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Journal Cover Journal of Cellular Physiology
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   Hybrid Journal Hybrid journal (It can contain Open Access articles)
     ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
     Published by John Wiley and Sons Homepage  [1603 journals]   [SJR: 1.608]   [H-I: 118]
  • Characterization of Cardiac Anoctamin1 Ca2+-activated Chloride Channels
           and Functional Role in Ischemia-Induced Arrhythmias
    • Authors: Zhen Ye; Ming-Ming Wu, Chun-Yu Wang, Yan-Chao Li, Chang-Jiang Yu, Yuan-Feng Gong, Jun Zhang, Qiu-Shi Wang, Bin-Lin Song, Kuai Yu, H. Criss Hartzell, Dayue Darrel Duan, Dan Zha, Zhi-Ren Zhang
      Pages: n/a - n/a
      Abstract: Anoctamin1 (ANO1) encodes a Ca2+-activated chloride (Cl-) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1-encoded CaCCs in ischemia-induced arrhythmias in the heart. Quantitative real-time RT-PCR, immunofluorescence staining assays and immunohistochemisrty identified the molecular expression, location and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch-clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca2+-activated Cl- current (ICl.Ca) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of ICl.Ca, which was inhibited by a specific ANO1 inhibitor, T16Ainh-A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia-induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper “spike and dome” compared to control cardiomyocytes from non-ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia-induced early phase 1 repolarization acceleration and caused a much shallower “spike and dome”. We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia-induced increase in ANO1 expression may be responsible for the increased density of ICl.Ca in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T10:20:25.073373-05:
      DOI: 10.1002/jcp.24709
       
  • Function of Latent TGFβ Binding Protein 4 and Fibulin 5 in
           Elastogenesis and Lung Development
    • Authors: Branka Dabovic; Ian B. Robertson, Lior Zilberberg, Melinda Vassallo, Elaine C. Davis, Daniel B Rifkin
      Pages: n/a - n/a
      Abstract: Mice deficient in Latent TGFβ Binding Protein 4 (Ltbp4) display a defect in lung septation and elastogenesis. The lung septation defect is normalized by genetically decreasing TGFβ2 levels. However, the elastic fiber assembly is not improved in Tgfb2-/-;Ltbp4S-/- compared to Ltbp4S-/- lungs. We found that decreased levels of TGFβ1 or TGFβ3 did not improve lung septation indicating that the TGFβ isoform elevated in Ltbp4S-/- lungs is TGFβ2. Expression of a form of Ltbp4 that could not bind latent TGFβ did not affect lung phenotype indicating that normal lung development does not require the formation of LTBP4-latent TGFβ complexes. Therefore, the change in TGFβ-level in the lungs is not directly related to Ltbp4 deficiency but probably is a consequence of changes in the extracellular matrix. Interestingly, combination of the Ltbp4S-/- mutation with a fibulin-5 null mutant in Fbln5-/-;Ltbp4S-/- mice improves the lung septation compared to Ltbp4S-/- lungs. Large globular elastin aggregates characteristic for Ltbp4S-/- lungs do not form in Fbln5-/-;Ltbp4S-/- lungs and EM studies showed that elastic fibers in Fbln5-/-;Ltbp4S-/- lungs resemble those found in Fbln5-/- mice. These results are consistent with a role for TGFβ2 in lung septation and for Ltbp4 in regulating fibulin-5 dependent elastic fiber assembly. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:59:02.003792-05:
      DOI: 10.1002/jcp.24704
       
  • M-CSF priming of osteoclast precursors can cause
           osteoclastogenesis-insensitivity, which can be prevented and overcome on
           bone
    • Authors: Teun J. de Vries; Ton Schoenmaker, David Aerts, Lilyanne C. Grevers, Pedro P.C. Souza, Kamran Nazmi, Mark van de Wiel, Bauke Ylstra, Peter L. van Lent, Pieter J.M. Leenen, Vincent Everts
      Pages: n/a - n/a
      Abstract: Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. Such M-CSF primed cells expressed the receptor RANK, but lacked the crucial osteoclastogenic transcription factor NFATc1. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. Osteoclastogenesis-insensitive precursors grown in the absence of bone regained their osteoclastogenic potential when transferred to bone. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-06-24T09:58:43.763942-05:
      DOI: 10.1002/jcp.24702
       
  • LPS promote the odontoblastic differentiation of human dental pulp stem
           cells via MAPK, but not NF‐KB signaling pathway
    • Abstract: Human dental pulp stem cells (hDPSCs) show significant potential for exploitation in novel regeneration strategies, although lack of understanding of their responses to bacterial challenge constrains their application. The present study aimed to investigate whether lipopolysaccharide (LPS), the major pathogenic factor of Gram‐negative bacteria, regulates the differentiation of hDPSCs and which intracellular signaling pathways may be involved. LPS treatment significantly promoted the differentiation of hDPSCs demonstrable by increased mineralized nodule formation and mRNA expression of several odontoblastic markers in a dose‐dependent manner. While inhibition of TLR4, p38, and ERK signaling markedly antagonized LPS‐mediated differentiation of hDPSCs. The inhibition of JNK and NF‐κB signaling had no detectable effect on LPS activation of hDPSCs. LPS stimulation resulted in phosphorylation of NF‐κB p65, IκB‐α, extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (MAPK) in DPSCs in a time‐dependent manner, which was markedly suppressed by their specific inhibitors, respectively. Data demonstrated that LPS promoted odontoblastic differentiation of hDPSCs via TLR4, ERK, and P38 MAPK signaling pathways, but not NF‐κB signaling. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
  • Detection of Phosphorylated Insulin Receptor in Colorectal Adenoma and
           Adenocarcinoma: Implications for Prognosis and Clinical Outcome
    • Abstract: Colorectal carcinoma remains among the most frequent causes of cancer death. Besides the well‐known genetic predisposition, a key role in colorectal adenoma and adenocarcinoma etio‐pathogenesis, mainly in sporadic cases, is played by definite risk factors, such as obesity, type 2 diabetes, insulin resistance, hyper‐insulinemia, and insulin therapy. These epidemiological data motivated us to determine, by means of immunohistochemistry, the amount of activated (phosphorylated) insulin receptor in archival samples from 22 colorectal adenoma and 117 adenocarcinoma patients, with the objective to estimate the role of this factor in colorectal epithelium transformation and cancer progression. Statistical analysis of the results clearly showed that positive staining for phosphorylated insulin receptor was significantly more frequent in adenomas than adenocarcinomas (P 
       
  • VEGF receptor 2 (VEGFR2) activation is essential for osteocyte survival
           induced by mechanotransduction
    • Abstract: Mechanical loading plays a key role in bone formation and maintenance. While unloading induces osteocyte apoptosis and bone loss in vivo, mechanical stimuli prevents osteocyte death through a mechanism involving β‐catenin accumulation and ERK nuclear translocation. Vascular endothelial growth factor (VEGF) has a crucial role in bone formation, but its interaction with osteocytes is not completely understood. Of interest, VEGF receptor 2 (VEGFR2) has recently been shown to mediate the mechanical response of endothelial cells. The present study aimed to evaluate the putative role of the VEGF system in osteocyte mechanosensing. We show that either short (10 min) mechanical stimulus by pulsatile fluid flow (FF) (10 dyn/cm2, 8 Hz) or exogenous VEGF165 (6 ng/ml) similarly stimulated cell viability, ERK phosphorylation, and β‐catenin membrane translocation. A VEGFR2 antagonist (SU5416) or transfection with specific VEGFR2 siRNAs (siVEGFR2) decreased these events. FF for 10 min increased VEGFR2 phosphorylation at both Tyr‐1059 and Tyr‐1175; an effect that was mimicked by VEGF165 but was unaffected by a VEGF neutralizing antibody. Subsequently (at 6 h), this mechanical stimulus induced VEGF gene overexpression, which was prevented by siVEGFR2 transfection. Depletion of the structural protein caveolin‐1 by using siRNA technology impaired FF‐induced VEGFR2 phosphorylation. In conclusion, these in vitro findings point to caveolin‐1‐dependent VEGFR2 activation as an important mechanism whereby mechanical stimuli promote osteocyte viability. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
  • Grb10 Deletion Enhances Muscle Cell Proliferation, Differentiation and
           GLUT4 Plasma Membrane Translocation
    • Abstract: Grb10 is an intracellular adaptor protein which binds directly to several growth factor receptors, including those for insulin and insulin‐like growth factor receptor‐1 (IGF‐1), and negatively regulates their actions. Grb10‐ablated (Grb10‐/‐) mice exhibit improved whole body glucose homeostasis and an increase in muscle mass associated specifically with an increase in myofiber number. This suggests that Grb10 may act as a negative regulator of myogenesis. In this study, we investigated in vitro, the molecular mechanisms underlying the increase in muscle mass and the improved glucose metabolism. Primary muscle cells isolated from Grb10‐/‐ mice exhibited increased rates of proliferation and differentiation compared to primary cells isolated from wild‐type mice. The improved proliferation capacity was associated with an enhanced phosphorylation of Akt and ERK in the basal state and changes in the expression of key cell cycle progression markers involved in regulating transition of cells from the G1 to S phase (e.g., retinoblastoma (Rb) and p21). The absence of Grb10 also promoted a faster transition to a myogenin positive, differentiated state. Glucose uptake was higher in Grb10‐/‐ primary myotubes in the basal state and was associated with enhanced insulin signaling and an increase in GLUT4 translocation to the plasma membrane. These data demonstrate an important role for Grb10 as a link between muscle growth and metabolism with therapeutic implications for diseases, such as muscle wasting and type 2 diabetes. J. Cell. Physiol. 229: 1753–1764, 2014. © 2014 Wiley Periodicals, Inc.
       
