ALBERT

Description: Similar to phosphorylation, transient conjugation of ubiquitin to target proteins (ubiquitination) mediated by the concerted action of ubiquitin ligases and de-ubiquitinating enzymes (DUBs) can affect substrate function. As obligate intracellular parasites, viruses rely on different cellular pathways for their own replication and the well conserved ubiquitin conjugating/de-conjugating system is not an exception. Viruses not only usurp the host proteins involved in the ubiquitination/de-ubiquitination process, but they also encode their own ubiquitin ligases and DUBs. Here we report that an N-terminal variant of the herpes simplex virus (HSV) type-1 large tegument protein VP1/2 (VP1/2 1–767 ), encompassing an active DUB domain (herpesvirus tegument ubiquitin specific protease, htUSP), and TSG101, a component of the endosomal sorting complex required for transport (ESCRT)-I, functionally interact. In particular, VP1/2 1–767 modulates TSG101 ubiquitination and influences its intracellular distribution. Given the role played by the ESCRT machinery in crucial steps of both cellular pathways and viral life cycle, the identification of TSG101 as a cellular target for the HSV-1 specific de-ubiquitinating enzyme contributes to the clarification of the still under debate function of viral encoded DUBs highly conserved throughout the Herpesviridae family. This article is protected by copyright. All rights reserved

Description: The transition to a pathological phenotype such as Barrett's esophagus occurs via induction of resistance upon repeated contact with gastric refluxate in esophagus. This study examined the molecular changes within normal esophageal epithelial cells (EECs) under short-term acid loading and the role of these changes in defensive resistance against acidic cytotoxicity. After primary cultured EECs were exposed to pH 4-acidified medium (AM4), cell viability was determined by the MTT assay. Reactive oxygen species (ROS) and NAD(P)H oxidase (NOX) activity were measured. Activation of the mitogen-activated protein kinases (MAPKs) MEK/ERK1/2, p38 and JNK; phosphoinositol-3-kinase (PI3K)/Akt, and nuclear factor-kappa B (NF-κB) were detected by Western blot analysis or immunofluorescence staining. AM4 incubation induced intracellular ROS generation accompanied by increase in NOX activity, which was further increased by Na + /H + exchange-1 (NHE1)-dependent inhibition but was prevented by inhibition of NOX or mitochondria complex I. AM4 also induced phosphorylation of MEK/ERK1/2, p38 MAPK, PI3K/Akt, and nuclear translocation of NF-κB, and all these effects, except for p38 MAPK phosphorylation, were abolished by inhibition of ROS. ROS-dependent PI3K/Akt activation, which mediates NF-κB nuclear translocation, was inhibited by protein tyrosine kinase (PTK) inhibitors and NHE1-specific inhibitor. All inhibitors of NHE, ROS, PTK, PI3K or NF-κB further decreased AM4-induced cell viability. Acid loading in the presence of NHE1-dependent protection induced ROS generation by activating NOX and mitochondria complex I, which stimulated PTK/PI3K/Akt/NF-κB-dependent survival in EEC. Our data indicate that normal EEC initially respond to acid loading through intrinsic survival activation. This article is protected by copyright. All rights reserved

Description: Osteolytic bone diseases are characterized by excessive osteoclast formation and activation. Protein kinase C (PKC)-dependent pathways regulate cell growth, differentiation and apoptosis in many cellular systems, and have been implicated in cancer development and osteoclast formation. A number of PKC inhibitors with anti-cancer properties have been developed, but whether they might also influence osteolysis (a common complication of bone invading cancers) is unclear. We studied the effects of the PKC inhibitor compound, GF109203X on osteoclast formation and activity, processes driven by receptor activator of NFκB ligand (RANKL). We found that GF109203X strongly and dose dependently suppresses osteoclastogenesis and osteoclast activity in RANKL-treated primary mouse bone marrow cells. Consistent with this GF109203X reduced expression of key osteoclastic genes, including cathepsin K, calcitonin receptor, tartrate resistant acid phosphatase (TRAP) and the proton pump subunit V-ATPase-d2 in RANKL-treated primary mouse bone marrow cells. Expression of these proteins is dependent upon RANKL-induced NF-κB and NFAT transcription factor actions; both were reduced in osteoclast progenitor populations by GF109203X treatment, notably NFATc1 levels. Furthermore, we showed that GF109203X inhibits RANKL-induced calcium oscillation. Together, this study shows GF109203X may block osteoclast functions, suggesting that pharmacological blockade of PKC-dependent pathways has therapeutic potential in osteolytic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Genomic and trascriptomic profiling has recently contributed details to the characterization of luminal B breast cancer. We explored the contribution of anthropometric, metabolic and molecular determinants to the multifaceted heterogeneity of this breast cancer subtype, with a specific focus on the association between body mass index (BMI), pre-treatment fasting glucose, hormone receptors and expression of human epidermal growth factor receptor 2 (HER2). Extensively annotated specimens were obtained from 154 women with luminal B breast cancer diagnosed at two Italian comprehensive cancer centres. Participants' characteristics