for Journals by Title or ISSN
for Articles by Keywords

Publisher: John Wiley and Sons   (Total: 1598 journals)

 A  B  C  D  E  F  G  H  I  J  K  L  M  N  O  P  Q  R  S  T  U  V  W  X  Y  Z  

  First | 9 10 11 12 13 14 15 16     

The end of the list has been reached. Please navigate to previous pages.

  First | 9 10 11 12 13 14 15 16     

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  [1598 journals]
  • Grp78 is Critical for Amelogenin‐Induced Cell Migration in a
           Multipotent Clonal Human Periodontal Ligament Cell Line
    • Authors: Kyosuke Toyoda; Takao Fukuda, Terukazu Sanui, Urara Tanaka, Kensuke Yamamichi, Ryo Atomura, Hidefumi Maeda, Atsushi Tomokiyo, Takaharu Taketomi, Takeshi Uchiumi, Fusanori Nishimura
      Abstract: Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose‐regulated protein 78 (Grp78) as a new amelogenin‐binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1‐17, which possesses the most typical characteristics of PDLSCs. Confocal co‐localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1‐17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration‐associated genes in 1‐17 cells. Moreover, Grp78 overexpression enhanced rM180‐induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin‐induced migration in 1‐17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin‐induced periodontal tissue regeneration. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-03T17:49:10.870409-05:
      DOI: 10.1002/jcp.25087
  • Alterations in Skeletal Muscle Oxidative Phenotype in Mice Exposed to
           Three Weeks of Normobaric Hypoxia
    • Authors: Ilse G.M. Slot; Annemie M.W.J. Schols, Chiel C. de Theije, Frank J.M. Snepvangers, Harry R. Gosker
      Abstract: Skeletal muscle of patients with chronic respiratory failure is prone to loss of muscle mass and oxidative phenotype. Tissue hypoxia has been associated with cachexia and emphysema in humans. Experimental research on the role of hypoxia in loss of muscle oxidative phenotype however has yielded inconsistent results. Animal studies are frequently performed in young animals, which may hinder translation to generally older aged patients. Therefore in this study we tested the hypothesis that hypoxia induces loss of skeletal muscle oxidative phenotype in a model of aged (52 weeks) mice exposed to three weeks of hypoxia. Additional groups of young (4 weeks) and adult (12 weeks) mice were included to examine age effects. To verify hypoxia‐induced cachexia, fat pad and muscle weights as well as muscle fiber cross‐sectional areas were determined. Muscle oxidative phenotype was assessed by expression and activity of markers of mitochondrial metabolism and fiber‐type distribution. A profound loss of muscle and fat was indeed accompanied by a slightly lower expression of markers of muscle oxidative capacity in the aged hypoxic mice. In contrast, hypoxia‐associated changes of fiber‐type composition were more prominent in the young mice. The differential response of the muscle of young, adult, and aged mice to hypoxia suggests that age matters and that the aged mouse is a better model for translation of findings to elderly patients with chronic respiratory disease. Furthermore, the findings warrant further mechanistic research into putative accelerating effects of hypoxia‐induced loss of oxidative phenotype on the cachexia process in chronic respiratory disease. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:54:30.354054-05:
      DOI: 10.1002/jcp.25083
  • TRPV3 Channel Negatively Regulates Cell Cycle Progression and Safeguards
           the Pluripotency of Embryonic Stem Cells
    • Authors: Iek Chi Lo; Hing Chung Chan, Zenghua Qi, Kwun Lam Ng, Chun So, Suk Ying Tsang
      Abstract: Embryonic stem cells (ESCs) have tremendous potential for research and future therapeutic purposes. However, the calcium handling mechanism in ESCs is not fully elucidated. Aims of this study are 1) to investigate if transient receptor potential vanilloid‐3 (TRPV3) channels are present in mouse ESCs (mESCs) and their subcellular localization; 2) to investigate the role of TRPV3 in maintaining the characteristics of mESCs. Western blot and immunocytochemistry showed that TRPV3 was present at the endoplasmic reticulum (ER) of mESCs. Calcium imaging showed that, in the absence of extracellular calcium, TRPV3 activators camphor and 6‐tert‐butyl‐m‐cresol increased the cytosolic calcium. However, depleting the ER store in advance of activator addition abolished the calcium increase, suggesting that TRPV3 released calcium from the ER. To dissect the functional role of TRPV3, TRPV3 was activated and mESC proliferation was measured by trypan blue exclusion and MTT assays. The results showed that TRPV3 activation led to a decrease in mESC proliferation. Cell cycle analysis revealed that TRPV3 activation increased the percentage of cells in G2/M phase; consistently, Western blot also revealed a concomitant increase in the expression of inactive form of cyclin‐dependent kinase 1, suggesting that TRPV3 activation arrested mESCs at G2/M phase. TRPV3 activation did not alter the expression of pluripotency markers Oct‐4, Klf4 and c‐Myc, suggesting that the pluripotency was preserved. Our study is the first study to show the presence of TRPV3 at ER. Our study also reveals the novel role of TRPV3 in controlling the cell cycle and preserving the pluripotency of ESCs. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:54:08.77255-05:0
      DOI: 10.1002/jcp.25086
  • Potential Role of Glycogen Synthase Kinase‐3β in Regulation of
           Myocardin Activity in Human Vascular Smooth Muscle Cells
    • Abstract: Glycogen synthase kinase (GSK)‐3β, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK‐3β results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co‐activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study therefore aimed to examine whether and how inhibition of GSK‐3β regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK‐3β inhibitors AR‐A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22) and ACTA2 (SM α‐actin). In human SMCs overexpressing myocardin through the T‐REx system, treatment with either GSK‐3β inhibitor also inhibited the expression of CNN1, TAGLN1 and ACTA2. These effects of GSK‐3β inhibitors were mimicked by transfection with GSK‐3β siRNA. Notably, both AR‐A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR‐A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant‐negative GSK‐3β mutant in myocardin‐overexpressing SMCs reduced the expression of calponin, SM22 and SM α‐actin. As expected, overexpression of constitutively‐active or wild‐type GSK‐3β in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK‐3β reduces myocardin transcriptional activity, suggesting a role for GSK‐3β in myocardin transcriptional activity and smooth muscle differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:53:47.238546-05:
      DOI: 10.1002/jcp.25084
  • Agents Which Inhibit NF‐κB Signaling Block Spontaneous
           Contractile Activity and Negatively Influence Survival of Developing
    • Authors: C. George Carlson; Lauren Stein, Elizabeth Dole, Ross M. Potter, David Bayless
      Abstract: Inhibiting the NF‐κB signaling pathway provides morphological and functional benefits for the mdx mouse, a model for Duchenne muscular dystrophy characterized by chronic elevations in the nuclear expression of p65, the transactivating component of the NF‐κB complex. The purpose of this study was to examine p65 expression in nondystrophic and mdx myotubes using confocal immunofluorescence, and determine whether inhibitors of the NF‐κB pathway alter myotube development. Primary cultures of nondystrophic and mdx myotubes had identical levels of nuclear and cytosolic p65 expression and exhibited equivalent responses to TNF‐α, thus excluding the hypothesis that the lack of dystrophin is sufficient to induce increases in NF‐κB signaling. The NF‐κB inhibitors pyrrolidine dithiocarbamate (PDTC) and sulfasalazine decreased spontaneous contractile activity and reduced myotube viability in a dose‐ and time‐dependent manner. Similarly, a vivo‐morpholino designed to block translation of murine p65 (m‐p65tb‐vivomorph1) rapidly abolished spontaneous contractile activity, reduced p65 expression measured by confocal immunofluorescence, and induced cell death in primary cultures of nondystrophic and mdx myotubes. Similar effects on p65 immunofluorescence and cell viability were observed following m‐p65tb‐vivomorph1 exposure to spontaneously inactive C2C12 myotubes, while exposure to a control scrambled vivo morpholino had no effect. These results indicate a direct role of the NF‐κB pathway in myotube development and identify a potential therapeutic limitation to the use of NF‐κB inhibitors in treating Duchenne and related muscular dystrophies. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:53:25.163707-05:
      DOI: 10.1002/jcp.25085
  • Pathophysiology of Corneal Dystrophies: From Cellular Genetic Alteration
           to Clinical Findings
    • Abstract: Corneal dystrophies are a heterogeneous group of bilateral, inherited, rare diseases characterized by slowly progressive corneal opacities, that lead to visual impairment. Most of them have an autosomal dominant pattern of inheritance with variable expressivity, but new mutations have been described. Many corneal dystrophies have been genetically characterized and the specific gene mutations identified, such as for the epithelial–stromal TGFBI dystrophies. Current classification systems identified four main groups of corneal dystrophies based on clinical, histologic and genetic information. Diagnosis is performed during a routine ophthalmic examination that shows typical cellular abnormalities of the corneal epithelium, stroma or endothelium. Disease progression should be carefully monitored to decide the proper clinical management. The treatment of corneal dystrophies is variable, depending on symptoms, clinical course, severity and type of dystrophy. Management aimed to reduce symptoms and to improve vision, includes different surgical approaches. Novel cellular and genetic therapeutic approaches are under evaluation. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-23T18:46:57.198342-05:
      DOI: 10.1002/jcp.25082
  • Is the Reg3α (HIP/PAP) Protein Really an Obesogenic Factor?