  • PTX3 Stimulates Osteoclastogenesis by Increasing Osteoblast RANKL
           Production
    • Abstract: Pentraxin‐3 (PTX3), also known as tumor necrosis factor‐stimulated gene 14 (TSG‐14), is produced by immune and vascular cells in response to pro‐inflammatory signals and is therefore a multipotent inflammatory mediator. The present study showed that during human osteoblast (OB) differentiation, precursor OBs (pOBs), but not mature OB, highly expressed PTX3. TNFα treatment elevated the PTX3 expression of pOBs. When mice were injected with lipopolysaccharide, which induces an inflammatory osteolytic condition characterized by trabecular bone destruction and high osteoclastogenesis, their bone marrow cells expressed elevated levels of PTX3 protein. Exogenous PTX3 did not directly affect osteoclast (OC) or OB differentiation. However, when pOBs and precursor OCs were co‐cultured, exogenous PTX3 significantly increased the number of tartrate‐resistant acid phosphatase‐positive multinucleated cells (i.e., OC cells) by increasing the pOB mRNA expression and protein secretion of RANK ligand (RANKL). This was accompanied with increased Runt‐related transcription factor 2 (Runx2) expression in the pOBs. Knock‐down of endogenous PTX3 with small‐interfering RNA did not change the osteogenic potential of pOBs but suppressed their production of RANKL and reduced osteoclastogenesis. Finally, TNFα treatment of the co‐culture elevated PTX3 expression by the pOBs and increased OC formation. This effect was suppressed by PTX3 knock‐down by decreasing RANKL expression. Thus, the PTX3‐driven increase in the osteoclastogenic potential of pOBs appears to be mediated by the effect of PTX3 on pOB RANKL production. These findings suggest that PTX3 is an inflammatory mediator that contributes to the deteriorating osteolytic condition of inflamed bone. J. Cell. Physiol. 229: 1744–1752, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Serum Biomarker for Diagnosis of Endometriosis
    • Abstract: Endometriosis is estimated to affect 10% of women during the reproductive years. The lack of a non‐invasive diagnostic test significantly contributes to the long delay between onset of the symptoms and definitive diagnosis of endometriosis. This case–control study was conducted to identify specific endometriosis antigens using 2D gel analysis in women with endometriosis (n = 5) and without endometriosis (n = 5). Differentially expresses spots were analyzed using matrix‐assisted laser desorption/ionization‐time‐of‐flight/mass spectrometry (nanoLC‐ESI‐MS/MS) with MASCOT analysis, in order to identify the corresponding proteins. ELISAs were performed on a different cohort of endometriosis (n = 120) and healthy patients (n = 20) in order to confirm the differential expression of the identified proteins. ROC analysis of ELISA results confirmed the statistical significance of the differential expression for one of these proteins: Zn‐alpha2‐glycoprotein (P = 0.019). We propose the analysis of the expression level of this protein in the serum as a new non‐invasive diagnostic test for endometriosis. J. Cell. Physiol. 229: 1731–1735, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Nemo‐Like Kinase Regulates Postnatal Skeletal Homeostasis
    • Abstract: Nemo‐like kinase (Nlk) is related to the mitogen‐activated protein (MAP) kinases and known to regulate signaling pathways involved in osteoblastogenesis. In vitro Nlk suppresses osteoblastogenesis, but the consequences of the Nlk inactivation in the skeleton in vivo are unknown. To study the function of Nlk, NlkloxP/loxP mice, where the Nlk exon2 is flanked by loxP sequences, were mated with mice expressing the Cre recombinase under the control of the paired‐related homeobox gene 1 (Prx1) enhancer (Prx1‐Cre), the Osterix (Osx‐Cre) or the osteocalcin/bone gamma carboxyglutamate protein (Bglap‐Cre) promoter. Prx1‐Cre;NlkΔ/Δ mice did not exhibit a skeletal phenotype except for a modest increase in trabecular number and connectivity observed only in 3‐month‐old male mice. Osx‐Cre;NlkΔ/Δ male and female mice exhibited an increase in trabecular bone volume secondary to an increased trabecular number at 3 months of age. Bone histomorphometry revealed a decrease in osteoclast number and eroded surface in male mice, and decreased osteoblast number and function in female mice. Expression of osteoprotegerin mRNA was increased in calvarial extracts, explaining the decreased osteoclast and osteoblast number. The conditional deletion of Nlk in mature osteoblasts (Bglap‐Cre;NlkΔ/Δ) resulted in no skeletal phenotype in 1‐ to 6‐month‐old male or female mice. In conclusion, when expressed in undifferentiated osteoblasts, Nlk is a negative regulator of skeletal homeostasis possibly by targeting signals that regulate osteoclastogenesis and bone resorption. J. Cell. Physiol. 229: 1736–1743, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Impaired Adipogenesis and Insulin Resistance in Epicardial
           Fat‐Mesenchymal Cells From Patients With Cardiovascular Disease
    • Abstract: The thickness of epicardial adipose tissue (EAT), which is an inflammatory source for coronary artery disease (CAD), correlates with insulin resistance. One trigger factor is impaired adipogenesis. Here, our aim was to clarify the underlying mechanisms of insulin resistance on EAT‐mesenchymal cells (MC). EAT and subcutaneous adipose tissue (SAT) were collected from 19 patients who were undergoing heart surgery. Their dedifferentiated adipocytes (DAs) and/or MCs were cultured. After the induction of adipogenesis or stimulation with insulin, the expression of adipokines was analyzed using real‐time polymerase chain reaction (PCR). Colorimetric assays were performed to measure glucose levels and proliferation rate. Proteins modifications were detected via the proteomic approach and Western blot. Our results showed lower adipogenic ability in EAT‐MCs than in SAT‐MCs. Maximum adiponectin levels were reached within 28–35 days of exposure to adipogenic inducers. Moreover, the adipogenesis profile in EAT‐MCs was dependent on the patients’ clinical characteristics. The low adipogenic ability of EAT‐MCs might be associated with an insulin‐resistant state because chronic insulin treatment reduced the inflammatory cytokine expression levels, improved the glucose consumption, and increased the post‐translational modifications (PTMs) of the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1). We found lower adipogenic ability in EAT‐MCs than in SAT‐MCs. This lower ability level was dependent on gender and the presence of diabetes, obesity, and CAD. Low adipogenesis ability and insulin resistance in EAT‐MCs might shed light on the association between EAT dysfunction and cardiovascular disease. J. Cell. Physiol. 229: 1722–1730, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Gating of the Kir2.1 Channel at the Bundle Crossing Region by
           Intracellular Spermine and Other Cations
    • Abstract: In the Kir2.1 channel, the flow‐dependent blocking effect of intracellular spermine (SPM) strongly indicates coupled movement of ions in a segment of the pore. We have shown that the bundle crossing region of M2 constitutes this critical segment of the pore. Moreover, this segment may undergo opening/closing conformational changes mimicking channel gating. In this study, we further investigate these “gating” conformational changes and relevant controlling mechanisms at this critical segment. We demonstrate that A184R mutation in the inner end of the bundle crossing region not only abolishes the inward rectifying features of SPM block but also tends to close the channel pore, which can then only be opened by intracellular (e.g., Na+, or equally effectively, K+) but not extracellular cations. We also found that the exit (back to the intracellular milieu) of the blocking in the deep site is facilitated rather than deterred by the presence of the other SPM in the superficial site. We conclude that intracellular SPM may bind to a deep site in the pore and serve as a flow‐dependent blocker. The SPM in the superficial site, on the other hand, serves both as a docking form ready for permeation to the deep site, and as a gating particle capable of opening the bundle crossing region. This inner end of the bundle crossing region of the Kir2.1 channel pore thus constitutes a pivotal segment, which, in collaboration with intracellular SPM and K+ ions, closely couple channel gating to (inward rectifying) ion permeation. J. Cell. Physiol. 229: 1703–1721, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Decreased Levels of BAG3 in a Family With a Rare Variant and in Idiopathic
           Dilated Cardiomyopathy
    • Abstract: The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole‐exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10‐nucleotide deletion in the BCL2‐associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C‐terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end‐stage HF undergoing cardiac transplantation when compared with non‐failing controls. Diminished levels of BAG3 protein may be associated with both familial and non‐familial forms of dilated cardiomyopathy. J. Cell. Physiol. 229: 1697–1702, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Nanotopography Directs Mesenchymal Stem Cells to Osteoblast Lineage
           Through Regulation of microRNA‐SMAD‐BMP‐2 Circuit
    • Abstract: The aim of this study was to investigate if chemically produced nanotopography on titanium (Ti) surface induces osteoblast differentiation of cultured human bone marrow mesenchymal stem cells (hMSCs) by regulating the expression of microRNAs (miRs). It was demonstrated that Ti with nanotopography induces osteoblast differentiation of hMSCs as evidenced by upregulation of osteoblast specific markers compared with untreated (control) Ti at day 4. At this time‐point, miR‐sequencing analysis revealed that 20 miRs were upregulated (>twofold) while 20 miRs were downregulated (>threefold) in hMSCs grown on Ti with nanotopography compared with control Ti. Three miRs, namely miR‐4448, ‐4708, and ‐4773, which were significantly downregulated (>fivefold) by Ti with nanotopography affect osteoblast differentiation of hMSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP‐2) osteogenic signal, which were upregulated by Ti with nanotopography. Overexpression of miR‐4448, ‐4708, and 4773 in MC3T3‐E1 pre‐osteoblasts noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and therefore repressed the gene expression of key bone markers. Additionally, it was observed that the treatment with BMP‐2 displayed a higher osteogenic effect on MC3T3‐E1 cells grown on Ti with nanotopography compared with control Ti, suggesting that the BMP‐2 signaling pathway was more effective on this surface. Taken together, these results indicate that a complex regulatory network involving a miR‐SMAD‐BMP‐2 circuit governs the osteoblast differentiation induced by Ti with nanotopography. J. Cell. Physiol. 229: 1690–1696, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Combined EGFR and Autophagy Modulation Impairs Cell Migration and Enhances
           Radiosensitivity in Human Glioblastoma Cells
    • Abstract: Glioblastoma (GBM) remains the most aggressive and lethal brain tumor due to its molecular heterogeneity and high motility and invasion capabilities of its cells, resulting in high resistance to current standard treatments (surgery, followed by ionizing radiation combined with Temozolomide chemotherapy administration). Locus amplification, gene overexpression, and genetic mutations of epidermal growth factor receptor (EGFR) are hallmarks of GBM that can ectopically activate downstream signaling oncogenic cascades such as PI3K/Akt/mTOR pathway. Importantly, alteration of this pathway, involved also in the regulation of autophagy process, can improve radioresistance in GBM cells, thus promoting the aggressive phenotype of this tumor. In this work, the endogenous EGFR expression profile and autophagy were modulated to increase radiosensitivity behavior of human T98G and U373MG GBM cells. Our results primarily indicated that EGFR interfering induced radiosensitivity according to a decrease of the clonogenic capability of the investigated cells, and an effective reduction of the in vitro migratory features. Moreover, EGFR interfering resulted in an increase of Temozolomide (TMZ) cytotoxicity in T98G TMZ‐resistant cells. In order to elucidate the involvement of the autophagy process as pro‐death or pro‐survival role in cells subjected to EGFR interfering, the key autophagic gene ATG7 was silenced, thereby producing a transient block of the autophagy process. This autophagy inhibition rescued clonogenic capability of irradiated and EGFR‐silenced T98G cells, suggesting a pro‐death autophagy contribution. To further confirm the functional interplay between EGFR and autophagy pathways, Rapamycin‐mediated autophagy induction during EGFR modulation promoted further impairment of irradiated cells, in terms of clonogenic and migration capabilities. Taken together, these results might suggest a novel combined EGFR‐autophagy modulation strategy, to overcome intrinsic GBM radioresistance, thus improving the efficacy of standard treatments. J. Cell. Physiol. 229: 1863–1873, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Ang II–AT1R Increases Cell Migration Through PI3K/AKT and
           NF‐κB Pathways in Breast Cancer
    • Abstract: Angiotensin II (Ang II), a biologically active peptide of the renin–angiotensin system (RAS), plays an important role in promoting cell migration via Angiotensin II type 1 receptor (AT1R). In this study, we examined the mechanisms by which Ang II affected cell migration in AT1R‐positive MDA‐MB‐231 human breast cancer cells. Ang II increased cell migration and expression of matrix metalloproteinase (MMP)‐2,‐9 in a dose‐dependent manner. Ang II‐mediated cell migration was reduced by specific blocking of MMP‐2 and MMP‐9, as well as with pretreatment with inhibitors of AT1R, phosphatidylinositol 3‐kinase (PI3K), Akt, and NF‐κB. Similarly, Ang II‐mediated expression of MMP‐2,‐9 was downregulated by pretreatment with inhibitors of AT1R and PI3K. In addition, Ang II treatment significantly induced phosphorylation of PI3K, Akt, and resulted in increased NF‐κB activity. These findings suggest that Ang II activates the AT1R/PI3K/Akt pathway, which further activates IKKα/β and NF‐κB, resulting in enhanced expression of MMP‐2,‐9 and migration in human breast cancer cells. Therefore, targeting Ang II/AT1R/PI3K/Akt/NF‐κB signaling could be a novel anti‐metastatic therapy for breast cancer. J. Cell. Physiol. 229: 1855–1862, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Role of Caspase‐3 Cleaved IP3R1 on Ca2+ Homeostasis and
           Developmental Competence of Mouse Oocytes and Eggs
    • Abstract: Apoptosis in most cell types is accompanied by altered Ca2+ homeostasis. During apoptosis, caspase‐3 mediated cleavage of the type 1 inositol 1,4,5‐trisphosphate receptor (IP3R1) generates a 95‐kDa C‐terminal fragment (C‐IP3R1), which represents the channel domain of the receptor. Aged mouse eggs display abnormal Ca2+ homeostasis and express C‐IP3R1, although whether or not C‐IP3R1 expression contributes to Ca2+ misregulation or a decrease in developmental competency is unknown. We sought to answer these questions by injecting in mouse oocytes and eggs cRNAs encoding C‐IP3R1. We found that: (1) expression of C‐IP3R1 in eggs lowered the Ca2+ content of the endoplasmic reticulum (ER), although, as C‐IP3R1 is quickly degraded at this stage, its expression did not impair pre‐implantation embryo development; (2) expression of C‐IP3R1 in eggs enhanced fragmentation associated with aging; (3) endogenous IP3R1 is required for aging associated apoptosis, as its down‐regulation prevented fragmentation, and expression of C‐IP3R1 in eggs with downregulated IP3R1 partly restored fragmentation; (4) C‐IP3R1 expression in GV oocytes resulted in persistent levels of protein, which abolished the increase in the ER releasable Ca2+ pool that occurs during maturation, undermined the Ca2+ oscillatory ability of matured eggs and their activation potential. Collectively, this study supports a role for IP3R1 and C‐IP3R1 in regulating Ca2+ homeostasis and the ER Ca2+ content during oocyte maturation. Nevertheless, the role of C‐IP3R1 on Ca2+ homeostasis in aged eggs seems minor, as in MII eggs the majority of endogenous IP3R1 remains intact and C‐IP3R1 undergoes rapid turnover. J. Cell. Physiol. 229: 1842–1854, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Heregulin Negatively Regulates Transcription of ErbB2/3 Receptors via an
           AKT‐Mediated Pathway
    • Abstract: Despite the importance of the ErbB2/3 heterodimer in breast cancer progression, the negative regulation of these receptors is still poorly understood. We demonstrate here for the first time that the ErbB3/4 ligand heregulin (HRG) reduced both ErbB2 and ErbB3 mRNA and protein levels in human breast cancer cell lines. In contrast, EGFR levels were unaffected by HRG treatment. The effect was rapid with a decline in steady‐state mRNA levels first noted 2 h after HRG treatment. HRG reduced the rate of transcription of ErbB2 and ErbB3 mRNA, but did not affect ErbB2 or ErbB3 mRNA stability. To test if ErbB2 kinase activity was required for the HRG‐induced downregulation, we treated cells with the ErbB2/EGFR inhibitor lapatinib. Lapatinib diminished the HRG‐induced decrease in ErbB2 and ErbB3 mRNA and protein, suggesting that the kinase activity of EGFR/ErbB2 is involved in the HRG‐induced receptor downregulation. Further, HRG‐mediated decreases in ErbB2/3 mRNA transcription are reversed by inhibiting the AKT but not MAPK pathway. To examine the functional consequences of HRG‐mediated decreases in ErbB receptor levels, we performed cell‐cycle analysis. HRG blocked cell‐cycle progression and lapatinib reversed this block. Our findings support a role for HRG in the negative regulation of ErbB expression and suggest that inhibition of ErbB2/3 signaling by ErbB2 directed therapies may interfere with this process. J. Cell. Physiol. 229: 1831–1841, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Probiotic L. reuteri Treatment Prevents Bone Loss in a Menopausal
           Ovariectomized Mouse Model
    • Abstract: Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post‐menopausal bone loss. Recent studies suggest an important role for gut‐bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri‐treated mice. Consistent with this, L. reuteri suppressed Ovx‐induced increases in bone marrow CD4+ T‐lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identified that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost‐effective approach to reduce post‐menopausal bone loss. J. Cell. Physiol. 229: 1822–1830, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Biological Effects of Insulin and Its Analogs on Cancer Cells With
           Different Insulin Family Receptor Expression
    • Abstract: Hyperinsulinemia is a likely cause of the increased cancer incidence and mortality in diabetic patients, but its role is difficult to define in vivo. Previous in vitro studies testing the mitogenic potential of insulin and its analogs provided incomplete and sometimes contradictory results. To better evaluate cancer cell responsiveness to insulin, to its analogs and to IGF‐I, we measured under identical experimental conditions cell proliferation, invasiveness, and foci formation in six cancer cell lines with different insulin receptor family expression levels. The cancer cells studied have a different expression of insulin receptor (IR), its isoforms (IR‐A and IR‐B), and of the IGF‐I receptor. The data indicate that insulin stimulates proliferation in all cancer cell lines, invasiveness in some, and foci formation in none. Cancer cell responses to insulin (and IGF‐I) are not related to receptor expression levels; moreover, hormone‐stimulated proliferation and invasiveness are not correlated. IGF‐I is a more potent stimulator than insulin in most but not all cancer cell lines. Insulin analogs including M1 and M2 Glargine metabolites stimulate cancer cells similar to insulin. However, exceptions occur for specific analogs in particular cancer cells. In conclusion, in vitro insulin is an effective growth factor for all cancer cells but the biological response to insulin cannot be predicted on the basis of receptor expression levels. In the clinical setting, these observations should be taken in account when deciding treatment for diabetic patients who are at risk of undiscovered cancer or survivors of oncological diseases. J. Cell. Physiol. 229: 1817–1821, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Independent Anti‐Angiogenic Capacities of Coagulation Factors X and
           Xa
    • Abstract: Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti‐angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti‐angiogenic properties but did not modify the anti‐angiogenic effect of FX. Furthermore, only the anti‐angiogenic activity of FXa is PAR‐1dependent. Using in vivo models, we show that FX and FXa are anti‐angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non‐hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX. J. Cell. Physiol. 229: 1673–1680, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Interleukin‑6 Contributes to the Paracrine Effects of Cardiospheres
           Cultured from Human, Murine and Rat Hearts
    • Abstract: Cardiosphere‐derived cells (CDCs) were cultured from human, murine, and rat hearts. Diluted supernatant (conditioned‐medium) of the cultures improved the contractile behavior of isolated rat cardiomyocytes (CMCs). This effect is mediated by the paracrine release of cytokines. The present study tested the hypothesis, that the cardiovascular state of the donor’s heart influences this effect on CMCs and tries to identify the responsible factors. CDCs were cultured from human tissue samples of cardiac surgery and from murine and rat hearts. The supernatants of cultured CDCs from hypertensive humans and rats showed a higher improvement of the contractile behavior of CMCs compared to CDCs of normotensive origin. Subsequently, the cytokine profile of the supernatants was analyzed. Among the cytokines elevated in supernatants originating from hypertensive humans or rats was Interleukin‑6. CDCs were also generated from Interleukin‑6−/−‐mice and their wildtype littermates. The supernatant of the cultured Interleukin‑6−/−‐CDCs had no effect on the contractile behavior, whereas the supernatant of the Interleukin‑6+/+‐CDCs showed a positive effect. To confirm the hypothesis that Interleukin‑6 contributes to the paracrine effects, CMCs were incubated with Interleukin‑6. It improved the contractile function in a concentration dependent way. Finally, the effect of the supernatant of cultured CDCs derived from a hypertensive human sample could be abolished by simultaneous incubation with a specific Interleukin‑6 antibody. CDCs release cytokines that improve the contractile behavior of CMCs. This effect is more intense in CDCs from hypertensive donors. Interleukin‑6 is involved in this phenomenon. J. Cell. Physiol. 229: 1681–1689, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Sox18 Preserves the Pulmonary Endothelial Barrier Under Conditions of
           Increased Shear Stress
    • Abstract: Shear stress secondary to increased pulmonary blood flow (PBF) is elevated in some children born with congenital cardiac abnormalities. However, the majority of these patients do not develop pulmonary edema, despite high levels of permeability inducing factors. Previous studies have suggested that laminar fluid shear stress can enhance pulmonary vascular barrier integrity. However, little is known about the mechanisms by which this occurs. Using microarray analysis, we have previously shown that Sox18, a transcription factor involved in blood vessel development and endothelial barrier integrity, is up‐regulated in an ovine model of congenital heart disease with increased PBF (shunt). By subjecting ovine pulmonary arterial endothelial cells (PAEC) to laminar flow (20 dyn/cm2), we identified an increase in trans‐endothelial resistance (TER) across the PAEC monolayer that correlated with an increase in Sox18 expression. Further, the TER was also enhanced when Sox18 was over‐expressed and attenuated when Sox18 expression was reduced, suggesting that Sox18 maintains the endothelial barrier integrity in response to shear stress. Further, we found that shear stress up‐regulates the cellular tight junction protein, Claudin‐5, in a Sox18 dependent manner, and Claudin‐5 depletion abolished the Sox18 mediated increase in TER in response to shear stress. Finally, utilizing peripheral lung tissue of 4 week old shunt lambs with increased PBF, we found that both Sox18 and Claudin‐5 mRNA and protein levels were elevated. In conclusion, these novel findings suggest that increased laminar flow protects endothelial barrier function via Sox18 dependent up‐regulation of Claudin‐5 expression. J. Cell. Physiol. 229: 1802–1816, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Functional Inhibition of Aquaporin‐3 With a Gold‐Based
           Compound Induces Blockage of Cell Proliferation
    • Abstract: AQP3 has been correlated with higher transport of glycerol, increment of ATP content, and larger proliferation capacity. Recently, we described the gold(III) complex Auphen as a very selective and potent inhibitor of AQP3's glycerol permeability (Pgly). Here we evaluated Auphen effect on the proliferation of various mammalian cell lines differing in AQP3 expression level: no expression (PC12), moderate (NIH/3T3) or high (A431) endogenous expression, cells stably expressing AQP3 (PC12‐AQP3), and human HEK293T cells transiently transfected (HEK‐AQP3) for AQP3 expression. Proliferation was evaluated in the absence or presence of Auphen (5 μM) by counting number of viable cells and analyzing 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation. Auphen reduced ≈50% the proliferation in A431 and PC12‐AQP3, ≈15% in HEK‐AQP3 and had no effect in PC12‐wt and NIH/3T3. Strong arrest in the S‐G2/M phases of the cell cycle, supported by analysis of cyclins (A, B1, D1, E) levels, was observed in AQP3‐expressing cells treated with Auphen. Flow‐cytometry of propidium iodide incorporation and measurements of mitochondrial dehydrogenases activity confirmed absence of cytotoxic effect of the drug. Functional studies evidenced ≈50% inhibition of A431 Pgly by Auphen, showing that the compound’s antiproliferative effect correlates with its ability to inhibit AQP3 Pgly. Role of Cys‐40 on AQP3 permeability blockage by Auphen was confirmed by analyzing the mutated protein (AQP3‐Ser‐40). Accordingly, cells transfected with mutated AQP3 gained resistance to the antiproliferative effect of Auphen. These results highlight an Auphen inhibitory effect on proliferation of cells expressing AQP3 and suggest a targeted therapeutic effect on carcinomas with large AQP3 expression. J. Cell. Physiol. 229: 1787–1801, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Autophagy Is Modulated in Human Neuroblastoma Cells Through Direct
           Exposition to Low Frequency Electromagnetic Fields
    • Abstract: In neurogenerative diseases, comprising Alzheimer’s (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low‐frequency EMF (LF‐EMF) on the modulation of autophagy was investigated in human neuroblastoma SH‐SY5Y cells, which were also subsequently exposed to Aβ peptides, key players in AD. The results primarily point that LF‐EMF induce a significant reduction of microRNA 30a (miR‐30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF‐EMF counteract the induced miR‐30a up‐regulation in the same cells transfected with miR‐30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy‐related markers (ATG7 and LC3B‐II) as well as the dynamics of autophagosome formation were also visualized after LF‐EMF exposition. Finally, different protocols of repeated LF‐EMF treatments were assayed to contrast the effects of Aβ peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF‐EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF‐EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776–1786, 2014. © 2014 Wiley Periodicals, Inc.
       