were descriptively analyzed overall and by HER2 status (positive vs negative). BMI (〈25 vs ≥25), pre-treatment fasting glucose (〈median value of 94 mg/dl vs ≥94) and percentage of hormone receptors were tested for association with HER2 expression in regression models. In univariate models, BMI, fasting glucose and, at a lesser extent, percentage of estrogen receptors (ER) were significantly and inversely associated with HER2 expression (OR: 0.32, 95%CI: 0.16-0.66; 0.43, 0.23-0.0.82; 0.96, 0.94-0.97, respectively). The multivariate models confirmed the protective role of BMI and ER on HER2 expression, with luminal B HER2 positive patients being significantly less frequent among women within the highest category of BMI and percentage expression of ER compared with their counterparts (OR: 0.22, 95%CI: 0.09-0.53; 0.95, 0.93-0.97). In conclusions, BMI and percentage of ER representation are inversely associated with HER2 expression in luminal B breast cancers. Upon confirmatory findings, this might help identify patient subgroups who may best benefit from the use of interventions targeting insulin resistance in well depicted breast cancer scenarios. This article is protected by copyright. All rights reserved

Description: Histone deacetylases (HDACs) play a crucial role in the regulation of gene expression through remodeling of chromatin structures. However, the molecular mechanisms involved in this event remain unknown. In this study, we sought to examine whether HDAC inhibition-mediated protective effects involved HDAC4 sumoylation, degradation, and the proteasome pathway. Isolated neonatal mouse ventricular myocytes (NMVM) and H9c2 cardiomyoblasts were subjected to 48 hours of hypoxia (H) (1% O 2 ) and 2 hours of reoxygenation (R). Treatment of cardiomyocytes with trichostatin A (TSA) attenuated H/R-elicited injury, as indicated by a reduction of lactate dehydrogenase (LDH) leakage, an increase in cell viability, and decrease in apoptotic positive cardiomyocytes. MG132, a potent proteasome pathway inhibitor, abrogated TSA-induced protective effects, which was associated with the accumulation of ubiquitinated HDAC4. NMVM transduced with adenoviral HDAC4 led to an exaggeration of H/R-induced injury. TSA treatment resulted in a decrease in HDAC4 in cardiomyocytes infected with adenoviral HDAC4, and HDAC4-induced injury was attenuated by TSA. HDAC inhibition resulted in a significant reduction in reactive oxygen species (ROS) in cardiomyoblasts exposed to H/R, which was attenuated by blockade of the proteasome pathway. Cardiomyoblasts carrying wild type and sumoylation mutation (K559R) were established to examine effects of HDAC4 sumoylation and ubiquitination on H/R injury. Disruption of HDAC4 sumoylation brought about HDAC4 accumulation and impairment of HDAC4 ubiquitination in association with enhanced susceptibility of cardiomyoblasts to H/R. Taken together, these results demonstrated that HDAC inhibition stimulates proteasome dependent degradation of HDAC4, which is associated with HDAC4 sumoylation to induce these protective effects. This article is protected by copyright. All rights reserved

Description: Endothelial cells play a major role in the initiation and perpetuation of the inflammatory process in health and disease, including their pivotal role in leukocyte recruitment. The role of pro-inflammatory transcription factors in this process has been well-described, including NF-κB. However, much less is known regarding transcription factors that play an anti-inflammatory role in endothelial cells. Myocyte enhancer factor 2 C (MEF2C) is a transcription factor known to regulate angiogenesis in endothelial cells. Here, we report that MEF2C plays a critical function as an inhibitor of endothelial cell inflammation. Tumor necrosis factor (TNF)-α inhibited MEF2C expression in endothelial cells. Knockdown of MEF2C in endothelial cells resulted in the upregulation of pro-inflammatory molecules and stimulated leukocyte adhesion to endothelial cells. MEF2C knockdown also resulted in NF-κB activation in endothelial cells. Conversely, MEF2C overexpression by adenovirus significantly repressed TNF-α induction of pro-inflammatory molecules, activation of NF-κB, and leukocyte adhesion to endothelial cells. This inhibition of leukocyte adhesion by MEF2C was partially mediated by induction of KLF2. In mice, lipopolysaccharide (LPS)-induced leukocyte adhesion to the retinal vasculature was significantly increased by endothelial cell-specific ablation of MEF2C. Taken together, these results demonstrate that MEF2C is a novel negative regulator of inflammation in endothelial cells and may represent a therapeutic target for vascular inflammation. This article is protected by copyright. All rights reserved