    • Abstract: We would like to point out an internal inconsistency in the data presented by V. Secq et al. in the article entitled “PAP/HIP protein is an obesogenic factor” published in the February 2014 issue of “Journal of Cellular Physiology”, which cast serious doubt on the validity of the statement serving as the title of the article. We, and others, including J. Iovanna and his collaborators, have long shown that the C‐type lectin PAP/HIP (also known as HIP/PAP or Reg3α) has anti‐inflammatory and antioxidant activities in a number of cell types, among which pancreatic cells, hepatocytes and neurons (Gironella et al., 2005; Closa et al., 2007; Moniaux et al., 2011; Kapur et al., 2012; Haldipur et al., 2014) and that, therefore, it might help cure, or prevent, inflammatory and metabolic diseases. Given its envisaged clinical uses, the question of whether PAP/HIP significantly modifies metabolism toward weight increase is essential and must be subjected to rigorous scrutiny. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-19T17:57:14.333248-05:
      DOI: 10.1002/jcp.25046
  • Critical Role of AMPK/FoxO3A Axis in Globular Adiponectin‐Induced
           Cell Cycle Arrest and Apoptosis in Cancer Cells
    • Abstract: Adiponectin predominantly secreted from adipose tissue has exhibited potent anti‐proliferative properties in cancer cells via modulating cell cycle and apoptosis. FoxO3A, a Forkhead box O member of the transcription factor, plays a critical role in modulating expression of genes involved in cell death and/or survival. In this study, we investigated the role of FoxO3A signaling in anti‐cancer activities of adiponectin. Herein, we have shown that treatment with globular adiponectin (gAcrp) increases p27 but decreases cyclinD1 expression in human hepatoma (HepG2) and breast (MCF‐7) cancer cells. Gene ablation of FoxO3A prevented gAcrp‐induced increase in p27 and decreased in cyclin D1 expression, and further ameliorated cell cycle arrest by gAcrp, indicating a critical role of FoxO3A in gAcrp‐induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase‐3/7 activation and increased Fas ligand (FasL) expression in both HepG2 and MCF‐7 cells. Transfection with FoxO3A siRNA inhibited gAcrp‐induced caspase‐3/7 activation and FasL expression, suggesting that FoxO3A signaling also plays an important role in gAcrp‐induced apoptosis of cancer cells. We also found that gene silencing of AMPK prevented gAcrp‐induced nuclear translocation of FoxO3A in HepG2 and MCF‐7 cells. In addition, suppression of AMPK also blocked gAcrp‐induced cell cycle arrest and further attenuated gAcrp‐induced caspase‐3/7 activation, indicating that AMPK signaling plays a pivotal role in both gAcrp‐induced cell cycle arrest and apoptosis via acting as an upstream signaling of FoxO3A. Taken together, our findings demonstrated that AMPK/FoxO3A axis plays a cardinal role in anti‐proliferative effect of adiponectin in cancer cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-18T18:54:36.875656-05:
      DOI: 10.1002/jcp.25080
  • Platelet‐Derived CCL5 Regulates CXC Chemokine Formation and
           Neutrophil Recruitment in Acute Experimental Colitis
    • Authors: Changhui Yu; Songen Zhang, Yongzhi Wang, Su Zhang, Lingtao Luo, Henrik Thorlacius
      Abstract: Accumulating data suggest that platelets not only regulate thrombosis and haemostasis but also inflammatory processes. Platelets contain numerous potent pro‐inflammatory compounds, including the chemokines CCL5 and CXCL4 although their role in acute colitis remains elusive. The aim of this study was to examine the role of platelets and platelet‐derived chemokines in acute colitis. Acute colitis was induced in female Balb/c mice by administration of 5% dextran sodium sulphate (DSS) for five days. Animals received a platelet‐depleting, anti‐CCL5, anti‐CXCL4 or a control antibody prior to DSS challenge. Colonic tissue was collected for quantification of myeloperoxidase (MPO) activity, CXCL5, CXCL2, interleukin‐6 (IL‐6) and CCL5 levels as well as morphological analyses. Platelet depletion reduced tissue damage and clinical disease activity index in DSS‐exposed animals. Platelet depletion not only reduced levels of CXCL2 and CXCL5 but also levels of CCL5 in the inflamed colon. Immunoneutralization of CCL5 but not CXCL4 reduced tissue damage, CXC chemokine expression and neutrophil recruitment in DSS‐treated animals. These findings show that platelets play a key role in acute colitis by regulating CXC chemokine generation, neutrophil infiltration and tissue damage in the colon. Moreover, our results suggest that platelet‐derived CCL5 is an important link between platelet activation and neutrophil recruitment in acute colitis. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-18T18:54:10.466808-05:
      DOI: 10.1002/jcp.25081
  • Genetic and Functional Analysis of Polymorphisms in the Human Dopamine
           Receptor and Transporter Genes in Small Cell Lung Cancer
    • Authors: Emanuela Cherubini; Arianna Di Napoli, Alessia Noto, Giorgia Amira Osman, Maria Cristina Esposito, Salvatore Mariotta, Rossella Sellitri, Luigi Ruco, Giuseppe Cardillo, Gennaro Ciliberto, Rita Mancini, Alberto Ricci
      Abstract: The regulatory role of dopamine (DA) in endocrine, cardiovascular and renal functions has been extensively studied and used for clinical purposes. More recently DA has been indicated as a regulatory molecule for immune cells and malignant cell proliferation. We assessed the expression and the functional role DA, DA receptors and transporters in primary small cell lung cancer (SCLC). By HPLC DA plasma levels were more elevated in SCLC patients in comparison with NSCLC patients and healthy controls. SCLC cell expressed DA D1‐ and D2‐like receptors and membrane and vesicular transporters at protein and mRNA levels. We also investigated the effects of independent D1‐ or D2‐like receptor stimulation on SCLC cell cultures. DA D1 receptor agonist SKF38393 induced the increase of cAMP levels and DARPP‐32 protein expression without affecting SCLC growth rate. Cell treatment with the DA D1 receptor antagonist SCH23390 inhibited SKF38393 effects. In contrast, the DA D2 receptor agonist quinpirole (10μM) counteracted, in a dose and time dependent way, SCLC cell proliferation, it did not affect cAMP levels and decreased phosphorylated AKT that was induced by DA D2 receptor antagonist sulpiride. However, in only one SCLC line, stimulation of DA D2 receptor failed to inhibit cell proliferation in vitro. This effect was associated to the existence of rs6275 and rs6277 polymorphisms in the D2 gene. These results gave more insight into DA control of lung cancer cell behavior and suggested the existence of different SCLC phenotypes. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-16T17:46:14.085232-05:
      DOI: 10.1002/jcp.25079
  • Aquaporin‐1 Plays Important Role in Proliferation by Affecting Cell
           Cycle Progression
    • Abstract: Aquaporin‐1 (AQP1) has been associated with tumor development. Here, we investigated how AQP1 may affect cell proliferation. The proliferative rate of adult carotid body (CB) cells, known to proliferate under chronic hypoxia, was analyzed in wild‐type (AQP1 +/ +) and knock out (AQP1 ‐/‐) mice, maintained in normoxia or exposed to hypoxia while BrdU was administered. Fewer numbers of total BrdU+ and TH‐BrdU+ cells were observed in AQP1 ‐/‐ mice, indicating a role for AQP1 in CB proliferation. Then, by flow cytometry, cell cycle state and proliferation of cells overexpressing AQP1 were compared to those of wild‐type cells. In the AQP1‐overexpressing cells, we observed higher cell proliferation and percentages of cells in phases S and G2/M and fewer apoptotic cells after nocodazole treatment were detected by annexin V staining. Also in these cells, proteomic assays showed higher expression of cyclin D1 and E1 and microarray analysis revealed changes in many cell proliferation‐related molecules, including, Zeb 2, Jun, NF‐kβ, Cxcl9, Cxcl10, TNF, and the TNF receptor. Overall, our results indicate that the presence of AQP1 modifies the expression of key cell cycle proteins apparently related to increases in cell proliferation. This contributes to explaining the presence of AQP1 in many different tumors. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-16T17:43:07.293339-05:
      DOI: 10.1002/jcp.25078
  • Dexamethasone Downregulates SLC7A5 Expression and Promotes Cell Cycle
           Arrest, Autophagy and Apoptosis in BeWo Cells
    • Authors: Bin He; Nana Zhang, Ruqian Zhao
      Abstract: Synthetic glucocorticoids (GCs) such as dexamethasone (Dex) are widely given to pregnant women to induce maturation and improve viability of preterm infants. Despite the beneficial effects, synthetic GCs have adverse effects on placental growth and nutrient transport system. However, the molecular mechanisms involved in these events remain unknown. Here we use a human placental choriocarcinoma cell line (BeWo) as model to explore the pathway linking amino acids transport with cell viability under Dex challenge. BeWo cells treated with Dex (100 nM) for 24 h demonstrated G1/S cell cycle arrest together with enhanced autophagy and apoptosis. Concurrently, the amino acid carrier SLC7A5 was down‐regulated in association with impaired cellular amino acids uptake and inhibition of mammalian target of rapamycin (mTOR) signaling. Similar cellular responses were observed in BeWo cells treated with BCH, a classical System L inhibitor which inactivates SLC7A5. The glucocorticoid receptor (GR) antagonist RU486 was able to diminish Dex‐induced translocation of GR into nucleus and to abolish these effects. Furthermore, Dex treatment significantly promoted the binding of GR to the proximal promoter sequence of SLC7A5 gene. Taken together, our results show that Dex downregulates SLC7A5 expression via GR‐mediated transrepression. The impaired amino acids uptake leads to inhibition of mTOR signaling which in turn causes inhibited proliferation and enhanced autophagy and apoptosis in BeWo cells. These findings indicate that SLC7A5 mediates the effect of Dex on cell viability, thus providing a novel molecular target for the prevention and treatment of Dex‐induced cell cycle arrest and apoptosis in placental cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:47:13.684676-05:
      DOI: 10.1002/jcp.25076
  • G‐CSF Contributes at the Healing of Tunica Media of
           Arteriotomy‐Injured Rat Carotids by Promoting Differentiation of
           Vascular Smooth Muscle Cells
    • Authors: Barbara Rinaldi; Mauro Finicelli, Maria Donniacuo, Giovanni Di Bernardo, Giulia Gritti, Stefania Del Gaudio, Amalia Forte, Gianfranco Peluso, Marilena Cipollaro, Francesco Rossi, Umberto Galderisi
      Abstract: Restenosis is a complex pathophysiological disease whose causative mechanisms are not fully understood. Previous studies allowed us to demonstrate the efficacy of bone marrow mesenchymal stromal cells (MSCs) transplantation in limiting the pathophysiological remodeling in a model of arteriotomy‐induced (re)stenosis. In the current research we studied the effectiveness of G‐CSF treatment on male rate rats that were subjected carotid arteriotomy in order to evaluate a potentially effective non‐invasive strategy that recapitulates the MSC‐mediated recovery of injured vessels. WKY male rats were subjected carotid arteriotomy and given a nine day treatment (3 days pre‐ to 6 days post‐arteriotomy) with G‐CSF or saline. Carotids were harvested 7 and 30 days following arteriotomy (early‐ and late‐phase, respectively). Although morphometrical analysis did not reveal differences in lumen narrowing between G‐CSF‐ and PBS‐carotids 30 days following arteriotomy, we detected a noticeable conservative effect of G‐CSF treatment on vascular wall morphology. Histological and molecular analysis revealed an increase in cellularity within the tunica media with a concomitant increase of the VSMCs differentiation markers both at early‐ and late‐phases of (re)stenotic response in G‐CSF‐treated carotids (Sm22‐alpha, Myocd and Smtn). These findings were accompanied by the downregulation of oxidative stress‐related genes in G‐CSF‐injured rats. The effect exerted by G‐CSF in our model of arteriotomy‐induced (re)stenosis seemed support the recovery of the architecture of the tunica media of injured vessels by: i) inducing VSMCs differentiation; and ii) limiting the oxidative‐stress response induced by arteriotomy. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:04:03.48673-05:0
      DOI: 10.1002/jcp.25074
  • Pathways Implicated in Tadalafil Amelioration of Duchenne Muscular
    • Authors: Valeria De Arcangelis; Georgios Strimpakos, Francesca Gabanella, Nicoletta Corbi, Siro Luvisetto, Armando Magrelli, Annalisa Onori, Claudio Passananti, Cinzia Pisani, Sophie Rome, Cinzia Severini, Fabio Naro, Elisabetta Mattei, Maria Grazia Di Certo, Lucia Monaco
      Abstract: Numerous therapeutic approaches for Duchenne and Becker Muscular Dystrophy (DMD and BMD), the most common X‐linked muscle degenerative disease, have been proposed. So far, the only one showing a clear beneficial effect is the use of corticosteroids. Recent evidence indicates an improvement of dystrophic cardiac and skeletal muscles in the presence of sustained cGMP levels secondary to a blocking of their degradation by phosphodiesterase 5 (PDE5). Due to these data, we performed a study to investigate the effect of the specific PDE5 inhibitor, tadalafil, on dystrophic skeletal muscle function. Chronic pharmacological treatment with tadalafil has been carried out in mdx mice. Behavioral and physiological tests, as well as histological and biochemical analyses, confirmed the efficacy of the therapy. We then performed a microarray‐based genomic analysis to assess the pattern of gene expression in muscle samples obtained from the different cohorts of animals treated with tadalafil. This scrutiny allowed us to identify several classes of modulated genes. Our results show that PDE5 inhibition can ameliorate dystrophy by acting at different levels. Tadalafil can lead to 1) increased lipid metabolism; 2) a switch towards slow oxidative fibers driven by the up‐regulation of PGC‐1α; 3) an increased protein synthesis efficiency; 4) a better actin network organization at Z‐disk. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:03:22.713185-05:
      DOI: 10.1002/jcp.25075
  • Tamoxifen Inhibits TGF‐β‐Mediated Activation of
           Myofibroblasts by Blocking Non‐Smad Signaling through ERK1/2
    • Abstract: TGF‐β is a multifunctional cytokine which stimulates the differentiation of fibroblasts into myofibroblasts. Myofibroblasts are critical for normal wound healing, but also accumulate pathologically in a number of chronic inflammatory conditions where they are key contributors to aberrant tissue remodeling and fibrosis, and in cancer stroma. In the current study, we identified a role for tamoxifen as a potent inhibitor of the TGF‐β‐mediated activation of primary human skin and breast fibroblasts. Our data indicate that tamoxifen does not interfere with canonical Smad signaling downstream of TGF‐β but rather blocks non‐Smad signaling through ERK1/2 MAP‐kinase and the AP‐1 transcription factor FRA2. We further demonstrate by siRNA‐mediated knockdown that FRA2 is critical for the induced expression of myogenic proteins in response to TGF‐β. Functionally, TGF‐β‐stimulated fibroblast‐mediated contraction of collagen gels was impaired in the presence of tamoxifen. Altogether, these data demonstrate that tamoxifen prevents myofibroblast differentiation and therefore may provide therapeutic benefits to patients suffering from chronic inflammatory conditions or cancer. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:02:58.161846-05:
      DOI: 10.1002/jcp.25049
  • S100A8, An Oocyte‐Specific Chemokine, Directs the Migration of
           Ovarian Somatic Cells During Mouse Primordial Follicle Assembly
    • 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. J. Cell. Physiol. 230: 2998–3008, 2015. © 2015 Wiley Periodicals, Inc.
