reduction in mitochondrial ROS production and stabilization of Tfam. In conclusion, lycopene protects cardiomyocytes from the oxidative damage of mtDNA induced by I/R‐injury. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:23:50.54021-05:0
      DOI: 10.1002/jcp.24941
       
  • MicroRNA‐214 is Up‐Regulated in Heart Failure Patients and
           Suppresses XBP1‐Mediated Endothelial Cells Angiogenesis
    • Authors: Quanlu Duan; Lei Yang, Wei Gong, Sandip chaugai, Feng Wang, Chen Chen, Peihua Wang, Ming‐Hui Zou, Dao Wen Wang
      Abstract: More and more miRNAs have been shown to regulate gene expression in the heart and dysregulation of their expression has been linked to cardiovascular diseases including the miR‐199a/214 cluster. However, the signature of circulating miR‐214 expression and its possible roles during the development of heart failure has been less well studied. In this study, we elucidated the biological and clinical significance of miR‐214 dysregulation in heart failure. Firstly, circulating miR‐214 was measured by quantitative PCR, and we found that miR‐214 was upregulated in the serum of chronic heart failure patients, as well as in hypertrophic and failing hearts of humans and mice. Adeno‐associated virus serotype 9 (AAV9)‐mediated miR‐214 silencing attenuates isoproterenol (ISO) infusion‐induced cardiac dysfunction and impairment of cardiac angiogenesis in mice. Mechanistically, miR‐214 overexpression reduces angiogenesis of HUVECs by targeting XBP1, an important transcription factor of unfolded protein response, and XBP1 silencing decreases HUVECs proliferation and angiogenesis similar to miR‐214 overexpression. Furthermore, ectopic expression of XBP1 enhances endothelial cells proliferation and tube formation, and reverses anti‐angiogenic effect of miR‐214 over expression. All these findings suggest that miR‐214 is an important regulator of angiogenesis in heart in vitro and in vivo, likely via regulating the expression of XBP1, and demonstrate that miR‐214 plays an essential role in the control/inhibition of cardiac angiogenesis. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-05T05:23:20.268101-05:
      DOI: 10.1002/jcp.24942
       
  • MNADK, a long‐awaited human mitochondrion‐localized NAD kinase
    • Authors: Ren Zhang
      Abstract: Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, play essential roles in numerous cellular processes in all organisms. NADP maintains a pool of its reducing equivalent, NADPH, which regenerates cellular oxidative defense systems to counteract oxidative damages. Mitochondria representa major source of oxidative stress, because the majority of superoxide, a reactive oxygen species, is generated from the mitochondrial respiratory chain. Therefore, as universal electron carriers in cellular electron transfer reactions, the pyridine nucleotides are required by mitochondria for both antioxidant protection and biosynthetic pathways.The NAD kinase (NADK) is the sole NADP biosynthetic enzyme. Because NADP is membrane‐impermeable,eukaryotes need compartment‐specific NADKs for different organelles. Consistently, in both yeast and plants, three compartment‐specific NADKs have been identified. In contrast, even though the first human NADK, a cytosolic one, was identified in 2001, the identity of a hypothesized mitochondrial NADK remained elusive, until a recent discovery that the uncharacterized human gene C5ORF33 encodes a mitochondrion‐localized NADK, referred to as MNADK. Three groups have characterized MNADK functions based on distinct systems involving yeast, mouse, and human studies, from aspects of both in vitro and in vivo evidence. MNADK is a mitochondrial NADK that is enriched and nutritionally‐regulated in mouse liver, and a MNADK‐deficient patient exhibits symptoms characteristic of mitochondrial disease. The identification of MNADKprovides a key clue to the mechanism involved in mitochondrial NADPH production and the maintenance of redox balance in mammalian cells. The roles of MNADK in physiological and pathological processes have yet to be discovered. This article is protected by copyright. All rights reserved
      PubDate: 2015-01-31T15:56:39.415358-05:
      DOI: 10.1002/jcp.24926
       
  • β2‐adrenoreceptor–mediated proliferation inhibition of
           embryonic pluripotent stem cells
    • Authors: Fan Sun; Xin‐Jie Yang, Hao‐Yu Lv, Ya‐Bin Tang, Shi‐Min An, Xu‐Ping Ding, Wen‐Bin Li, Lin Teng, Ying Shen, Hong‐Zhuan Chen, Liang Zhu
      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
      PubDate: 2015-01-29T16:28:50.876519-05:
      DOI: 10.1002/jcp.24937
       
  • Y‐Box binding protein‐1 is part of a complex molecular network
           linking ΔNp63α to the PI3K/AKT pathway in cutaneous squamous
           cell carcinoma
    • Authors: Annaelena Troiano; Irene Schiano Lomoriello, Orsola di Martino, Sabato Fusco, Alessandra Pollice, Maria Vivo, Girolama La Mantia, Viola Calabrò
      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
      PubDate: 2015-01-29T16:28:33.824329-05:
      DOI: 10.1002/jcp.24934
       
  • Hairless up‐regulates Tgf‐β2 expression via
           down‐regulation of miR‐31 in the skin of
           ‘Hairpoor’ (HrHp) mice
    • Authors: Bong‐Kyu Kim; Sungjoo Kim Yoon
      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
      PubDate: 2015-01-29T16:28:17.39703-05:0
      DOI: 10.1002/jcp.24935
       
  • MechanosensitiveStore‐Operated Calcium Entry Regulatesthe Formation
           of Cell Polarity
    • Authors: Yi Weihuang; Shu‐Jing Chang, Hans I‐Chen Harn, Hui‐Ting Huang, Hsi‐Hui Lin, Meng‐Ru Shen, Ming‐Jer Tang, Wen‐Tai Chiu
      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
      PubDate: 2015-01-29T16:28:01.095486-05:
      DOI: 10.1002/jcp.24936
       
  • SIRT1 protects against oxidative stress‐induced endothelial
           progenitor cells apoptosis by inhibiting FOXO3a via FOXO3a ubiquitination
           and degradation
    • Authors: Yu‐Qiang Wang; Qing Cao, Fei Wang, Li‐Ya Huang, Tian‐Tian Sang, Fang Liu, Shu‐Yan Chen
      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
      PubDate: 2015-01-29T16:27:42.808505-05:
      DOI: 10.1002/jcp.24938
       
  • Dynamically regulated CFTR expression and its functional role in cutaneous
           wound healing
    • Authors: Jianda Dong; Xiaohua Jiang, Xiaohu Zhang, Kai Sheng Liu, Jieting Zhang, Jing Chen, Mei Kuen Yu, Lai Ling Tsang, Yiu Wa Chung, Yanrong Wang, Wen‐liang Zhou, Hsiao Chang Chan
      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
      PubDate: 2015-01-29T16:23:27.755214-05:
      DOI: 10.1002/jcp.24931
       