Description: Trehalose-6-phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti-stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43-fold purification. The protein sequence, determined by N-terminal sequencing and MALDI-TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length gene sequence of TPP of Candida utilis was identified using rapid amplification of cDNA ends - PCR reaction (RACE-PCR). The gene was cloned and expressed in E. coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steady state fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. K m was determined to be ∼0.8 mM. Optimum temperature and pH was found to be 30 °C and 8.5 respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylation mediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations towards understanding the physico-chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalose synthesis and survivability of the organism under thermal stress was established. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: The microvascular network of the microcirculation works in tight communication with surrounding tissues to control blood supply and exchange of solutes. In cerebral circulation, microvascular endothelial cells constitute a selective permeability barrier that controls the environment of parenchymal brain tissue, which is known as the blood-brain barrier (BBB). Connexin- and pannexin-formed channels (gap junctions and hemichannels) play a central role in the coordination of endothelial and smooth muscle cell function and connexin-mediated signaling in endothelial cells is essential in the regulation of BBB permeability. Likewise, gap junction communication between astrocyte-end feet also contributes to maintain the BBB integrity, but the participation of hemichannels in this process cannot be discarded. Sympathetic and sensory perivascular nerves are also involved in the control and coordination of vascular function through the release of vasoconstrictor or vasodilator signals and by the regulation of gap junction communication in the vessel wall. Conversely, ATP release through pannexin-1-formed channels mediates the α1-adrenergic signaling. Furthermore, here we show that capsaicin-induced CGRP release from mesenteric perivascular sensory nerves induces pannexin-1-formed channel opening, which in turn leads to reduction of pannexin-1 and endothelial nitric oxide synthase (eNOS) expression along the time. Interestingly, blockade of CGRP receptors with CGRP 8–37 increased eNOS expression by ∼5 fold, suggesting that capsaicin-sensitive sensory nerves are involved in the control of key signaling proteins for vascular function. In this review, we discuss the importance of connexin-based channels in the control of BBB integrity and the functional interaction of vascular connexins and pannexins with the peripheral nervous system.

Description: We previously established a role for cancer-associated fibroblasts (CAFs) in enhancing the self-renewal and differentiation potentials of putative prostate cancer stem cells (CSCs). Our published work focused on androgen-dependent prostate cancer (ADPC) using the conditional Pten deletion mouse model. Employing the same model, we now describe the interaction of CAF and CSC in castration-resistant prostate cancer (CRPC). CAF isolated from ADPC (ADPCAF) and from CRPC (CRPCAF) were compared in terms of their ability to support organoid formation and tumor initiation by CSC from CRPC (CRPCSC) in vitro and in vivo. CRPCSC formed spheroids in vitro and well-differentiated glandular structures under the renal capsules of recipient mice in vivo more effectively in the presence of CRPCAF compared to ADPCAF. Furthermore, whereas CSC with CAF from ADPC formed mostly well-differentiated tumors in our previous study, we now show that CRPCSC, when combined with CRPCAF (but not ADPCAF) can form aggressive, poorly differentiated tumors. The potential of CRPCAF to support organoid/tumor formation by CRPCSC remained greater even when compared to 10-fold more ADPCAF, suggesting that paracrine factors produced specifically by CRPCAF preferentially potentiate the stemness and tumorigenic properties of the corresponding CSC. This apparently unique property of CRPCAF was notable when the CAF and CSC were grafted in either intact or castrated recipient mice. In both environments, CRPCAF induced in the epithelial compartment higher proliferative activity compared to ADPCAF, indicated by a higher Ki67 index. Factors released by CRPCAF to regulate CRPCSC may be targeted to develop novel therapeutic approaches to manage prostate cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Tat transactivating activity regulated by NAD + -dependent histone deacetylase sirtuin1 (SIRT1) connects HIV transcription with the metabolic state of the cell. Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the mammalian NAD + biosynthesis. Nampt, SIRT1 and AMPK were involved in inhibiting HIV-1 transactivation through redox-regulated pathway. Tanshinone IIA is a main lipid-soluble monomer derivative from the root of Salvia miltiorrhiza (Danshen) and tanshinone ⅡA possess a variety of biological activities through redox signaling pathway. Here we investigated the effect of tanshinone ⅡA on Tat-induced HIV-1 transactivation and the redox signaling pathway involved in it. As the results were shown, tanshinone ⅡA reversed Tat-induced reactive oxygen species (ROS) production and down-regulation of glutathione (GSH) levels in TZM-bl cells through up-regulation of Nrf2 expression. Tanshinone ⅡA reversed Tat-induced inhibition of SIRT1 activity but not SIRT1 protein expression. Tanshinone ⅡA reversed Tat-induced inhibition of Nampt protein expression and depletion of NAD + levels in TZM-bl cells in a dose-dependent manner. Tanshinone ⅡA-evoked Nampt expression was mediated by AMPK signaling pathway. Tanshinone ⅡA inhibited Tat-induced HIV-1 LTR transactivation dependent on AMPK-Nampt pathway. Collectively, our data provide new insights into understanding of the molecular mechanisms of tanshinone ⅡA inhibited Tat-regulated transcription, suggesting that targeting AMPK/Nampt/SIRT1 pathway could serve as new anti-HIV-1 agents.

