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Journal Cover   Journal of Cellular Physiology
  [SJR: 1.948]   [H-I: 125]   [3 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  [1607 journals]
  • EGF‐Induced Connexin43 Negatively Regulates Cell Proliferation in
           Human Ovarian Cancer
    • Authors: Xin Qiu; Jung‐Chien Cheng, Christian Klausen, Hsun‐Ming Chang, Qianlan Fan, Peter C.K. Leung
      Abstract: Connexin43 (Cx43) has been shown to regulate cell proliferation and its down‐regulation is correlated with poor prognosis and survival in several types of human cancer. Cx43 expression levels are frequently down‐regulated in human ovarian cancer, suggesting a potential role for Cx43 in regulating the progression of this disease. Epidermal growth factor (EGF) is a well‐characterized hormone that stimulates ovarian cancer cell proliferation. Although EGF is able to regulate Cx43 expression in other cell types, it is unclear whether EGF can regulate Cx43 expression in ovarian cancer cells. Additionally, it remains unknown whether Cx43 is involved in EGF‐stimulated ovarian cancer cell proliferation. In the present study, we demonstrate that treatment with EGF up‐regulates Cx43 expression in two ovarian cancer cell lines, SKOV3 and OVCAR4. Although treatment with EGF activates both ERK1/2 and Akt signaling pathways, pharmacological inhibition and siRNA‐mediated knockdown suggest that only the activation of Akt1 is required for EGF‐induced Cx43 up‐regulation. Functionally, Cx43 knockdown enhanced basal and EGF‐induced cell proliferation, whereas the proliferative effects of EGF were reduced by Cx43 overexpression. Co‐treatment with the gap junction inhibitor carbenoxolone did not alter the suppressive effects of Cx43 overexpression on EGF‐induced cell proliferation, suggesting a gap junction‐independent mechanism. This study reveals an important role for Cx43 as a negative regulator of EGF‐induced human ovarian cancer cell proliferation. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:44:55.234757-05:
      DOI: 10.1002/jcp.25058
  • Cell‐Penetrating and Endoplasmic Reticulum‐Locating
           TAT‐IL‐24‐KDEL Fusion Protein Induces Tumor Apoptosis
    • Authors: Jian Zhang; Aiyou Sun, Rui Xu, Xinyi Tao, Yuguo Dong, Xinxin Lv, Dongzhi Wei
      Abstract: Interleukin‐24 (IL‐24) is a unique IL‐10 family cytokine that could selectively induce apoptosis in cancer cells without harming normal cells. Previous research demonstrated that intracellular IL‐24 protein induces an endoplasmic reticulum (ER) stress response only in cancer cells, culminating in apoptosis. In this study, we developed a novel recombinant fusion protein to penetrate into cancer cells and locate on ER. It is composed of three distinct functional domains, IL‐24, and the targeting domain of transactivator of transcription (TAT) and an ER retention four‐peptide sequence KDEL (Lys‐Asp‐Glu‐Leu) that link at its NH2 and COOH terminal, respectively. The in vitro results indicated that TAT‐IL‐24‐KDEL inhibited growth in bladder cancer cells, as well as in non‐small cell lung cancer cell line and breast cancer cell line, but the normal human lung fibroblast cell line was not affected, indicating the cancer specificity of TAT‐IL‐24‐KDEL. Western blot analysis showed that apoptosis activation was induced by TAT‐IL‐24‐KDEL through the ER stress‐mediated cell death pathway. Treatment with TAT‐IL‐24‐KDEL significantly inhibited the growth of human H460 xenografts in nude mice, and the tumor growth inhibition was correlated with increased hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) staining. These findings suggest that the artificially designed recombinant fusion protein TAT‐IL‐24‐KDEL may be highly effective in cancer therapy and worthy of further evaluation and development. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:44:35.492967-05:
      DOI: 10.1002/jcp.25054
  • Error‐Prone Repair of DNA Double‐Strand Breaks
    • Authors: Kasey Rodgers; Mitch McVey
      Abstract: Preserving the integrity of the DNA double helix is crucial for the maintenance of genomic stability. Therefore, DNA double‐strand breaks represent a serious threat to cells. In this review, we describe the two major strategies used to repair double strand breaks: non‐homologous end joining and homologous recombination, emphasizing the mutagenic aspects of each. We focus on emerging evidence that homologous recombination, long thought to be an error‐free repair process, can in fact be highly mutagenic, particularly in contexts requiring large amounts of DNA synthesis. Recent investigations have begun to illuminate the molecular mechanisms by which error‐prone double‐strand break repair can create major genomic changes, such as translocations and complex chromosome rearrangements. We highlight these studies and discuss proposed models that may explain some of the more extreme genetic changes observed in human cancers and congenital disorders. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:44:16.245325-05:
      DOI: 10.1002/jcp.25053
  • p53 Modulates Notch Signaling in MCF‐7 Breast Cancer Cells by
           Associating with the Notch Transcriptional Complex via MAML1
    • Authors: Jieun Yun; Ingrid Espinoza, Antonio Pannuti, Damian Romero, Luis Martinez, Mary Caskey, Adina Stanculescu, Maurizio Bocchetta, Paola Rizzo, Vimla Band, Hamid Band, Hwan Mook Kim, Song‐Kyu Park, Keon Wook Kang, Maria Laura Avantaggiati, Christian R. Gomez, Todd Golde, Barbara Osborne, Lucio Miele
      Abstract: p53 and Notch‐1 play important roles in breast cancer biology. Notch‐1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T‐cells but stimulates it in human keratinocytes. Notch co‐activator MAML1 binds p53 and functions as a p53 co‐activator. We studied the regulation of Notch signaling by p53 in MCF‐7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin‐3 inhibits Notch‐dependent transcriptional activity and Notch target expression in a dose‐dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co‐immunoprecipitation experiments readily detected a complex containing p53 and Notch‐1 in MCF‐7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN‐MAML1) and stimulated by wild‐type MAML1. Standard and quantitative far‐Western experiments showed a complex including p53, Notch‐1 and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch‐dependent HEY1 promoter and this association is inhibited by DN‐MAML1 and stimulated by wild‐type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1‐dependent fashion, most likely through a p53‐MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch‐dependent transcription. Our data suggest that p53‐null breast cancers may lack this Notch‐modulatory mechanism, and that therapeutic strategies that activate wild‐type p53 can indirectly cause inhibition of Notch transcriptional activity. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:43:59.77372-05:0
      DOI: 10.1002/jcp.25052
  • microRNA as Biomarkers and Diagnostics
    • Authors: Jin Wang; Jinyun Chen, Subrata Sen
      Abstract: MicroRNAs (miRNAs) are a group of small non‐coding RNAs that are involved in regulating a range of developmental and physiological processes; their dysregulation has been associated with development of diseases including cancer. Circulating miRNAs and exosomal miRNAs have also been proposed as being useful in diagnostics as biomarkers for diseases and different types of cancer. In this review, miRNAs are discussed as biomarkers for cancer and other diseases, including viral infections, nervous system disorders, cardiovascular disorders, and diabetes. We summarize some of the clinical evidence for the use of miRNAs as biomarkers in diagnostics and provide some general perspectives on their use in clinical situations. The analytical challenges in using miRNAs in cancer and disease diagnostics are evaluated and discussed. Validation of specific miRNA signatures as biomarkers is a critical milestone in diagnostics. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:43:44.603437-05:
      DOI: 10.1002/jcp.25056
  • Endotoxin‐Stimulated Rat Hepatic Stellate Cells Induce Autophagy in
           Hepatocytes as a Survival Mechanism
    • Authors: Anil Dangi; Chao Huang, Ashish Tandon, Donna Stolz, Tong Wu, Chandrashekhar R. Gandhi
      Abstract: Bacterial lipopolysaccharide (LPS)‐stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/ml) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS‐stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6h. In vitro, HSCs were treated with 100 ng/ml LPS till 24h. The medium was transferred to hepatocytes, and determinations were made at 0‐12h. Controls were HSC‐conditioned medium or medium‐containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c‐Jun NH2 terminal kinase (JNK) and apoptosis of ∼4‐5% of hepatocytes. In vitro, LPS‐conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ∼10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS‐stimulated HSCs produced IFNβ, and LPS/HSC‐induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti‐IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS‐stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:43:27.71238-05:0
      DOI: 10.1002/jcp.25055
  • Enhanced Chemotherapeutic Behavior of Open‐Caged DNA@Doxorubicin
           Nanostructures for Cancer Cells
    • Authors: Vinit Kumar; Samer Bayda, Mohamad Hadla, Isabella Caligiuri, Concetta Russo Spena, Stefano Palazzolo, Susanne Kempter, Giuseppe Corona, Giuseppe Toffoli, Flavio Rizzolio
      Abstract: In cancer therapy, it is imperative to increase the efficacy and reduce side effects of chemotherapeutic drugs. Nanotechnology offers the unique opportunity to overcome these barriers. In particular, in the last few years DNA nanostructures have gained attention for their biocompatibility, easy customized synthesis and ability to deliver drugs to cancer cells. Here, an open‐caged pyramidal DNA@Doxorubicin (Py‐Doxo) nanostructure was constructed with 10 DNA sequences of 26–28 nucleotides for drug delivery to cancer cells. The synthesized DNA nanostructures are sufficiently stable in biological medium. Py‐Doxo exhibited significantly enhanced cytotoxicity of the delivered doxorubicin to breast and liver cancer cells up to two fold compared to free doxorubicin. This study demonstrates the importance of the shape and structure of the designed transporter DNA nanostructures for biomedical applications. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:43:10.761567-05:
      DOI: 10.1002/jcp.25057
  • MicroRNAs Provide Feedback Regulation of Epithelial‐Mesenchymal
           Transition Induced by Growth Factors
    • Authors: Zailong Qin; Wei He, Jingqun Tang, Qiurong Ye, Wei Dang, Yuanjun Lu, Jia Wang, Guiyuan Li, Qun Yan, Jian Ma
      Abstract: As regulators in gene expression, microRNAs take part in most biological processes including cell differentiation, apoptosis, cell cycle and epithelial‐to‐mesenchymal transition (EMT). In order to evaluate their roles in EMT process, microRNA expression profile changes induced by EGF or TGF‐β treatment on nasopharyngeal carcinoma cell HK‐1 were analyzed, and miR‐21, miR‐148a, miR‐505 and miR‐1207‐5p were found to be upregulated in growth factors‐induced EMT process. miR‐21 is already known as an oncogenic miRNA to promote metastasis, however, the exact functions of other three miRNAs in EMT are unclear. To our surprise, we found that miR‐148a, miR‐505 and miR‐1207‐5p can suppress EMT and metastasis phenotypes in HK‐1 cells both in vitro and in vivo, which may relate to their inhibition on EMT and Wnt signaling molecules. MiRNAs confer robustness to biological processes by posttranscriptional repression of key transcriptional programs that are related to previous developmental stages or to alternative cell fates. Our findings indicate that miRNA feedback circuit is tuned to respond to growth factors‐induced EMT, and we suggested a new negative feedback loop which may be an important element of the EMT process and confer biological robustness. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:42:52.178496-05:
      DOI: 10.1002/jcp.25060
  • Bioregulation
    • Authors: Arthur B. Pardee
      Abstract: Regulation is at the heart of biology. What bioregulatory mechanisms finely tune and coordinate our cell's numerous complicated biochemical processes, maintaining constancy of the cell internal environment when external conditions change and it differentiates during development? Knowledge of the biochemistry of small molecules in normal cells progressed greatly in the early 1900s. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-29T18:42:35.629388-05:
      DOI: 10.1002/jcp.25059
  • Acidosis Is a Key Regulatior of Osteoblast Ecto‐Nucleotidase
           Pyrophosphatase/Phosphodiesterase 1 (NPP1) Expression and Activity
    • Authors: Isabel R Orriss; Michelle L Key, Mark OR Hajjawi, José Luis Millán, Timothy R Arnett
      Abstract: Previous work has shown that acidosis prevents bone nodule formation by osteoblasts in vitro by inhibiting mineralisation of the collagenous matrix. The ratio of phosphate (Pi) to pyrophosphate (PPi) in the bone microenvironment is a fundamental regulator of bone mineralisation. Both Pi and PPi, a potent inhibitor of mineralisation, are generated from extracellular nucleotides by the actions of ecto‐nucleotidases. This study investigated the expression and activity of ecto‐nucleotidases by osteoblasts under normal and acid conditions. We found that osteoblasts express mRNA for a number of ecto‐nucleotidases including NTPdase 1‐6 (ecto‐nucleoside triphosphate diphosphohydrolase) and NPP1‐3 (ecto‐nucleotide pyrophosphatase/phosphodiesterase). The rank order of mRNA expression in differentiating rat osteoblasts (day 7) was Enpp1 > NTPdase 4 > NTPdase 6 > NTPdase 5 > alkaline phosphatase > ecto‐5‐nucleotidase > Enpp3 > NTPdase 1 > NTPdase 3 > Enpp2 > NTPdase 2. Acidosis (pH 6.9) upregulated NPP1 mRNA (2.8‐fold) and protein expression at all stages of osteoblast differentiation compared to physiological pH (pH 7.4); expression of other ecto‐nucleotidases was unaffected. Furthermore, total NPP activity was increased up to 53% in osteoblasts cultured in acid conditions (p 