  • Molecular Mechanisms of Helicobacter Pylori Pathogenesis
    • Authors: Maria De Falco; Angela Lucariello, Salvatore Iaquinto, Vincenzo Esposito, Germano Guerra, Antonio De Luca
      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
      PubDate: 2015-01-29T16:23:08.911896-05:
      DOI: 10.1002/jcp.24933
       
  • Identification of genes selectively regulated in human hepatoma cells by
           treatment with dyslipidemic sera and PUFAs
    • Authors: Maria Caterina De Rosa; Mariella Caputo, Hylde Zirpoli, Tania Rescigno, Roberta Tarallo, Giorgio Giurato, Alessandro Weisz, Gaetano Torino, Mario Felice Tecce
      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
      PubDate: 2015-01-29T16:22:50.40305-05:0
      DOI: 10.1002/jcp.24932
       
  • Epoxyeicosatrienoic Acids Regulate Macrophage Polarization and Prevent
           LPS‐Induced Cardiac Dysfunction
    • Authors: Meiyan Dai; Lujin Wu, Zuowen He, Shasha Zhang, Chen Chen, Xizhen Xu, Peihua Wang, Artiom Gruzdev, Darryl C. Zeldin, Dao Wen Wang
      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
      PubDate: 2015-01-28T05:06:44.10661-05:0
      DOI: 10.1002/jcp.24939
       
  • The Na+/HCO3‐ Co‐Transporter SLC4A4 Plays a Role in Growth and
           Migration of Colon and Breast Cancer Cells
    • Authors: Scott K. Parks; Jacques Pouyssegur
      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
      PubDate: 2015-01-22T05:29:26.182955-05:
      DOI: 10.1002/jcp.24930
       
  • Quantifying Heterogeneity and Dynamics of Clonal Fitness in Response to
           Perturbation
    • Authors: Peter Frick; Bishal Paudel, Darren Tyson, Vito Quaranta
      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
      PubDate: 2015-01-20T06:26:35.650196-05:
      DOI: 10.1002/jcp.24888
       
  • Biphasic Role of Calcium in Mouse Sperm Capacitation Signaling Pathways
    • Authors: Felipe A. Navarrete; Francisco A. García‐Vázquez, Antonio Alvau, Jessica Escoffier, Dario Krapf, Claudia Sánchez‐Cárdenas, Ana M. Salicioni, Alberto Darszon, Pablo E. Visconti
      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
      PubDate: 2015-01-17T13:40:03.87002-05:0
      DOI: 10.1002/jcp.24873
       
  • Bisphosphonate treatment of type I diabetic mice prevents early bone loss
           but accentuates suppression of bone formation
    • Authors: Lindsay M. Coe; Srinivasan Arjun Tekalur, Yutian Shu, Melissa J. Baumann, Laura R. McCabe
      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
      PubDate: 2015-01-12T06:29:00.872838-05:
      DOI: 10.1002/jcp.24929
       
  • Acetate supplementation as a means of inducing glioblastoma
           stem‐like cell growth arrest
    • Authors: Patrick M. Long; Scott W. Tighe, Heather E. Driscoll, Karen A. Fortner, Mariano S. Viapiano, Diane M. Jaworski
      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
      PubDate: 2015-01-09T00:54:25.413983-05:
      DOI: 10.1002/jcp.24927
       
  • Activation of Nfatc2 in Osteoblasts Causes Osteopenia
    • Authors: Stefano Zanotti; Ernesto Canalis
      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
      PubDate: 2015-01-09T00:54:07.960412-05:
      DOI: 10.1002/jcp.24928
       
  • TGF‐β1 up‐regulates connexin43 expression: a potential
           mechanism for human trophoblast cell differentiation
    • Authors: Jung‐Chien Cheng; Hsun‐Ming Chang, Lanlan Fang, Ying‐Pu Sun, Peter C.K. Leung
      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
      PubDate: 2015-01-05T05:44:26.110414-05:
      DOI: 10.1002/jcp.24902
       
  • Impaired expression of HIF ‐2α induces compensatory expression
           of HIF‐1α for the recovery from anemia
    • Authors: Ikki Tsuboi; Toshiharu Yamashita, Masumi Nagano, Kenichi Kimura, Georgina To'a Salazar, Osamu Ohneda
      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
      PubDate: 2015-01-03T05:40:55.278977-05:
      DOI: 10.1002/jcp.24899
       
  • Endochondral ossification model system: Designed cell fate of human
           epiphyseal chondrocytes during long‐term implantation
    • Authors: Michiyo Nasu; Shinichiro Takayama, Akihiro Umezawa
      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
      PubDate: 2015-01-03T05:37:07.447824-05:
      DOI: 10.1002/jcp.24882
       
  • The anti‐spasticity drug baclofen alleviates collagen‐induced
           arthritis and regulates dendritic cells
    • Authors: Shichao Huang; Jianxin Mao, Bin Wei, Gang Pei
      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
      PubDate: 2015-01-03T05:29:31.322947-05:
      DOI: 10.1002/jcp.24884
       
  • Baicalein decreases hydrogen peroxide‐induced damage to
           NG108‐15 cells via upregulation of Nrf2
    • Authors: Chao‐Hung Yeh; Kuo‐Hsing Ma, Pei‐Shan Liu, Jung‐Kuei Kuo, Sheau‐Huei Chueh
      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
      PubDate: 2015-01-03T05:28:16.413918-05:
      DOI: 10.1002/jcp.24900
       
  • Angiotensin receptor I stimulates osteoprogenitor proliferation through
           tgfβ ‐mediated signaling
    • Authors: Francesca Querques; Bruno Cantilena, Carmine Cozzolino, Maria Teresa Esposito, Fabiana Passaro, Silvia Parisi, Barbara Lombardo, Tommaso Russo, Lucio Pastore
      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
      PubDate: 2015-01-03T05:28:01.77571-05:0
      DOI: 10.1002/jcp.24887
       
  • Biosafety evidence for human dedifferentiated adipocytes
    • Authors: Antonella Poloni; Giulia Maurizi, Domenico Mattiucci, Elena Busilacchi, Stefania Mancini, Giancarlo Discepoli, Augusto Amici, Massimo Falconi, Saverio Cinti, Pietro Leoni
      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
      PubDate: 2015-01-03T05:26:56.603278-05:
      DOI: 10.1002/jcp.24898
       
  • Exploration of molecular pathways mediating electric field‐directed
           Schwann cell migration by RNA‐Seq
    • Authors: Li Yao; Yongchao Li, Jennifer Knapp, Peter Smith
      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.
      PubDate: 2015-01-03T05:25:52.38867-05:0
      DOI: 10.1002/jcp.24897
       