  • Brg1 Controls the Expression of Pax7 to Promote Viability and
           Proliferation of Mouse Primary Myoblasts
    • Abstract: Brg1 (Brahma‐related gene 1) is a catalytic component of the evolutionarily conserved mammalian SWI/SNF ATP‐dependent chromatin remodeling enzymes that disrupt histone‐DNA contacts on the nucleosome. While the requirement for the SWI/SNF enzymes in cell differentiation has been extensively studied, its role in precursor cell proliferation and survival is not as well defined. Muscle satellite cells constitute the stem cell pool that sustains and regenerates myofibers in adult skeletal muscle. Here, we show that deletion of Brg1 in primary mouse myoblasts derived from muscle satellite cells cultured ex vivo leads to a cell proliferation defect and apoptosis. We determined that Brg1 regulates cell proliferation and survival by controlling chromatin remodeling and activating transcription at the Pax7 promoter, which is expressed during somite development and is required for controlling viability of the satellite cell population. Reintroduction of catalytically active Brg1 or of Pax7 into Brg1‐deficient satellite cells rescued the apoptotic phenotype and restored proliferation. These data demonstrate that Brg1 functions as a positive regulator for cellular proliferation and survival of primary myoblasts. Therefore, the regulation of gene expression through Brg1‐mediated chromatin remodeling is critical not just for skeletal muscle differentiation but for maintaining the myoblast population as well. J. Cell. Physiol. 230: 2990–2997, 2015. © 2015 Wiley Periodicals, Inc.
  • Possible Involvement of Palmitate in Pathogenesis of Periodontitis
    • 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. J. Cell. Physiol. 230: 2981–2989, 2015. © 2015 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
  • Anti Proliferative and Pro Apoptotic Effects of Flavonoid Quercetin Are
           Mediated by CB1 Receptor in Human Colon Cancer Cell Lines
    • 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. J. Cell. Physiol. 230: 2973–2980, 2015. © 2015 Wiley Periodicals, Inc.
  • Ephrin reverse signaling mediates palatal fusion and
           epithelial‐to‐mesenchymal transition independently of
    • 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 ephrin reverse signaling. J. Cell. Physiol. 9999: 1–12, 2015. © 2015 Wiley Periodicals, Inc. J. Cell. Physiol. 230: 2961–2972, 2015. © 2015 Wiley Periodicals, Inc.
  • Regulation of Osteoblast Migration Involving Receptor Activator of Nuclear
           Factor‐kappa B (RANK) Signaling
    • 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. J. Cell. Physiol. 230: 2951–2960, 2015. © 2015 Wiley Periodicals, Inc.
  • Functional Characterization of Nupr1L, A Novel p53‐Regulated Isoform
           of the High‐Mobility Group (HMG)‐Related Protumoral Protein
    • Abstract: We have previously demonstrated a crucial role of nuclear protein 1 (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 downregulates 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 downregulating 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. J. Cell. Physiol. 230: 2936–2950, 2015. © 2015 Wiley Periodicals, Inc.
  • Para‐cresyl sulfate acutely impairs vascular reactivity and induces
           vascular remodeling
    • 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. J. Cell. Physiol. 230: 2927–2935, 2015. © 2015 Wiley Periodicals, Inc.
  • Impairment of the Vascular KATP Channel Imposes Fatal Susceptibility to
           Experimental Diabetes Due to Multi‐Organ Injuries
    • 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. J. Cell. Physiol. 230: 2915–2926, 2015. © 2015 Wiley Periodicals, Inc.
  • Anandamide drives cell cycle progression through CB1 receptors in a rat
           model of synchronized liver regeneration
    • Abstract: The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti‐proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation‐promoting functions of endocannabinoids through CB1 receptors when pro‐growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor, the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid‐like molecules oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals. J. Cell. Physiol. 230: 2905–2914, 2015. © 2015 Wiley Periodicals, Inc.
  • Bioregulation
    • Abstract: During the 20th century great progress was made in genetics and biochemistry, and these were combined into a molecular biological understanding of functions of macromolecules. Further great discoveries will be made about bioregulations, applicable to scientific problems such as cell development and evolution, and to illnesses including heart disease through defective control of cholesterol production, and to neurological cell‐based diseases. The “War Against Cancer” is still far from won. The present generation of scientists can develop clinical applications from recent basic science discoveries. J. Cell. Physiol. 230: 2898–2902, 2015. © 2015 Wiley Periodicals, Inc.
  • The role of mitochondrial derived peptides (MDPs) in metabolism
  • The Many Faces of the A2b Adenosine Receptor in Cardiovascular and
           Metabolic Diseases
    • 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. J. Cell. Physiol. 230: 2891–2897, 2015. © 2015 Wiley Periodicals, Inc.
  • Coalition of Nuclear Receptors in the Nervous System
    • Abstract: A universal signaling module has been described which utilizes the nuclear form of Fibroblast growth Factor Receptor 1 (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. J. Cell. Physiol. 230: 2875–2880, 2015. © 2015 Wiley Periodicals, Inc.
  • Control of the Normal and Pathological Development of Neural Stem and
           Progenitor Cells by the PC3/Tis21/Btg2 and Btg1 Genes
    • 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. J. Cell. Physiol. 230: 2881–2890, 2015. © 2015 Wiley Periodicals, Inc.
  • Journal of Cellular Physiology: Volume 230, Number 12, December 2015
    • Abstract: Cover: Proliferating primary mouse myoblasts labeled with an anti‐Brg1 antibody. See article by Padilla‐Benavides et al. on pages 2990–2997.
  • Table of Contents: Volume 230, Number 12
  • Editor's Choice
  • Highlights: Volume 230, Number 12
  • Revisiting Chaos Theorem to Understand the Nature of miRNAs in Response to
           Drugs of Abuse
    • 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 miRNAs related to substance use disorders (SUD), general patterns of miRNA signatures are compared among studies for single or multiple 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. J. Cell. Physiol. 230: 2857–2868, 2015. © 2015 Wiley Periodicals, Inc.
  • Animal Models for Progressive Multifocal Leukoencephalopathy
    • Abstract: Progressive multifocal leukoencephalopathy (PML) is a severe demyelinating disease of the CNS caused by the human polyomavirus JC (JCV). JCV replication occurs only in human cells and investigation of PML has been severely hampered by the lack of an animal model. The common feature of PML is impairment of the immune system. The key to understanding PML is working out the complex mechanisms that underlie viral entry and replication within the CNS and the immunosurveillance that suppresses the virus or allows it to reactivate. Early models involved the simple inoculation of JCV into animals such as monkeys, hamsters, and mice. More recently, mouse models transgenic for the gene encoding the JCV early protein, T‐antigen, a protein thought to be involved in the disruption of myelin seen in PML, have been employed. These animal models resulted in tumorigenesis rather than demyelination. Another approach is to use animal polyomaviruses that are closely related to JCV but able to replicate in the animal such as mouse polyomavirus and SV40. More recently, novel models have been developed that involve the engraftment of human cells into the animal. Here, we review progress that has been made to establish an animal model for PML, the advances and limitations of different models and weigh future prospects. J. Cell. Physiol. 230: 2869–2874, 2015. © 2015 Wiley Periodicals, Inc.
  • Cell Surface Human Airway Trypsin‐Like Protease Is Lost During
           Squamous Cell Carcinogenesis
    • Abstract: Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro‐cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin‐like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non‐proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas. This article is protected by copyright. All rights reserved
  • Stable Incretin Mimetics Counter Rapid Deterioration of Bone Quality in
           Type 1 Diabetes Mellitus
    • Abstract: 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. 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. [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. 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. J. Cell. Physiol. 230: 3009–3018, 2015. © 2015 Wiley Periodicals, Inc.
  • Kinin B1 Receptor Deletion Affects Bone Healing in Type 1 Diabetic Mice
    • 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 immunolabeling 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 immunolabeling 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. J. Cell. Physiol. 230: 3019–3028, 2015. © 2015 Wiley Periodicals, Inc.
  • GLP‐2 as Beneficial Factor in the Glucose Homeostasis in Mice Fed a
           High Fat Diet
    • 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 10th 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 4 weeks (from the 6th to the 10th 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. J. Cell. Physiol. 230: 3029–3036, 2015. © 2015 Wiley Periodicals, Inc.
  • Deficiency in the α1 subunit of Naa+/K+‐ARPase enhances the
           anti‐proliferative effect of high osmolality in nucleus pulposus
           intervertebral disc cells
    • 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. J. Cell. Physiol. 230: 3037–3048, 2015. © 2015 Wiley Periodicals, Inc.
  • Acidosis Is a key regulator of osteoblast ecto‐nucleotidase
           pyrophosphatase/phosphodiesterase 1 (NPP1) expression and activity
    • 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 
  • Differential Cellular Effects of Plk1 Inhibitors Targeting the
           ATP‐binding Domain or Polo‐box Domain
    • 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 (BI 2536, GSK 461364) and PBD inhibitors (poloxin, thymoquinone) to determine their mechanisms of Plk1 inhibition. Our data show that BI 2536 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 BI 2536 or GSK461364, but not by treatment with poloxin or thymoquinone. Furthermore, treatment with BI 2536 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. J. Cell. Physiol. 230: 3057–3067, 2015. © 2015 Wiley Periodicals, Inc.