      PubDate: 2015-05-29T18:31:24.934646-05:
      DOI: 10.1002/jcp.25041
  • Stable Incretin Mimetics Counter Rapid Deterioration of Bone Quality in
           Type 1 Diabetes Mellitus
    • Authors: Sity Aishah Mansur; Aleksandra Mieczkowska, Béatrice Bouvard, Peter R Flatt, Daniel Chappard, Nigel Irwin, Guillaume Mabilleau
      Abstract: Aims Type 1 diabetes mellitus is associated with a high risk for bone fractures. Although bone mass is reduced, bone quality is also dramatically altered in this disorder. However, recent evidences suggest a beneficial effect of the glucose‐dependent insulinotropic polypeptide (GIP) and glucagon‐like peptide 1 (GLP‐1) pathways on bone quality. The aims of the present study were to conduct a comprehensive investigation of bone strength at the organ and tissue level; and to ascertain whether enzyme resistant GIP or GLP‐1 mimetic could be beneficial in preventing bone fragility in type 1 diabetes mellitus. Materials and methods Streptozotocin‐treated mice were used as a model of type 1 diabetes mellitus. Control and streptozotocin‐diabetic animals were treated for 21 days with an enzymatic‐resistant GIP peptide ([D‐Ala2]GIP) or with liraglutide (each at 25 nmol/kg bw, ip). Bone quality was assessed at the organ and tissue level by microCT, qXRI, 3‐point bending, qBEI, nanoindentation and Fourier‐transform infrared microspectroscopy. Results [D‐Ala2]GIP and liraglutide treatment did prevent loss of whole bone strength and cortical microstructure in the STZ‐injected mice. However, tissue material properties were significantly improved in STZ‐injected animals following treatment with [D‐Ala2]GIP or liraglutide. Conclusions Treatment of STZ‐diabetic mice with [D‐Ala2]GIP or liraglutide was capable of significantly preventing deterioration of the quality of the bone matrix. Further studies are required to further elucidate the molecular mechanisms involved and to validate whether these findings can be translated to human patients. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-27T12:56:27.572693-05:
      DOI: 10.1002/jcp.25033
  • The Effects of miR‐20a on p21: Two Mechanisms Blocking Growth Arrest
           in TGF‐β Responsive Colon Carcinoma
    • Authors: Viktorija Sokolova; Antonio Fiorino, Eugenio Zoni, Elisabetta Crippa, James F Reid, Manuela Gariboldi, Marco A Pierotti
      Abstract: Loss of response to TGF‐β is a central event in the genesis of colorectal cancer (CRC), a disease that, in the majority cases, is refractory to growth inhibition induced by this cytokine. However, inactivating mutations at receptors and transducers from the TGF‐β cascade occur only in approximately half of CRCs, suggesting the involvement of additional mechanisms altering the response to the cytokine. We have recently described the amplification of the 13q31 locus, where the miR‐17‐92 cluster maps, associated with overexpression of members of the cluster. In this study, we address the potential role of miR‐20a, from the miR‐17‐92 cluster, in the suppression of TGF‐β cytostatic response in CRC. Using the poorly tumorigenic and TGF‐β sensitive FET cell line that expresses low miR‐20a levels, we first confirmed that miR‐20a down‐modulated CDKN1A expression, both at mRNA and protein level, through direct binding to its 3'‐UTR. We demonstrated that miR‐20a significantly diminished cell response to TGF‐β by preventing its delay of G1/S transition and promoting progression into cell cycle. Moreover, besides modulating CDKN1A, miR‐20a blocked TGF‐β‐induced transactivation of its promoter without affecting the post‐receptor activation of Smad3/4 effectors. Finally, miR‐20a abrogated the TGF‐β mediated c‐Myc repression, a direct inhibitor of the CDKN1A promoter activation, most likely by reducing the expression of specific MYC‐regulating genes from the Smad/E2F‐based core repressor complex. Collectively, our experiments indicate that miR‐20a interferes with the colonic epithelium homeostasis by disrupting the regulation of Myc/p21 by TGF‐β, which is essential for its malignant transformation. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-26T17:52:50.46159-05:0
      DOI: 10.1002/jcp.25051
  • Editor's Choice
    • PubDate: 2015-05-26T11:27:44.947009-05:
      DOI: 10.1002/jcp.25030
  • Highlights: Volume 230, Number 9
    • PubDate: 2015-05-26T11:27:41.114061-05:
      DOI: 10.1002/jcp.24764
  • Journal of Cellular Physiology: Volume 230, Number 9, September 2015
    • Abstract: Cover: Dynamic association of n‐CHD9 with the nucleolus during the cell cycle. A–C: During mitosis pre‐rRNA processing factors are separated from the RNA Pol I transcription apparatus, which remains attached to nucleolar organizing regions (NORs) on the metaphase plate. D: During late anaphase both n‐CHD9 and upstream binding factor (UBF) are present in nucleolar structures. E,F: At early telophase n‐CHD9 and UBF are present in mature nucleolar structures (green arrow), but n‐CHD9 is also uniquely present in pre‐nucleolar bodies (white arrow). During late telophase both n‐CHD9 and UBF are assembled into mature nucleoli. See article by Salomon‐Kent et al. on pages 2270–2280.
      PubDate: 2015-05-26T11:27:41.063693-05:
      DOI: 10.1002/jcp.24762
  • Table of Contents: Volume 230, Number 9
    • PubDate: 2015-05-26T11:27:39.106897-05:
      DOI: 10.1002/jcp.24763
  • Dual Effect of Adenosine A1 Receptor Activation on Renal O2 Consumption
    • Authors: Victor Babich; Komal Vadnagara, Francesca Di Sole
      Abstract: The high requirement of O2 in the renal proximal tubule stems from a high rate of Na+ transport. Adenosine A1 receptor (A1R) activation regulates Na+ transport in this nephron segment. Thus, the effect of the acute activation and the mechanisms of A1R on the rate of O2 consumption were evaluated. The A1R‐antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (CPX) and adenosine deaminase (ADA), which metabolize endogenous adenosine, reduced O2 consumption (40‐50%). Replacing Na+ in the buffer reversed the ADA‐ or CPX‐mediated reduction of O2 consumption. Blocking the Na/H‐exchanger activity, which decreases O2 usage per se, did not enhance the ADA‐ or CPX‐induced inhibition of O2 consumption. These data indicate that endogenous adenosine increases O2 usage via the activation of Na+ transport. In the presence of endogenous adenosine, A1R was further activated by the A1R‐agonist N6‐cyclopentyladenosine (CPA); CPA inhibited O2 usage (30%) and this effect also depended on Na+ transport. Moreover, a low concentration of CPA activated O2 usage in tissue pretreated with ADA, whereas a high concentration of CPA inhibited O2 usage; both effects depended on Na+. Protein kinase C signaling mediated the inhibitory effect of A1R, while adenylyl cyclase mediated its stimulatory effect on O2 consumption. In summary, increasing the local concentrations of adenosine can either activate or inhibit O2 consumption via A1R, and this mechanism depends on Na+ transport. The inhibition of O2 usage by A1R activation might restore the compromised balance between energy supply and demand under pathophysiological conditions, such as renal ischemia, which results in high adenosine production. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-23T17:47:44.209202-05:
      DOI: 10.1002/jcp.25050
  • Differential Hematopoietic Activity in White Adipose Tissue Depending on
           its Localization
    • Authors: Elodie Luche; Coralie Sengenès, Emmanuelle Arnaud, Patrick Laharrague, Louis Casteilla, Beatrice Cousin
      Abstract: White adipose tissue (WAT) can be found in different locations in the body, and these different adipose deposits exhibit specific physiopathological importance according to the subcutaneous or abdominal locations. We have shown previously the presence of functional hematopoietic stem/progenitor cells (HSPC) in subcutaneous adipose tissue (SCAT). These cells exhibit a specific hematopoietic activity that contributes to the renewal of the immune cell compartment within this adipose deposit. In this study we investigated whether HSPC can be found in visceral adipose tissue (VAT) and whether a putative difference in in situ hematopoiesis may be related to anatomical location and to site‐specific immune cell content in VAT compared to SCAT. Therein, we identified for the first time the presence of HSPC in VAT. Using both in vitro assays and in vivo competitive repopulation experiments with sorted HSPC from VAT or SCAT, we showed that the hematopoietic activity of HSPC was lower in VAT, compared to SCAT. In addition, this altered hematopoietic activity of HSPC in VAT was due to their microenvironment, and may be related to a specific combination of secreted factors and extracellular matrix molecules expressed by adipose derived stromal cells. Our results indicate that WAT specific hematopoietic activity may be generalized to all adipose deposits, although with specificity according to the fat pad location. Considering the abundance of WAT in the body, this emphasizes the potential importance of this hematopoietic activity in physiopathological situations. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-18T17:51:56.231608-05:
      DOI: 10.1002/jcp.25045
  • Involvement of a P2 × 7 Receptor in the Acrosome Reaction
           Induced by ATP in Rat Spermatozoa
    • Authors: Jorge L. Torres‐Fuentes; Mariana Rios, Ricardo D. Moreno
      Abstract: The acrosome reaction (AR) is the exocytosis of the acrosomal vesicle in response to different physiological and non‐physiological stimuli. Particularly in mammals, the AR is needed for sperm to fuse with the oocyte plasma membrane, and it occurs only in capacitated sperm. Previous evidence in the literature indicates that extracellular ATP induces the AR in capacitated human and bovine spermatozoa, but its receptor has not yet been identified. The aim of this work was to define a putative ATP receptor in rat spermatozoa using pharmacological and biochemical approaches. We found that ATP induced the AR only in capacitated rat spermatozoa, which was inhibited in the presence of two general inhibitors of ATP receptors (P2 receptors), Suramin, and oxidized ATP (oATP), and one inhibitor of P2X receptor (pyridoxalphosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS)). In addition, the AR induced by ATP in capacitated rat spermatozoa was inhibited by brilliant blue‐G (BB‐G) and 17‐β‐oestradiol, two blockers of P2 × 7 receptors. Moreover, the ATP analog 2'(3')‐O‐(4‐Benzoylbenzoyl) ATP (BzATP) was almost 500 times more potent than ATP to induce the AR, which agrees with the pharmacology of a P2 × 7 receptor. Here we show the presence of P2 × 7 receptor by Western blot and its localization in the tail and acrosome by indirect immunofluorescence. Finally, we quantify the presence of ATP in the rat oviduct during the estrous cycle. We found that the ATP concentration within the lumen of the oviduct is similar to those required to induce acrosome reaction, which agree with its role during in vivo fertilization. Therefore, our results strongly suggest that ATP induces the AR in capacitated rat spermatozoa through a P2 × 7 receptor, which may be functional during in vivo fertilization. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-18T17:50:53.03108-05:0
      DOI: 10.1002/jcp.25044
  • The Many Faces of the A2b Adenosine Receptor in Cardiovascular and
           Metabolic Diseases
    • Authors: Anna Eisenstein; Shenia Patterson, Katya Ravid
      Abstract: Modulation of the low affinity adenosine receptor subtype, the A2b adenosine receptor (A2bAR), has gained interest as a therapeutic target in various pathologic areas associated with cardiovascular disease. The actions of the A2bAR are diverse and at times conflicting depending on cell and tissue type and the timing of activation or inhibition of the receptor. The A2bAR is a promising and exciting pharmacologic target, however, a thorough understanding of A2bAR action is necessary to reach the therapeutic potential of this receptor. This review will focus on the role of the A2bAR in various cardiovascular and metabolic pathologies in which the receptor is currently being studied. We will illustrate the complexities of A2bAR signaling and highlight areas of research with potential for therapeutic development. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-14T18:05:22.807075-05:
      DOI: 10.1002/jcp.25043
  • Differential Cellular Effects of Plk1 Inhibitors Targeting the
           ATP‐Binding Domain or Polo‐Box Domain
    • Authors: Sol‐Bi Shin; Sang‐Uk Woo, Hyungshin Yim
      Abstract: The expression of polo‐like kinase 1 (Plk1) correlates with malignancy and is thus recognized as a target for cancer therapy. In addition to the development of ATP‐competitive Plk1 inhibitors, the polo‐box domain (PBD), a unique functional domain of PLKs, is being targeted to develop Plk1‐specific inhibitors. However, the action mechanisms of these two classes of Plk1 inhibitors have not been thoroughly evaluated. Here, we evaluate the differences in cellular effects of ATP‐binding domain inhibitors (BI2536, GSK461364) and PBD inhibitors (poloxin, thymoquinone) to determine their mechanisms of Plk1 inhibition. Our data show that BI2536 and GSK461364 increased the population of cells in the G2/M phase compared with controls, while treatment with poloxin and thymoquinone increased cell population in the S phase as well as in G2/M, in a p53‐independent manner. The population of cells staining positively for p‐Histone H3 and MPM2, mitotic index, was increased by treatment with BI2536 or GSK461364, but not by treatment with poloxin or thymoquinone. Furthermore, treatment with BI2536 or GSK461364 resulted in activation of the BubR1 spindle checkpoint kinase, suggesting that treatment with ATP‐binding domain inhibitors induces metaphase arrest. However, the administration of poloxin and thymoquinone resulted in an increase in p21WAF1 and S arrest, indicating that PBD inhibitors also affected interphase before mitotic entry. Taken together, these data suggest that the PDB of Plk1 plays a role in S phase progression through interaction with other proteins, while its ATP‐binding domain is important for regulating mitotic progression mediated by its catalytic activity involving consumption of ATP. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-14T18:04:58.96938-05:0