  • Generation of tumor‐specific cytotoxic T‐lymphocytes from the
           peripheral blood of colorectal cancer patients for adoptive T‐cell
           transfer
    • Authors: Silvia Carluccio; Serena Delbue, Lucia Signorini, Elisabetta Setola, Anna Bagliani, Alberto Della Valle, Andrea Galli, Pasquale Ferrante, Marco Bregni
      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
      PubDate: 2015-01-03T05:25:37.383123-05:
      DOI: 10.1002/jcp.24886
       
  • FADD phosphorylation impaired islet morphology and function
    • Authors: Chun Yao; Hongqin Zhuang, Wei Cheng, Yan Lin, Pan Du, Bingya Yang, Xiaofeng Huang, Sheng Chen, Qingang Hu, Zi‐Chun Hua
      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.
      PubDate: 2015-01-03T05:25:23.116862-05:
      DOI: 10.1002/jcp.24885
       
  • Functional role of the KCa3.1 potassium channel in synovial fibroblasts
           from rheumatoid arthritis patients
    • Authors: Kristin Friebel; Roland Schönherr, Raimund W. Kinne, Elke Kunisch
      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
      PubDate: 2014-12-29T04:17:58.425054-05:
      DOI: 10.1002/jcp.24924
       
  • Chronic Oxidative Stress Leads to Malignant Transformation Along with
           Acquisition of Stem Cell Characteristics, and Epithelial to Mesenchymal
           Transition in Human Renal Epithelial Cells
    • Authors: Prathap Kumar S Mahalingaiah; Logeswari Ponnusamy, Kamaleshwar P Singh
      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
      PubDate: 2014-12-29T04:14:13.089495-05:
      DOI: 10.1002/jcp.24922
       
  • Role of the Unfolded Protein Response, GRP78 and GRP94 in Organ
           Homeostasis
    • Authors: Genyuan Zhu; Amy S. Lee
      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
      PubDate: 2014-12-29T04:12:50.14875-05:0
      DOI: 10.1002/jcp.24923
       
  • Bmp2 deletion causes an amelogenesis imperfecta phenotype via regulating
           enamel gene expression
    • Authors: Feng Guo; Junsheng Feng, Feng Wang, Wentong Li, Qingping Gao, Zhuo Chen, Lisa Shoff, Kevin J. Donly, Jelica Gluhak‐Heinrich, Yong Hee Patricia Chun, Stephen E. Harris, Mary MacDougall, Shuo Chen
      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
      PubDate: 2014-12-24T05:25:23.654276-05:
      DOI: 10.1002/jcp.24915
       
  • Down‐regulated CFTR during aging contributes to benign prostatic
           hyperplasia
    • Authors: Chen Xie; Xiao Sun, Jing Chen, Chi Fai Ng, Kin Mang Lau, Zhiming Cai, Xiaohua Jiang, Hsiao Chang Chan
      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
      PubDate: 2014-12-24T05:23:56.516951-05:
      DOI: 10.1002/jcp.24921
       
  • Angiopoietin‐1 protects the endothelial cells against advanced
           glycation end product injury by strengthening cell junctions and
           inhibiting cell apoptosis
    • Authors: Jingling Zhao; Lei Chen, Bin Shu, Jinming Tang, Lijun Zhang, Julin Xie, Xusheng Liu, Yingbin Xu, Shaohai Qi
      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
      PubDate: 2014-12-24T05:19:14.623404-05:
      DOI: 10.1002/jcp.24920
       
  • Cannabinoid receptor 1 but not 2 mediates macrophage phagocytosis by
           G(α)i/o/RhoA/ROCK signaling pathway
    • Authors: Ping Mai; Lei Tian, Le Yang, Lin Wang, Lin Yang, Liying Li
      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
      PubDate: 2014-12-24T05:18:11.573044-05:
      DOI: 10.1002/jcp.24911
       
  • Pantethine Alters Lipid Composition and Cholesterol Content of Membrane
           Rafts, With Down‐Regulation of CXCL12‐Induced T Cell Migration
           
    • Abstract: Pantethine, a natural low‐molecular‐weight thiol, shows broad activity in a large range of essential cellular pathways. It has been long known as a hypolipidemic and hypocholesterolemic agent. We showed recently that it exerts a neuroprotective action in mouse models of cerebral malaria and Parkinson's disease through multiple mechanisms. In the present study we looked at its effects on membrane lipid rafts that serve as platforms for molecules engaged in cell activity, therefore providing a target against inappropriate cell response leading to chronic inflammation. We found that pantethine‐treated cells showed a significant change in raft fatty acid composition and cholesterol content, with ultimate downregulation of cell adhesion, CXCL12‐driven chemotaxis and transendothelial migration of various T cell types, including human Jurkat cell line and circulating effector T cells. The mechanisms involved include the alteration of the following: i) CXCL12 binding to its target cells; ii) membrane dynamics of CXCR4 and CXCR7, the two CXCL12 receptors; iii) cell redox status, a crucial determinant in the regulation of the chemokine system. In addition, we considered the linker for activation of T cells (LAT) molecule to show that pantethine effects were associated with the displacement from the rafts of the acylated signaling molecules which palmitoylation level was reduced. In conclusion, the results presented here, together with previously published findings, indicate that, due to its pleiotropic action, pantethine can down‐regulate the multifaceted process leading to pathogenic T cell activation and migration. This article is protected by copyright. All rights reserved
       
  • A New Medical Device Rigeneracons Allows to Obtain Viable
           Micro‐Grafts from Mechanical Disaggregation of Human Tissues
    • Abstract: Autologous graft is considered the gold standard of graft materials, however, this approach is still limited due to both small amount of tissue that can be collected and to reduced cell viability of cells that can be obtained. The aim of this preliminary study was to demonstrate the efficacy of an innovative medical device called Rigeneracons® (CE certified Class I) to provide autologous micro‐grafts immediately available to be used in the clinical practice. Moreover, Rigeneracons® is an instrument able to create micro‐grafts enriched of progenitors cells which maintain their regenerative and differentiation potential. We reported preliminary data about viability cell of samples derived from different kind of human tissues, such as periosteum, cardiac atrial appendage biopsy and lateral rectus muscle of eyeball and disaggregated by Rigeneracons®. In all cases we observed that micro‐grafts obtained by Rigeneracons® displayed high cell viability. Furthermore, by cell characterization of periosteum samples, we also evidenced an high positivity to mesenchymal cell markers, suggesting an optimal regenerative potential. This article is protected by copyright. All rights reserved
       