  • N‐Cadherin/Wnt Interaction Controls Bone Marrow Mesenchymal Cell
           Fate and Bone Mass During Aging
    • Abstract: Age‐related bone loss is characterized by reduced osteoblastogenesis and excessive bone marrow adipogenesis. The mechanisms governing bone marrow mesenchymal stromal cell (BMSC) differentiation into adipocytes or osteoblasts during aging are unknown. We show here that overexpressing N‐cadherin (Cadh2) in osteoblasts increased BMSC adipocyte differentiation and reduced osteoblast differentiation in young transgenic (Tg) mice whereas this phenotype was fully reversed with aging. The reversed phenotype with age was associated with enhanced Wnt5a and Wnt10b expression in osteoblasts and a concomitant increase in BMSC osteogenic differentiation. Consistent with this mechanism, conditioned media from young wild type osteoblasts inhibited adipogenesis and promoted osteoblast differentiation in BMSC from old Cadh2 Tg mice, and this response was abolished by Wnt5a and Wnt10b silencing. Transplantation of BMSC from old Cadh2 Tg mice into young Tg recipients increased Wnt5a and Wnt10b expression and rescued BMSC osteogenic differentiation. In senescent osteopenic mice, blocking the CADH2–Wnt interaction using an antagonist peptide increased Wnt5a and Wnt10b expression, bone formation, and bone mass. The data indicate that Cadh2/Wnt interaction in osteoblasts regulates BMSC lineage determination, bone formation, and bone mass and suggest a therapeutic target for promoting bone formation in the aging skeleton. J. Cell. Physiol. 229: 1765–1775, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Pharmacological Strategies in Lung Cancer‐Induced Cachexia: Effects
           on Muscle Proteolysis, Autophagy, Structure, and Weakness
    • Abstract: Cachexia is a relevant comorbid condition of chronic diseases including cancer. Inflammation, oxidative stress, autophagy, ubiquitin–proteasome system, nuclear factor (NF)‐κB, and mitogen‐activated protein kinases (MAPK) are involved in the pathophysiology of cancer cachexia. Currently available treatment is limited and data demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF‐κB, and MAPK inhibitors improve muscle mass and function loss through several molecular mechanisms. Body and muscle weights, limb muscle force, protein degradation and the ubiquitin–proteasome system, signaling pathways, oxidative stress and inflammation, autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing cachectic mice (BALB/c), with and without concomitant treatment with NF‐κB (sulfasalazine), MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins, autophagy, myostatin, protein oxidation, FoxO‐1, NF‐κB and MAPK signaling pathways, and muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow‐ and fast‐twitch muscle fiber size were decreased. Pharmacological inhibition of NF‐κB and MAPK, but not the proteasome system, induced in cancer cachectic animals, a substantial restoration of muscle mass and force through a decrease in muscle protein oxidation and catabolism, myostatin, and autophagy, together with a greater content of myogenin, and contractile and functional proteins. Attenuation of MAPK and NF‐κB signaling pathway effects on muscles is beneficial in cancer‐induced cachexia. J. Cell. Physiol. 229: 1660–1672, 2014. © 2014 Wiley Periodicals, Inc.
       