  • Involvement of a P2X7 Receptor in the Acrosome Reaction Induced by ATP in
           Rat Spermatozoa
    • 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 P2X7 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 P2X7 receptor. Here, we show the presence of P2X7 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 P2X7 receptor, which may be functional during in vivo fertilization. J. Cell. Physiol. 230: 3068–3075, 2015. © 2015 Wiley Periodicals, Inc.
  • Differential Hematopoietic Activity in White Adipose Tissue Depending on
           its Localization
    • 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. J. Cell. Physiol. 230: 3076–3083, 2015. © 2015 Wiley Periodicals, Inc.
  • Dual Effect of Adenosine A1 Receptor Activation on Renal O2 Consumption
    • 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. J. Cell. Physiol. 230: 3093–3104, 2015. © 2015 Wiley Periodicals, Inc.
  • The Effects of miR‐20a on p21: Two Mechanisms Blocking Growth Arrest
           in TGF‐β‐Responsive Colon Carcinoma
    • 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 its members. 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 downmodulated 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 directly. 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. 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. J. Cell. Physiol. 230: 3105–3114, 2015. © 2015 Wiley Periodicals, Inc.
  • p53 Modulates Notch Signaling in MCF‐7 Breast Cancer Cells by
           Associating With the Notch Transcriptional Complex Via MAML1
    • 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. J. Cell. Physiol. 230: 3115–3127, 2015. © 2015 Wiley Periodicals, Inc.
  • Defining Plasma MicroRNAs Associated With Cognitive Impairment in
           Hiv‐Infected Patients
    • Abstract: Human Immunodeficiency Virus (HIV)‐infected individuals are at increased risk for developing neurocognitive disorders and depression. These conditions collectively affect more than 50% of people living with HIV/AIDS and adversely impact adherence to HIV therapy. Thus, identification of early markers of neurocognitive impairment could lead to interventions that improve psychosocial functioning and slow or reverse disease progression through improved treatment adherence. Evidence has accumulated for the role and function of microRNAs in normal and pathological conditions. We have optimized a protocol to profile microRNAs in body fluids. Using this methodology, we have profiled plasma microRNA expression for 30 age‐matched, HIV‐infected (HIV+) patients and identified highly sensitive and specific microRNA signatures distinguishing HIV+ patients with cognitive impairment from those without cognitive impairment. These results justify follow‐on studies to determine whether plasma microRNA signatures can be used as a screening or prognostic tool for HIV+ patients with neurocognitive impairment. This article is protected by copyright. All rights reserved
  • Peripheral Blood Mononuclear Cells Spontaneous Osteoclastogenesis:
    • Abstract: In vitro peripheral blood mononuclear cells differentiate into osteoclasts under the influence of osteoclast‐stimulating factors. However, accumulating evidence suggests spontaneous osteoclasts formation and activity in patients affected by local or systemic bone remodeling diseases in comparison with healthy controls. Therefore, within this review, we summarize the studies where spontaneous osteoclastogenesis of peripheral blood mononuclear cells was observed in pathological conditions of the skeletal system. We indicate a linkage between immunoregulation by T cells and spontaneous osteoclasts formation with increased levels of tumor necrosis factors‐α, receptor activator of nuclear factor kappa‐B ligand and inteleukin‐7 production. In the light of these results, it would be of crucial importance to deepen the correlation between systemic bone remodeling diseases and spontaneous osteoclastogenesis as well as to investigate in detail the mechanisms underlying this phenomenon and the clinical relevance in bone remodeling disease diagnosis and monitoring. This article is protected by copyright. All rights reserved
  • MDA‐9/Syntenin Control
    • Abstract: MDA‐9/Syntenin is a small PDZ domain containing scaffolding protein with diverse array of function regulating membrane trafficking, cell adhesion, neural and synaptic development, ubiquitination and exosome biogenesis. An appreciable number of studies also established a pivotal role of MDA‐9/Syntenin in cancer development and progression. In this review, we will discuss the dynamic role of MDA‐9/Syntenin in regulating normal and abnormal fate of various cellular processes. This article is protected by copyright. All rights reserved
  • Systematic Review and Meta‐Analysis: Circulating miRNAs for
           Diagnosis of Hepatocellular Carcinoma
    • Abstract: Because early‐stage hepatocellular carcinoma (HCC) is difficult to diagnose using the existing techniques, identifying better biomarkers would likely improve the patients' prognoses. We performed a systematic review and meta‐analysis of published studies to appraise the utility of microRNAs (miRNAs) for the early diagnosis of HCC. Pertinent literature was collected from the Medline, Embase, and Chinese National Knowledge Infrastructure databases. We analyzed 50 studies that included 3423 cases of HCC, 2403 chronic hepatic disease (CH) patients, and 1887 healthy controls in 16 articles. Summary receiver operating characteristic analyses of all miRNAs showed an area under the curve (AUC) of 0.82, with 75.8% sensitivity and 75.0% specificity in discriminating patients with HCC from healthy controls. miR‐21 and miR‐122 individually distinguished patients with HCC from healthy controls, with an AUC of 0.88 for miR‐21 and 0.77 for miR‐122. The sensitivity and specificity for miR‐21 were 86.6% and 79.5%, respectively; those for miR‐122 were 68.0% and 73.3%. We conclude that circulating miRNAs, particularly miR‐21 and miR‐122, are promising biomarkers for the early diagnosis of HCC. This article is protected by copyright. All rights reserved
  • Response to “Is the Reg3α (HIP/PAP) Protein Really an
           Obesogenic Factor'”
    • Abstract: We are grateful to Gonzalez and colleagues for their comments and remarks, which give us the opportunity to clarify a few points that were probably not sufficiently detailed in the manuscript. In our work we demonstrate that TgPAP/HIP mice, which express the PAP/HIP transgene in their liver and have a high level of circulating PAP/HIP, are obese. This statement is based on the augmented body weight but not only: circulating leptin, which is known to correlate with the amount of adipose tissue, is significantly elevated in these mice as presented in Figure 2. This article is protected by copyright. All rights reserved
  • Vitamin D Induces Cyclooxygenase 2 Dependent Prostaglandin E2 Synthesis in
           HaCaT keratinocytes
    • Abstract: The active metabolite of vitamin D calcitriol and its analogs are well‐known for their anti‐inflammatory action in the skin, while their main side effect associated with topical treatment of inflammatory disorders is irritant contact dermatitis. Prostaglandin E2 (PGE2) is pro‐inflammatory at the onset of inflammation and anti‐inflammatory at its resolution. We hypothesized that induction of PGE2 synthesis by calcitriol in epidermal keratinocytes may contribute both to its pro‐inflammatory and anti‐inflammatory effects on the skin. Treatment of human immortalized HaCaT keratinocytes with calcitriol (3–100 nM, 2–24 h) increased PGE2 production due to increased mRNA and protein expression of COX‐2, but not to increase of COX‐1 or release of arachidonic acid. The effect of calcitriol on COX‐2 mRNA was observed also in primary human keratinocytes. The increase in COX‐2 mRNA is associated with COX‐2 transcript stabilization. Calcitriol exerts this effect by a rapid (2 h) and protein synthesis independent mode of action that is dependent on PKC and Src kinase activities. Treatment with a COX‐2 inhibitor partially prevented the attenuation of the keratinocyte inflammatory response by calcitriol. We conclude that up‐regulation of COX‐2 expression with the consequent increase in PGE2 synthesis may be one of the mechanisms explaining the Janus face of calcitriol as both a promoter and attenuator of cutaneous inflammation. This article is protected by copyright. All rights reserved
  • G Protein‐Coupled Receptor 120 Signaling Negatively Regulates
           Osteoclast Differentiation, Survival, and Function
    • Abstract: G protein‐coupled receptor 120 (GPR120) plays an important role in the regulation of inflammation and lipid metabolism. In this study, we investigated the role of GPR120 in osteoclast development and found that GPR120 regulates osteoclast differentiation, survival and function. We observed that GPR120 was highly expressed in osteoclasts compared to their precursors, bone marrow‐derived macrophages (BMMs). Activation of GPR120 by its ligand GW9508 suppressed receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast differentiation and the expression of nuclear factor of activated T cells c1 (NFATc1), a key modulator of osteoclastogenesis. GPR120 activation further inhibited the RANKL‐stimulated phosphorylation of IκBα and JNK. In addition to osteoclast differentiation, GPR120 activation increased the apoptosis of mature osteoclasts by inducing caspase‐3 and Bim expression. Activation of GPR120 also interfered with cell spreading and actin cytoskeletal organization mediated by M‐CSF but not by RANKL. Coincident with the impaired cytoskeletal organization, GPR120 activation blocked osteoclast bone resorbing activity. Furthermore, knockdown of GPR120 using small hairpin RNA abrogated all these inhibitory effects on osteoclast differentiation, survival, and function. Together, our findings identify GPR120 as a negative modulator of osteoclast development that may be an attractive therapeutic target for bone‐destructive diseases. This article is protected by copyright. All rights reserved
  • The Histone‐Deacetylase‐Inhibitor Suberoylanilide Hydroxamic
           Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix
           Metalloproteinase‐13 Activity
    • Abstract: Direct application of histone‐deacetylase‐inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular‐reparative events. We have previously demonstrated that HDACis (Valproic acid, Trichostatin A) increase mineralization in dental papillae‐derived cell‐lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 μM); while low concentrations (1 μM) SAHA did not increase apoptosis. HDACi‐exposure for 24 h induced mineralization‐per‐cell dose‐dependently after 2 weeks; however, constant 14d SAHA‐exposure inhibited mineralization. Microarray analysis (24 h and 14d) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0‐fold change at 24 h, which reduced to 36 genes at 14d. 59% of genes were down‐regulated at 24 h and 36% at 14d, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA‐supplementation increased MMP‐13 protein expression (7d, 14 d) and enzyme activity (48 h, 14d). Selective MMP‐13‐inhibition (MMP‐13i) dose‐dependently accelerated mineralization in both SAHA‐treated and non‐treated cultures. MMP‐13i‐supplementation promoted expression of several mineralization‐associated markers, however, HDACi‐induced cell migration and wound healing were impaired. Data demonstrate that short‐term low‐dose SAHA‐exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP‐13i further increased mineralization‐associated events, but decreased HDACi cell migration indicating a specific role for MMP‐13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit. This article is protected by copyright. All rights reserved
  • Autophagy‐Induced Apoptosis in Lung Cancer Cells By a Novel
           Digitoxin Analog
    • Abstract: We have synthesized a novel derivative of Digitoxin, termed “MonoD”, which demonstrates cytotoxic effects in lung cancer cells with much higher potency as compared to Digitoxin. Our data show that within one hour of MonoD treatment, H460 cells showed increased oxidative stress, increased formation of autophagic vacuoles and increased expression of pro‐autophagic markers Beclin‐1 and LC3‐II. Cells pretreated with MnTBAP, a superoxide scavenger not only lowered superoxide production, but also had lower levels of LC3‐II and Beclin‐1. Prolonged treatment with MonoD induced apoptosis in lung cancer cells. We investigated MonoD‐dependent regulation of Akt and Bcl2, proteins that are known regulators of both autophagy and apoptosis. Molecular and pharmacologic inhibitors of Bcl2 and Akt, when combined with MonoD, led to higher expression of LC3‐II and Beclin‐1 as compared to MonoD alone, suggesting a repressive effect for these proteins in MonoD‐dependent autophagy. Pretreatment of cells with an autophagy inhibitor repressed the apoptotic potential of MonoD, confirming that early autophagic flux is important to drive apoptosis. Therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis may prove advantageous over current therapies that have unimodal basis for action and may drive sustained tumor regression, which is highly desirable. This article is protected by copyright. All rights reserved