Description: Osteoblast differentiation is tightly regulated by several factors including microRNAs (miRNAs). In this paper we report that pre-mir-15b is highly expressed in differentiated osteoblasts. The functional role of miR-15b in osteoblast differentiation was determined using miR-15b mimic/inhibitor and the expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP), type I collagen genes was decreased by miR-15b inhibitor. Runx2, a bone specific transcription factor is generally required for expression of osteoblast differentiation marker genes and in response to miR-15b inhibitor treatment, Runx2 mRNA expression was not changed; whereas its protein expression was decreased. Even though Smurf1 (SMAD specific E3 ubiquitin protein ligase 1), HDAC4 (histone deacetylase 4), Smad7, and Crim1 were found to be few of miR-15b's putative target genes, there was increased expression of only Smurf1 gene at mRNA and protein levels by miR-15b inhibitor. miR-15b mimic treatment significantly increased and decreased expressions of Runx2 and Smurf1 proteins, respectively. We further identified that the Smurf1 3’UTR is directly targeted by miR-15b using the luciferase reporter gene system. This is well documented that Smurf1 interacts with Runx2 and degrades it by proteasomal pathway. Hence, based on our results we suggest that miR-15b promotes osteoblast differentiation by indirectly protecting Runx2 protein from Smurf1 mediated degradation. Thus, this study identified that miR-15b can act as a positive regulator for osteoblast differentiation.

Description: Expression of smooth muscle alpha-actin (SMαA) is essential for myofibroblast-mediated wound contraction following tissue injury. The Pur α/β and YB-1 transcriptional repressors govern the DNA-binding activity of serum response factor (SRF) and phosphorylated Smad3 (pSmad3) transcriptional activators during induction of SMαA gene expression in human pulmonary myofibroblasts. In quiescent fibroblasts, Pur α exhibited a novel function in enhancing stability of pre-existing SRF complexes with SMαA core promoter DNA whereas Pur β was more effective in disrupting SRF-DNA interaction. Pur proteins were less efficient competitors of pre-existing, core-promoter complexes containing both SRF and pSmad3 in nuclear extracts from TGFβ1-activated myofibroblasts. TGFβ1 signaling dissociated a SRF/Pur protein complex with concurrent formation of a transient pSmad3/MRTF-A/Pur β complex during early phase myofibroblast differentiation. Pur β was replaced by Pur α in the pSmad3/MRTF-A complex in mature myofibroblasts. Combining all three repressors potently inhibited SRF and pSmad3 binding to promoter DNA in quiescent fibroblasts and TGFβ1-activated myofibroblasts, respectively. The results point to dynamic interplay between transcriptional activators and repressors in regulating SMαA gene output during myofibroblast differentiation. Therapeutic targeting of nucleoprotein complexes regulating the SMαA promoter may prevent excessive myofibroblast accumulation associated with chronic cardiopulmonary fibrosis and dysfunctional tissue remodeling.