  • In vivo Sodium Tungstate Treatment Prevents E‐Cadherin Loss Induced
           by Diabetic Serum in HK‐2 Cell Line
    • Abstract: Diabetic nephropathy (DN) is characterized by interstitial inflammation and fibrosis, which is the result of chronic accumulation of extracellular matrix produced by activated fibroblasts in the renal tubulointerstitium. Renal proximal tubular epithelial cells (PTECs), through the process of epithelial‐to‐mesenchymal transition (EMT), are the source of fibroblasts within the interstitial space, and loss of E‐cadherin has shown to be one of the earliest steps in this event. Here, we studied the effect of the anti‐diabetic agent sodium tungstate (NaW) in the loss of E‐cadherin induced by transforming growth factor (TGF) β‐1, the best‐characterized in vitro EMT promoter, and serum from untreated or NaW‐treated diabetic rats in HK‐2 cell line, a model of human kidney PTEC. Our results showed that both TGFβ‐1 and serum from diabetic rat induced a similar reduction in E‐cadherin expression. However, E‐cadherin loss induced by TGFβ‐1 was not reversed by NaW, whereas sera from NaW‐treated rats were able to protect HK‐2 cells. Searching for soluble mediators of NaW effect, we compared secretion of TGFβ isoforms and vascular endothelial growth factor (VEGF)‐A, which have opposite actions on EMT. 1mM NaW alone reduced secretion of both TGFβ‐1 and ‐2, and stimulated secretion of VEGF‐A after 48 h. However, these patterns of secretion were not observed after diabetic rat serum treatment, suggesting that protection from E‐cadherin loss by serum from NaW‐treated diabetic rats originates from an indirect rather than a direct effect of this salt on HK‐2 cells, via a mechanism independent of TGFβ and VEGF‐A functions. This article is protected by copyright. All rights reserved
       
  • Transplanted adipose‐derived stem cells ameliorate testicular
           dysfunction in a D‐galactose–induced aging rat model
    • Abstract: Glycation product accumulation during aging of slowly renewing tissues may be an important mechanism underlying aging of the testis. Adipose‐derived stem cells (ADSCs) have shown promise in a novel tissue regenerative technique and may have utility in treating sexual dysfunction. ADSCs have also been found to be effective in antiaging therapy, although the mechanism underlying their effects remains unknown. This study was designed to investigate the anti‐aging effect of ADSCs in a D‐galactose (D‐gal)–induced aging animal model and to clarify the underlying mechanism. Randomly selected 6‐week‐old male Sprague–Dawley rats were subcutaneously injected with D‐gal daily for 8 weeks. Two weeks after completion of treatment, D‐gal–induced aging rats were randomized to receive caudal vein injections of 3 × 106 5‐bromo 2′deoxy­uridine–labeled ADSCs or an equal volume of phosphate‐buffered saline. Serum testosterone level, steroidogenic enzymes (3‐beta‐hydroxysteroid dehydrogenase), and superoxide dismutase (SOD) activity decreased significantly in aging rats compared with the control group; serum lipid peroxidation, spermatogenic cell apoptosis, and methane dicarboxylic aldehyde (MDA) expression increased significantly. ADSCs increased the SOD level and reduced the MDA level in the aging animal model and restored levels of serum testosterone, steroidogenic enzymes, and spermatogenic cell apoptosis. These results demonstrate that ADSCs can contribute to testicular regeneration during aging. ADSCs also provide functional benefits through glycation suppression and antioxidant effects in a rat model of aging. Although some ADSCs differentiated into Leydig cells, the paracrine pathway seems to play a main role in this process, resulting in the reduction of apoptosis. This article is protected by copyright. All rights reserved
       
  • Silencing angiopoietin‐like protein 4 (ANGPTL4) protects against
           lipopolysaccharide‐induced acute lung injury via regulating SIRT1
           /NF‐kB pathway
    • Abstract: Lung inflammation and alveolar epithelial cell death are critical events in the development and progression of acute lung injury (ALI). Although angiopoietin‐like protein 4 (ANGPTL4) participates in inflammation, whether it plays important roles in ALI and alveolar epithelial cell inflammatory injury remains unclear. We therefore investigated the role of angptl4 in lipopolysaccharide (LPS)‐induced ALI and the associated mechanisms. Lentivirus‐mediated short interfering RNA targeted to the mouse angptl4 gene (AngsiRNA) and a negative control lentivirus (NCsiRNA) were intranasally administered to mice. Lung inflammatory injury and the underlying mechanisms for regulation of angptl4 on the LPS‐induced ALI were subsequently determined. We reported that angptl4 levels were increased both in human alveolar epithelial A549 cells and lung tissues obtained from a mouse model of LPS‐induced ALI. Angptl4 expression was induced by LPS in alveolar epithelial cells, whereas LPS‐induced lung inflammation (neutrophils infiltration in the lung tissues, tumor necrosis factor α, interleukin 6), lung permeability (lung wet/dry weight ratio and bronchoalveolar lavage fluid (BALF) protein concentration), tissue damage (caspase3 activation) and mortality rates were attenuated in AngsiRNA‐treated mice. The inflammatory reaction (tumor necrosis factor α, interleukin 6) and apoptosis rates were reduced in AngsiRNA(h)‐treated A549 cells. Moreover, angptl4 promoted NF‐kBp65 expression and suppressed SIRT1 expression both in mouse lungs and A549 cells. Additionally, SIRT1 antagonist nicotinamide (NAM) attenuated the inhibitory effects of AngsiRNA both on LPS‐induced NF‐kBp65 expression and IL6 expression. These findings suggest that silencing angptl4 protects against LPS‐induced ALI via regulating SIRT1/NF‐kB signaling pathway. This article is protected by copyright. All rights reserved
       
  • A SMYD3 Small‐Molecule Inhibitor Impairing Cancer Cell Growth
    • Abstract: SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi‐mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI‐121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 levels. Of note, the extent of cell growth inhibition by BCI‐121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment. This article is protected by copyright. All rights reserved
       