  • MicroRNAs in Control of Plant Development
    • Abstract: In the long evolutionary history, plant has evolved elaborate regulatory network to control functional gene expression for surviving and thriving, such as transcription factor‐regulated transcriptional programming. However, plenty of evidences from the past decade studies demonstrate that the 21‐24 nucleotides small RNA molecules, majorly microRNAs (miRNAs) play dominant roles in post‐transcriptional gene regulation through base pairing with their complementary mRNA targets, especially prefer to target transcription factors in plants. Here, we review current progresses on miRNA‐controlled plant development, from miRNA biogenesis dysregulation‐caused pleiotropic developmental defects to specific developmental processes, such as SAM regulation, leaf and root system regulation, plant floral transition. We also summarize some miRNAs that are experimentally proved to greatly affect crop plant productivity and quality. In addition, recent reports show that a single miRNA usually display multiple regulatory roles, such as organ development, phase transition and stresses responses. Thus, we infer that miRNA may act as a node molecule to coordinate the balance between plant development and environmental clues, which may shed the light on finding key regulator or regulatory pathway for uncovering the mysterious molecular network. This article is protected by copyright. All rights reserved
  • Toll‐Like Receptors and Tissue Remodeling: The Pro/Cons Recent
    • Abstract: The Toll‐like Receptor (TLR) family ensures prompt response towards pathogens, protecting the host against infections, and guarantees a realistic balance between protective and detrimental activities. Multiple regulating mechanisms characterize TLR activity that is not limited to innate and adaptive antimicrobial immune responses, as observed in the inflammatory (either infective, allergic or autoimmune) responses associated with tissue remodeling. Following the insult and the arise of inflammatory response, tissue remodeling takes place and might develop in fibrosis, depending on microenvironment as a result of imbalanced fibroblasts (FBs) and myofibroblasts (myoFBs) activation/survival. The process is driven by an epithelial‐fibroblast‐immune cell cross‐talk. While the main FB function is the matrix metabolism for tissue homeostasis or repair, the myoFB differentiation represents a crucial step in attempting repair of injury. FBs/myoFBs provide more than structural support at site of injury, synthesizing and/or reacting to different cytokines, growth factors, neuromediators and soluble/lipid mediators. TLR‐bearing FBs/myoFBs might contribute at the innate immune level, providing a second line of protection/defense as well as being a target/effector cell of tissue remodeling. TLRs might also interfere with acute inflammation as well as with established fibrosis, triggering structural/functional changes in agreement with the genetic background, the site of lesion, the entity of associated infection, the poor blood circulation or the pharmacological treatments, all together strictly influencing tissue repair/remodeling process. This review will focus on the recent findings on TLRs at launch and long‐lasting tissue remodeling process, that strongly suggest TLRs as optional targets for future therapies. This article is protected by copyright. All rights reserved
  • Ca2+ Signalling in Endothelial Progenitor Cells: Friend or Foe'
    • Abstract: Endothelial progenitor cells (EPCs) are mobilized either from the bone marrow and/or the arterial to replace dysfunctional endothelial cells and rescue blood perfusion in ischemic tissues. In addition, they may contribute to the angiogenic switch, thereby sustaining tumour growth and metastatization. Understanding the molecular mechanisms utilized by vascular endothelial growth factor (VEGF) to stimulate EPCs might unveil novel targets to enhance their clinical outcome in regenerative medicine and to adverse tumour vascularisation. VEGF stimulates peripheral blood‐derived EPCs to undergo repetitive Ca2+ oscillations shaped by the interaction between inositol‐1,4,5‐trisphosphate (InsP3)‐dependent Ca2+ release and store‐operated Ca2+ entry (SOCE). However, the Ca2+ machinery underlying VEGF‐induced Ca2+ spikes changes in umbilical cord blood‐derived EPCs, which require TRPC3‐mediated Ca2+ entry to trigger the interplay between InsP3 and SOCE. Surprisingly, VEGF fails to elicit pro‐angiogenic Ca2+ signals when EPCs derive from renal cellular carcinoma patients, thus questioning the suitability of VEGFR‐2 as a target for anti‐angiogenic treatments in these individuals. The lack of response to VEGF is likely due to the dramatic rearrangement of the Ca2+ toolkit occurring in RCC‐derived EPCs. Finally, primary myelofibrosis‐derived EPCs display a further pattern of reorganization of the Ca2+ machinery and proliferate independently of SOCE. Thus, the Ca2+ machinery in human ECFCs is extremely plastic and may change depending on the physio‐pathological background of the donor. As a consequence, the Ca2+ toolkit could properly be used to enhance the regenerative outcome of cell‐based therapy or adverse tumor vascularisation. This article is protected by copyright. All rights reserved
  • CD44 Influences Fibroblast Behaviors Via Modulation of Cell‐Cell and
           Cell‐Matrix Interactions, Affecting Survivin and Hippo Pathways
    • Abstract: CD44 has been studied in a wide variety of cell types, in a diverse array of cell behaviors and in a diverse range of signaling pathways. We now document a role for CD44 in mediating fibroblast behaviors via regulation of N‐cadherin, extracellular matrix expression, Survivin and the Hippo pathway. Here we report our findings on the roles of CD44 in modulating proliferation, apoptosis, migration and invasion of murine wild‐type (WT‐FB) and CD44 knockout dermal fibroblasts (CD44KO‐FB). As we have documented in microvascular endothelial cells lacking CD44, we found persistent increased proliferation, reduced activation of cleaved caspase 3, increased initial attachment, but decreased strength of cell attachment in high cell density, post confluent CD44KO‐FB cultures. Additionally, we found that siRNA knock‐down of CD44 mimicked the behaviors of CD44KO‐FB, restoring the decreases in N‐cadherin, collagen type I, fibronectin, Survivin, nuclear fractions of YAP and phospho‐YAP and decreased levels of cleaved caspase 3 to the levels observed in CD44KO‐FB. Interestingly, plating CD44KO‐FB on collagen type I or fibronectin resulted in significant decreases in secondary proliferation rates compared to plating cells on non‐coated dishes, consistent with increased cell adhesion compared to their effects on WT‐FB. Lastly, siRNA knockdown of CD44 in WT‐FB resulted in increased fibroblast migration compared to WT‐FB, albeit at reduced rates compared to CD44KO‐FB. These results are consistent with CD44's pivotal role in modulating several diverse behaviors important for adhesion, proliferation, apoptosis, migration and invasion during development, growth, repair, maintenance and regression of a wide variety of mesenchymal tissues. This article is protected by copyright. All rights reserved
  • Nuclear Translocation of p65 is Controlled by Sec6 Via the Degradation of
    • Abstract: Nuclear factor‐κB (NF‐κB) is an inducible transcription factor that mediates immune and inflammatory responses. NF‐κB pathways are also involved in cell adhesion, differentiation, proliferation, autophagy, senescence, and protection against apoptosis. The deregulation of NF‐κB activity is found in a number of disease states, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease. The 90 kDa ribosomal S6 kinase (p90RSK) family, which is serine/threonine kinases, is phosphorylated by extracellular signal‐regulated kinase1/2 (ERK1/2) and is related to NF‐κB pathways. Our previous studies revealed that Sec6, a component of the exocyst complex, plays specific roles in cell‐cell adhesion and cell cycle arrest. However, the mechanism by which Sec6 regulates the NF‐κB signaling pathway is unknown. We demonstrated that Sec6 knockdown inhibited the degradation of IκBα and delayed the nucleus‐cytoplasm translocation of p65 in HeLa cells transfected with Sec6 siRNAs after treatment with tumor necrosis factor alpha (TNF‐α). Furthermore, the binding of p65 and cAMP response element binding protein (CREB) binding protein (CBP) or p300 decreased and NF‐κB related genes which were inhibitors of NF‐κB alpha (IκBα), A20, B cell lymphoma protein 2 (Bcl‐2), and monocyte chemoattractant protein‐1 (MCP‐1) were low in cells transfected with Sec6 siRNAs in response to TNF‐α stimulation. Sec6 knockdown decreased the expression of p90RSKs and the phosphorylation of ERK or p90RSK1 at Ser380 or IκBα at Ser32. The present study suggests that Sec6 regulates NF‐κB transcriptional activity via the control of the phosphorylation of IκBα, p90RSK1, and ERK. This article is protected by copyright. All rights reserved
  • Fucoidan Suppresses the Growth of Human Acute Promyelocytic Leukemia Cells
           In Vitro and In Vivo
    • Abstract: Fucoidan, a natural component of seaweeds, is reported to have immunomodulatory and anti‐tumor effects. The mechanisms underpinning these activities remain poorly understood. In this study, the cytotoxicity and anti‐tumor activities of fucoidan were investigated in acute myeloid leukemia (AML) cells. The human AML cell lines NB4, KG1a, HL60 and K562 were treated with fucoidan and cell cycle, cell proliferation and expression of apoptotic pathways molecules were analyzed. Fucoidan suppressed the proliferation and induced apoptosis through the intrinsic and extrinsic pathways in the acute promyelocytic leukemia (APL) cell lines NB4 and HL60, but not in KG1a and K562 cells. In NB4 cells, apoptosis was caspase‐dependent as it was significantly attenuated by pre‐treatment with a pan‐caspase inhibitor. P21/WAF1/CIP1 was significantly up‐regulated leading to cell cycle arrest. Fucoidan decreased the activation of ERK1/2 and down‐regulated the activation of AKT through hypo‐phosphorylation of Thr(308) residue but not Ser(473). In vivo, a xenograft model using the NB4 cells was employed. Mice were fed with fucoidan and tumor growth was measured following inoculation with NB4 cells. Subsequently, splenic natural killer (NK) cell cytotoxic activity was also examined. Oral doses of fucoidan significantly delayed tumor growth in the xenograft model and increased cytolytic activity of NK cells. Taken together, these data suggest that the selective inhibitory effect of fucoidan on APL cells and its protective effect against APL development in mice warrant further investigation of fucoidan as a useful agent in treatment of certain types of leukemia. This article is protected by copyright. All rights reserved
  • Comparison of Dasatinib, Nilotinib and Imatinib in the Treatment of
           Chronic Myeloid Leukemia
    • Abstract: To overcome the drug resistance phenomenon induced by Imatibib (IM), in clinical practice, are often used second generation of tyrosine kinase inhibitors as Nilotinib (NIL) a such potent inhibitor of the BCR/ABL kinase and Dasatinib (DAS) a inhibitor of BCR/ABL kinase and inhibitor SrC family kinase. In this study we evaluated the in vivo effect of DAS, NIL and IM on intracellular calcium concentration, oxidative stress and apoptosis in peripheral blood leukocytes of 45 newly diagnosed patients with chronic myeloid leukaemia (CML‐PBM). Our data demonstrated that treatment with DAS and NIL showed an higher modulating potential than IM on intracellular calcium concentration by inhibiting the thapsigargin, a sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor and Lithium (Li) an inositol 1,4,5‐triphosphate (InsP3) receptor inhibitor activities. Moreover our data demonstrated that NIL and DAS have significantly increased apoptosis more than IM by involving both intracellular calcium signaling as well as oxidative stress. The acquisition of the oxidative stress and calcium channels receptors values data could help the hematologist to modulate and improve the treatment of chronic myeloid leukaemia (CML) pathology. This article is protected by copyright. All rights reserved
  • Impaired Hematopoiesis and Disrupted Monocyte/Macrophage Homeostasis in
           Mucopolysaccharidosis Type I Mice