Description: 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 that directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (BMP-2) osteogenic signal, 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. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: Recent technological advances allow for high throughput profiling of biological systems in a cost-efficient manner. The low cost of data generation is leading us to the “big data” era. The availability of big data provides unprecedented opportunities, but it also brings out challenges in data mining and analysis. In this review, we introduce key concepts in the analysis of big data, including both “machine learning” algorithms as well as “unsupervised” and “supervised” examples of each. We note packages for the R programming language that are available perform machine learning analyses. In addition to programming based solutions, we review webservers that allow users with limited or no programming background to perform these analyses on large data compendia. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to 〉100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation → cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: Myocardial infarction (MI) is defined as cardiac cell death due to prolonged ischemia. Although necrotic cell death was considered to be solely responsible for myocyte death during MI, it was recently revealed that apoptosis also plays its part in this death process. Our laboratory has recently shown that endoplasmic reticulum (ER) stress-induced apoptosis is the predominant route for apoptosis during MI and the conventional mitochondrial pathway is bypassed by activation of a small heat shock protein α-crystallin B (CRYAB). Since CRYAB is a direct target of P38 mitogen-activated protein kinase (MAPK) cascade, we were prompted to check the role of P38 MAPK in 20-week-old male Wister rats immediately after infarct formation. Interestingly, parallel activation of mitochondrial apoptotic pathway with an increase in ER stress-induced apoptotic load was observed along with decreased activation of CRYAB and Nrf2 (a pro-survival protein activated in response to ER stress) in MI rats treated with SB203580, a specific inhibitor of P38α and P38β compared to the MI alone. As a cumulative effect, this inhibitor treatment also resulted in significant increase in the levels of caspase3 activity and TUNEL positivity, the end point apoptotic markers. Furthermore, SB203580-treated hypoxic adult cardiomyocytes showed formation of desmin aggregates which were previously associated with impaired cardiac function. Thus, this study shows for the first time the precise mechanism by which P38 MAPK plays a pro-survival role and confers protection of cardiomyocytes, during infarct formation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Colon cancer remains one of the lethal malignancies in the world. Aberrant activation of canonical Wnt/β-catenin signaling pathway has been observed in colon cancer. In contrast, the non-canonical Wnt signaling functions remain obscure. Wnt5a is a representative non-canonical Wnt ligand which has gained extensive attention nowadays. Wnt5a has been shown to play an important role in EMT in prostate cancer and melanoma, but its role in colon cancer is still ambiguous. Here we have evaluated Wnt5a expression in a large cohort of 217 colon cancers by immunohistochemistry and analyzed its correlation with clinicopathologic characteristics. We found that expression of Wnt5a was diminished significantly in majority of primary colon cancers and negatively related with EMT biomarkers. To further enlighten the mechanism which Wnt5a regulates EMT in vitro , we established ectopic Wnt5a expression models. Protein analysis demonstrated that Wnt5a inhibited EMT and antagonized canonical Wnt signaling in colon cancer cells. Overexpression of Wnt5a impaired cell motility and invasion and inhibited cell proliferation by manipulating Bax. Moreover, Wnt5a suppressed the tumor growth in nude mice and impaired tumorigenicity in vivo . Wnt5a also induced intracellular calcium and activated non-canonical Wnt/Ca 2+ signaling in colon cancer. In summary, although Wnt5a was down-regulated in majority of colon cancers, enhanced Wnt5a expression predict preferable outcome in colon cancer patients. Our findings indicate that Wnt5a might act as tumor suppressor by inhibiting cell proliferation and attenuating EMT in colon cancer cells. Wnt5a could be used as a novel prognostic marker and/or therapeutic target for colon cancer in the future. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A 2A and A 2B are expressed at the highest level among the four receptor subtypes (A 2B  〉 A 2A  〉 A 1  〉 A 3 ) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A 2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A 2A receptor and by the A 2A -specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1, and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1, and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Dental pulp originating from the neural crest is considered a better source of postnatal stem cells for cell-based therapies in neurodegenerative diseases. Dental Pulp Stem Cells (DPSCs) have been shown to differentiate into cell-types of cranial neural crest ontology; however, their ability to differentiate to functional neurons of the central nervous system remains to be studied. We hypothesized that midbrain cues might commit DPSCs to differentiate to functional dopaminergic cell-type. As expected, DPSCs in their naïve state spontaneously expressed early and mature neuronal markers like nestin, musashi12, β tubulin III and Map2ab. On exposure to midbrain cues (sonic hedgehog, fibroblast growth factor 8 and basic fibroblast growth factor), DPSCs showed upregulation of dopaminergic neuron-specific transcription factors Nuclear