  • TGF‐β1 and FGF2 Stimulate the Epithelial–Mesenchymal
           Transition of HERS Cells Through a MEK‐Dependent Mechanism
    • Abstract: Hertwig’s epithelial root sheath (HERS) cells participate in cementum formation through epithelial–mesenchymal transition (EMT). Previous studies have shown that transforming growth factor beta 1 (TGF‐β1) and fibroblast growth factor 2 (FGF2) are involved in inducing EMT. However, their involvement in HERS cell transition remains elusive. In this study, we confirmed that HERS cells underwent EMT during the formation of acellular cementum. We found that both TGF‐β1 and FGF2 stimulated the EMT of HERS cells. The TGF‐β1 regulated the differentiation of HERS cells into periodontal ligament fibroblast‐like cells, and FGF2 directed the differentiation of HERS cells into cementoblast‐like cells. Treatment with TGF‐β1 or FGF2 inhibitor could effectively suppress HERS cells differential transition. Combined stimulation with both TGF‐β1 and FGF‐2 did not synergistically accelerate the EMT of HERS. Moreover, TGF‐β1/FGF2‐mediated EMT of HERS cells was reversed by the MEK1/2 inhibitor U0126. These results suggest that TGF‐β1 and FGF2 induce the EMT of HERS through a MAPK/ERK‐dependent signaling pathway. They also exert their different tendency of cellular differentiation during tooth root formation. This study further expands our knowledge of tooth root morphogenesis and provides more evidence for the use of alternative cell sources in clinical treatment of periodontal diseases. J. Cell. Physiol. 229: 1647–1659, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Thymosin Beta‐4 Knockdown in IEC‐6 Normal Intestinal
           