    • Abstract: Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disease caused by alpha‐L‐iduronidase deficiency in which heparan and dermatan sulfate degradation is compromised. Besides primary lysosomal glycosaminoglycan accumulation, further changes in cellular functions have also been described in several murine MPS models. Herein, we evaluated alterations in hematopoiesis and its implications on the production of mature progeny in a MPS I murine model. Despite the significant increase in hematopoietic stem cells, a reduction in common myeloid progenitors and granulocyte‐macrophage progenitor cells was observed in Idua ‐/‐ mice bone marrow. Furthermore, no alterations in number, viability nor activation of cell death mechanisms were observed in Idua ‐/‐ mice mature macrophages but they presented higher sensitivity to apoptotic induction after staurosporine treatment. In addition, changes in Ca2+ signaling and a reduction in phagocytosis ability were also found. In summary, our results revealed significant intracellular changes in mature Idua ‐/‐ macrophages related to alterations in Idua ‐/‐ mice hematopoiesis, revealing a disruption in cell homeostasis. These results provide new insights into physiopathology of MPS I. This article is protected by copyright. All rights reserved
  • Retinoblastoma Protein Knockdown Favors Oxidative Metabolism and Glucose
           and Fatty Acid Disposal in Muscle Cells
    • Abstract: Deficiency in the retinoblastoma protein (Rb) favors leanness and a healthy metabolic profile in mice largely attributed to activation of oxidative metabolism in white and brown adipose tissues. Less is known about Rb modulation of skeletal muscle metabolism. This was studied here by transiently knocking down Rb expression in differentiated C2C12 myotubes using small interfering RNAs. Compared with control cells transfected with non‐targeting RNAs, myotubes silenced for Rb (by 80–90%) had increased expression of genes related to fatty acid uptake and oxidation such as Cd36 and Cpt1b (by 61% and 42%, respectively), increased Mitofusin 2 protein content (∼2.5‐fold increase), increased mitochondrial to nuclear DNA ratio (by 48%), increased oxygen consumption (by 65%) and decreased intracellular lipid accumulation. Rb silenced myotubes also displayed up‐regulated levels of glucose transporter type 4 expression (∼5‐fold increase), increased basal glucose uptake, and enhanced insulin‐induced Akt phosphorylation. Interestingly, exercise in mice led to increased Rb phosphorylation (inactivation) in skeletal muscle as evidenced by immunohistochemistry analysis. In conclusion, the silencing of Rb enhances mitochondrial oxidative metabolism and fatty acid and glucose disposal in skeletal myotubes, and changes in Rb status may contribute to muscle physiological adaptation to exercise. This article is protected by copyright. All rights reserved
  • Virtual Reality in Health System: Beyond Entertainment. A
           Mini‐Review on the Efficacy of VR During Cancer Treatment
    • Abstract: Virtual Reality (VR), a computer‐generated virtual environment, has been increasingly used in the entertainment world becoming a very new evolving field, but VR technology has also found a variety of applications in the biomedical field. VR can offer to subjects a safe environment within which to carry on different interventions ranging from the rehabilitation of discharged patients directly at home, to the support of hospitalized patients during different procedures and also of oncological inpatient subjects. VR appears as a promising tool for support and monitoring treatments in cancer patients influencing psychological and physiological functions. The aim of this systematic review is to provide an overview of all the studies that used VR intervention on cancer patients and analyze their main findings. Nineteen studies across nearly a thousand articles were identified that explored effects of VR interventions on cancer patients. Although these studies varied greatly in setting and design, this review identified some overarching themes. Results found that VR improved patients' emotional well‐being, and diminished cancer related psychological symptoms. The studies explored various relevant variables including different types of settings (i.e. during chemotherapy, during pain procedures, during hospitalization). Here, we point to the need of a global and multi‐disciplinary approach aimed at analyzing the effects of VR taking advantage of the new technology systems like bio sensors as well as electroencephalogram monitoring pre‐during and after intervention. Devoting more attention to bio physiological variables, standardized procedures, extending duration to longitudinal studies and adjusting for motion sickness related to VR treatment need to become standard of this research field. This article is protected by copyright. All rights reserved
  • Regulation of Spermatogenic Cell T‐Type Ca2+ Currents by Zn2+:
           Implications in Male Reproductive Physiology
    • Abstract: Zn2+ is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis and prostate contain high Zn2+ concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express CaV3.2‐encoded T‐type Ca2+ currents (ICaT) which are positively or negatively modulated by Zn2+ in other tissues. To explore whether ICaT could be regulated by Zn2+ and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell ICaT in the absence or presence of different Zn2+ concentrations. Zn2+ decreased ICaT in a concentration‐dependent manner (IC50= 2 μM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, ICaT did not show a tonic inhibition by Zn2+. Further analysis showed that Zn2+ did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady‐state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong ICaT reduction. Using non‐stationary fluctuation analysis, we found that Zn2+ mainly reduced the number of available Ca2+ channels without changing the single channel current amplitude. ICaT modulation by Zn2+ could be relevant for spontaneous Ca2+ oscillations during spermatogenesis and in pathophysiological conditions such as diabetes. This article is protected by copyright. All rights reserved
  • IPSCs, a Promising Tool to Restore Muscle Atrophy
    • Abstract: Induced pluripotent stem cells (iPSCs) are a promising tool for regenerative medicine in chronic conditions associated with muscle atrophy since iPSCs are easier to obtain, pose less ethical limitations and can better capture human genetic diversity compared with human embryonic stem cells. We highlight the potentiality of iPSCs for treating muscle‐affecting conditions for which no effective cure is yet available, notably aging sarcopenia and inherited neurometabolic conditions. This article is protected by copyright. All rights reserved
  • Hypoxia Promotes the Inflammatory Response and Stemness Features in
           Visceral Fat Stem Cells from Obese Subjects
    • Abstract: Low‐grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of 2 subpopulations of adipose‐derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of 2 subpopulations of ASC from the same isolation sites of non‐obese individuals. In particular, the behavior of ASC of obese vs non‐obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV‐ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation‐related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue‐specific NF‐kB activation and an NF‐kB‐mediated increase in inflammatory and fibrogenic responses. Moreover, obV‐ASC, which showed a less stem‐like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV‐ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF‐kB inhibitors. These novel findings suggest that the deregulated hyper‐responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low‐grade chronic inflammation, which has been implicated in obesity‐related morbidity. This article is protected by copyright. All rights reserved
  • BMP‐2 Induced Expression of PLCβ1 That Is a Positive Regulator
           of Osteoblast Differentiation
    • Abstract: Bone morphogenetic protein 2 (BMP‐2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide‐phospholipase Cβ 1 (PLCβ1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCβ1. Here, the function of PLCβ1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP‐2 we assist to a remarkable increase in PLCβ1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCβ1 promotes osteogenic differentiation by up‐regulating alkaline phosphatase (ALP). Moreover, PLCβ1 is present in the nuclear compartment of these cells and overexpression of a cytosolic‐PLCβ1mutant (cyt‐PLCβ1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR‐214 is a regulator of Osterix (Osx) which is an osteoblast‐specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCβ1 could be a potential target of miR‐214 in the control of osteogenic differentiation by gain‐ and loss‐ of function experiment. The results indicated that inhibition of miR‐214 in C2C12 cells significantly enhances the protein level of PLCβ1 and promotes C2C12 BMP‐2‐induced osteogenesis by targeting PLCβ1. This article is protected by copyright. All rights reserved
  • Sustained Inhibition of Proliferative Response After Transient FGF
           Stimulation is Mediated by Interleukin 1 Signaling
    • Abstract: Transient FGF stimulation of various cell types results in FGF memory – a sustained blockage of efficient proliferative response to FGF and other growth factors. FGF memory establishment requires HDAC activity, indicating its epigenetic character. FGF treatment stimulates proinflammatory NFκB signaling, which is also critical for FGF memory formation. The search for FGF‐induced mediators of FGF memory revealed that FGF stimulates HDAC‐dependent expression of the inflammatory cytokine IL1α. Similarly to FGF, transient cell treatment with recombinant IL1α inhibits the proliferative response to further FGF and EGF stimulation, but does not prevent FGF receptor‐mediated signaling. Interestingly, like cells pretreated with FGF1, cells pretreated with IL1α exhibit enhanced restructuring of actin cytoskeleton and increased migration in response to FGF stimulation. IRAP, a specific inhibitor of IL1 receptor, and a neutralizing anti‐IL1α antibody prevent the formation of FGF memory and rescue an efficient proliferative response to FGF restimulation. A similar effect results following treatment with the anti‐inflammatory agents aspirin and dexamethasone. Thus, FGF memory is mediated by proinflammatory IL1 signaling. It may play a role in the limitation of proliferative response to tissue damage and prevention of wound‐induced hyperplasia. This article is protected by copyright. All rights reserved
  • Failure to Target RANKL Signaling Through p38‐MAPK Results in
           Defective Osteoclastogenesis in the Microphthalmia Cloudy‐eyed
    • Abstract: The microphthalmia‐associated transcription factor (MITF) is a basic helix‐loop‐helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of receptor activator of NFκB ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C‐terminal end. Here we show that the Mitfce allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitfce/ce mice. The loss of the leucine zipper domain in Mitfce/ce mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitfce. This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site‐directed mutations in the docking site interfered with the interaction between MITF and its co‐factors FUS and BRG1. MITF‐ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/ p38 MAPK interactions in osteoclast differentiation. This article is protected by copyright. All rights reserved
  • Leptin Effect on Acetylation and Phosphorylation of PGC1α in Muscle
           Cells Associated With AMPK and Akt Activation in High‐Glucose Medium
    • Abstract: Leptin is crucial in energy metabolism, including muscle regulation. Peroxisome proliferator activated receptor gamma co‐activator 1α (PGC1α) orchestrates energy metabolism and is tightly controlled by post‐translational covalent modifications such as phosphorylation and acetylation. We aimed to further the knowledge of PGC1α control by leptin (at physiological levels) in muscle cells by time‐sequentially analysing the activation of AMP activated protein kinase (AMPK), p38 mitogen‐activated protein kinase (p38 MAPK) and Akt (Protein kinase B) – all known to phosphorylate PGC1α and to be involved in the regulation of its acetylation status – in C2C12 myotubes placed in a high‐glucose serum‐free medium. We also studied the protein levels of PGC1α, Sirtuin 1, adiponectin, COX IV, mitofusin 2 (Mfn2) and pyruvate dehydrogenase kinase 4 (PDK4). Our main findings suggest an important role of leptin regulating AMPK and Akt phosphorylation, mitofusin 2 induction and PGC1α acetylation status, with the novelty that the latter in transitorily increased in response to leptin, an effect dependent, at least in part, on AMPK regulation. These post‐translational reversible changes in PGC1α in response to leptin, especially the increase in acetylation status, may be related to the physiological role of the hormone in modulating muscle cell response to the physiological/nutritional status. This article is protected by copyright. All rights reserved
  • Correction of the FSHD Myoblast Differentiation Defect by Fusion With
           Healthy Myoblasts
    • Abstract: Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context. This article is protected by copyright. All rights reserved