  • Absence of Bone Sialoprotein (BSP) Alters Profoundly Hematopoiesis and
           Upregulates Osteopontin
    • 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 hematopoietic component. In this respect, osteopontin (OPN) has been implicated in the hematopoietic stem cell (HSC) niche as negative regulator of the HSC function. We investigated the impact on hematopoietic regulation of the absence of the cognate bone sialoprotein (BSP). BSP knockout (−/−) mice display increased bone marrow cellularity, and an altered commitment of hematopoietic 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 toward 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 hematopoietic system 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. J. Cell. Physiol. 230: 1342–1351, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • ­­Depletion of Amyloid Precursor Protein (APP) Causes G0 Arrest
           in Non‐Small Cell Lung Cancer (NSCLC) Cells
    • 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. J. Cell. Physiol. 230: 1332–1341, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Transcription Factor MEF2C Suppresses Endothelial Cell Inflammation via
           Regulation of NF‐κB and KLF2
    • Abstract: Endothelial cells play a major role in the initiation and perpetuation of the inflammatory process in health and disease, including their pivotal role in leukocyte recruitment. The role of pro‐inflammatory transcription factors in this process has been well‐described, including NF‐κB. However, much less is known regarding transcription factors that play an anti‐inflammatory role in endothelial cells. Myocyte enhancer factor 2 C (MEF2C) is a transcription factor known to regulate angiogenesis in endothelial cells. Here, we report that MEF2C plays a critical function as an inhibitor of endothelial cell inflammation. Tumor necrosis factor (TNF)‐α inhibited MEF2C expression in endothelial cells. Knockdown of MEF2C in endothelial cells resulted in the upregulation of pro‐inflammatory molecules and stimulated leukocyte adhesion to endothelial cells. MEF2C knockdown also resulted in NF‐κB activation in endothelial cells. Conversely, MEF2C overexpression by adenovirus significantly repressed TNF‐α induction of pro‐inflammatory molecules, activation of NF‐κB, and leukocyte adhesion to endothelial cells. This inhibition of leukocyte adhesion by MEF2C was partially mediated by induction of KLF2. In mice, lipopolysaccharide (LPS)‐induced leukocyte adhesion to the retinal vasculature was significantly increased by endothelial cell‐specific ablation of MEF2C. Taken together, these results demonstrate that MEF2C is a novel negative regulator of inflammation in endothelial cells and may represent a therapeutic target for vascular inflammation. J. Cell. Physiol. 230: 1310–1320, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • HDAC4 Degradation Mediates HDAC Inhibition‐Induced Protective
           Effects Against Hypoxia/Reoxygenation Injury
    • Abstract: Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression through remodeling of chromatin structures. However, the molecular mechanisms involved in this event remain unknown. In this study, we sought to examine whether HDAC inhibition‐mediated protective effects involved HDAC4 sumoylation, degradation, and the proteasome pathway. Isolated neonatal mouse ventricular myocytes (NMVM) and H9c2 cardiomyoblasts were subjected to 48 h of hypoxia (H) (1% O2) and 2 h of reoxygenation (R). Treatment of cardiomyocytes with trichostatin A (TSA) attenuated H/R‐elicited injury, as indicated by a reduction of lactate dehydrogenase (LDH) leakage, an increase in cell viability, and decrease in apoptotic positive cardiomyocytes. MG132, a potent proteasome pathway inhibitor, abrogated TSA‐induced protective effects, which was associated with the accumulation of ubiquitinated HDAC4. NMVM transduced with adenoviral HDAC4 led to an exaggeration of H/R‐induced injury. TSA treatment resulted in a decrease in HDAC4 in cardiomyocytes infected with adenoviral HDAC4, and HDAC4‐induced injury was attenuated by TSA. HDAC inhibition resulted in a significant reduction in reactive oxygen species (ROS) in cardiomyoblasts exposed to H/R, which was attenuated by blockade of the proteasome pathway. Cardiomyoblasts carrying wild type and sumoylation mutation (K559R) were established to examine effects of HDAC4 sumoylation and ubiquitination on H/R injury. Disruption of HDAC4 sumoylation brought about HDAC4 accumulation and impairment of HDAC4 ubiquitination in association with enhanced susceptibility of cardiomyoblasts to H/R. Taken together, these results demonstrated that HDAC inhibition stimulates proteasome dependent degradation of HDAC4, which is associated with HDAC4 sumoylation to induce these protective effects. J. Cell. Physiol. 230: 1321–1331, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Rac1 Regulates Myosin II Phosphorylation Through Regulation of Myosin
           Light Chain Phosphatase
    • 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 novel 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. J. Cell. Physiol. 230: 1352–1364, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • An Alternative Long‐Term Culture System for Highly‐Pure Mouse
           Spermatogonial Stem Cells
    • Abstract: Increasing evidence suggests that spermatogonial stem cells (SSCs) have great clinical potential to give rise to a variety of cell types besides all spermatogenic lineage cells. The development of an efficient method for long‐term culture of highly‐pure SSCs is essential for further studies related to SSC biological events. Here, we describe an in vitro culture system obtaining mouse SSC cultures of high purity, viability, and proliferation. For establishing long‐term cultures of SSCs, we mainly focused on isolation procedures and culture conditions. These included co‐coating of extracellular substrates, that is, poly‐L‐lysine (PLL) and laminin, as well as combinatiorial use of three milder enzymes and simultaneously less trypsin to minimize enzyme‐mediated degradation of SSCs. Furthermore, a unique purification procedure was performed to effectively eliminate contaminating non‐SSCs. Finally, a critical step is to ensure SSC maintenance and expansion by utilizing optimal culture medium. Obtained data suggest that applying our optimally modified method, SSCs can be cultured for over 90 days with high purity (around 93.5%). Moreover, SSCs isolated and expanded using our protocol fulfills all criteria of SSCs without losing their stemness‐characterized by SSC‐phenotypic gene expression and long‐term self‐renewal. This study describes for the first time a protocol allowing isolation and expansion of SSCs suitable for numerous studies related to SSC‐based clinical therapies of various diseases. J. Cell. Physiol. 230: 1365–1375, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Galectin‐1 Triggers Epithelial‐Mesenchymal Transition in Human
           Hepatocellular Carcinoma Cells
    • Abstract: Galectin‐1 (Gal1), a β‐galactoside‐binding protein abundantly expressed in tumor microenvironments, is associated with the development of metastasis in hepatocellular carcinomas (HCC). However, the precise roles of Gal1 in HCC cell invasiveness and dissemination are uncertain. Here, we investigated whether Gal1 mediate epithelial‐mesenchymal transition (EMT) in HCC cells, a key process during cancer progression. We used the well‐differentiated and low invasive HepG2 cells and performed ‘gain‐of‐function' and ‘loss‐function' experiments by transfecting cells with Gal1 cDNA constructs or by siRNA strategies, respectively. Epithelial and mesenchymal markers expression, changes in apico‐basal polarity, independent‐anchorage growth, and activation of specific signaling pathways were studied using Western blot, fluorescence microscopy, soft‐agar assays, and FOP/TOP flash reporter system. Gal1 up‐regulation in HepG2 cells induced down‐regulation of the adherens junction protein E‐cadherin and increased expression of the transcription factor Snail, one of the main inducers of EMT in HCC. Enhanced Gal1 expression facilitated the transition from epithelial cell morphology towards a fibroblastoid phenotype and favored up‐regulation of the mesenchymal marker vimentin in HCC cells. Cells overexpressing Gal1 showed enhanced anchorage‐independent growth and loss of apico‐basal polarity. Remarkably, Gal1 promoted Akt activation, β‐catenin nuclear translocation, TCF4/LEF1 transcriptional activity and increased cyclin D1 and c‐Myc expression, suggesting activation of the Wnt pathway. Furthermore, Gal1 overexpression induced E‐cadherin downregulation through a PI3K/Akt‐dependent mechanism. Our results provide the first evidence of a role of Gal1 as an inducer of EMT in HCC cells, with critical implications in HCC metastasis. J. Cell. Physiol. 230: 1298–1309, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • High Levels of Gpr30 Protein in Human Testicular Carcinoma In Situ and