    • Abstract: Thymosin β4 (Tβ4) is a multifunctional protein already used clinically to treat various diseases; however, the promoting effect of this protein on tumor malignancy should not be neglected. Here, we assessed whether Tβ4 alteration influences normal intestinal epithelial cells because Tβ4 is deemed a novel target for treating colorectal cancer (CRC). For this purpose, we examined the consequences of shRNA‐mediated knockdown of Tβ4 in IEC‐6 normal rat small intestinal cells and found that inhibiting Tβ4 expression significantly suppressed their growth and induced apoptosis in some cells. Flow cytometric analysis further revealed a marked decrease of G0/G1 population but a drastic increase of polyploid ones in these cells. The increase of polyploidy likely resulted from DNA re‐replication because not only the de novo DNA synthesis was greatly increased but also the expression levels of Cdc6 (a replication‐licensing factor), cyclin A, and phosphorylated‐checkpoint kinase 1 were all dramatically elevated. Moreover, marked reductions in both RNA and protein levels of Emi1 (early mitotic inhibitor 1) were also detected in Tβ4‐downregulated IEC‐6 cells which might be accounted by the downregulation of E2F1, a transcription factor capable of inducing Emi1 expression, mediated by glycogen synthase‐3β (GSK‐3β). To our best knowledge, this is the first report showing that inhibiting Tβ4 expression triggers DNA re‐replication in normal intestinal epithelial cells, suggesting that this G‐actin sequester may play a crucial role in maintaining genome stability in these cells. More importantly, clinical oncologists should take this novel activity into consideration when design CRC therapy based on targeting Tβ4. J. Cell. Physiol. 229: 1639–1646, 2014. © 2014 Wiley Periodicals, Inc.
       
  • hsa‐miR‐4516 Mediated Downregulation of STAT3/CDK6/UBE2N Plays
           a Role in PUVA Induced Apoptosis in Keratinocytes
    • Abstract: Psoriasis is a chronic inflammatory skin disorder mediated by cross‐talk occurring between epidermal keratinocytes, dermal vascular cells and immunocytes. Literature reveals that Signal transducer and activator of transcription 3 (STAT3), a protein involved in transmitting extracellular signals to the nucleus, is a possible important link between keratinocytes and immunocytes and is crucial to the development of psoriasis. Although photochemotherapy using UV in combination with 8 methoxypsoralen is one of the most effective therapy for moderate to severe plaque psoriasis, its mechanism of action is largely unknown. Herein, we studied the change in miRNA profiles of cultured human keratinocytes (HaCaT cells) before and after in vitro PUVA treatment by 8 methoxypsoralen and found significant up regulation of hsa‐miR‐4516. We for the first time demonstrate that ectopic expression of hsa‐miR‐4516 directly targets STAT3 protein by binding to its 3′UTR in HaCaT cells as confirmed by Luciferase reporter assays and Western blot analysis. We further show that overexpression of hsa‐miR‐4516 downregulates STAT3, p‐STAT3, CDK6, and UBE2N proteins that are consistently upregulated in psoriasis and induces apoptosis in HaCaT cells. We also observed that anti‐miR‐4516 treatment was able to partially inhibit PUVA‐induced apoptosis, suggesting that miR‐4516 is involved in PUVA‐induced apoptosis. Taken together, these results not only indicate the mechanistic involvement of hsa‐miR‐4516 in PUVA mediated effects by down‐regulating STAT3 in HaCaT keratinocytes, but also highlight the potential of hsa‐miR‐4516 in development of novel therapeutic strategies. J. Cell. Physiol. 229: 1630–1638, 2014. © 2014 Wiley Periodicals, Inc.
       
  • IGF‐1 Alleviates NMDA‐Induced Excitotoxicity in Cultured
           Hippocampal Neurons Against Autophagy via the
           NR2B/PI3K‐AKT‐mTOR Pathway
    • Abstract: Insulin‐like growth factor‐1 (IGF‐1) is a brain‐specific multifunctional protein involved in neuronal polarity and axonal guidance. Mature IGF‐1 triggers three enzymes, mitogen‐activated protein kinase (MAPK), phosphatidylinositol 3‐kinase (PI3K), and phosphoinositide phospholipase C‐γ (PLC‐γ), which are its predominant downstream regulators. The PI3K‐AKT signaling pathway is upstream of the mammalian target of rapamycin (mTOR), which is of great importance in the induction of autophagy. However, whether the neuroprotective effect of IGF‐1 against excitotoxicity is mediated by autophagy through the PI3K/AKT/mTOR pathway remains to be elucidated. The induction of autophagy following NMDA treatment was determined by microtubule‐associated protein light chain 3 (LC3) conversion and the result of this autophagy was assessed by monitoring the cleavage of caspase 3 in cultured hippocampal neurons. Cell viability was determined using MTT and LDH assay, and PI‐staining was used to estimate the fate of autophagy and the protective effect of IGF‐1. In addition, IGF‐1 was found to decrease autophagy induced by NMDA using transmission electron microscopy and MDC staining. The protective effect of IGF‐1 against autophagy was accompanied with up‐regulation of phospho‐AKT (p‐AKT) and phospho‐mTOR (p‐mTOR), which was blocked by the inhibitor of PI3K. At the same time, the activation of NR2B resulting in the down‐regulation of p‐AKT and p‐mTOR was blocked by IGF‐1. Together, these data suggest that NMDA induces the autophagy, followed by apoptosis in cultured hippocampal neurons, and that IGF‐1 can block this effect via inhibition of NR2B receptors and activation of the PI3K‐AKT‐mTOR pathway. J. Cell. Physiol. 229: 1618–1629, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Loss of Jab1 in Osteochondral Progenitor Cells Severely Impairs Embryonic
           Limb Development in Mice
    • Abstract: The transcriptional cofactor Jab1 controls cell proliferation, apoptosis, and differentiation in diverse developmental processes by regulating the activity of various transcription factors. To determine the role of Jab1 during early limb development, we developed a novel Jab1flox/flox; Prx1‐Cre conditional Knockout (cKO) mutant mouse model in which Jab1 was deleted in the osteochondral progenitor cells of the limb buds. Jab1 cKO mutant mice displayed drastically shortened limbs at birth. The short‐limb defect became apparent in Jab1 cKO mutants at E15.5 and increasingly worsened thereafter. By E18.5, Jab1 cKO mutant mice exhibited significantly shorter limbs with: very few hypertrophic chondrocytes, disorganized chondrocyte columns, much smaller primary ossification centers, and significantly increased apoptosis. Real‐time RT‐PCR analysis showed decreased expression of Sox9, Col2a1, Ihh, and Col10a1 in Jab1 cKO mutant long bones, indicating impaired chondrogenesis. Furthermore, in a micromass culture model of early limb mesenchyme cells, alcian blue staining showed a significant decrease in chondrogenesis in Jab1 cKO limb bud cells. The expression of Sox9 and its downstream targets Col2a1 and Aggrecan, as well as BMP signaling downstream targets, Noggin, Id1, and Ihh, were significantly decreased in Jab1 cKO micromass cultures. Moreover, over‐expression of SOX9 in Jab1 cKO micromass cultures partially restored Col2a1and Aggrecan expression. Jab1‐deficient micromass cultures also exhibited decreased BMP signaling response and reduced BMP‐specific reporter activity ex vivo. In summary, our study demonstrates that Jab1 is an essential regulator of early embryonic limb development in vivo, likely in part by co‐activating Sox9 and BMP signaling. J. Cell. Physiol. 229: 1607–1617, 2014. © 2014 Wiley Periodicals, Inc.
       