  • Androgen Receptor Coactivators in Regulation of Growth and Differentiation
           in Prostate Cancer
    • Abstract: Androgen receptor (AR) is a key factor in regulation of growth and differentiation in normal and malignant prostate. Endocrine therapies for prostate cancer include inhibition of androgen production either by analogues of luteinizing hormone releasing hormone or abiraterone acetate and/or use of anti‐androgens such as hydroxyflutamide, bicalutamide, and enzalutamide. Castration therapy‐resistant cancer develops inevitably in patients who undergo treatment. AR coactivators are proteins which interact with one or more regions of the AR thus enhancing its function. Although several functions of AR coactivators may be redundant, specific functions have been identified and analyzed. The p160 group of coactivators, SRC‐1, ‐2, and ‐3 not only potentiate the activation of the AR, but are also implicated in potentiation of function of insulin‐like growth factor‐I and activation of the Akt pathway. Transcriptional integrators p300 and CBP are up‐regulated by androgen ablation and may influence antagonist/agonist balance of non‐steroidal anti‐androgens. A therapy approach designed to target p300 in prostate cancer revealed its role in regulation of proliferation of migration of androgen‐sensitive and –insensitive prostate cancer cells. Coactivators p300 and SRC‐1 are required for AR activation by interleukin‐6 (IL‐6), a cytokine that is overexpressed in castration‐therapy resistant prostate cancer. Some coactivators, such as Vav3 are involved in regulation of transcriptional activity of truncated AR, which emerge during endocrine thrapy. Stimulation of cellular migration and invasion by AR coactivators has also been described. Translational studies with aim to introduce anti‐AR coactivator therapy have not been successfuly implemented in the clinic so far. This article is protected by copyright. All rights reserved
  • Endoplasmic Reticulum Stress Interacts with Inflammation in Human Diseases
    • Abstract: The endoplasmic reticulum (ER) is a critical organelle for normal cell function and homeostasis. Disturbance in the protein folding process in the ER, termed ER stress, leads to the activation of unfolded protein response (UPR) that encompasses a complex network of intracellular signaling pathways. The UPR can either restore ER homeostasis or activate pro‐apoptotic pathways depending on the type of insults, intensity and duration of the stress, and cell types. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune, infectious, neurodegenerative, and metabolic disorders. In the cell, ER stress and inflammatory signaling share extensive regulators and effectors in a broad spectrum of biological processes. In spite of different etiologies, the two signaling pathways have been shown to form a vicious cycle in exacerbating cellular dysfunction and causing apoptosis in many cells and tissues. However, the interaction between ER stress and inflammation in many of these diseases remains poorly understood. Further understanding of the biochemistry, cell biology, and physiology may enable the development of novel therapies that spontaneously target these pathogenic pathways. This article is protected by copyright. All rights reserved
  • Selective Life‐Long Skeletal Myofiber – Targeted VEGF Gene
           Ablation Impairs Exercise Capacity in Adult Mice
    • Abstract: Exercise is dependent on adequate oxygen supply for mitochondrial respiration in both cardiac and locomotor muscle. To determine whether skeletal myofiber VEGF is critical for regulating exercise capacity, independent of VEGF function in the heart, ablation of the VEGF gene was targeted to skeletal myofibers (skmVEGF‐/‐) during embryogenesis (∼ E9.5), leaving intact VEGF expression by all other cells in muscle. In adult mice, VEGF levels were decreased in the soleus (by 65%), plantaris (94%), gastrocnemius (74%), EDL (99%) and diaphragm (64%) (p 
  • Reciprocal Control of Osteogenic and Adipogenic Differentiation by ERK/MAP
           Kinase Phosphorylation of Runx2 and PPARγ Transcription Factors
    • Abstract: In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPARγ, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPARγ. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPARγ phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPARγ‐dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant‐negative suppression of activity had the opposite effect. In rescue experiments using Runx2‐/‐ mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation‐deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation‐resistant S112A mutant PPARγ strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg ‐/‐ MEFs, while S112E mutant PPARγ was less active. Competition between RUNX2 and PPARγ was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPARγ phosphorylation in the control of osteoblast and adipocyte lineages. This article is protected by copyright. All rights reserved
  • CyclinD1 Down Regulation and Increased Apoptosis Are Common Features of
    • Abstract: Genetic variants within components of the cohesin complex (NIPBL, SMC1A, SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a spectrum of human syndromes known as “cohesinopathies” that includes Cornelia de Lange Syndrome (CdLS). CdLS is a multiple malformation syndrome affecting almost any organ and causing severe developmental delay. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in cohesin components. However, it is still unclear how mutations in different components of the cohesin complex affect the output of gene regulation. In this study, zebrafish embryos and SMC1A‐mutated patient‐derived fibroblasts were used to analyze abnormalities induced by SMC1A loss of function. We show that the knockdown of smc1a in zebrafish impairs neural development, increases apoptosis and specifically down‐regulates Ccnd1 levels. The same down‐regulation of cohesin targets is observed in SMC1A‐mutated patient fibroblasts. Previously, we have demonstrated that haploinsufficiency of NIPBL produces similar effects in zebrafish and in patients fibroblasts indicating a possible common feature for neurological defects and mental retardation in cohesinopathies. Interestingly, expression analysis of Smc1a and Nipbl in developing mouse embryos reveals a specific pattern in the hindbrain, suggesting a role for cohesin in neural development in vertebrates. This article is protected by copyright. All rights reserved
  • Osteogenic Potential of Human Oral‐Periosteal Cells (PCs) Isolated
           from Different Oral Origin: An In Vitro Study
    • Abstract: The periosteum is a specialized connective tissue containing multipotent stem cells capable of bone formation. In this study, we aimed at demonstrating that human oral periosteal cells derived from three different oral sites (upper vestibule, lower vestibule and hard palate) represent an innovative cell source for maxillo‐facial tissue engineering applications in terms of accessibility and self‐commitment towards osteogenic lineage. Periosteal cells (PCs) were isolated from patients with different ages (20‐30 yy, 40‐50 yy, 50‐60 yy); we then analyzed the in vitro proliferation capacity and the bone self‐commitment of cell clones culturing them without any osteogenic supplement to support their differentiation. We found that oral PCs, independently of their origin and age of patients, are mesenchymal stem cells with stem cell characteristics (clonogenical and proliferative activity) and that, even in absence of any osteogenic induction, they undertake the osteoblast lineage after 45 days of culture. These results suggest that oral periosteal cells could replace mesenchymal cells from bone marrow in oral tissue‐engineering applications. This article is protected by copyright. All rights reserved
  • Cellular and Molecular Mechanisms of Phenotypic Switch in Gastrointestinal
           Smooth Muscle
    • Abstract: As a general rule, smooth muscle cells (SMC) are able to switch from a contractile phenotype to a less mature synthetic phenotype. This switch is accompanied by a loss of differentiation with decreased expression of contractile markers, increased proliferation as well as the synthesis and the release of several signaling molecules such as pro‐inflammatory cytokines, chemotaxis‐associated molecules and growth factors. This SMC phenotypic plasticity has extensively been investigated in vascular diseases but interest is also emerging in the field of gastroenterology. It has in fact been postulated that altered micro‐environmental conditions, including the composition of microbiota, could trigger the remodeling of the enteric SMC, with phenotype changes and consequent alterations of contraction and impairment of gut motility. Several molecular actors participate in this phenotype remodeling. These include extracellular molecules such as cytokines and extracellular matrix proteins, as well as intracellular proteins, e.g. transcription factors. Epigenetic control mechanisms and miRNA have also been suggested to participate. In this review key roles and actors of smooth muscle phenotypic switch, mainly in GI tissue, are described and discussed in the light of literature data available so far. This article is protected by copyright. All rights reserved
  • The PPARβ/δ Agonist GW0742 Induces Early Neuronal Maturation of
           Cortical Post‐Mitotic Neurons: Role of PPARβ/δ in Neuronal
    • Abstract: Increasing evidences support that signalling lipids participate in synaptic plasticity and cell survival, and that the lipid signalling is closely associated with neuronal differentiation, learning and memory and with pathologic events such as epilepsy and Alzheimer's disease. The Peroxisome Proliferator‐Activated Receptors (PPAR) are strongly involved in the fatty acid cell signalling, as many of the natural lypophylic compounds are PPAR ligands. We have previously shown that PPARβ/δ is the main isotype present in cortical neuron primary cultures and that during neuronal maturation, PPARβ/δ is gradually increased and activated. To get more insight into the molecular mechanism by which PPARβ/δ may be involved in neuronal maturation processes, in this work a specific PPARβ/δ agonist, GW0742 was used administered alone or in association with a specific PPARβ/δ antagonist, the GSK0660 and the parameters involved in neuronal differentiation and maturation were assayed. The data obtained demonstrated the strong involvement of PPARβ/δ in neuronal maturation, triggering the agonist an anticipation of neuronal differentiation and the antagonist abolishing the observed effects. These effects appear to be mediated by the activation of both BDNF canonical (BDNF7TrkB) pathway. This article is protected by copyright. All rights reserved
  • IL‐6 Activates PI3K and PKCζ Signaling and Determines Cardiac
           Differentiation in Rat Embryonic H9c2 Cells
    • Abstract: Introduction IL‐6 influences several biological processes, including cardiac stem cell and cardiomyocyte physiology. Although JAK‐STAT3 activation is the defining feature of IL‐6 signaling, signaling molecules such as PI3K, PKCs and ERK1/2 are also activated and elicit different responses. Moreover, most studies on the specific role of these signaling molecules focus on the adult heart, and few studies are available on the biological effects evoked by IL‐6 in embryonic cardiomyocytes. Aim The aim of this study was to clarify the biological response of embryonic heart derived cells to IL‐6 by analyzing the morphological modifications and the signaling cascades evoked by the cytokine in H9c2 cells. Results IL‐6 stimulation determined the terminal differentiation of H9c2 cells, as evidenced by the increased expression of cardiac transcription factors (NKX2.5 and GATA4), structural proteins (α‐myosin heavy chain and cardiac Troponin T) and the gap junction protein Connexin 43. This process was mediated by the rapid modulation of PI3K, Akt, PTEN and PKCζ phosphorylation levels. PI3K recruitment was an upstream event in the signaling cascade and when PI3K was inhibited, IL‐6 failed to modify PKCζ, PTEN and Akt phosphorylation. Blocking PKCζ activity affected only PTEN and Akt. Finally, the overexpression of a constitutively active form of PKCζ in H9c2 cells largely mimicked the morphological and molecular effects evoked by IL‐6. Conclusion This study demonstrated that IL‐6 induces the cardiac differentiation of H9c2 embryonic cells though a signaling cascade that involves PI3K, PTEN and PKCζ activities. This article is protected by copyright. All rights reserved
  • RhoA‐Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate
    • Abstract: Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho‐family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M) and nanotopography (N) to evaluate the effect of surface topography on RhoA‐mediated functions (cell spreading, adhesion, migration and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S< M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness‐dependent (S 