           Seminomas Correlate with Low Levels of Estrogen Receptor‐Beta and
           Indicate a Switch in Estrogen Responsiveness
    • Abstract: The G protein‐coupled estrogen receptor (GPR30) is suggested to be involved in non‐nuclear estrogen signalling and is expressed in a variety of hormone dependent cancer entities. It is well established that oestrogens are involved in pathological germ cell proliferation including testicular germ cell tumours. This study was performed to further elucidate the role of this receptor and the possible correlation with the estrogen receptor β in human testicular carcinoma in situ (CIS), seminomas and in GC1 and TCam‐2 germ cell lines; in addition, a Tissue Micro‐Array was built using the most representative areas from 25 cases of human testicular seminomas and 20 cases of CIS. The expression of ERβ and GPR30 were observed by using Western blot analysis in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that down regulation of ERβ associates with GPR30 over‐expression both in human testicular CIS and seminomas. In addition, we show that 17β‐oestradiol induces the ERK1/2 activation and increases c‐Fos expression through GPR30 associated with ERβ down‐regulation in TCam‐2 cell line. The present results suggest that exposure to oestrogens or oestrogen‐mimics, in some as of yet undefined manner, diminishes the ERβ‐mediated growth restraint in CIS and in human testicular seminoma, probably due to ERβ down‐regulation associated to GPR30 increased expression indicating that GPR30 could be a potential therapeutic target to design specific inhibitors. J. Cell. Physiol. 230: 1290–1297, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • The Synergistic Effect of SAHA and Parthenolide in MDA‐MB231 Breast
           Cancer Cells
    • Abstract: The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA‐MB231 cells, a triple‐negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA‐MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down‐regulation of DNMT1 expression induced by PN. Inhibition of the DNA‐binding activity of NF‐kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. 230: 1276–1289, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • A Tissue Specific Magnetic Resonance Contrast Agent, Gd‐AMH, for
           Diagnosis of Stromal Endometriosis Lesions: A Phase I Study
    • Abstract: The anti‐mullerian hormone (AMH) is a homodimeric glycoprotein member of the transforming growth factor β (TGF‐β) superfamily, is secreted by Sertoli cells in the embryonic testes and is responsible of the regression of the mullerian duct. The physiological functions of this protein remain largely unknown, and its expression in human tissues has yet to be completely determined. Firstly, we analyzed AMH expression in human tissues by immunohistochemistry. AMH was distributed in many organs, although with different tissue and cell localization and various expression levels; we also demonstrated strong AMH expression in endometriosis tissues. Secondly, we demonstrated the ability of an anti‐AMH antibody, labeled with gadiolinium, to be directly detected by magnetic resonance in small endometriosis lesions (5 mm in diameter) in vivo in a mouse model. In conclusion, our data suggest that based on its expression pattern, AMH may serve to maintain physiological cellular homeostasis in different human tissues and organs. Moreover, it is strongly expressed in endometriosis lesions as a selective tissue specific contrast agent for in vivo detection of stromal endometriosis lesions. The potential significance of these findings could be further validated in a clinical setting. J. Cell. Physiol. 230: 1270–1275, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Anti‐Inflammatory/Tissue Repair Macrophages Enhance the
           Cartilage‐Forming Capacity of Human Bone Marrow‐Derived
           Mesenchymal Stromal Cells
    • Abstract: Macrophages are key players in healing processes. However, little is known on their capacity to modulate the differentiation potential of mesenchymal stem/stromal cells (MSC). Here we investigated whether macrophages (Mf) with, respectively, pro‐inflammatory and tissue‐remodeling traits differentially modulate chondrogenesis of bone marrow derived‐MSC (BM‐MSC). We demonstrated that coculture in collagen scaffolds of BM‐MSC with Mf derived from monocytes polarized with M‐CSF (M‐Mf), but not with GM‐CSF (GM‐Mf) resulted in significantly higher glycosaminoglycan (GAG) content than what would be expected from an equal number of BM‐MSC alone (defined as chondro‐induction). Moreover, type II collagen was expressed at significantly higher levels in BM‐MSC/M‐Mf as compared to BM‐MSC/GM‐Mf constructs, while type X collagen expression was unaffected. In order to understand the possible cellular mechanism accounting for chondro‐induction, developing monoculture and coculture tissues were digested and the properties of the isolated BM‐MSC analysed. We observed that as compared to monocultures, in coculture with M‐Mf, BM‐MSC decreased less markedly in number and exhibited higher clonogenic and chondrogenic capacity. Despite their chondro‐inductive effect in vitro, M‐Mf did not modulate the cartilage tissue maturation in subcutaneous pockets of nude mice, as evidenced by similar accumulation of type X collagen and calcified tissue. Our results demonstrate that coculture of BM‐MSC with M‐Mf results in synergistic cartilage tissue formation in vitro. Such effect seems to result from the survival of BM‐MSC with high chondrogenic capacity. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of cartilage repair. J. Cell. Physiol. 230: 1258–1269, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Pigment Epithelium Derived Factor Suppresses Expression of Sost/Sclerostin
           by Osteocytes: Implication for Its Role in Bone Matrix Mineralization
    •  
  • In Type 2 Diabetes Mellitus Glycated Albumin Alters Macrophage Gene
           Expression Impairing ABCA1‐Mediated Cholesterol Efflux
    • Abstract: Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE‐albumin induces oxidative stress, which is linked to a reduction in ABCA‐1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2‐HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C‐ or DM2‐HSA to measure the 14C‐cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA‐1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real‐time RT‐PCR. An increased mean mass was observed in DM2‐HSA compared with C‐HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3, and HDL2 was impaired in DM2‐HSA‐treated cells, which was related to greater intracellular lipid accumulation. DM2‐HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA‐1 protein content decreased. Compared with C‐HAS‐treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2‐HSA treatment. Stearoyl‐Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2‐HSA. The level of glycation that occurs in vivo in DM2‐HSA‐treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2. J. Cell. Physiol. XXXX: XX–XX, 2015. © 2015 Wiley Periodicals, Inc. J. Cell. Physiol. 230: 1250–1257, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Protein Kinase C Inhibitor, GF109203X Attenuates Osteoclastogenesis, Bone
           Resorption and RANKL‐Induced NF‐κB and NFAT Activity
    • Abstract: Osteolytic bone diseases are characterized by excessive osteoclast formation and activation. Protein kinase C (PKC)‐dependent pathways regulate cell growth, differentiation and apoptosis in many cellular systems, and have been implicated in cancer development and osteoclast formation. A number of PKC inhibitors with anti‐cancer properties have been developed, but whether they might also influence osteolysis (a common complication of bone invading cancers) is unclear. We studied the effects of the PKC inhibitor compound, GF109203X on osteoclast formation and activity, processes driven by receptor activator of NFκB ligand (RANKL). We found that GF109203X strongly and dose dependently suppresses osteoclastogenesis and osteoclast activity in RANKL‐treated primary mouse bone marrow cells. Consistent with this GF109203X reduced expression of key osteoclastic genes, including cathepsin K, calcitonin receptor, tartrate resistant acid phosphatase (TRAP) and the proton pump subunit V‐ATPase‐d2 in RANKL‐treated primary mouse bone marrow cells. Expression of these proteins is dependent upon RANKL‐induced NF‐κB and NFAT transcription factor actions; both were reduced in osteoclast progenitor populations by GF109203X treatment, notably NFATc1 levels. Furthermore, we showed that GF109203X inhibits RANKL‐induced calcium oscillation. Together, this study shows GF109203X may block osteoclast functions, suggesting that pharmacological blockade of PKC‐dependent pathways has therapeutic potential in osteolytic diseases. J. Cell. Physiol. 230: 1235–1242, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Autophagy Mediates HBx‐Induced Nuclear Factor‐κB
           