  • PPARγ and RXR Ligands Disrupt the Inflammatory Cross‐talk in
           the Hypoxic Breast Cancer Stem Cells Niche
    • Abstract: Cancer stem cells (CSCs) are affected by the local micro‐environment, the niche, in which inflammatory stimuli and hypoxia act as steering factors. Here, two nuclear receptors (NRs) agonists, i.e. pioglitazone (PGZ), a ligand of peroxisome proliferator activated receptor‐γ, and 6‐OH‐11‐O‐hydroxyphenanthrene (IIF), a ligand of retinoid X receptors, were investigated for their capability to interference with the cross‐talk between breast CSCs and the niche compartment. We found that IIF potentiates the ability of PGZ to hamper the mammospheres‐forming capability of human breast tumours and MCF7 cancer cells, reducing the expression of CSCs regulatory genes (Notch3, Jagged1, SLUG, Interleukin‐6, Apolipoprotein E, Hypoxia inducible factor‐1α and Carbonic anhydrase IX). Notably, these effects are not observed in normal‐MS obtained from human breast tissue. Importantly, NRs agonists abolish the capability of hypoxic MCF7 derived exosomes to induce a pro‐inflammatory phenotype in mammary glands fibroblasts. Moreover, NRs agonist also directly acts on breast tumour associated fibroblasts to downregulate nuclear factor‐κB pathway and metalloproteinases (MMP2 and MMP9) expression and activity. In conclusion, NRs agonists disrupt the inflammatory cross‐talk of the hypoxic breast CSCs niche. J. Cell. Physiol. 229: 1595–1606, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Ribosome Biogenesis: Emerging Evidence for a Central Role in the
           Regulation of Skeletal Muscle Mass
    • Abstract: The ribosome is a supramolecular ribonucleoprotein complex that functions at the heart of the translation machinery to convert mRNA into protein. Ribosome biogenesis is the primary determinant of translational capacity of the cell and accordingly has an essential role in the control of cell growth in eukaryotes. Cumulative evidence supports the hypothesis that ribosome biogenesis has an important role in the regulation of skeletal muscle mass. The purpose of this review is to, first, summarize the main mechanisms known to regulate ribosome biogenesis and, second, put forth the hypothesis that ribosome biogenesis is a central mechanism used by skeletal muscle to regulate protein synthesis and control skeletal muscle mass in response to anabolic and catabolic stimuli. The mTORC1 and Wnt/β‐catenin/c‐myc signaling pathways are discussed as the major pathways that work in concert with each of the three RNA polymerases (RNA Pol I, II, and III) in regulating ribosome biogenesis. Consistent with our hypothesis, activation of these two pathways has been shown to be associated with ribosome biogenesis during skeletal muscle hypertrophy. Although further study is required, the finding that ribosome biogenesis is altered under catabolic states, in particular during disuse atrophy, suggests that its activation represents a novel therapeutic target to reduce or prevent muscle atrophy. Lastly, the emerging field of ribosome specialization is discussed and its potential role in the regulation of gene expression during periods of skeletal muscle plasticity. J. Cell. Physiol. 229: 1584–1594, 2014. © 2014 Wiley Periodicals, Inc.
       
  • p16INK4a, NAD+, and Sestrins: New Targets for Combating
           Aging‐Related Chronic Illness'
    • Abstract: Aging‐related chronic illness is a price we have to pay to live longer. Prevalent among the oldest old, the condition limits their functional independence and also aggravates the course of several age‐related chronic diseases. Thus, the search is on for efficient therapies that will mitigate age‐related pathologies. In this article, we point out the potential clinical implications of recent provocative basic research in the field. New possible targets have been recently discovered, are clearly involved in age‐related pathologies and might benefit the treatment of other age‐related conditions, particularly metabolic diseases. J. Cell. Physiol. 229: 1575–1576, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Disease Pathways in Proliferative Vitreoretinopathy: An Ongoing Challenge
    • Abstract: Despite remarkable advances in vitreoretinal surgery, proliferative vitreoretinopathy (PVR) remains a common cause of severe visual loss or blindness. One of the critical reasons for PVR‐induced blindness is tractional retinal detachment due to the formation of contractile preretinal fibrous membranes. This membrane formation is characterized by the proliferation and migration of cells and the excessive synthesis and deposition of extracellular matrix proteins. Herein we present the disease pathways of PVR, reviewing the role of both systemic and intraocular cells as well as molecular mediators. A chronological sequence of events leading to PVR is also hypothesized. Better understanding of the pathogenesis of PVR is needed in order to improve disease management. Efforts should be oriented towards greater cooperation between basic researchers and clinicians, aimed at matching the different clinical scenarios with the biological markers of the disease. J. Cell. Physiol. 229: 1577–1583, 2014. © 2014 Wiley Periodicals, Inc.
       
  • Journal of Cellular Physiology: Volume 229, Number 11, November 2014
    • Abstract: Cover: Epigenetic reader domains in the MOZ HAT complex recognize histone post‐translational modifications. Model illustrating how recognition of histone modifications by various subunits within the MOZ HAT directs the complex to chromatin and regulates enzymatic activity. See article by Carlson and Glass on pages 1571–1574.
       
  • Table of Contents: Volume 229, Number 11
    •  
  • Editor's Choice
    •  
  • Highlights: Volume 229, Number 11
    •  
  • The MOZ Histone Acetyltransferase in Epigenetic Signaling and Disease
    • Abstract: The monocytic leukemic zinc finger (MOZ) histone acetyltransferase (HAT) plays a role in acute myeloid leukemia (AML). It functions as a quaternary complex with the bromodomain PHD finger protein 1 (BRPF1), the human Esa1‐associated factor 6 homolog (hEAF6), and the inhibitor of growth 5 (ING5). Each of these subunits contain chromatin reader domains that recognize specific post‐translational modifications (PTMs) on histone tails, and this recognition directs the MOZ HAT complex to specific chromatin substrates. The structure and function of these epigenetic reader modules has now been elucidated, and a model describing how the cooperative action of these domains regulates HAT activity in response to the epigenetic landscape is proposed. The emerging role of epigenetic reader domains in disease, and their therapeutic potential for many types of cancer is also highlighted. J. Cell. Physiol. 229: 1571–1574, 2014. © 2014 Wiley Periodicals, Inc.
       
  • C/EBPα and the Vitamin D Receptor Cooperate in the Regulation of
           Cathelicidin in Lung Epithelial Cells
    • Abstract: 1,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) and vitamin D receptor (VDR) have been reported to have an important role in the regulation of innate immunity. We earlier reported that the antimicrobial peptide cathelicidin is induced by 1,25(OH)2D3 in normal human bronchial epithelial cells with a resultant increase in antimicrobial activity against airway pathogens. In this study we demonstrate that C/EBP alpha (C/EBPα) is a potent enhancer of human cathelicidin antimicrobial peptide (CAMP) gene transcription in human lung epithelial cells. In addition we found that C/EBPα functionally cooperates with VDR in the regulation of CAMP transcription. A C/EBP binding site was identified at ‐627/‐619 within the CAMP promoter, adjacent to the vitamin D response element (VDRE; ‐615/‐600). Mutation of this site markedly attenuated the transcriptional response to C/EBPα as well as to 1,25(OH)2D3, further indicating cooperation between these two factors in the regulation of CAMP. ChIP analysis using 1,25(OH)2D3 treated human lung epithelial cells showed C/EBPα and VDR binding to the CAMP promoter. C/EBPα has previously been reported to cooperate with Brahma (Brm), an ATPase that is component of the SWI/SNF chromatin remodeling complex. We found that dominant negative Brm significantly inhibited C/EBPα as well as 1,25(OH)2D3 mediated induction of CAMP transcription, suggesting the functional involvement of Brm. These findings define novel mechanisms involving C/EBPα, SWI/SNF and 1,25(OH)2D3 in the regulation of CAMP in lung epithelial cells. These mechanisms of enhanced activation of the CAMP gene in lung epithelial cells suggest potential candidates for the development of modulators of innate immune responses for adjunct therapy in the treatment of airway infections. © 2014 Wiley Periodicals, Inc.
       