      DOI: 10.1002/jcp.25042
  • Electrospun Type 1 Collagen Matrices Using a Novel Benign Solvent for
           Cardiac Tissue Engineering
    • Authors: Alan M. Punnoose; Anuradha Elamparithi, Sarah Kuruvilla
      Abstract: Electrospinning is a well‐established technique that uses a high electric field to fabricate ultra fine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers due to its biocompatibility and widespread occurrence in nature. Electrospinning of Collagen alone has been reported with fluoroalcohols such as Hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE), which are toxic to the environment. In this study we describe the use of a novel benign binary solvent to generate nanofibers of Collagen type 1, which is non‐toxic and economical. Transmission electron microscopy (TEM) analysis revealed the characteristic feature of native collagen namely the 67 nm banding pattern, confirming that the triple helical structure was maintained. Scanning Electron Microscopy (SEM) analysis showed the fiber diameters to be in the 200‐800 nm range. Biocompatibility of the three dimensional (3D) scaffolds was established by MTT assays using skeletal myoblasts and Confocal Microscopic analysis of immunofluorescent stained sections for muscle specific markers such as Desmin and Actin. Primary neonatal rat ventricular cardiomyocytes seeded onto the scaffolds were able to maintain their contractile function for a period of 17 days. Our work provides evidence that Collagen 1 can be electrospun without combining with other polymers using a novel benign solvent and we are currently exploring the potential of this approach for cardiac and skeletal muscle tissue engineering. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:48:51.922438-05:
      DOI: 10.1002/jcp.25035
  • Deficiency in the α1 Subunit of Na+/K+‐ATPase Enhances the
           Anti‐Proliferative Effect of High Osmolality in Nucleus Pulposus
           Intervertebral Disc Cells
    • Authors: Eleni Mavrogonatou; Konstantinos Papadimitriou, Jill P. Urban, Vassilios Papadopoulos, Dimitris Kletsas
      Abstract: Intervertebral disc cells are constantly exposed to a hyperosmotic environment. Among cellular responses towards this stress is the inhibition of proliferation through the activation of p38 MAPK and p53. In an effort to further elucidate the biochemical pathways triggered by hyperosmotic stress, we assessed the high osmolality‐induced transcriptional changes of bovine nucleus pulposus cells using whole‐genome arrays. A 5‐ and a 24‐h hyperosmotic treatment led to the differential expression of >100 and >200 genes, respectively, including nine genes encoding transporters (SLC4A11, SLC5A3, ATP1A1, SLC38A2, KCNK17, KCTD20, KCTD11, SLC7A5 and CLCA2). Differences in the transcriptional profile of these selected genes, as indicated by the microarrays experiments, were validated by qRT‐PCR in 2D and 3D cell cultures, under hyperosmolar salt and sorbitol conditions, revealing the presence of a common triggering signal for osmotic adaptation. The key signaling molecules p38 MAPK and p53 were demonstrated to differently participate in the regulation of the aforementioned transporters. Finally, siRNA‐mediated knocking‐down of each one of the three transporters with the highest and sustained over‐expression (i.e. SLC4A11, SLC5A3 and ATP1A1) had a distinct outcome on the transcriptional profile of the other transporters, on p38 MAPK and p53 phosphorylation and consequently on cell cycle progression. The inhibition of ATP1A1 had the most prominent effect on the transcription of the rest of the transporters and was found to enhance the anti‐proliferative effect of hyperosmotic conditions through an increased G2/M cell cycle block, ascribing to this pump a central role in the osmoregulatory response of nucleus pulposus cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:48:07.380527-05:
      DOI: 10.1002/jcp.25040
  • Coalition of Nuclear Receptors in the Nervous System
    • Authors: Benjamin Förthmann; John M. Aletta, Yu‐Wei Lee, Chris Terranova, Barbara Birkaya, Ewa K. Stachowiak, Michal K. Stachowiak, Peter Claus
      Abstract: A universal signaling module has been described which utilizes the nuclear form of Fibroblast growth Factor Receptor (FGFR1) in a central role directing the post‐mitotic development of neural cells through coordinated gene expression. In this review, we discuss in detail the current knowledge of FGFR1 nuclear interaction partners in three scenarios: (i) Engagement of FGFR1 in neuronal stem cells and regulation of neuronal differentiation; (ii) interaction with the orphan receptor Nurr1 in development of mesencephalic dopaminergic neurons; (iii) modulation of nuclear FGFR1 interactions downstream of nerve growth factor (NGF) signaling. These coalitions demonstrate the versatility of non‐canonical, nuclear tyrosine kinase signaling in diverse cellular differentiation programs of neurons. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:43:09.403151-05:
      DOI: 10.1002/jcp.25036
  • Control of the Normal and Pathological Development of Neural Stem and
           Progenitor Cells by the PC3/Tis21/Btg2 and Btg1 Genes
    • Authors: Laura Micheli; Manuela Ceccarelli, Stefano Farioli‐Vecchioli, Felice Tirone
      Abstract: The PC3/Tis21/Btg2 and Btg1 genes are transcriptional cofactors belonging to the Btg/Tob family, which regulate the development of several cell types, including neural precursors. We summarize here the actions of these genes on neural precursors in the adult neurogenic niches and the cognitive defects associated when their expression is altered. We consider also recent findings implicating them in neural and non‐neural tumors, since common developmental mechanisms are involved. PC3/Tis21 is required for the regulation of the maturation of stem and progenitor cells in the adult dentate gyrus and subventricular zone (SVZ), by controlling both their exit from the cell cycle and the ensuing terminal differentiation. Such actions are effected by regulating the expression of several genes, including cyclin D1, BMP4, Id3. In cerebellar precursors, however, PC3/Tis21 regulates chiefly their migration rather than proliferation or differentiation, with important implications for the onset of medulloblastoma, the cerebellar tumor. In fact PC3/Tis21 is a medulloblastoma‐suppressor, as its overexpression in cerebellar precursors inhibits this tumor; PC3/Tis21 shows anti‐tumor activity also in non‐neural tumors. Btg1 presents a different functional profile, as it controls proliferation in adult stem/progenitor cells of dentate gyrus and SVZ, where is required to maintain their self‐renewal and quiescence, but is apparently devoid of a direct control of their terminal differentiation or migration. Notably, physical exercise in Btg1‐null mice rescues the loss of proliferative capability occurring in older stem cells. Both genes could be further investigated as therapeutical targets, namely, Btg1 in the process of aging and PC3/Tis21 as a tumor‐suppressor. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:42:51.81523-05:0
      DOI: 10.1002/jcp.25038
  • Revisiting Chaos Theorem to Understand the Nature of Mirnas in Response to
           Drugs of Abuse
    • Authors: Faten A. Taki; Xiaoping Pan, Baohong Zhang
      Abstract: Just like Matryoshka dolls, biological systems follow a hierarchical order that is based on dynamic bidirectional communication among its components. In addition to the convoluted inter‐relationships, the complexity of each component spans several folds. Therefore, it becomes rather challenging to investigate phenotypes resulting from these networks as it requires the integration of reductionistic and holistic approaches. One dynamic system is the transcriptome which comprises a variety of RNA species. Some, like microRNAs, have recently received a lot of attention. miRNAs are very pleiotropic and have been considered as therapeutic and diagnostic candidates in the biomedical fields. In this review, we survey miRNA profiles in response to drugs of abuse (DA) using 118 studies. After providing a summary of substance use disorders (Pandurangaiah et al.)‐related miRNAs, general patterns of miRNA signatures are compared among studies for each drug or for all drugs of abuse. Then, current challenges and drawbacks in the field are discussed. Finally, we provide support for considering miRNAs as a chaotic system in normal versus disrupted states particularly in SUD and propose an integrative approach for studying and analyzing miRNA data. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:42:33.44171-05:0
      DOI: 10.1002/jcp.25037
  • GLP‐2 as Beneficial Factor in the Glucose Homeostasis in Mice Fed a
           High Fat Diet
    • Authors: Sara Baldassano; Francesca Rappa, Antonella Amato, Francesco Cappello, Flavia Mulè
      Abstract: Glucagon like peptide‐2 (GLP‐2) is a gastrointestinal hormone released in response to dietary nutrients, which acts through a specific receptor, the GLP‐2 receptor (GLP‐2R). The physiological effects of GLP‐2 are multiple, involving also the intestinal adaptation to high fat diet (HFD). In consideration of the well‐known relationship between chronic HFD and impaired glucose metabolism, in the present study we examined if the blocking of the GLP‐2 signaling by chronic treatment with the GLP‐2R antagonist, GLP‐2 (3–33), leads to functional consequences in the regulation of glucose metabolism in HFD‐fed mice. Compared with animals fed standard diet (STD), mice at the tenth week of HFD showed hyperglycaemia, glucose intolerance, high plasma insulin level after glucose load, increased pancreas weight and β cell expansion, but not insulin resistance. In HFD fed mice, GLP‐2 (3‐33) treatment for four weeks (from the sixth to the tenth week of diet) did not affect fasting glycaemia, but it significantly increased the glucose intolerance, both fasting and glucose‐induced insulin levels, and reduced the sensitivity to insulin leading to insulin‐resistance. In GLP‐2 (3‐33)‐treated HFD mice pancreas was significantly heavier and displayed a significant increase in β‐cell mass in comparison with vehicle‐treated HFD mice. In STD mice, the GLP‐2 (3‐33) treatment did not affect fasted or glucose‐stimulated glycemia, insulin, insulin sensitivity, pancreas weight and beta cell mass. The present study suggests that endogenous GLP‐2 may act as a protective factor against the dysregulation of the glucose metabolism that occurs in HFD mice, because GLP‐2 (3‐33) worsens glucose metabolism disorders. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:42:23.527174-05:
      DOI: 10.1002/jcp.25039
  • Kinin B1 Receptor Deletion Affects Bone Healing in Type 1 Diabetic Mice
    • Authors: Natália P. Cignachi; João B. Pesquero, Rogério B. Oliveira, Adriana Etges, Maria M. Campos
      Abstract: The effects of kinin B1 receptor (B1R) deletion were examined on femur bone regeneration in streptozotocin (STZ)‐type 1 diabetes. Diabetes induction in wild‐type C57/BL6 (WTC57BL6) mice led to decrease in body weight and hyperglycemia, compared to the non‐diabetic group of the same strain. The lack of B1R did not affect STZ‐elicited body weight loss, but partially prevented hyperglycemia. Diabetic mice had a clear delay in bone regeneration, and displayed large areas of loose connective tissue within the defects, with a reduced expression of the mineralization‐related protein osteonectin, when compared to the non‐diabetic WTC57/BL6. The non‐diabetic and diabetic B1R knockout (B1RKO) mice had bone regeneration levels and osteonectin expression comparable to that seen in control WTC57/BL6 mice. WTC57/BL6 STZ‐diabetic mice also showed a marked reduction of collagen contents, with increased immunolabelling for the apoptosis marker caspase‐3, whereas diabetic B1RKO had collagen levels and caspase‐3 activity comparable to those observed in non‐diabetic WTC57/BL6 or B1RKO mice. No significant difference was detected in the number of tartrate‐resistant acid phosphatase (TRAP)‐stained cells, or in RANK/RANKL/OPG system immunolabelling throughout the experimental groups. Data bring novel evidence on the relevance of kinin B1R under type 1 diabetes with regards to its role in bone regeneration. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:26:09.52783-05:0
      DOI: 10.1002/jcp.25034
  • Protein Kinase C Is Involved in the Induction of ATP‐Binding
           Cassette Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor
           Agonist in Human Macrophages
    • Authors: Etimad A. Huwait; Nishi N. Singh, Daryn R. Michael, Thomas S. Davies, Joe W. E. Moss, Dipak P. Ramji
      Abstract: The transcription of the ATP‐binding cassette transporter A1 (ABCA1) gene, which plays a key anti‐atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c‐Jun N‐terminal kinase (JNK) and phosphoinositide 3‐kinase (PI3K) in the LXRs‐mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have therefore investigated a potential role for PKC in the action of LXR/RXR agonist 22‐(R)‐hydroxycholesterol (22‐(R)‐HC)/9‐cis‐retinoic acid (9cRA) in THP‐1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein‐1 (AP‐1) DNA binding activity in macrophages treated with 22‐(R)‐HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype‐specific inhibitors. These studies therefore reveal a potentially important role for PKC in the action of 22‐(R)‐HC and 9cRA in human macrophages. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-07T18:01:38.140641-05:
      DOI: 10.1002/jcp.25157
  • S100A8, an Oocyte‐Specific Chemokine, Directs the Migration of
           Ovarian Somatic Cells During Mice Primordial Follicle Assembly