  • D‐Aspartate Induces Proliferative Pathways in Spermatogonial
           GC‐1 Cells
    • Abstract: D‐aspartate (D‐Asp) is an endogenous amino acid present in vertebrate tissues, with particularly high levels in the testis. In vivo studies indicate that D‐Asp indirectly stimulates spermatogenesis through the hypothalamic‐pituitary‐gonadal axis. Moreover, in vitro studies have demonstrated that D‐Asp up‐regulates testosterone production in Leydig cells by enhancing expression of the steroidogenic acute regulatory protein. In this study, a cell line derived from immortalized type‐B mousespermatogonia retaining markers of mitotic germ cells (GC‐1) was employed to explore more direct involvement of D‐Asp in spermatogenesis.Activity and protein expression of markers of cell proliferation were determined at intervals during incubation in D‐Asp‐containing medium. D‐Asp induced phosphorylation of ERK and Akt proteins, stimulated expression of PCNA and Aurora B, and enhanced mRNA synthesis and protein expression of P450 aromatase and protein expressionof Estrogen Receptor β (ERβ). These results are the first demonstration of a direct effect of D‐Asp on spermatogonial mitotic activity. Considering thatspermatogonia express the NR1 subunit of the N‐Methyl‐D‐Aspartic Acid receptor (NMDAR), we suggest that their response to D‐Asp depends on NMDAR‐mediated activation of the ERK and Akt pathways and is further enhanced by activation of the P450 aromatase/ERβ pathway. This article is protected by copyright. All rights reserved
  • Hydroxytyrosol Inhibits Cannabinoid CB1 Receptor Gene Expression in
           3T3‐L1 Preadipocyte Cell Line
    • Abstract: The 3T3‐L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3‐L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR‐γ receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity‐associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3‐L1 pre‐adipocytes the effects of n‐3‐PUFA, α‐Linolenic acid (OM‐3), n‐6‐PUFA, Linoleic acid (OM‐6) and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis‐related genes PPAR‐γ, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3‐L1 cell differentiation by down‐regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti‐adipogenic effects, whereas OM‐3 and OM‐6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM‐3 and OM‐6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription. This article is protected by copyright. All rights reserved
  • Beta Adrenergic Receptor Stimulation Suppresses Cell Migration in
           Association With Cell Cycle Transition in Osteoblasts – Live Imaging
           Analyses Based on FUCCI System
    • Abstract: Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed‐ridden conditions in patients suffering from aging‐associated diseases including cardiovascular, neurological and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed‐ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis is still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppresses bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, FUCCI (Fluorescent Ubiquitination‐based Cell Cycle Indicator), in osteoblast biology and examined isoproterenol regulation of cell cycle transition cell migration in osteoblasts. Isoproterenol treatment suppresses the first entry peak of quiescent osteoblastic cells into cell cycle phase from G1/G0 to S/G2/M and also suppresses second major peak population size that enters into S/G2/M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1/S nor in G2/M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system. This article is protected by copyright. All rights reserved
  • Nitric Oxide Up‐Regulates RUNX2 in LNCaP Prostate Tumours:
           Implications for Tumour Growth In Vitro and In Vivo
    • Abstract: Background Aberrant expression of the transcription factor RUNX2 in prostate cancer has a number of important consequences including increased resistance to apoptosis, invasion and metastasis to bone. We previously demonstrated that hypoxia up‐regulated RUNX2 in tumour cells, which in turn up‐regulated the anti‐apoptotic factor Bcl‐2. Here, we investigate the impact of nitric oxide (NO) on RUNX2 and Bcl‐2 expression in prostate cancer and further, how RUNX2 over‐expression can impact tumour growth, angiogenesis and oxygenation in vivo. Methods The effect of NO levels on RUNX2 and thus Bcl‐2 expression was examined in prostate cancer cells in vitro using methods including gene and protein expression analyses, nitrite quantitation, protein‐DNA interaction assays (ChIP) and viability assays (XTT). The effect of RUNX2 over‐expression on tumour physiology (growth, oxygenation and angiogenesis) was also assessed in vivo using LNCaP xenografts. Results A low (but not high) concentration of NO (10µM) induced expression of RUNX2 and Bcl‐2, conferring resistance to docetaxel. These effects were induced via the ERK and PI3K pathways and were dependent on intact AP‐1 binding sites in the RUNX2 promoter. RUNX2 over‐expression in LNCaP tumours in vivo decreased the time to tumour presentation and increased tumour growth. Moreover, these tumours exhibited improved tumour angiogenesis and oxygenation. Conclusion Low levels of NO increase expression of RUNX2 and Bcl‐2 in LNCaP prostate tumour cells, and in vivo up‐regulation of RUNX2 created tumours with a more malignant phenotype. Collectively, our data reveals the importance of NO‐regulation of key factors in prostate cancer disease progression. This article is protected by copyright. All rights reserved
  • Selective β2‐AR Blockage Suppresses Colorectal Cancer Growth
           through Regulation of EGFR‐Akt/ERK1/2 Signaling, G1‐phase
           Arrest, and Apoptosis
    • Abstract: The stress‐upregulated catecholamines‐activated β1‐ and β2‐adrenergic receptors (β1/2‐ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of β1/2‐ARs signaling for the treatment of CRC and elucidated the significance of β2‐AR expression in CRC in vitro and in clinical samples. The impacts of β1/2‐AR antagonists in CRC in vitro and CRC‐xenograft in vivo were examined. We found that repression of β2‐AR but not β1‐AR signaling selectively suppressed cell viability, induced G1‐phase cell cycle arrest, caused both intrinsic and extrinsic pathways‐mediated apoptosis of specific CRC cells and inhibited CRC‐xenograft growth in vivo. Moreover, the expression of β2‐AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidences that the expression profiles, signaling, and blockage of β2‐AR have a unique pattern in CRC comparing to other cancers. β2‐AR antagonism selectively suppresses the growth of CRC accompanying active β2‐AR signaling, which potentially carries wild‐type KRAS, in vitro and in vivo via the inhibition of β2‐AR transactivated EFGR‐Akt/ERK1/2 signaling pathway. Thus, β2‐AR blockage might be a potential therapeutic strategy for combating the progressions of β2‐AR‐dependent CRC. This article is protected by copyright. All rights reserved
  • Intravenous Immunoglobulin (IVIG) Attenuates TNF‐Induced Pathologic
           Bone Resorption and Suppresses Osteoclastogenesis by Inducing A20
    • Abstract: Investigations on the therapeutic effects of intravenous immunoglobulin (IVIG) have focused on the suppression of autoantibody‐ and immune complex‐mediated inflammatory pathogenesis. Inflammatory diseases such as rheumatoid arthritis are often accompanied by excessive bone erosion but the effect of IVIG on osteoclasts, bone‐resorbing cells, has not been studied. Here, we investigate whether IVIG directly regulates osteoclast differentiation and has therapeutic potential for suppressing osteoclast‐mediated pathologic bone resorption. IVIG or cross‐linking of Fcγ receptors with plate‐bound IgG suppressed receptor activator of nuclear factor‐kappa B ligand (RANKL)‐induced osteoclastogenesis and expression of osteoclast‐related genes such as integrin β3 and cathepsin K in a dose‐dependent manner. Mechanistically, IVIG or plate‐bound IgG suppressed osteoclastogenesis by downregulating RANKL‐induced expression of NFATC1, the master regulator of osteoclastogenesis. IVIG suppressed NFATC1 expression by attenuating RANKL‐induced NF‐κB signaling, explained in part by induction of the inflammatory signaling inhibitor A20. IVIG administration attenuated in vivo osteoclastogenesis and suppressed bone resorption in the tumor necrosis factor (TNF)‐induced calvarial osteolysis model. Our findings show that, in addition to suppressing inflammation, IVIG directly inhibits osteoclastogenesis through a mechanism involving suppression of RANK signaling. Direct suppression of osteoclast differentiation may provide beneficial effects on preserving bone mass when IVIG is used to treat rheumatic disorders. This article is protected by copyright. All rights reserved
  • Distinct Roles for Intestinal Epithelial Cell‐Specific Hdac1 and
           Hdac2 in the Regulation of Murine Intestinal Homeostasis
    • Abstract: The intestinal epithelium responds to and transmits signals from the microbiota and the mucosal immune system to insure intestinal homeostasis. These interactions are in part conveyed by epigenetic modifications, which respond to environmental changes. Protein acetylation is an epigenetic signal regulated by histone deacetylases, including Hdac1 and Hdac2. We have previously shown that villin‐Cre‐inducible intestinal epithelial cell (IEC)‐specific Hdac1 and Hdac2 deletions disturb intestinal homeostasis. To determine the role of Hdac1 and Hdac2 in the regulation of IEC function and the establishment of the dual knockout phenotype, we have generated villin‐Cre murine models expressing one Hdac1 allele without Hdac2, or one Hdac2 allele without Hdac1. We have also investigated the effect of short‐term deletion of both genes in naphtoflavone‐inducible Ah‐Cre and tamoxifen‐inducible villin‐CreER mice. Mice with one Hdac1 allele displayed normal tissue architecture, but increased sensitivity to DSS‐induced colitis. In contrast, mice with one Hdac2 allele displayed intestinal architecture defects, increased proliferation, decreased goblet cell numbers as opposed to Paneth cells, increased immune cell infiltration associated with fibrosis, and increased sensitivity to DSS‐induced colitis. In comparison to dual knockout mice, intermediary activation of Notch, mTOR and Stat3 signaling pathways was observed. While villin‐CreER Hdac1 and Hdac2 deletions led to an impaired epithelium and differentiation defects, Ah‐Cre‐mediated deletion resulted in blunted proliferation associated with the induction of a DNA damage response. Our results suggest that IEC determination and intestinal homeostasis are highly dependent on Hdac1 and Hdac2 activity levels, and that changes in the IEC acetylome may alter the mucosal environment. This article is protected by copyright. All rights reserved
  • S‐Adenosylmethionine Affects ERK1/2 and STAT3 Pathways and Induces
           Apoptosis in Osteosarcoma Cells
    • Abstract: Osteosarcoma is a very aggressive bone tumor. Its clinical outcome remains discouraging despite intensive surgery, radiotherapy and chemotherapy. Thus, novel therapeutic approaches are demanded. S‐Adenosylmethionine (AdoMet) is a naturally occurring molecule that is synthesized in our body by methionine adenosyltransferase isoenzymes and is also available as a nutritional supplement. AdoMet is the principal methyl donor in numerous methylation reactions and is involved in many biological functions. Interestingly, AdoMet has been shown to exert antiproliferative action in various cancer cells. However, the underlying molecular mechanisms are just starting to be studied. Here, we investigated the effects of AdoMet on the proliferation of osteosarcoma U2OS cells and the underlying mechanisms. We carried out direct cell number counting, MTT and flow cytometry‐based assays, and immunoblotting experiments in response to AdoMet treatment. We found that AdoMet strongly inhibits proliferation of U2OS cells by slowing‐down cell cycle progression and by inducing apoptosis. We also report that AdoMet consistently causes an increase of p53 and p21 cell‐cycle inhibitor, a decrease of cyclin A and cyclin E protein levels, and a marked increase of pro‐apoptotic Bax/Bcl‐2 ratio, with caspase‐3 activation and PARP cleavage. Moreover, the AdoMet‐induced antiproliferative effects were dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels. Altogether, our data enforce the evidence of AdoMet acting as a biomolecule with antiproliferative action in osteosarcoma cells, capable of down‐regulating ERK1/2 and STAT3 pathways leading to cell cycle inhibition and apoptosis, and provide a rationale for the possible use of AdoMet in osteosarcoma therapy. This article is protected by copyright. All rights reserved
School of Mathematical and Computer Sciences
Heriot-Watt University
Edinburgh, EH14 4AS, UK
Tel: +00 44 (0)131 4513762
Fax: +00 44 (0)131 4513327
About JournalTOCs
News (blog, publications)
JournalTOCs on Twitter   JournalTOCs on Facebook

JournalTOCs © 2009-2015