    • Abstract: HBV and one of its encoded proteins, HBV X protein (HBx), have been shown to induce autophagy in hepatoma cells. Substantial evidence indicates that autophagy is a potent suppressor of inflammation. However, sporadic reports suggest that autophagy could promote pro‐inflammatory cytokine expression and inflammation in some biological contexts. Here we show that overexpression of HBx induces LC3B‐positive autophagosome formation, increases autophagic flux and enhances the expression of ATG5, ATG7, and LC3B‐II in normal hepatocytes. Abrogation of autophagy by small interfering RNA against ATG5 and ATG7 prevents HBx‐induced formation of autophagosomes. Autophagy inhibition also abrogates HBx‐induced activation of nuclear factor‐κB and production of interleukin‐6 (IL‐6), IL‐8 and CXCL2. These findings suggest that autophagy is required for HBx‐induced nuclear factor‐κB activation and pro‐inflammatory cytokine production and could shed new light on the complex role of autophagy in the modulation of inflammation. This article is protected by copyright. All rights reserved
       
  • Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of
           Breast Cancer Cells
    • Abstract: The tumor‐suppressor protein Fhit exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho‐MAPK, and phospho‐STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation. This article is protected by copyright. All rights reserved
       
  • Glucose Concentration and Streptomycin Alter In Vitro Muscle Function and
           Metabolism
    • Abstract: Cell culture conditions can vary between laboratories and have been optimised for 2D cell culture. In this study, engineered muscle was cultured in 5.5 mM low glucose (LG) or 25 mM high glucose (HG) and in the absence or presence (+S) of streptomycin and the effect on C2C12 tissue‐engineered muscle function and metabolism was determined. Following 2 weeks differentiation, streptomycin (3‐fold) and LG (0.5‐fold) significantly decreased force generation. LG and/or streptomycin resulted in upward and leftward shifts in the force‐frequency curve and slowed time‐to‐peak tension and half‐relaxation time. Despite changes in contractile dynamics, no change in myosin isoform was detected. Instead, changes in troponin isoform, calcium sequestering proteins (CSQ and parvalbumin) and the calcium uptake protein SERCA predicted the changes in contractile dynamics. Culturing in LG and/or streptomycin resulted in increased fatigue resistance despite no change in the mitochondrial enzymes SDH, ATPsynthase and cytochrome C. However, LG resulted in increases in the β‐oxidation enzymes LCAD and VLCAD and the fatty acid transporter CPT‐1, indicative of a greater capacity for fat oxidation. In contrast, HG resulted in increased GLUT4 content and the glycolytic enzyme PFK, indicative of a more glycolytic phenotype. These data suggest that streptomycin has negative effects on force generation and that glucose can be used to shift engineered muscle phenotype via changes in calcium‐handling and metabolic proteins. J. Cell. Physiol. 230: 1226–1234, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • TGFβ3 Regulates Periderm Removal Through ΔNp63 in the Developing
           Palate
    • Abstract: The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor β3 (TGFβ3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (−/−) TGFβ3, interferon regulatory factor 6 (IRF6) (−/−), and truncated p63 (ΔNp63) (−/−) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFβ3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFβ3 (−/−), ΔNp63 (−/−), and wild‐type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFβ3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFβ3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFβ3 (−/−) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (−/−) palates because of premature fusion. J. Cell. Physiol. 230: 1212–1225, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Enhanced Phenotypic Alterations of Alveolar Type II Cells in Response to
           Aflatoxin G1‐Induced Lung Inflammation
    • Abstract: Recently, we discovered that Aflatoxin G1 (AFG1) induces chronic lung inflammatory responses, which may contribute to lung tumorigenesis in Balb/C mice. The cancer cells originate from alveolar type II cells (AT‐II cells). The activated AT‐II cells express high levels of MHC‐II and COX‐2, may exhibit altered phenotypes, and likely inhibit antitumor immunity by triggering regulatory T cells (Tregs). However, the mechanism underlying phenotypic alterations of AT‐II cells caused by AFG1‐induced inflammation remains unknown. In this study, increased MHC‐II expression in alveolar epithelium was observed and associated with enhanced Treg infiltration in mouse lung tissues with AFG1‐induced inflammation. This provides a link between phenotypically altered AT‐II cells and Treg activity in the AFG1‐induced inflammatory microenvironment. AFG1‐activated AT‐II cells underwent phenotypic maturation since AFG1 upregulated MHC‐II expression on A549 cells and primary human AT‐II cells in vitro. However, mature AT‐II cells may exhibit insufficient antigen presentation, which is necessary to activate effector T cells, due to the absence of CD80 and CD86. Furthermore, we treated A549 cells with AFG1 and TNF‐α together to mimic an AFG1‐induced inflammatory response in vitro, and we found that TNF‐α and AFG1 coordinately enhanced MHC‐II, CD54, COX‐2, IL‐10, and TGF‐β expression levels in A549 cells compared to AFG1 alone. The phenotypic alterations of A549 cells in response to the combination of TNF‐α and AFG1 were mainly regulated by TNF‐α‐mediated induction of the NF‐κB pathway. Thus, enhanced phenotypic alterations of AT‐II cells were induced in response to AFG1‐induced inflammation. Thus, AT‐II cells are likely to suppress anti‐tumor immunity by triggering Treg activity. J. Cell. Physiol. 230: 1199–1211, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Vitamin D Attenuates Cytokine‐Induced Remodeling in Human Fetal
           Airway Smooth Muscle Cells
    • Abstract: Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25‐dihydroxyvitamin D3; 1,25(OH)2D3), modulates responses to inflammation; however, this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro‐inflammatory cytokines. Cells were pre‐treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP‐9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ‐induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood. J. Cell. Physiol. 230: 1189–1198, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Regulation of Ribosomal Gene Expression in Cancer
    • Abstract: The ability of a cell to undergo malignant transformation is both associated with and dependent on a concomitant increase in protein synthesis due to increased cell division rates and biosynthetic activities. Protein synthesis, in turn, depends upon the synthesis of ribosomes and thus ultimately on the transcription of ribosomal RNA by RNA polymerase I that occurs in the nucleolus. Enlargement of nucleoli has long been considered a hallmark of the malignant cell, but it is only recently that the rate of synthesis of rRNA in the nucleolus has been recognized as both a critical regulator of cellular proliferation and a potential target for therapeutic intervention. As might be expected, the factors regulating rRNA synthesis are both numerous and complex. It is the objective of this review to highlight recent advances in understanding how rRNA synthesis is perturbed in transformed mammalian cells and to consider the impact of these findings on the development of new approaches to the treatment of malignancies. In‐depth analysis of the process of rRNA transcription itself may be found in several recently published reviews (Drygin et al., 2010, Annu Rev Pharmacol Toxicol 50:131–156; Bywater et al., 2013,Cancer Cell 22: 51–65; Hein et al., 2013,Trends Mol Med 19:643–654). J. Cell. Physiol. 230: 1181–1188, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Table of Contents: Volume 230, Number 6
    •  
  • Editor's Choice
    •  
  • Highlights: Volume 230, Number 6
    •  
  • Novel Insights Into TRPM7 Function in Fibrotic Diseases: A Potential
           Therapeutic Target
    • Abstract: “Transient receptor potential (TRP) channels are cellular sensors for a wide spectrum of physical and chemical stimuli. Activation of TRP channels changes the membrane potential, translocates important signaling ions crossing the cell membrane, alters enzymatic activity, and initiates endocytosis/exocytosis (Zheng, 2013).” Fibrosis is the leading cause of organ dysfunction in diseases, which is characterized by an imbalance in the turnover of extracellular matrix components. Accumulating evidence has demonstrated that TRPM7, a member of TRP channels superfamily, participates in the development and pathogenesis of fibrotic diseases, such as hepatic, pulmonary and cardiac fibrosis. In this review, we discuss the comprehensive role of TRPM7 in modulating profibrotic response and its potential as therapeutic target for fibrotic diseases. J. Cell. Physiol. 230: 1163–1169, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Keeping Wnt Signalosome in Check by Vesicular Traffic
    • Abstract: Wg/Wnts are paracrine and autocrine ligands that activate distinct signaling pathways while being internalized through surface receptors. Converging and contrasting views are shaping our understanding of whether, where, and how endocytosis may modulate Wnt signaling. We gather considerable amount of evidences to elaborate the point that signal‐receiving cells utilize distinct, flexible, and sophisticated vesicular trafficking mechanisms to keep Wnt signaling activity in check. Same molecules in a highly context‐dependent fashion serve as regulatory hub for various signaling purposes: amplification, maintenance, inhibition, and termination. Updates are provided for the regulatory mechanisms related to the three critical cell surface complexes, Wnt‐Fzd‐LRP6, Dkk1‐Kremen‐LRP6, and R‐spondin‐LGR5‐RNF43, which potently influence Wnt signaling. We pay particular attentions to how cells achieve sustained and delicate control of Wnt signaling strength by employing comprehensive aspects of vesicular trafficking. J. Cell. Physiol. 230: 1170–1180, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Journal of Cellular Physiology: Volume 230, Number 6, June 2015
    • Abstract: Cover: Cellular and molecular characterization of spermatogonial stem cells (SSCs) maintained under the modified culture system. See article by He et al. on pages 1365–1375.
       