  • Perlecan Heparan Sulfate is Required for the Inhibition of Smooth Muscle
           Cell Proliferation by all‐trans‐Retinoic Acid
    • Abstract: Smooth muscle cell (SMC) proliferation is a key process in stabilization of atherosclerotic plaques, and during restenosis after interventions. A clearer understanding of SMC growth regulation is therefore needed to design specific anti‐proliferative therapies. Retinoic acid has been shown to inhibit proliferation of SMCs both in vitro and in vivo and to affect the expression of extracellular matrix molecules. To explore the mechanisms behind the growth inhibitory activity of retinoic acid, we hypothesized that retinoids may induce the expression of perlecan, a large heparan sulfate proteoglycan with anti‐proliferative properties. Perlecan expression and accumulation was induced in murine SMC cultures by all‐trans‐retinoic acid (AtRA). Moreover, the growth inhibitory effect of AtRA on wild‐type cells was greatly diminished in SMCs from transgenic mice expressing heparan sulfate‐deficient perlecan, indicating that the inhibition is perlecan heparan sulfate‐dependent. In addition, AtRA influenced activation and phosphorylation of PTEN and Akt differently in wild‐type and mutant SMCs, consistent with previous studies of perlecan‐dependent SMC growth inhibition. We demonstrate that AtRA regulates perlecan expression in SMCs and that the inhibition of SMC proliferation by AtRA is, at least in part, secondary to an increased expression of perlecan and dependent upon its heparan sulfate‐chains. © 2014 Wiley Periodicals, Inc.
       
  • Runx1 Activities in Superficial Zone Chondrocytes, Osteoarthritic
           Chondrocyte Clones and Response to Mechanical Loading
    • Abstract: Objective Runx1, the hematopoietic lineage determining transcription factor, is present in perichondrium and chondrocytes. Here we addressed Runx1 functions, by examining expression in cartilage during mouse and human osteoarthritis (OA) progression and in response to mechanical loading. Methods Spared and diseased compartments in knees of OA patients and in mice with surgical destabilization of the medial meniscus were examined for changes in expression of Runx1 mRNA (Q‐PCR) and protein (immunoblot, immunohistochemistry). Runx1 levels were quantified in response to static mechanical compression of bovine articular cartilage. Runx1 function was assessed by cell proliferation (Ki67, PCNA) and cell type phenotypic markers. Results Runx1 is enriched in superficial zone (SZ) chondrocytes of normal bovine, mouse, and human tissues. Increasing loading conditions in bovine cartilage revealed a positive correlation with a significant elevation of Runx1. Runx1 becomes highly expressed at the periphery of mouse OA lesions and in human OA chondrocyte 'clones' where Runx1 co‐localizes with Vcam1, the mesenchymal stem cell (MSC) marker and lubricin (Prg4), a cartilage chondroprotective protein. These OA induced cells represent a proliferative cell population, Runx1 depletion in MPCs decreases cell growth, supporting Runx1 contribution to cell expansion. Conclusion The highest Runx1 levels in SZC of normal cartilage suggest a function that supports the unique phenotype of articular chondrocytes, reflected by upregulation under conditions of compression. We propose Runx1 co‐expression with Vcam1 and lubricin in murine cell clusters and human 'clones' of OA cartilage, participate in a cooperative mechanism for a compensatory anabolic function. © 2014 Wiley Periodicals, Inc.
       
  • Neoadjuvant chemotherapy in breast cancer patients induces miR‐34a
           and miR‐122 expression
    • Abstract: Circulating microRNAs (miRNAs) have been extensively studied in cancer as biomarkers but little is known regarding the influence of anti‐cancer drugs on their expression levels. In this article, we describe the modifications of circulating miRNAs profile after neoadjuvant chemotherapy (NAC) for breast cancer. The expression of 188 circulating miRNAs was assessed in the plasma of 25 patients before and after NAC by RT‐qPCR. Two miRNAs, miR‐ 34a and miR‐122, that were significantly increased after NAC, were measured in tumor tissue before and after chemotherapy in 7 patients with pathological partial response (pPR) to NAC. These 2 chemotherapy‐induced miRNAs were further studied in the plasma of 22 patients with adjuvant chemotherapy (AC) as well as in 12 patients who did not receive any chemotherapy. Twenty‐five plasma miRNAs were modified by NAC. Among these miRNAs, miR‐34a and miR‐122 were highly upregulated, notably in pPR patients with aggressive breast cancer. Furthermore, miR‐34a level was elevated in the remaining tumor tissue after NAC treatment. Studying the kinetics of circulating miR‐34a and miR‐122 expression during NAC revealed that their levels were especially increased after anthracycline‐based chemotherapy. Comparisons of the plasma miRNA profiles after NAC and AC suggested that chemotherapy‐induced miRNAs originated from both tumoral and non‐tumoral compartments. This study is the first to demonstrate that NAC specifically induces miRNA expression in plasma and tumor tissue, which might be involved in the anti‐tumor effects of chemotherapy in breast cancer patients. © 2014 Wiley Periodicals, Inc.
       
  • Non‐random Patterns in the Distribution of NOR‐bearing
           Chromosome Territories in Human Fibroblasts: A Network Model of
           Interactions
    • Abstract: We present a 3‐D mapping in WI38 human diploid fibroblast cells of chromosome territories (CT) 13,14,15,21, and 22, which contain the nucleolar organizing regions (NOR) and participate in the formation of nucleoli. The nuclear radial positioning of NOR‐CT correlated with the size of chromosomes with smaller CT more interior. A high frequency of pairwise associations between NOR‐CT ranging from 52% (CT13‐21) to 82% (CT15‐21) was detected as well as a triplet arrangement of CT15‐21‐22 (72%). The associations of homologous CT were significantly lower (24‐36%). The arrangements of each pairwise CT varied from CT13‐14 and CT13‐22, which had a majority of cells with single associations, to CT13‐15 and CT13‐21 where a majority of cells had multiple interactions. In cells with multiple nucleoli, one of the nucleoli (termed “dominant”) always associated with a higher number of CT. Moreover, certain CT pairs more frequently contributed to the same nucleolus than to others. This nonrandom pattern suggests that a large number of the NOR‐chromsomes are poised in close proximity during the postmitotic nucleolar recovery and through their NORs may contribute to the formation of the same nucleolus. A global data mining program termed the chromatic median determined the most probable interchromosomal arrangement of the entire NOR‐CT population. This interactive network model was significantly above randomized simulation and was composed of 13 connections among the NOR‐CT. We conclude that the NOR‐CT form a global interactive network in the cell nucleus that may be a fundamental feature for the regulation of nucleolar and other genomic functions. © 2014 Wiley Periodicals, Inc.
       
  • Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane
           Reveals Roles of Acrosomal Proteins in ZP3 Binding
    • Abstract: The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm‐ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non‐capacitated and capacitated sperm. The results revealed that ZP‐binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far‐western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non‐capacitated and capacitated sperm, but the complexes (~750‐1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome‐intact sperm during capacitation. Co‐immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with antizonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm‐ZP binding during capacitation. © 2014 Wiley Periodicals, Inc.
       
  • Fibronectin Peptides as Potential Regulators of Hepatic Fibrosis through
           apoptosis of hepatic stellate cells
    • Abstract: The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX‐2 based on oligonucleosomal DNA fragmentation, caspase‐3 and ‐9 activation, Bcl‐2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease‐9 (MMP‐9) expression in human monocytes. © 2014 Wiley Periodicals, Inc.
       
  • Distinct effects of inorganic phosphate on cell cycle and apoptosis in
           human vascular smooth muscle cells
    • Abstract: Objective Inorganic phosphate (Pi) is an essential nutrient to all living organisms. Nevertheless, hyperphosphatemia is now recognized as a risk factor for cardiovascular events and mortality in chronic kidney disease (CKD) patients. To our knowledge, the mechanisms by which elevated Pi alters smooth muscle cells proliferation have been poorly addressed. Therefore, in this study, we investigated the effects of Pi on cell cycle regulation and apoptosis in human aortic smooth muscle cells (HAoSMC). Approach and Results HAoSMC were treated with physiologic (1 mM) or high (2 and 3 mM) Pi concentrations. We showed that Pi not only decreased significantly cell viability (p 
       
  • Membrane‐Type 1 Matrix Metalloproteinase Downregulates Fibroblast
           Growth Factor‐2 Binding to the Cell Surface and Intracellular
           Signaling
    • Abstract: Membrane‐type 1 matrix metalloproteinase (MT1‐MMP, MMP‐14), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades extracellular matrix components and controls diverse cell functions through proteolytic and non‐proteolytic interactions with extracellular, intracellular and transmembrane proteins. Here we show that in tumor cells MT1‐MMP downregulates fibroblast growth factor‐2 (FGF‐2) signaling by reducing the amount of FGF‐2 bound to the cell surface with high and low affinity. FGF‐2 induces weaker activation of ERK1/2 MAP kinase in MT1‐MMP expressing cells than in cells devoid of MT1‐MMP. This effect is abolished in cells that express proteolytically inactive MT1‐MMP but persists in cells expressing MT1‐MMP mutants devoid of hemopexin‐like or cytoplasmic domain, showing that FGF‐2 signaling is downregulated by MT1‐MMP proteolytic activity. MT1‐MMP expression results in downregulation of FGFR‐1 and ‐4, and in decreased amount of cell surface‐associated FGF‐2. In addition, MT1‐MMP strongly reduces the amount of FGF‐2 bound to the cell surface with low affinity. Because FGF‐2 association with low‐affinity binding sites is a prerequisite for binding to its high‐affinity receptors, downregulation of low‐affinity binding to the cell surface results in decreased FGF‐2 signaling. Consistent with this conclusion, FGF‐2 induction of tumor cell migration and invasion in vitro is stronger in cells devoid of MT1‐MMP than in MT1‐MMP expressing cells. Thus, MT1‐MMP controls FGF‐2 signaling by a proteolytic mechanism that decreases the cell's biological response to FGF‐2. © 2014 Wiley Periodicals, Inc.
       