    • Authors: Zhen Teng; Chao Wang, Yijing Wang, Hua Zhang, Kun Huang, Xi Xiang, Wanbao Niu, Lizhao Feng, Lihua Zhao, Hao Yan, Guoliang Xia
      Abstract: In the mammalian ovaries, the primordial follicle pool determines the reproductive capability over the lifetime of a female. The primordial follicle is composed of two cell members, namely the oocyte and the pre‐granulosa cells that encircle the oocyte. However, it is unclear what factors are involved in the reorganization of the two distinct cells into one functional unit. This study was performed to address this issue. Firstly, in an in vitro reconstruction system, dispersed ovarian cells from murine fetal ovaries at 19.0 days post coitum (dpc) reassembled into follicle‐like structures, independent of the physical distance between the cells, implying that either oocytes or ovarian somatic cells (OSCs) were motile. We then carried out a series of transwell assay experiments, and determined that it was in fact 19.0 dpc OSCs (as opposed to oocytes), which exhibited a significant chemotactic response to both fetal bovine serum and oocytes themselves. We observed that S100A8, a multi‐functional chemokine, may participate in the process as it is mainly expressed in oocytes within the cysts/plasmodia. S100A8 significantly promoted the number of migrating OSCs by 2.5 times in vitro, of which 66.9% were FOXL2 protein‐positive cells, implying that the majority of motile OSCs were pre‐granulosa cells. In addition, an S100A8‐specific antibody inhibited the formation of follicle‐like reconstruction cell mass in vitro. And, the primordial follicle formation was reduced when S100a8‐specific siRNA was applied onto in vitro cultured 17.5 dpc ovary. Therefore, S100A8 could be a chemokine of oocyte origin, which attracts OSCs to form the primordial follicles. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-06T17:18:24.367503-05:
      DOI: 10.1002/jcp.25032
  • Possible Involvement of Palmitate in Pathogenesis of Periodontitis
    • Authors: Yosuke Shikama; Yasusei Kudo, Naozumi Ishimaru, Makoto Funaki
      Abstract: Type 2 diabetes (T2D) is characterized by decreased insulin sensitivity and higher concentrations of free fatty acids (FFAs) in plasma. Among FFAs, saturated fatty acids (SFAs), such as palmitate, have been suggested to promote inflammatory responses. Although many epidemiological studies have shown a link between periodontitis and T2D, little is known about the clinical significance of SFAs in periodontitis. In this study, we showed that gingival fibroblasts have cell‐surface expression of CD36, which is also known as FAT/fatty acid translocase. Moreover, CD36 expression was increased in gingival fibroblasts of high‐fat diet‐induced T2D model mice, compared with gingival fibroblasts of mice fed a normal diet. DNA microarray analysis revealed that palmitate increased mRNA expression of pro‐inflammatory cytokines and chemokines in human gingival fibroblasts (HGF). Consistent with these results, we confirmed that palmitate induced interleukin (IL)‐6, IL‐8, and CXCL1 secretion in HGF, using a cytokine array and ELISA. SFAs, but not an unsaturated fatty acid, oleate, induced IL‐8 production. Docosahexaenoic acid (DHA), which is one of the omega‐3 polyunsaturated fatty acids, significantly suppressed palmitate‐induced IL‐6 and IL‐8 production. Treatment of HGF with a CD36 inhibitor also inhibited palmitate‐induced pro‐inflammatory responses. Finally, we demonstrated that Porphyromonas gingivalis (P.g.) lipopolysaccharide and heat‐killed P.g. augmented palmitate‐induced chemokine secretion in HGF. These results suggest a potential link between SFAs in plasma and the pathogenesis of periodontitis. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-29T06:27:24.646609-05:
      DOI: 10.1002/jcp.25029
  • c‐Src‐dependent transactivation of EGFR mediates
           CORM‐2‐induced HO‐1 expression in human tracheal smooth
           muscle cells
    • Authors: Chuen‐Mao Yang; Chih‐Chung Lin, I‐Ta Lee, Chih‐Kai Hsu, Yu‐Chen Tai, Hsi‐Lung Hsieh, Pei‐Ling Chi, Andli‐Der Hsiao
      Abstract: Carbon monoxide (CO), a reaction product of the cytoprotective heme oxygenase (HO)‐1, displays an anti‐inflammatory effect in various cellular injuries, but the precise mechanisms of HO‐1 expression remain unknown. We used the transition metal carbonyl compound carbon monoxide‐releasing molecule‐2 (CORM‐2) that acts as carbon monoxide donor. The effects of CORM‐2 on expression of HO‐1 in human tracheal smooth muscle cells (HTSMCs) were determined by Western blot, real‐time PCR, and promoter activity assay. In HTSMCs, CORM‐2 activated Nrf2 through the activation of a c‐Src/EGFR/PI3K/Akt‐dependent pathway, resulting in HO‐1 expression. We showed that CORM‐2‐induced HO‐1 protein and mRNA levels were inhibited by the inhibitor of c‐Src (PP1 or SU6656), EGFR (AG1478), PI3K (LY294002), Akt (SH‐5), JNK1/2 (SP600125), or p38 MAPK (SB202190) and transfection with siRNA of c‐Src, EGFR, Akt, p38, JNK2, or Nrf2 in HTSMCs. We also showed that CORM‐2 stimulated c‐Src, EGFR, Akt, p38 MAPK, and JNK1/2 phosphorylation. CORM‐2 also enhanced Nrf2 translocation from the cytosol to the nucleus and antioxidant response element (ARE) promoter activity. Moreover, CORM‐2 mediated p38 MAPK and JNK1/2 activation via a c‐Src/EGFR/PI3K/Akt pathway, which further enhanced Nrf2 activation and translocation. Finally, we observed that CORM‐2 induced in vivo binding of Nrf2 to the HO‐1 promoter. CORM‐2 activates the c‐Src/EGFR/PI3K/Akt/JNK1/2 and p38 MAPK pathways, which in turn trigger Nrf2 activation and ultimately induces HO‐1 expression in HTSMCs. Thus, the HO‐1/CO system might be potential therapeutics in airway diseases. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-29T06:27:07.927948-05:
      DOI: 10.1002/jcp.24912
  • The Immunomodulatory and Therapeutic Effects of Mesenchymal Stromal Cells
           for Acute Lung Injury and Sepsis
    • Authors: Mirabelle S.H. Ho; Shirley H.J. Mei, Duncan J. Stewart
      Abstract: It is increasingly recognized that immunomodulation represents an important mechanism underlying the benefits of many stem cell therapies, rather than the classical paradigm of transdifferentiation and cell replacement. In the former paradigm, the beneficial effects of cell therapy result from paracrine mechanism(s) and/or cell‐cell interaction as opposed to direct engraftment and repair of diseased tissue and/or dysfunctional organs. Depending on the cell type used, components of the secretome, including microRNA (miRNA) and extracellular vesicles, may be able to either activate or suppress the immune system even without direct immune cell contact. Mesenchymal stromal cells (MSCs), also referred to as mesenchymal stem cells, are found not only in the bone marrow, but in a wide variety of organs and tissues. In addition to any direct stem cell activities, MSCs were the first stem cells recognized to modulate immune response, and therefore they will be the focus of this review. Specifically, MSCs appear to be able to effectively attenuate acute and protracted inflammation via interactions with components of both innate and adaptive immune systems. To date, this capacity has been exploited in a large number of pre‐clinical studies and MSC immunomodulatory therapy has been attempted with various degrees of success in a relatively large number of clinical trials. Here we will explore the various mechanism employed by MSCs to effect immunosuppression as well as review the current status of its use to treat excessive inflammation in the context of acute lung injury and sepsis in both pre‐clinical and clinical settings. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-25T06:46:25.357218-05:
      DOI: 10.1002/jcp.25028
  • Treg(s) in Cancer: Friends or Foe'
    • Authors: Dominik Wolf; Sieghart Sopper, Andreas Pircher, Guenther Gastl, Anna Maria Wolf
      Abstract: Immune escape is a hallmark of cancer. Regulatory T cells (Treg) have been described to maintain peripheral tolerance. The role of Treg in cancer is ambiguous, as they are central inhibitory regulators in solid tumors, whereas during inflammation‐driven tumorigenesis they prevent cancer initiation by restraining inflammation. As a consequence, under conditions with chronic inflammation that may initiate malignant transformation, application rather than depletion of Treg may be helpful. In solid tumors, however, the success story of immune‐activating antibodies targeting checkpoint molecules of T cell activation fuels the hope that Treg inactivation or depletion may additionally boost anti‐tumor immune response. In this review we summarize important aspects on the dual role of Treg in cancer to provide a rationale for future Treg targeting attempts. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-25T06:46:07.296271-05:
      DOI: 10.1002/jcp.25016
  • Para‐Cresyl Sulfate Acutely Impairs Vascular Reactivity and Induces
           Vascular Remodeling
    • Authors: Priscilla Gross; Ziad A. Massy, Lucie Henaut, Cédric Boudot, Joanna Cagnard, Cécilia March, Saïd Kamel, Tilman B. Drueke, Isabelle Six
      Abstract: Chronic kidney disease (CKD) is characterized by vascular remodeling and the retention of uremic toxins, several of which are independently associated with the high cardiovascular mortality rate in CKD patients. Whether the association between these uremic toxins and cardiovascular mortality is due to induction of vascular dysfunction and resulting vascular remodeling remains to be determined. This study evaluates the effects of para‐cresyl sulfate (PCS), a newly identified uremic toxin, on vascular function and remodeling. PCS acutely induced oxidative stress in both endothelial and vascular smooth muscle cells, with a maximal effect at 0.15 mM, corresponding to the mean ‘uremic’ concentration found in dialysis patients. PCS significantly increased within 30 min phenylephrine‐induced contraction of mouse thoracic aorta, through direct activation of rho‐kinase, independently of oxidative stress induction, as demonstrated by the capacity of rho‐kinase inhibitor Y‐27632 to abolish this effect. After exposure of the aorta to PCS for 48 h, we observed inward eutrophic remodeling, a hallmark of uremic vasculopathy characterized by a reduction of the area of both lumen and media, with unchanged media/lumen ratio. In conclusion, elevated PCS concentrations such as those observed in CKD patients, by promoting both vascular dysfunction and vascular remodeling, may contribute to the development of hypertension and to cardiovascular mortality in CKD. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-22T00:19:35.852625-05:
      DOI: 10.1002/jcp.25018
  • Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation in
           Vivo and Osteoprogenitor Differentiation ex Vivo
    • Authors: Nagat Frara; Samir M. Abdelmagid, Gregory R. Sondag, Fouad M. Moussa, Vanessa R. Yingling, Thomas A. Owen, Steven N. Popoff, Mary F. Barbe, Fayez F. Safadi
      Abstract: Initial identification of osteoactivin (OA)/glycoprotein non‐melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased 3‐fold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post‐fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV‐promoter (OA‐Tg). Western blot analysis showed increased OA/gpnmb in OA‐Tg osteoblasts, compared to wild‐type (WT). In OA‐Tg mouse femurs versus WT littermates, micro‐CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA‐Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over‐expressed in osteoclasts in OA‐Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX‐1 and RANK‐L, and decreased osteoclast numbers in OA‐Tg, compared to WT mice, indicating decreased bone remodeling in OA‐Tg mice. The proliferation rate of OA‐Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA‐Tg osteoprogenitors. Quantitative RT‐PCR analysis showed increased TGF‐β1 and TGF‐β receptors I and II expression in OA‐Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-22T00:19:18.454348-05:
      DOI: 10.1002/jcp.25020
  • Myeloid Zinc Finger 1 (MZF‐1) Regulates Expression of the CCN2/CTGF
           and CCN3/NOV Genes in the Hematopoietic Compartment
    • Authors: Richard T. Piszczatowski; Brian J. Rafferty, Andre Rozado, James V. Parziale, Nathan H. Lents
      Abstract: Connective Tissue Growth Factor (CCN2/CTGF) and Nephroblastoma Overexpressed (CCN3/NOV) execute key functions within the hematopoietic compartment. Both are abundant in the bone marrow stroma, which is a niche for hematopoiesis and supports marrow function. Roles for 1,25‐dihydroxyvitamin D3 (calcitriol) and all‐trans retinoic acid in the bone marrow have also been elucidated. Interestingly, some of the annotated roles of these vitamins overlap with established functions of CCN2 and CCN3. Yet, no factor has been identified that unifies these observations. In this study, we report the regulation of the CTGF and NOV genes by Myeloid Zinc Finger‐1 (MZF‐1), a hematopoietic transcription factor. We show the interaction of MZF‐1 with the CTGF and NOV promoters in several cell types. Up‐regulation of MZF‐1 via calcitriol and vitamin A induces expression of CTGF and NOV, implicating a role for these vitamins in the functions of these two genes. Lastly, knockdown of MZF1 reduces levels of CTGF and NOV. Collectively, our results argue that MZF‐1 regulates the CTGF and NOV genes in the hematopoietic compartment, and may be involved in their respective functions in the stroma. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-22T00:18:59.642223-05:
      DOI: 10.1002/jcp.25021
  • Functional Characterization of Nupr1L, a novelp53‐Regulated Isoform
           of the High Mobility Group (HMG)‐Related Protumoral Protein Nupr1
    • Authors: Maria Belen Lopez; Maria Noé Garcia, Daniel Grasso, Jennifer Bintz, Maria Inés Molejon, Gabriel Velez, Gwen Lomberk, Jose Luis Neira, Raul Urrutia, Juan Iovanna