  • Phospholipase C Epsilon (PLCε) Induced TRPC6 Activation: A Common but
           Redundant Mechanism in Primary Podocytes
    • 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. J. Cell. Physiol. 230: 1389–1399, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
       
  • Protective Effect of the Y220C Mutant p53 Against Steatosis: Good
           News'
    •  
  • Prostaglandin E2 Receptor EP1 Transactivates EGFR/MET Receptor Tyrosine
           
    •  
  • New face for Chromatin‐Related Mesenchymal Modulator: n‐CHD9
           localizes to nucleoli and interacts with ribosomal genes
    • Abstract: Mesenchymal stem cells' differentiation into several lineages is coordinated by a complex of transcription factors and co‐regulators which bind to specific gene promoters. The Chromatin‐Related Mesenchymal Modulator, CHD9 demonstrated in vitro its ability for remodeling activity to reposition nucleosomes in an ATP‐dependent manner. Epigenetically, CHD9 binds with modified H3‐(K9me2/3 and K27me3). Previously we presented a role for CHD9 with RNA Polymerase II (Pol II)‐dependent transcription of tissue specific genes. Far less is known about CHD9 function in RNA Polymerase I (Pol I) related transcription of the ribosomal locus that also drives specific cell fate. We here describe a new form, the nucleolar CHD9 (n‐CHD9) that is dynamically associated with Pol I, fibrillarin and Upstream Binding Factor (UBF) in the nucleoli, as shown by imaging and molecular approaches. Inhibitors of transcription disorganized the nucleolar compartment of transcription sites where rDNA is actively transcribed. Collectively, these findings link n‐CHD9 with RNA pol I transcription in fibrillar centers. Using chromatin immunoprecipitation (ChIP) and tilling arrays (ChIP–chip), we find an association of n‐CHD9 with Pol I related to rRNA biogenesis. Our new findings support the role for CHD9 in chromatin regulation and association with rDNA genes, in addition to its already known function in transcription control of tissue specific genes. This article is protected by copyright. All rights reserved
       
  • Nexavar/Stivarga and Viagra Interact to Kill Tumor Cells
    • Abstract: We determined whether the multi‐kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as Viagra (sildenafil) to kill tumor cells. PDE5 and PDGFRα/β were over‐expressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death, regardless of whether cells were grown in 10% or 100% human serum. Knock down of PDE5 or of PDGFRα/β recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. Inhibition of CD95 / FADD / caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK‐1, Beclin1 or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas re‐expression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib / regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. From multiplex assays on tumor tissue and plasma we discovered that increased FGF levels and ERBB1 and AKT phosphorylation were biomarkers that were directly associated with lower levels of cell killing by ‘rafenib + sildenafil. Our data is now being translated into the clinic for further determination as to whether this drug combination is a useful anti‐tumor therapy for solid tumor patients. This article is protected by copyright. All rights reserved
       
  • Anandamide Drives Cell Cycle Progression through CB1 Receptors in a Rat
           Model of Synchronized Liver Regeneration
    • Abstract: The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti‐proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation‐promoting functions of endocannabinoids through CB1 receptors when pro‐growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid‐like molecules OEA and PEA in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals. This article is protected by copyright. All rights reserved
       
 
 
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