  • Beta‐Adrenoceptor Activation Reduces Both Dermal Microvascular
           Endothelial Cell Migration Via a cAMP‐Dependent Mechanism and Wound
           Angiogenesis
    • Abstract: Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar‐free fetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta‐adrenoceptors (β‐AR) are G protein‐coupled receptors expressed on all skin cell‐types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β‐AR‐mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β‐AR activation reduced HDMEC migration via cAMP‐dependent and PKA‐independent mechanisms as demonstrated through use of an EPAC agonist that auto‐inhibited the cAMP‐mediated β‐AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β‐AR activation reduced pro‐angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β‐AR‐mediated autocrine and paracrine anti‐angiogenic mechanisms. In more complex environments, β‐AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β‐AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β‐AR agonists could be promising anti‐angiogenic modulators in skin. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
  • S‐nitrosylation of Cofilin‐1 Serves as a Novel Pathway for
           VEGF‐stimulated Endothelial Cell Migration
    • Abstract: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) mediates vascular endothelial growth factor (VEGF)‐stimulated endothelial cytoskeleton remodeling and migration; however, the underlying mechanisms are elusive. Covalent adduction of a NO moiety (NO•) to cysteines called S‐nitrosylation (SNO) is a key NO signaling pathway. The small actin‐binding protein cofilin‐1 (CFL1) is essential for actin cytoskeleton remodeling. We investigated whether S‐nitrosylation regulates CFL1 function and endothelial cytoskeleton remodeling and migration upon VEGF stimulation. VEGF rapidly stimulated S‐nitrosylation of CFL1, which was blocked by NO Synthase inhibition and eNOS knockdown by specific eNOS‐siRNA. Cys80 and Cys139 were identified as the major SNO‐sites in CFL1 by LC‐MS/MS. The actin severing activity of recombinant SNO‐mimetic CFL1 (C80/139A DMA‐CFL1), but not SNO‐deficient CFL1 (C80/139S DMS‐CFL1), was significantly greater than that of wild‐type CFL1 (wt‐CFL1). When wt‐CFL1 and its mutants were overexpressed in endothelial cells, basal actin bound wt‐CFL1 was undetectable but significantly increased by VEGF; basal actin bound DMA‐CFL1 was readily high and basal actin bound DMS‐CFL1 was detectable but low, and both were unresponsive to VEGF. Treatment with VEGF significantly increased filamentous (F‐) actin and filopodium formation and cell migration in endothelial cells. Overexpression of wt‐CFL1 inhibited VEGF‐induced F‐actin formation. Overexpression of DMA but not DMS CFL1 decreased basal but not VEGF‐stimulated F‐actin formation. Overexpression of DMA but not DMS CFL1 suppressed VEGF‐stimulated filopodium formation and migration in endothelial cells. Thus, S‐nitrosylation of CFL1 provides a novel signaling pathway post‐NO biosynthesis via eNOS‐derived NO for endothelial cytoskeleton remodeling and migration upon VEGF stimulation. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
       
  • Regulation of Osteoclast Multinucleation by the Actin Cytoskeleton
           Signaling Network
    • Abstract: Although it is known that osteoclasts are multinucleated cells that are responsible for bone resorption, the mechanism by which their size is regulated is unclear. We previously reported that an actin‐rich superstructure, termed the zipper‐like structure, specifically appears during the fusion of large osteoclast‐like cells (OCLs). Actin cytoskeleton reorganization in osteoclasts is regulated by a signaling network that includes the macrophage colony‐stimulating factor (M‐CSF) receptor, a proto‐oncogene, Src, and small GTPases. Here, we examined the role of actin reorganization in the multinucleation of OCLs differentiated from RAW 264.7 cells using various pharmacological agents. Jasplakinolide, which stabilizes actin stress fibers, induced the development of small OCLs, and the Src inhibitor SU6656 and the dynamin inhibitor dynasore impaired the maintenance of the podosome belt and the zipper‐like structure. These inhibitors decreased the formation of large OCLs but increased the number of small OCLs. M‐CSF is known to stimulate osteoclast fusion. M‐CSF signaling via Src up‐regulated Rac1 activity but down‐regulated Rho activity. Rac1 and Rho localized to the center of the zipper‐like structure. Rho activator II promoted the formation of small OCLs, whereas the Rho inhibitor Y27632 promoted the generation of large OCLs. These results suggest that the status of the actin cytoskeleton signaling network determines the size of OCLs during cell fusion. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
       
  • Hydrogen Sulfide Epigenetically Attenuates Homocysteine‐Induced
           Mitochondrial Toxicity Mediated through NMDA Receptor in Mouse Brain
           Endothelial (bEnd3) Cells
    • Abstract: Previously we have showed that homocysteine (Hcy) caused oxidative stress and altered mitochondrial function. Hydrogen sulphide (H2S) has potent anti‐inflammatory, anti‐oxidative and anti‐apoptotic effects. Therefore, in the present study we examined whether H2S ameliorates Hcyinduced mitochondrial toxicity which led to endothelial dysfunction in part, by epigenetic alterations in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 100μM Hcy treatment in the presence or absence of 30μM NaHS (donor of H2S) for 24hrs. Hcy‐activate NMDA receptor and induced mitochondrial toxicity by increased levels of Ca2+,NADPH‐oxidase‐4 (NOX‐4) expression, mitochondrial dehydrogenase activity and decreased the level of nitrate, superoxide dismutase (SOD‐2) expression, mitochondria membrane potentials, ATP production. To confirm the role of epigenetic, 5'‐azacitidine (an epigenetic modulator) treatment was given to the cells. Pretreatment with NaHS (30μM) attenuated the Hcy‐induced increased expression of DNMT1, DNMT3a, Ca2+ and decreased expression of DNMT3b in bEND3 cells. Furthermore, NaHS treatment also enhanced mitochondrial oxidative stress (NOX4, ROS, and NO) and restored ATP that indicates its protective effects against mitochondrial toxicity. Additional, NaHS significantly alleviated Hcyinduced LC3‐I/II, CSE, Atg3/7 and low p62 expression which confirm its effect on mitophagy. Likewise, NaHS also restored level of eNOS, CD31, VE‐Cadherin and ET‐1 and maintains endothelial function in Hcy treated cells. Molecular inhibition of NMDA receptor by using small interfering RNA showed protective effect whereas inhibition of H2S production by propargylglycine (PG) (inhibitor of enzyme CSE) showed mitotoxic effect. Taken together, results demonstrate that, administration of H2S protected the cells from HHcy‐induced mitochondrial toxicity and endothelial dysfunction. J. Cell. Biochem. © 2014 Wiley Periodicals, Inc.
       
  • Inflammation‐induced down‐regulation of butyrate uptake and
           oxidation is not caused by a reduced gene expression
    • Abstract: In ulcerative colitis (UC) the butyrate metabolism is impaired, leading to energy‐deficiency in the colonic cells. The effect of inflammation on the butyrate metabolism was investigated. HT‐29 cells were incubated with pro‐inflammatory cytokines (TNF‐α and/or IFN‐γ) for 1h and 24h. Cells were additionally stimulated with butyrate to investigate its anti‐inflammatory potential. Butyrate uptake and oxidation were measured using 14C‐labelled butyrate. Gene expression of the butyrate metabolism enzymes, interleukin 8 (IL‐8; inflammatory marker) and villin‐1 (VIL‐1; epithelial cell damage marker) was measured via quantitative RT‐PCR. Significantly increased IL‐8 expression and decreased VIL‐1 expression after 24h incubation with TNF‐α and/or IFN‐γ confirmed the presence of inflammation. These conditions induced a decrease of both butyrate uptake and oxidation, whereas the gene expression was not reduced. Simultaneous incubation with butyrate counteracted the reduced butyrate oxidation. In contrast, 1h incubation with TNF‐α induced a significant increased IL‐8 expression and decreased butyrate uptake. Incubation with TNF‐α and/or IFN‐γ for 1h did not induce cell damage nor influence butyrate oxidation. The inflammation‐induced down‐regulation of the butyrate metabolism was not caused by a reduced gene expression, but appeared consequential to a decreased butyrate uptake. Increasing the luminal butyrate levels might have therapeutic potential in UC. © 2014 Wiley Periodicals, Inc.
       
 
 
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