      Abstract: We have previously demonstrated a crucial role of NUPR1 in tumor development and progression. In this work we report the functional characterization of a novel Nupr1‐like isoform (NUPR1L) and its functional interaction with the protumoral factor NUPR1. Through the use of primary sequence analysis, threading, and homology‐based molecular modeling as well as expression and immunolocalization, studies reveal that NUPR1L displays properties which are similar to member of the HMG‐like family of chromatin regulators including its ability to translocate to the cell nucleus and bind to DNA. Analysis of the NUPR1L promoter showed the presence of two p53‐response elements at positions ‐37 and ‐7 respectively. Experiments using reporter assays combined with site‐directed mutagenesis and using cells with controllable p53 expression demonstrate that both of these sequences are responsible for the regulation of NUPR1L expression by p53. Congruently, NUPR1L gene expression is activated in response to DNA damage induced by Oxaliplatin treatment or cell cycle arrest induced by serum starvation, two well‐validated methods to achieve p53 activation. Interestingly, expression of NUPR1L down‐regulates the expression of NUPR1, its closely related protumoral isoform, by a mechanism that involves the inhibition of its promoter activity. At the cellular level, overexpression of NUPR1L induces G1 cell cycle arrest and a decrease in their cell viability, an effect that is mediated, at least in part, by down‐regulating NUPR1 expression. Combined these experiments constitute the first functional characterization of NUPR1L as a new p53‐induced gene which negatively regulates the protumoral factor NUPR1. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-22T00:18:41.462726-05:
      DOI: 10.1002/jcp.25022
  • Melanoma Treatments: Advances and Mechanisms
    • Authors: Alexander Marzuka; Laura Huang, Nicholas Theodosakis, Marcus Bosenberg
      Abstract: Advances in the understanding of the molecular pathogenesis of melanoma and in cancer immunology have led to the rational design and recent clinical implementation of a variety of novel therapies for metastatic melanoma. BRAF and MEK inhibitors that target the MAPK pathway have high rates of clinical response in BRAF‐mutant melanoma. Therapies that modulate the immune response to melanoma, including monoclonal antibodies that interfere with pathways that inhibit T‐cell function, have been shown to be effective in melanoma. Lessons learned from these encouraging results are driving the development of new treatments for melanoma and other cancers. This review will focus on the science and clinical findings related to targeted therapies that inhibit BRAF or MEK as well as the immunotherapies that block the CTLA‐4 or PD‐1 pathways. Other experimental and combinatorial therapeutic approaches will also be discussed. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-22T00:18:24.092711-05:
      DOI: 10.1002/jcp.25019
  • TRPC1 Regulates Calcium‐Activated Chloride Channels in Salivary
           Gland Cells
    • Authors: Yuyang Sun; Lutz Birnbaumer, Brij B Singh
      Abstract: Calcium‐activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca2+ influx that activates ion channels such as CaCC to initiate Cl‐ efflux. However direct evidence as well as the molecular identity of the Ca2+ channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl‐ current was activated by increasing [Ca2+]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh‐A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store‐depletion and activates TRPC1‐mediated Ca2+ entry, potentiated the Cl‐ current, which was inhibited by the addition of a non‐specific TRPC channel blocker SKF96365 or removal of external Ca2+. Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca2+ entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl‐ currents upon increasing [Ca2+]i. These Cl‐ currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh‐A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl‐ currents without decreasing TMEM16a expression. Together the data suggests that Ca2+ entry via the TRPC1 channels is essential for the activation of CaCC. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-21T06:44:39.122861-05:
      DOI: 10.1002/jcp.25017
  • The Role of Mitochondrial Derived Peptides (MDPs) in Metabolism
    • Authors: Rafael Alis; Alejandro Lucia, Jose R Blesa, Fabian Sanchis‐Gomar
      Abstract: The term “mitokines” refers to signals derived from mitochondria that have an impact on other cells or tissues (Durieux et al., 2011). Rather than being simply a set of DNA composed by 37 genes, the mitochondrial DNA (mtDNA) is quite complex and includes small RNAs (Mercer et al., 2011). Mitochondrial‐derived peptides (MDPs) are encoded by functional short open reading frames (sORFs) in the mtDNA.
      PubDate: 2015-04-20T05:55:13.8841-05:00
      DOI: 10.1002/jcp.25023
  • Ephrin Reverse Signaling Mediates Palatal Fusion and
           Epithelial‐to‐Mesenchymal Transition Independently of
    • Authors: Maria J. Serrano; Jingpeng Liu, Kathy K. H. Svoboda, Ali Nawshad, M. Douglas Benson
      Abstract: The mammalian secondary palate forms from shelves of epithelia‐covered mesenchyme that meet at midline and fuse. The midline epithelial seam (MES) is thought to degrade by apoptosis, epithelial‐to‐mesenchymal transition (EMT), or both. Failure to degrade the MES blocks fusion and causes cleft palate. It was previously thought that transforming growth factor ß3 (Tgfß3) is required to initiate fusion. Members of the Eph tyrosine kinase receptor family and their membrane‐bound ephrin ligands are expressed on the MES. We demonstrated that treatment of mouse palates with recombinant EphB2/Fc to activate ephrin reverse signaling (where the ephrin acts as a receptor and transduces signals from its cytodomain) was sufficient to cause mouse palatal fusion when Tgfß3 signaling was blocked by an antibody against Tgfß3 or by an inhibitor of the TgfßrI serine/threonine receptor kinase. Cultured palatal epithelial cells traded their expression of epithelial cell markers for that of mesenchymal cells and became motile after treatment with EphB2/Fc. They concurrently increased their expression of the EMT‐associated transcription factors Snail, Sip1, and Twist1. EphB2/Fc did not cause apoptosis in these cells. These data reveal that ephrin reverse signaling directs palatal fusion in mammals through a mechanism that involves EMT but not apoptosis and activates a gene expression program not previously associated with reverse signaling. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-20T05:54:48.211525-05:
      DOI: 10.1002/jcp.25025
  • Regulation of Osteoblast Migration Involving Receptor Activator of Nuclear
           Factor (NF)‐Kappa B (RANK) Signaling
    • Authors: Diana Golden; Elizabeth A. Saria, Marc F. Hansen
      Abstract: Bone remodeling requires osteoclast activation, resorption and reversal, prior to osteoblast migration into the bone pit. The Receptor Activator of NF‐κB (RANK) signaling pathway plays an important role in bone remodeling. Two components of the RANK signaling pathway, RANK Ligand (RANKL) and the decoy receptor Osteoprotegerin (OPG), are expressed predominantly on the surface of osteoblasts, while RANK is principally expressed on the surface of osteoclasts. However, RANK has also been reported to be expressed on the surface of osteoblasts and osteosarcoma tumor cells. Treatment with soluble RANKL (sRANKL) of both normal osteoblasts and osteosarcoma tumor cells activated phosphorylation of ERK, p38MAPK, Akt and p65NF‐κB. However, modified Boyden chamber assays and wound repair assays showed differential response to sRANKL‐induced chemotactic migration in normal osteoblasts and osteosarcoma tumor cells. In contrast to previously published results, both normal osteoblasts and osteosarcoma tumor cells responded to sRANKL‐induced chemotactic migration but the normal osteoblasts did so only in the presence of an ERK pathway inhibitor. For both normal and tumor cells, the chemotactic response could be blocked by inhibiting the PI3K/Akt or p65NF‐κB pathway. Response to sRANKL in normal and tumor cells suggests a role for RANK/ERK‐mediated signaling in normal osteoblasts chemotactic migration during bone remodeling that is altered or lost during osteosarcoma tumorigenesis. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-20T05:54:29.876563-05:
      DOI: 10.1002/jcp.25024
  • Anti Proliferative and Pro Apoptotic Effects of Flavonoid Quercetin Are
           Mediated by CB1 Receptor in Human Colon Cancer Cell Lines
    • Authors: Maria Grazia Refolo; Rosalba D'Alessandro, Natascia Malerba, Chiara Laezza, Maurizio Bifulco, Caterina Messa, Maria Gabriella Caruso, Maria Notarnicola, Valeria Tutino
      Abstract: Quercetin, the major constituent of flavonoid and widely present in fruits and vegetables, is an attractive compound for cancer prevention due to its beneficial anti proliferative effects, showing a crucial role in the regulation of apoptosis and cell cycle signaling. In vitro studies have demonstrated that quercetin specifically influences colon cancer cell proliferation. Our experiments, using human colon adenocarcinoma cells, confirmed the anti proliferative effect of quercetin and gave intriguing new insight in to the knowledge of the mechanisms involved. We observed a significant increase in the expression of the endocannabinoids receptor (CB1‐R) after quercetin treatment. CB1‐R can be considered an estrogen responsive receptor and quercetin, having a structure similar to that of the estrogens, can interact with CB1‐R leading to the regulation of cell growth. In order to clarify the contribution of the CB1‐R to the quercetin action, we investigated some of the principal molecular pathways that are inhibited or activated by this natural compound. In particular we detected the inhibition of the major survival signals like the PI3K/Akt/mTOR and an induction of the pro apoptotic JNK/JUN pathways. Interestingly, the metabolism of β‐catenin was modified by flavonoid both directly and through activated CB1‐R. In all the experiments done, the quercetin action has proven to be reinforced by anandamide (Met‐F‐AEA), a CB1‐R agonist, and partially counteracted by SR141716, a CB1‐R antagonist. These findings open new perspectives for anticancer therapeutic strategies. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-20T05:53:36.630868-05:
      DOI: 10.1002/jcp.25026
  • GRP78 / Dna K is a Target for Nexavar / Stivarga / Votrient in the
           Treatment of Human Malignancies, Viral Infections and Bacterial Diseases
    • Authors: Jane L. Roberts; Mehrad Tavallai, Aida Nourbakhsh, Abigail Fidanza, Tanya Cruz‐Luna, Elizabeth Smith, Paul Siembida, Pascale Plamondon, Kelly A. Cycon, Christopher D. Doern, Laurence Booth, Paul Dent
      Abstract: Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi‐kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug‐treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10 and cyclophilin A. Prolonged ‘rafenib / sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola / Hepatitis A and B viruses, respectively. Pre‐treatment with the ‘Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib / pazopanib and sildenafil was much more potent than sorafenib / pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever and Enterovirus 71 infection and reproduction. ‘Rafenib drugs / pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of ‘Rafenib drugs / pazopanib restored antibiotic sensitivity in pan‐antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-09T17:56:44.887878-05:
      DOI: 10.1002/jcp.25014
  • Development of an Experimental Animal Model for Lower Back Pain by
           Percutaneous Injury‐Induced Lumbar Facet Joint Osteoarthritis
    • Authors: Jae‐Sung Kim; Kasra Ahmadinia, Xin Li, John L Hamilton, Steven Andrews, Chris A. Haralampus, Guozhi Xiao, Hong‐Moon Sohn, Jae‐Won You, Yo‐Seob Seo, Gary S. Stein, Andre J Van Wijnen, Su‐Gwan Kim, Hee‐Jeong Im
      Abstract: We report generation and characterization of pain‐related behavior in a minimally‐invasive facet joint degeneration (FJD) animal model in rats. FJD was produced by a non‐open percutaneous puncture‐induced injury on the right lumbar FJs at three consecutive levels. Pressure hyperalgesia in the lower back was assessed by measuring the vocalization response to pressure from a force transducer. After hyperalgesia was established, pathological changes in lumbar FJs and alterations of intervertebral foramen size were assessed by histological and imaging analyses. To investigate treatment options for lumber FJ osteoarthritis‐induced pain, animals with established hyperalgesia were administered with analgesic drugs, such as morphine, a selective COX‐2 inhibitor, a non‐steroidal anti‐inflammatory drug (NSAID) (ketorolac), or pregabalin. Effects were assessed by behavioral pain responses. One week after percutaneous puncture‐induced injury of the lumbar FJs, ipsilateral primary pressure hyperalgesia developed and was maintained for at least 12 weeks without foraminal stenosis. Animals showed decreased spontaneous activity, but no secondary hyperalgesia in the hind paws. Histopathological and microfocus X‐ray computed tomography analyses demonstrated that the percutaneous puncture injury resulted in osteoarthritis‐like structural changes in the FJs cartilage and subchondral bone. Pressure hyperalgesia was completely reversed by morphine. The administration of celecoxib produced moderate pain reduction with no statistical significance while the administration of ketorolac and pregabalin produced no analgesic effect on FJ osteoarthritis‐induced back pain. Our animal model of non‐open percutanous puncture‐induced injury of the lumbar FJs in rats shows similar characteristics of low back pain produced by human facet arthropathy. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-09T17:56:26.532772-05:
      DOI: 10.1002/jcp.25015
  • Role of Contraction Duration in Inducing Fast‐To‐Slow
           Contractile and Metabolic Protein and Functional Changes in Engineered
    • Authors: Alastair Khodabukus; Leslie M. Baehr, Sue C. Bodine, Keith Baar
      Abstract: The role of factors such as frequency, contraction duration and active time in the adaptation to chronic low‐frequency electrical stimulation (CLFS) is widely disputed. In this study we explore the ability of contraction duration (0.6, 6, 60 and 600 secs) to induce a fast‐to‐slow shift in engineered muscle while using a stimulation frequency of 10Hz and keeping active time constant at 60%. We found that all contraction durations induced similar slowing of time‐to‐peak tension. Despite similar increases in total myosin heavy (MHC) levels with stimulation, increasing contraction duration resulted in progressive decreases in total fast myosin. With contraction durations of 60 and 600 secs, MHC IIx levels decreased and MHC IIa levels increased. All contraction durations resulted in fast‐to‐slow shifts in TnT and TnC but increased both fast and slow TnI levels. Half‐relaxation slowed to a greater extent with contraction durations of 60 and 600 secs despite similar changes in the calcium sequestering proteins calsequestrin and parvalbumin and the calcium uptake protein SERCA. All CLFS groups resulted in greater fatigue resistance than control. Similar increases in GLUT4, mitochondrial enzymes (SDH and ATPsynthase), the fatty acid transporter CPT‐1 and the metabolic regulators PGC‐1α and MEF2 were found with all contraction durations. However, the mitochondrial enzymes cytochrome C and citrate synthase were increased to greater levels with contraction durations of 60 and 600 seconds. These results demonstrate that contraction duration plays a pivotal role in dictating the level of CLFS‐induced contractile and metabolic adaptations in tissue engineered skeletal muscle. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-09T17:56:06.872202-05:
      DOI: 10.1002/jcp.24985
  • The Other Side of the RAAS ‐ Aldosterone Improves Migration of
           Cardiac Progenitor Cells
    • Authors: Stephanie Könemann; Kristin Wenzel, Sabine Ameling, Karina Grube, Elke Hammer, Raik Könemann, Rasmita Samal, Uwe Völker, Stephan B. Felix
      Abstract: Stem cell therapy is a promising new option for patients suffering from heart failure. Though many clinical studies show encouraging results, little is known about the signals which cause stem cells to home to diseased but not to healthy hearts. We hypothesized that aldosterone as one of the main players of heart failure functions as an attractant for progenitor cells and stimulates their migration. Stem cell antigen‐1 (Sca‐1) positive cells were isolated from the hearts of wild type FVB mice via magnetic cell sorting. The migration rate of the cells was determined using aldosterone as an attractant in a modified Boyden chamber (n = 5). Aldosterone led to a dose dependent increase in migration rate and this effect could be prevented by adding its blocker eplerenone. The mineralocorticoid receptor could be detected on Sca‐1+ cells via western blot and immunofluorescence. Therefore, aldosterone seems to play a role in stem cell migration and there the effect is most likely mediated by the mineralocorticoid receptor. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-08T17:47:27.86942-05:0
      DOI: 10.1002/jcp.25013
  • Effect of Diabetes Mellitus on Adipocyte‐Derived Stem Cells in Rat
    • Authors: Medet Jumabay; Jeremiah H. Moon, Huwate Yeerna, Kristina I. Boström
      Abstract: Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein (BMP)4 is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte‐derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co‐localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-08T17:47:10.011655-05:
      DOI: 10.1002/jcp.25012
  • Glycation of Wild‐Type Apomyoglobin Induces Formation of Highly
           Cytotoxic Species
    • Authors: Clara Iannuzzi; Vincenzo Carafa, Lucia Altucci, Gaetano Irace, Margherita Borriello, Roberto Vinciguerra, Ivana Sirangelo
      Abstract: Protein glycation is a non‐enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild type apomyoglobin have been also investigated. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-03T04:17:52.492318-05:
      DOI: 10.1002/jcp.25011
  • Lovastatin‐Mediated Changes in Human Tendon Cells
    • Authors: Maria Kuzma‐Kuzniarska; Hannah R. Cornell, Michael C. Moneke, Andrew J. Carr, Philippa A. Hulley
      Abstract: Statins are among the most widely prescribed drugs worldwide. Numerous studies have shown their beneficial effects in prevention of cardiovascular disease through cholesterol‐lowering and anti‐atherosclerotic properties. Although some statin patients may experience muscle‐related symptoms, severe side effects of statin therapy are rare, primarily due to extensive first‐pass metabolism in the liver. Skeletal muscles appear to be the main site of side effects, however recently some statin‐related adverse effects have been described in tendon. The mechanism behind these side effects remains unknown. This is the first study that explores tendon‐specific effects of statins in human primary tenocytes. The cells were cultured with different concentrations of lovastatin for up to 1 week. No changes in cell viability or morphology were observed in tenocytes incubated with therapeutic doses. Short‐term exposure to lovastatin concentrations outside the therapeutic range had no effect on tenocyte viability, however cell migration was reduced. Simvastatin and atorvastatin, two other drug family members, also reduced the migratory properties of the cells. Prolonged exposure to high concentrations of lovastatin induced changes in cytoskeleton leading to cell rounding and decreased levels of mRNA for matrix proteins, but increased BMP‐2 expression. Gap junctional communication was impaired but due to cell shape change and separation rather than direct gap junction inhibition. These effects were accompanied by inhibition of prenylation of Rap1a small GTPase. Collectively, we showed that statins in a dose‐dependent manner decrease migration of human tendon cells, alter their expression profile and impair the functional network, but do not inhibit gap junction function. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-02T02:04:27.795551-05:
      DOI: 10.1002/jcp.25010
  • Reflections on Cellular Physiology as a Catalytic Framework for Medicine
    • Authors: Arthur Perelman
      Abstract: These are the reflections of a retired physician who was introduced to cellular physiology in 1947 at the University of Pennsylvania. I majored in Zoology. I took the required major courses, which were interesting but not exciting. I was a premedical student at the time. Fortunately I was introduced to cellular physiology in 1947. The teacher was Professor Louis Heilbrunn, an amazing man dedicated to teaching and the love of his students. He had a number of graduate students at the time. The undergraduates were well integrated with these advanced students. We used his textbook of physiology and we were all proud of being his students. He gave us a new way of thinking about biology. It was exciting and we were treated as mature students. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-01T07:24:16.511292-05:
      DOI: 10.1002/jcp.25007
  • Increased Linear Bone Growth by GH in the Absence of SOCS2 is Independent
           of IGF‐1
    • Authors: R. Dobie; S.F. Ahmed, K.A. Staines, C. Pass, S. Jasim, V.E. MacRae, C. Farquharson
      Abstract: Growth hormone (GH) signalling is essential for postnatal linear bone growth, but the relative importance of GHs actions on the liver and/or growth plate cartilage remains unclear. The importance of liver‐derived insulin like‐growth factor‐1 (IGF‐1) for endochondral growth has recently been challenged. Here we investigate linear growth in Suppressor of Cytokine Signalling (SOCS2)‐2 knockout mice, which have enhanced growth despite normal systemic GH/IGF‐1 levels. Wild‐type embryonic ex vivo metatarsals failed to exhibit increased linear growth in response to GH, but displayed increased Socs2 transcript levels (p 
      PubDate: 2015-04-01T07:23:05.871481-05:
      DOI: 10.1002/jcp.25006
  • Immortalized Mouse Floxed Fam20c Dental Papillar Mesenchymal and
           Osteoblast Cell Lines Retain Their Primary Characteristics
    • Authors: Chao Liu; Xiaofang Wang, Hua Zhang, Xiaohua Xie, Peihong Liu, Ying Liu, Priyam H Jani, Yongbo Lu, Shuo Chen, Chunlin Qin
      Abstract: Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T‐antigen (SV40 T‐Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long‐term passages and genomic integration of SV40 T‐Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-01T07:22:46.98116-05:0
      DOI: 10.1002/jcp.25008
  • TGF‐Beta Suppresses Ift88 Expression in Chondrocytic ATDC5 Cells
    • Authors: Makiri Kawasaki; Yoichi Ezura, Tadayoshi Hayata, Takuya Notomi, Yayoi Izu, Masaki Noda
      Abstract: IFT88 is an intraflagella transport protein, critical for the cilium and has been shown to be required for the maintenance of chondrocytes and cartilage. However, how IFT88 is controlled by cytokines that play a role in osteoarthritis is not well understood. Therefore, we examined the effects of TGF beta on the expression of IFT88. We used ATDC5 cells as chondrocytes and analyzed the effects of TGF beta on gene expression. TGF beta treatment suppresses the levels of Ift88 mRNA in a dose‐dependent manner starting from as low as 0.5 ng/ml and reaching the nadir at around 2 ng/ml. TGF beta treatment also suppresses the protein levels of Ift88. TGF beta suppression of Ift88 is still observed when the cells are cultured in the presence of a transcriptional inhibitor while the TGF beta suppression is weakened in the presence of a protein synthesis inhibitor, cycloheximide. TGF beta treatment suppresses the levels of Ift88 mRNA stability suggesting the presence of posttranscriptional regulation. TGF beta treatment reduces the number of cilia positive cells and suppresses average length of cilia. Knockdown of Ift88 by siRNA enhances TGF beta‐induced increase in type II collagen mRNA expression in ATDC5 cells revealing the suppressive role of Ift88 on TGF beta‐induced regulation of extracellular matrix protein expression. TGF beta also suppresses Ift88 mRNA expression in primary culture of chondrocytes. These data indicate that TGF beta regulates Ift88 gene expression at least in part via posttrascriptional manner. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-31T09:06:55.347463-05:
      DOI: 10.1002/jcp.25005
  • Impairments of the Vascular KATP Channel Imposes Fatal Susceptibility to
           Experimental Diabetes Due to Multi‐Organ Injuries
    • Authors: Shan‐Shan Li; Ningren Cui, Yang Yang, Timothy C Trower, Yu‐Min Wei, Yang Wu, Shuang Zhang, Xin Jin, Chun Jiang
      Abstract: The vascular isoform of ATP‐sensitive K+ (KATP) channels regulates blood flow to all organs. The KATP channel is strongly inhibited by reactive oxygen and carbonyl species produced in diabetic tissue inflammation. To address how such channel inhibition impacts vascular regulation as well as tissue viability, we performed studies in experimental diabetic mice. Strikingly, we found that knockout of the Kcnj8 encoding Kir6.1 subunit (Kcnj8‐KO) caused mice to be fatally susceptible to diabetes. Organ perfusion studies suggested that the lack of this vascular K+ channel handicapped activity‐dependent vasodilation, leading to hypoperfusion, tissue hypoxia and multi‐organ failure. Morphologically, Kcnj8‐KO mice showed greater inflammatory cell infiltration, higher levels of expression of inflammation indicator proteins, more severe cell apoptosis and worse tissue disruptions. These were observed in the kidney, liver and heart under diabetic condition in parallel comparison to tissues from WT mice. Patch clamping and molecular studies showed that the KATP channel was S‐glutathionylated in experimental diabetes contributing to the inhibition of channel activity as well as the reduced arterial responses to vasodilators. These results suggest that the vascular KATP channel is organ‐protective in diabetic condition, and since the channel is suppressed by diabetic oxidative stress, therapeutical interventions to the maintenance of functional KATP channels may help to lower or prevent diabetic organ dysfunction. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-30T01:48:09.247148-05:
      DOI: 10.1002/jcp.25003
  • Hyperosmolarity‐Induced Down‐Regulation of Claudin‐2
           Mediated by Decrease in PKCβ‐Dependent GATA‐2 in MDCK
    • Authors: Akira Ikari; Naoko Fujii, Shinya Hahakabe, Hisayoshi Hayashi, Masahiko Yamaguchi, Yasuhiro Yamazaki, Satoshi Endo, Toshiyuki Matsunaga, Junko Sugatani
      Abstract: Hyperosmolarity decreases claudin‐2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity‐induced decrease in claudin‐2 expression is inhibited by Go6983, a non‐selective protein kinase C (PKC) inhibitor, and PKCβ specific inhibitor in Madin‐Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca2+ concentration and phosphorylated PKCβ level, which were inhibited by RN‐1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12‐myristate 13‐acetate, a PKC activator, decreased claudin‐2 expression. These results indicate hyperosmolarity decreases claudin‐2 expression mediated by the activation of RN‐1734‐sensitive channel and PKCβ. Hyperosmolarity decreased promoter activity of claudin‐2, which was inhibited by Go6983 and PKCβ inhibitor similar to those in real‐time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of ‐469/‐6, a deletion mutant. Claudin‐2 has hyperosmolarity‐sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA‐2, which was inhibited by Go6983 and PKCβ inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity‐induced decrease in reporter activity and claudin‐2 expression were rescued by over‐expression of wild type GATA‐2. Chromatin immunoprecipitation assay showed that GATA‐2 bound to promoter region of claudin‐2. These results suggest that hyperosmolarity decreases the expression level of claudin‐2 via a decrease in PKCβ‐dependent GATA‐2 transcriptional activity in renal tubular epithelial cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-30T01:46:57.244021-05:
      DOI: 10.1002/jcp.25004
  • Lactogenic Hormone Stimulation and Epigenetic Control of L‐Amino
           Acid Oxidase Expression in Lactating Mammary Glands
    • Authors: Kazuki Fujii; Haolin Zhang, Kento Usuda, Gen Watanabe, Kentaro Nagaoka
      Abstract: L‐amino acid oxidase (LAO), a classic flavoprotein, shows antibacterial activity by producing hydrogen peroxide. LAO exists in many tissues such as salivary gland, thymus, spleen, small intestine, and testis. In particular, LAO was highly expressed in mice milk and plays an important factor in innate immunity of mammary glands. However, the mechanism which LAO expression is regulated spatially and temporally in lactating mammary glands has been unclear. In this study, we showed the contribution of lactogenic hormone and epigenetic control on LAO gene expression. In monolayer of mammary epithelial cells, treatment of lactogenic hormone mixture, dexamethasone, insulin and prolactin, did not induce LAO mRNA expression and its promoter activity, even though one of milk protein β‐casein expression was stimulated. However, increase of LAO expression was observed when the cells were treated with lactogenic hormones in a 3‐dimensional culture. The results of chromatin immunoprecipitation analysis revealed that histone H3K18 acetylation increased and histone H3K27 tri‐methylation decreased with lactation, which is associated with a period of high LAO expression. Moreover, the treatment of histone methylation inhibitor (DZNep) as well as histone deacetylation inhibitor (Trichostatine A) induced LAO expression in monolayer of mammary cells. Taken together, this is the first demonstration showing that LAO expression is induced in cell culture, and stimulation of lactogenic hormone and change of histone modification are promising signals to show highly expression of LAO in lactating mammary glands. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:58:17.84328-05:0
      DOI: 10.1002/jcp.25000
  • Nuclear Translocation of Calpain‐2 Mediates Apoptosis of
           Hypertrophied Cardiomyocytes in Transverse Aortic Constriction Rat
    • Authors: Juan‐Juan Sheng; Hui Chang, Zhi‐Bin Yu
      Abstract: Apoptosis of cardiomyocytes plays an important role in the transition from cardiac hypertrophy to heart failure. Hypertrophied cardiomyocytes show enhanced susceptibility to apoptosis. Therefore, the aim of this study was to determine the susceptibility to apoptosis and its mechanism in hypertrophied cardiomyocytes using a rat model of transverse abdominal aortic constriction (TAC). Sixteen weeks of TAC showed compensatory and pathological hypertrophy in the left ventricle. TUNEL‐positive nuclei were significantly increased in TAC with angiotensin II (Ang II) treatment. Calpain inhibitor, PD150606, effectively inhibited Ang II‐induced apoptosis of hypertrophied cardiomyocytes. Ang II increased nuclear translocation of intracellular Ca2+ activated calpain‐2 in hypertrophied cardiomyocytes. Ang II enhanced the interaction between activated calpain‐2 and Ca2+/calmodulin‐dependent protein kinase II δB (CaMKIIδB), and promoted the degradation of CaMKIIδB by calpain‐2 in the nuclei of hypertrophied cardiomyocytes. Consequently, the depressed CaMKIIδB downregulated the expression of antiapoptotic Bcl‐2 leading to mitochondrial depolarization and release of cytochrome c led to apoptosis of hypertrophied cardiomyocytes. In conclusion, hypertrophied cardiomyocytes show increased susceptibility to apoptosis during Ang II stimulation via nuclear calpain‐2 and CaMKIIδB pathway. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:58:02.592822-05:
      DOI: 10.1002/jcp.24999
  • VEGF Enhances the Migration of MSCs in Neural Differentiation by
           Regulating Focal Adhesion Turnover
    • Authors: Huihui Wang; Xingkai Wang, Jing Qu, Qing Yue, Ya'nan Hu, Huanxiang Zhang
      Abstract: Mesenchymal stem cells (MSCs) hold great promise in neural regeneration, due to their intrinsic neuronal potential and migratory tropism to damaged nervous tissues. However, the chemotactic signals mediating the migration of MSCs remain poorly understood. Here, we investigated the regulatory roles for focal adhesion kinase (FAK) and Rac1 in vascular endothelial growth factor (VEGF)‐stimulated migration of MSCs in neural differentiation. We found that MSCs in various differentiation states show significant different chemotactic responses to VEGF and cells in 24‐h preinduction state possess the highest migration speed and efficiency. FAK, as the downstream signaling molecule, is involved in the VEGF‐induced migration by regulating the assembly and distribution of focal adhesions (FAs) and reorganization of F‐actin. The features of FAs and cytoskeletons and the ability of lamellipodia formation are closely related to the neural differentiation states of MSCs. VEGF promotes FA formation with an asymmetric distribution of FAs and induces the activation of Y397‐FAK and Y31/118‐paxillin of undifferentiated and 24‐h preinduced MSCs in a time‐dependent manner. Inhibition of FAK by PF‐228 or expressing FAK‐Y397F mutant impairs the dynamics of FAs in MSCs during VEGF‐induced migration. Furthermore, Rac1 regulates FA formation in a FAK‐dependent manner. Overexpression of constitutive activated mutants of Rac1 increases the number of FAs in undifferentiated and 24‐h preinduced MSCs, while VEGF‐induced increase of FA formation is decreased by inhibiting FAK by PF‐228. Collectively, these results demonstrate that FAK and Rac1 signalings coordinately regulate the dynamics of FAs during VEGF‐induced migration of MSCs in varying neural differentiation states. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:57:44.911676-05:
      DOI: 10.1002/jcp.24997
  • Determinants of Muscle and Bone Aging
    • Authors: E Curtis; A Litwic, C Cooper, E Dennison
      Abstract: Loss of bone and muscle with advancing age represent a huge threat to loss of independence in later life. “Osteoporosis represents a major public health problem through its association with fragility fractures, primarily of the hip, spine and distal forearm” (Moselhy et al., 2012). Sarcopenia, the age related loss of muscle mass and function, may add to fracture risk by increasing falls risk. In the context of muscle aging, it is important to remember that it is not just a decline in muscle mass which contributes to the deterioration of muscle function. Other factors underpinning muscle quality come into play, including muscle composition, aerobic capacity and metabolism, fatty infiltration, insulin resistance, fibrosis and neural activation. Genetic, developmental, endocrine and lifestyle factors, such as physical activity, smoking and poor diet have dual effects on both muscle and bone mass in later life and these will be reviewed here. These include poor nutrition, lack of physical activity and cigarette smoking, comorbidities or medication use. Recent work has highlighted a possible role for the early environment. Inflammaging is an exciting emerging research field that is likely to prove relevant to future work, including interventions designed to retard to reverse bone and muscle loss with age. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:57:24.088441-05:
      DOI: 10.1002/jcp.25001
  • Angiogenic Role of MMP‐2/9 Expressed on the Cell Surface of Early
           Endothelial Progenitor Cells/Myeloid Angiogenic Cells
    • Authors: Toshie Kanayasu‐Toyoda; Takeshi Tanaka, Akiko Ishii‐Watabe, Hiroko Kitagawa, Akifumi Matsuyama, Eriko Uchida, Teruhide Yamaguchi
      Abstract: Since the introduction of angiogenic cell therapy using early endothelial progenitor cells (EPCs), myeloid angiogenic cells (MACs) have been expected to be useful in treating ischemic diseases. In order to elucidate the angiogenic properties of MACs/EPCs, we clarified the characteristics of MACs as compared to M2 macrophages (Mϕs). Comparison of the gene expression profiles of MACs and late EPCs revealed that MACs expressed greater amounts of metalloproteinase (MMP)‐9. It should be noted that the profile of MMP‐2/9 expression on the cell surface of MACs was similar to that of M2 Mϕs, and that cell surface MMP‐2/9 might be an active form based on molecular size. In addition, the invasion of MACs was prohibited not only by MMP‐2/9 inhibitor, but also by the hyaluronidase treatment that caused the down‐regulation of MMP‐9 on the cell surface of MACs and inhibited their invasion activity. These results indicate that cell surface MMP‐2/9 plays an important role in the high invasion ability of MACs. The conditioned medium of both MACs and M2 Mϕs stimulated tube formation of endothelial cells in vitro. MACs caused an increase in vessel formation in in vivo models through the production of IL‐8. We propose that the role of MACs with cell surfaces expressing MMP‐2/9 is rapidly invading ischemic tissue. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:57:10.704862-05:
      DOI: 10.1002/jcp.25002
  • Loss of microRNA‐101 Promotes Epithelial to Mesenchymal Transition
           in Hepatocytes
    • Authors: Shuhua Zhao; Yuanyuan Zhang, Xiuxiu Zheng, Xiaolong Tu, Huanan Li, Jiangning Chen, Yuhui Zang, Junfeng Zhang
      Abstract: Epithelial‐to‐mesenchymal transition (EMT) has been implicated in embryonic development and various pathological events. However, the involvement of microRNA in the process of EMT remains to be fully defined in hepatocyte. ZEB1 is a well‐known transcriptional repressor of E‐cadherin and plays a major role in triggering EMT during organ fibrosis and cancer cell metastasis. Computational microRNA target predictions detect a conserved sequence matching to miR‐101 in the 3'UTR of ZEB1 mRNA. Our results confirm that miR‐101 suppresses ZEB1 expression by targeting the predicted site of ZEB1 3'UTR. Subsequent investigations show that miR‐101 is significantly downregulated in the cultured hepatocytes undergoing EMT and in the hepatocytes isolated from fibrotic liver. Along with the loss of miR‐101, the ZEB1 expression increases simultaneously in hepatocytes. In addition, miR‐101 levels in HCC cell lines are negatively associated with the ZEB1 productions and the metastatic potentials of tumor cells. Mechanistically, we demonstrate that miR‐101 significantly inhibits the TGF‐β1‐induced EMT in hepatocytes, whereas inhibition of miR‐101 promotes the EMT process as indicated by the changes of morphology, cell migration and the expression profiles of EMT markers. In the fibrotic liver, ectopic expression of miR‐101 can significantly downregulate ZEB1 in the hepatocyte and thereby reduces the mesenchymal marker expression. Moreover, miR‐101 significantly inhibits the proliferation and migration of HCC cell. Our results demonstrate that miR‐101 regulates HCC cell phenotype by upregulating the epithelial marker genes and suppressing the mesenchymal ones. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-25T02:51:19.937133-05:
      DOI: 10.1002/jcp.24995
  • Establishment of Four New Human Primary Cell Cultures from
           Chemo‐Naïve Italian Osteosarcoma Patients
    • Authors: Marcella Laschi; Giulia Bernardini, Michela Geminiani, Lorenzo Ghezzi, Loredana Amato, Daniela Braconi, Lia Millucci, Bruno Frediani, Adriano Spreafico, Alessandro Franchi, Domenico Campanacci, Rodolfo Capanna, Annalisa Santucci
      Abstract: Osteosarcoma (OS) is a primary highly malignant tumor of bone, affecting predominately adolescents and young adults between 10 and 20 years of age. OS is characterized by an extremely aggressive clinical course, with a rapid development of metastasis to the lung and distant bones. The relative rarity of the tumor contributes significantly to the difficulty in studying the molecular contributions to the disease process and thereby in the development of novel targeted therapeutic approaches for the cure of OS. Moreover, individual and ethnic differences in OS onset and in the responsiveness to irradiation and chemotherapy, makes restrictive and not effective the use of the most frequently studied OS commercial cell lines (SaOS‐2, U2OS, MG63). Actual experimental protocols should be applied on a variety of human primary cell cultures generated from OS patient tumors before the administration of a chemotherapeutic treatment. With this aim, we established four novel OS primary cell culture models based on chemo‐naïve osteoblastic, chondroblastic and small cell high grade OS tumors from adolescent patients of Italian origin. Several phenotypic features of these cells were biochemically characterized and compared with those of the original tumors, such as with those of established OS commercial cell lines and primary not transformed cells. The cultured cells appear to have retained the peculiar characteristics of the original tumors and could be used as new culture models for in vitro OS studies, aimed not only at a better understanding of the OS cellular origin and malignant progression but also at the identification of diagnostic markers and new therapeutic targets for OS. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-25T02:51:03.223684-05:
      DOI: 10.1002/jcp.24996
  • Concise Review: The Role of Oxygen in Hematopoietic Stem Cell Physiology
    • Authors: Mojca Jež; Primož Rožman, Zoran Ivanović, Tuba Bas
      Pages: 1999 - 2005
      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 the 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 the 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. J. Cell. Physiol. 230: 1999–2005, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:38.595843-05:
      DOI: 10.1002/jcp.24953
  • Targeting Osteopontin, the Silent Partner of Na+/H+ Exchanger Isoform 1 in
           Cardiac Remodeling
    • Authors: Iman A. Mohamed; Fatima Mraiche
      Pages: 2006 - 2018
      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. J. Cell. Physiol. 230: 2006–2018, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:41.224131-05:
      DOI: 10.1002/jcp.24958
  • The Role of Auxiliary Subunits for the Functional Diversity of
           Voltage‐Gated Calcium Channels
    • Authors: Marta Campiglio; Bernhard E. Flucher
      First page: 2019
      Abstract: Voltage‐gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore‐forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice‐variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre‐existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macro‐molecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein‐protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein‐protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:58:40.644318-05:
      DOI: 10.1002/jcp.24998
  • 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: 2032 - 2037
      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 2 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. J. Cell. Physiol. 230: 2032–2037, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:44.438192-05:
      DOI: 10.1002/jcp.24954
  • KMUP‐1 Promotes Osteoblast Differentiation Through cAMP and cGMP
           Pathways and Signaling of BMP‐2/Smad1/5/8 and
    • Authors: Shu‐Fen Liou; Jong‐Hau Hsu, Hsin‐Chieh Chu, Hung‐Hong Lin, Ing‐Jun Chen, Jwu‐Lai Yeh
      Pages: 2038 - 2048
      Abstract: Phosphodiesterase (PDE) inhibitors have been suggested as a possible candidate for the treatment of osteopenia, including osteoporosis. KMUP‐1 is a novel xanthine derivative with inhibitory activities on the PDE 3, 4, and 5 iso‐enzymes to suppress the degradation of cAMP and cGMP. This study aimed to investigate the effect of KMUP‐1 on osteoblast differentiation and the underlying cellular and molecular mechanisms. Primary osteoblasts and osteoblastic MC3T3‐E1 cells were examined. KMUP‐1 enhanced alkaline phosphatase (ALP) activity and mineralization compared to untreated controls in primary osteoblasts and MC3T3‐E1 cells. KMUP‐1 also increased the mRNA expression of the osteoblastic differentiation markers, including collagen type 1a, ALP, osteocalcin, osteoprotegerin, BMP‐2, and Runx2, a key transcription regulator for osteoblastic differentiation. The osteogenic effect of KMUP‐1 was abolished by BMP signaling inhibitor, noggin. Furthermore, we found that KMUP‐1 upregulated Smad1/5/8 phosphorylations with subsequent BRE‐Luc activation confirmed by transient transfection assay. In addition, KMUP‐1 inactivated glycogen synthase kinase‐3β (GSK‐3β), with associated nuclear translocation of β‐catenin. Co‐treatment with H89 and KT5823, cAMP and cGMP pathway inhibitors, respectively, reversed the KMUP‐1‐induced activations of Smad1/5/8, β‐catenin, and Runx2. The findings demonstrate for the first time that KMUP‐1 can promote osteoblast maturation and differentiation in vitro via BMP‐2/Smad1/5/8 and Wnt/β‐catenin pathways. These effects are mediated, in part, by the cAMP and cGMP signaling. Thus, KMUP‐1 may be a novel osteoblast activator and a potential new therapy for osteoporosis. J. Cell. Physiol. 230: 2038–2048, 2015. © 2014 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:34.369764-05:
      DOI: 10.1002/jcp.24904
  • 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
      Pages: 2049 - 2058
      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. J. Cell. Physiol. 230: 2049–2058, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:35.381288-05:
      DOI: 10.1002/jcp.24931
  • 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
      Pages: 2059 - 2066
      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. J. Cell. Physiol. 230: 2059–2066, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:49.890662-05:
      DOI: 10.1002/jcp.24932
  • 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
      Pages: 2067 - 2074
      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. J. Cell. Physiol. 230: 2067–2074, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:34.882836-05:
      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
      Pages: 2075 - 2085
      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. J. Cell. Physiol. 230: 2075–2085, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:36.021695-05:
      DOI: 10.1002/jcp.24935
  • Mechanosensitive store‐operated calcium entry regulates the
           formation of cell polarity
    • Authors: Yi‐Wei Huang; Shu‐Jing Chang, Hans I‐Chen Harn, Hui‐Ting Huang, Hsi‐Hui Lin, Meng‐Ru Shen, Ming‐Jer Tang, Wen‐Tai Chiu
      Pages: 2086 - 2097
      Abstract: Ca2+‐mediated formation of cell polarity is essential for directional migration which plays an important role in physiological 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, TRPC1, and 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. J. Cell. Physiol. 230: 2086–2097, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:45.55665-05:0
      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
      Pages: 2098 - 2107
      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 is 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, whereas 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 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. J. Cell. Physiol. 230: 2098–2107, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:47.237188-05:
      DOI: 10.1002/jcp.24938
  • 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
      Pages: 2108 - 2119
      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 lipopolysaccharide (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 peroxisome proliferator‐activated receptors (PPARα/γ) and heme oxygenase 1 (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 diseases. J. Cell. Physiol. 230: 2108–2119, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:49.211469-05:
      DOI: 10.1002/jcp.24939
  • 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
      Pages: 2120 - 2127
      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. J. Cell. Physiol. 230: 2120–2127, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:37.257464-05:
      DOI: 10.1002/jcp.24940
  • Mitochondrial DNA Oxidative Damage Contributes to Cardiomyocyte
           Ischemia/Reperfusion‐Injury in Rats: Cardioprotective Role of
    • Authors: Rongchuan Yue; Xuewei Xia, Jiahui Jiang, Dezhong Yang, Yu Han, Xiongwen Chen, Yue Cai, Liangpeng Li, Wei Eric Wang, Chunyu Zeng
      Pages: 2128 - 2141
      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. J. Cell. Physiol. 230: 2128–2141, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:40.505459-05:
      DOI: 10.1002/jcp.24941
  • 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
      Pages: 2142 - 2151
      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 mitogen‐activated protein kinases, Janus kinase/signal transducer and activator of transcription, and nuclear factor‐kappaB 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, 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. J. Cell. Physiol. 230: 2142–2151, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:39.201476-05:
      DOI: 10.1002/jcp.24943
  • 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
      Pages: 2152 - 2163
      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. J. Cell. Physiol. 230: 2152–2163, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:39.90092-05:0
      DOI: 10.1002/jcp.24944
  • 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
      Pages: 2164 - 2173
      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. J. Cell. Physiol. 230: 2164–2173, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:36.690639-05:
      DOI: 10.1002/jcp.24945
  • 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
      Pages: 2174 - 2183
      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 h:12 h LD cycle (12:12 LD) or exposed to weekly 12 h 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. J. Cell. Physiol. 230: 2174–2183, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:31.9167-05:00
      DOI: 10.1002/jcp.24946
  • 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
      Pages: 2184 - 2201
      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 (OP). Some studies have investigated the possible beneficial effects of ALA on OP 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/receptor activator of nuclear factor‐κB (NF‐κB) ligand (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 be a candidate for clinical OP treatment. J. Cell. Physiol. 230: 2184–2201, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:37.803299-05:
      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
      Pages: 2202 - 2211
      Abstract: 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. 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. 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. 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. J. Cell. Physiol. 230: 2202–2211, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:32.748334-05:
      DOI: 10.1002/jcp.24948
  • 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
      Pages: 2212 - 2223
      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. J. Cell. Physiol. 230: 2212–2223, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:42.365449-05:
      DOI: 10.1002/jcp.24949
  • The SRE Motif in the Human PNPLA3 Promoter (−97 to
           −88 bp) 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
      Pages: 2224 - 2232
      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. J. Cell. Physiol. 230: 2224–2232, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:44.991491-05:
      DOI: 10.1002/jcp.24951
  • TSH/TSHR Signaling Suppresses Fatty Acid Synthase (FASN) Expression in
    • Authors: Jicui Chen; Jianmin Ren, Qingping Jing, Sumei Lu, Yuchao Zhang, Yuantao Liu, Cong Yu, Peng Gao, Chen Zong, Xia Li, Xiangdong Wang
      Pages: 2233 - 2239
      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 downregulation 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. J. Cell. Physiol. 230: 2233–2239, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:33.943813-05:
      DOI: 10.1002/jcp.24952
  • Inhibition of PKC‐induced COX‐2 and IL‐8 Expression in
           Human Breast Cancer Cells by Glucosamine
    • Authors: Wan‐Yu Chou; Kun‐Han Chuang, David Sun, Yu‐Hsiu Lee, Pu‐Hong Kao, Yen‐Yu Lin, Hsei‐Wei Wang, Yuh‐Lin Wu
      First page: 2240
      Abstract: Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase‐2 (COX‐2) and interleukin‐8 (IL‐8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti‐inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC‐regulation of COX‐2/IL‐8 expression and the associated impact on breast cancer. In MCF‐7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX‐2 and IL‐8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E2 (PGE2) and IL‐8. Glucosamine is able to promote COX‐2 protein degradation in a calpain‐dependent manner and IL‐8 protein degradation in calpain‐dependent and proteasome‐dependent manners. The MAPK and NF‐κB pathways are involved in PKC‐induced COX‐2 expression, but only the NF‐κB pathway is involved in PKC‐induced IL‐8 expression. Glucosamine attenuates PKC‐mediated IκBα phosphorylation, nuclear NF‐κB translocation, and NF‐κB reporter activation. Both PGE2 and IL‐8 promote cell proliferation and IL‐8 induces cell migration; thus glucosamine appears to suppress PKC‐induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX‐2 and IL‐8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF‐κB signaling pathway, resulting in an inhibition of breast cancer cell growth. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-09T00:21:52.1074-05:00
      DOI: 10.1002/jcp.24955
  • Oral squamous cancer cell exploits hnRNP A1 to regulate cell cycle and
    • Authors: Cheng Yu; Jihua Guo, Yu Liu, Jun Jia, Rong Jia, Mingwen Fan
      Pages: 2252 - 2261
      Abstract: Oral squamous cell carcinoma (OSCC) is a common human malignant tumor with high mortality. So far, the molecular pathogenesis of OSCC remains largely unclear. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an important multi‐function splicing factor and closely related to tumorigenesis. hnRNP A1 is overexpressed in various tumors, and promotes aerobic glycolysis and elongation of telomere, but the function of hnRNP A1 in cell cycle and proliferation remains unclear. We found that hnRNP A1 was overexpressed in OSCC tissues, and was required for the growth of OSCC cells. Moreover, hnRNP A1 was highly expressed in the G2/M cell cycle phase. Knockdown of hnRNP A1 induced G2/M arrest. DNA microarray assay result showed that hnRNP A1 regulated the expression of a number of target genes associated with G2/M phase. Moreover, hnRNP A1 controlled the alternative splicing of CDK2 exon 5. These findings suggested that hnRNP A1 plays key roles in the regulation of cell cycle progression and pathogenesis of OSCC. J. Cell. Physiol. 230: 2252–2261, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:47.985874-05:
      DOI: 10.1002/jcp.24956
  • 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: 2262 - 2269
      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. J. Cell. Physiol. 230: 2262–2269, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:42.954101-05:
      DOI: 10.1002/jcp.24957
  • New Face for Chromatin‐Related Mesenchymal Modulator: n‐CHD9
           Localizes to Nucleoli and Interacts With Ribosomal Genes
    • Authors: Ronit Salomon‐Kent; Ronit Marom, Sam John, Miroslav Dundr, Louis R. Schiltz, Jose Gutierrez, Jerry Workman, Dafna Benayahu, Gordon L. Hager
      Pages: 2270 - 2280
      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. J. Cell. Physiol. 230: 2270–2280, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:48.615465-05:
      DOI: 10.1002/jcp.24960
  • Nexavar/Stivarga and Viagra Interact to Kill Tumor Cells
    • Authors: Mehrad Tavallai; Hossein A. Hamed, Jane L. Roberts, Nichola Cruickshanks, John Chuckalovcak, Andrew Poklepovic, Laurence Booth, Paul Dent
      Pages: 2281 - 2298
      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 are 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. J. Cell. Physiol. 230: 2281–2298, 2015. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-05-26T11:27:46.272627-05:
      DOI: 10.1002/jcp.24961
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