Receptor related protein 1 (Nurr1), Engrailed 1 (En1) and paired-like homeodomain transcription factor 3 (Pitx3) as revealed by real-time RT-PCR. Immunofluorescence and flow cytometry analysis showed enhanced expression of mature neuronal marker Map2ab and dopaminergic-neuronal markers [tyrosine hydroxylase (TH), En1, Nurr1 and Pitx3], with nearly 77% of the induced DPSCs positive for TH. Functional studies indicated that the induced DPSCs could secrete dopamine constitutively and upon stimulation with potassium chloride (KCl) and adenosine triphosphate (ATP), as measured by dopamine ELISA. Additionally, the induced DPSCs showed intracellular Ca 2+ influx in the presence of KCl, unlike control DPSCs. ATP-stimulated Ca 2+ influx was observed in control and induced DPSCs, but only the induced cells secreted dopamine. Our data clearly demonstrates for the first time that DPSCs in the presence of embryonic midbrain cues show efficient propensity towards functional dopaminergic cell-type. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The chromatin remodeling complex SWI/SNF and the transcription factor C/EBPβ play critical roles in osteoblastic cells as they jointly control transcription of a number of bone-related target genes. The largest C/EBPβ isoform, LAP*, possesses a short additional N-terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N-terminus in C/EBPβ-LAP* for binding SWI/SNF and for recruiting this complex to the Ric-8B gene to mediate transcriptional repression. We find that both C/EBPβ-LAP* and SWI/SNF simultaneously bind to the Ric-8B promoter in differentiating osteoblasts that repress Ric-8B expression. This decreased expression of Ric-8B is not accompanied by significant changes in histone acetylation at the Ric-8B gene promoter sequence. A single aminoacid change at the C/EBPβ-LAP* N-terminus (R3L) that inhibits C/EBPβ-LAP*-SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric-8B promoter as well as C/EBPβ-LAP*-dependent repression of the Ric-8B gene. Inducible expression of the C/EBPβ-LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPβ-LAP*-target promoters and competes with the endogenous C/EBPβ factor. Together our results indicate that a functional N-terminus in C/EBPβ-LAP* is required for interacting with SWI/SNF and for Ric-8B gene repression in osteoblasts. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: Despite a regain of interest recently in ERK3 kinase signaling, the molecular regulations of both ERK3 gene expression and protein kinase activity are still largely unknown. While it is shown that disruption of ERK3 gene causes neonatal lethality, cell type-specific functions of ERK3 signaling remain to be explored. In this study, we report that ERK3 gene expression is upregulated by cytokines through c-Jun in endothelial cells; c-Jun binds to the ERK3 gene and regulates its transcription. We further reveal a new role for ERK3 in regulating endothelial cell migration, proliferation and tube formation by upregulating SRC-3/SP-1-mediated VEGFR2 expression. The underlying molecular mechanism involves ERK3-stimulated formation of a transcriptional complex involving coactivator SRC-3, transcription factor SP-1 and the secondary coactivator CBP. Taken together, our study identified a molecular regulatory mechanism of ERK3 gene expression and revealed a previously unknown role of ERK3 in regulating endothelial cell functions. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: Core Binding Factor β (CBFβ) is complexed with the RUNX family of transcription factors in the nucleus to support activation or repression of genes related to bone (RUNX2), hematopoiesis (RUNX1) and gastrointestinal (RUNX3) development. Furthermore, RUNX proteins contribute to the onset and progression of different types of cancer. Although CBFβ localizes to cytoskeletal architecture, its biological role in the cytoplasmic compartment remains to be established. Additionally, the function and localization of CBFβ during the cell cycle are important questions relevant to its biological role. Here we show that CBFβ dynamically distributes in different stages of cell division and importantly is present during telophase at the midbody, a temporal structure important for successful cytokinesis. A functional role for CBFβ localization at the midbody is supported by striking defects in cytokinesis that include polyploidy and abscission failure following siRNA-mediated downregulation of endogenous CBFβ or overexpression of the inv(16) fusion protein CBFβ-SMMHC. Our results suggest that CBFβ retention in the midbody during cytokinesis reflects a novel function that contributes to epigenetic control. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: Our previous work showed that the expression of bone morphogenetic protein-4 (BMP4) was up-regulated in pathological cardiac hypertrophy models and BMP4 induced cardiomyocyte hypertrophy and apoptosis. Bone morphogenetic protein-2 (BMP2) and BMP4 share greater than 80% amino acid homology and there exists an interaction between BMP2 and BMP4, so the aim of the present study was to elucidate the changes of BMP2 in the cardiac hypertrophy models and the effects of BMP2 on BMP4-induced cardiomyocyte hypertrophy and apoptosis. The in vivo cardiac hypertrophy models were induced by pressure-overload and swimming exercise in mice. BMP2 mRNA and protein expressions increased in pressure-overload and swimming-exercise induced cardiac hypertrophy. BMP2 itself did not elicit cardiomyocyte hypertrophy and apoptosis, but antagonized BMP4-induced cardiomyocyte hypertrophy and apoptosis. BMP2 stimulated Akt in cardiomyocytes and Akt inhibitor prevented the antagonism of BMP2 on BMP4-induced cardiomyocyte apoptosis. Furthermore, BMP2 inhibited BMP4-induced JNK activation in cardiomyocytes. In conclusion, BMP2 antagonizes BMP4-induced cardiomyocyte hypertrophy and apoptosis. The anti-apoptotic effects of BMP2 on BMP4-induced cardiomyocyte apoptosis might be through activating Akt and inhibiting JNK activation. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: Expression studies have implicated FLRT2 in cranial neural crest cell migration and prechondrogenic cell condensation during craniofacial skeletogenesis. We aimed to determine whether FLRT2 was involved in mediating cell-matrix interactions in the ATDC5 chondroprogenitor cell line. Immunolocalization experiments of ATDC5 cells revealed that FLRT2 was present on the cell membrane as well as extracellularly, where it colocalized with Fibronectin (Fn). After cell extraction of the matrix, FLRT2 was identified in the ATDC5-derived extracellular matrix (ECM) and was further found to be associated with Fn-coated beads in cell cultures. Blockage of Fn fibril formation via a blocking peptide resulted in a concomitant decrease in extracellular FLRT2 accumulation. Over a 7-day period following the replenishment of the Fn blocking peptide to the cultures, there was a partial rebound in Fn fibril formation that was accompanied by a concomitant reappearance of FLRT2 co-expression. Co-immunoprecipitation confirmed that FLRT2 and Fn interacted, either directly or indirectly. Immunoprecipitation and Western blot analyses with antibodies recognizing epitopes located on the extra- and intracellular domains of FLRT2 further revealed the presence of different sized bands, suggesting that FLRT2 may exist in both membrane-bound and shed forms. Our data therefore provide evidence that FLRT2 and/or its cleavage products may be cooperating with Fn and other ECM proteins to regulate critical cellular events. Further studies will be necessary in delineate more precisely the roles of FLRT2 in mediating cell- and cell-matrix interactions during normal development. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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, Nlk loxP/loxP mice, where the Nlk exon2 is flanked by lox P 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;Nlk Δ/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;Nlk Δ/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;Nlk Δ/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. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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/cm 2 ), 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.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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 (P gly ). 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 wt-PC12 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 P gly by Auphen, showing that the compound's anti-proliferative effect correlates with its ability to inhibit AQP3 P gly . 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. © 2014 Wiley Periodicals, Inc.

Description: There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET-based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti-fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The proper organization and segregation of chromosomes during cell division is essential to the preservation of genomic integrity. To understand the mechanisms that spatially control the arrangement and dynamics of mitotic chromosomes requires imaging assays to quantitatively resolve their positions and movements. Here, we will discuss analytical approaches to investigate the position-dependent control of mitotic chromosomes in cultured cells. These methods can be used to dissect the specific contributions of mitotic proteins to the molecular control of chromosome dynamics. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: 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. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: 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. © 2014 Wiley Periodicals, Inc.

Description: Apoptosis in most cell types is accompanied by altered Ca 2+ homeostasis. During apoptosis, caspase-3 mediated cleavage of the type 1 inositol 1,4,5-trisphosphate receptor (IP 3 R1) generates a 95-kDa C-terminal fragment (C-IP 3 R1), which represents the channel domain of the receptor. Aged mouse eggs display abnormal Ca 2+ homeostasis and express C-IP 3 R1, although whether or not C-IP 3 R1 expression contributes to Ca 2+ 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-IP 3 R1. We found that: (1) expression of C-IP 3 R1 in eggs lowered the Ca 2+ content of the endoplasmic reticulum (ER), although, as C-IP 3 R1 is quickly degraded at this stage, its expression did not impair pre-implantation embryo development; (2) expression of C-IP 3 R1 in eggs enhanced fragmentation associated with aging; (3) endogenous IP 3 R1 is required for aging associated apoptosis, as its down-regulation prevented fragmentation, and expression of C-IP 3 R1 in eggs with downregulated IP 3 R1 partly restored fragmentation; (4) C-IP 3 R1 expression in GV oocytes resulted in persistent levels of protein, which abolished the increase in the ER releasable Ca 2+ pool that occurs during maturation, undermined the Ca 2+ oscillatory ability of matured eggs and their activation potential. Collectively, this study supports a role for IP 3 R1 and C-IP 3 R1 in regulating Ca 2+ homeostasis and the ER Ca 2+ content during oocyte maturation. Nevertheless, the role of C-IP 3 R1 on Ca 2+ homeostasis in aged eggs seems minor, as in MII eggs the majority of endogenous IP 3 R1 remains intact and C-IP 3 R1 undergoes rapid turnover. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Increasing evidence supports the role of epigenetics in the development of autoimmune disorders and the possibility of using epigenetic modifying drugs in the context of MS has not yet been investigated. We have explored the effect of the hypomethylating agent 5-aza-2′-deoxycytidine (DAC) in two murine models of experimental allergic encephalomyelitis (EAE). DAC treatment was associated with a significant amelioration of the clinical and histological hallmarks of EAE in both models. These effects were observed both in prophylactic and therapeutic regimens. The milder course of the disease was associated with a reduction in the number of spinal cord infiltrating lymphocytes and amelioration of the histopathological signs associated with EAE. In addition, increased transcript levels of anti-inflammatory cytokines and decreased mRNA expression of pro-inflammatory mediators were also observed. Finally, DAC treatment increased the percentage of circulating regulatory T cells by inducing Foxp3 expression via demethylation of a CpG island in Foxp3. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Primary cilia are sensory organelles that provide a feedback mechanism to restrict Wnt signaling in the absence of endogenous Wnt activators. Abnormal Wnt signaling has been shown to result in polycystic kidney disease (PKD) although the exact mechanism has been debated. Previously, we reported that the calcium channel CaV1.2 functions in primary cilia. In this study, we show that CaV1.2 expression level is regulated by Wnt signaling. This occurs through modulation of mitochondrial mass and activity resulting in increased reactive oxygen species which generate oxidative DNA lesions. We found that the subsequent cellular DNA damage response triggers increased CaV1.2 expression. In the absence of primary cilia where Wnt signaling is upregulated, we found that CaV1.2 is overexpressed as a compensatory mechanism. We show for the first time that CaV1.2 knockdown in zebrafish results in classic primary cilia defects including renal cyst formation, hydrocephalus, and left-right asymmetry defects. Our study shows that suppressed Wnt signaling prevents CaV1.2 expression ultimately resulting in PKD phenotypes. Thus, CaV1.2 expression is tightly regulated through Wnt signaling and plays an essential sensory role in primary cilia necessary for cellular homeostasis. J. Cell. Physiol. 9999: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

Description: The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a reactive oxygen species-sensitive multiprotein complex that regulates IL-1β maturation via caspase-1. It also plays an important role in the pathogenesis of inflammation-related disease. Cyclooxygenase-2 (COX-2) is induced by inflammatory stimuli and contributes to the pathogenesis of inflammation-related diseases. However, there is currently little known about the relationship between COX-2 and the NLRP3 inflammasome. Here, we describe a novel role for COX-2 in regulating the activation of the NLRP3 inflammasome. NLRP3 inflammasome-derived IL-1β secretion and pyroptosis in macrophages were reduced by pharmaceutical inhibition or genetic knockdown of COX-2. COX-2 catalyzes the synthesis of prostaglandin E 2 and increases IL-1β secretion. Conversely, pharmaceutical inhibition or genetic knockdown of prostaglandin E 2 receptor 3 reduced IL-1β secretion. The underlying mechanisms for the COX-2-mediated increase in NLRP3 inflammasome activation were determined to be the following: (1) enhancement of lipopolysaccharide-induced proIL-1β and NLRP3 expression by increasing NF-κB activation and (2) enhancement of the caspase-1 activation by increasing damaged mitochondria, mitochondrial reactive oxygen species production and release of mitochondrial DNA into cytosol. Furthermore, inhibition of COX-2 in mice in vivo with celecoxib reduced serum levels of IL-1β and caspase-1 activity in the spleen and liver in response to lipopolysaccharide (LPS) challenge. These findings provide new insights into how COX-2 regulates the activation of the NLRP3 inflammasome and suggest that it may be a new potential therapeutic target in NLRP3 inflammasome-related diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Estrogen receptor (ER) activation has complex effects on bone cells, and loss of circulating estradiol adversely affects skeletal status in women. Hormone replacement therapy effectively circumvents bone loss after menopause, but enhances disease risk in other tissues. Here we show that prostaglandin E2 (PGE2) augments the activity of an osteoblast-derived selective ER modulator, ObSERM. The stimulatory effect of PGE2 is replicated in part by either the PG receptor EP3 agonist 17-phenyl trinor PGE2 or by the PG receptor FP agonist PGF2α. Whereas activation of the various PG receptors induces multiple downstream signals, the response to PGE2 was mimicked by activators of protein kinase C, and suppressed by inhibition of protein kinase C but not by inhibition of protein kinase A. Moreover, inhibition of nitric oxide synthesis and activation of the PTH and Wnt pathways increases ObSERM activity. Our studies therefore reveal that ObSERM activity is controlled in distinct ways and revise our understanding of ER activation within bone by agents or events associated with PG expression. They also predict ways to sustain or improve bone formation, fracture repair, and surgical healing without adding the risk of disease in other tissues where ER activation also has important biological functions. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a critical role in non-homologous end-joining repair of DNA double-strand breaks (DSB) induced by ionizing radiation (IR). Little is known, however, regarding the relationship between DNA-PKcs and IR-induced angiogenesis; thus, in this study we aimed to further elucidate this relationship. Our findings revealed that lack of DNA-PKcs expression or activity sensitized glioma cells to radiation due to the defective DNA DSB repairs and inhibition of phosphorylated Akt Ser473 . Moreover, DNA-PKcs deficiency apparently mitigated IR-induced migration, invasion and tube formation of human microvascular endothelial cell (HMEC-1) in conditioned media derived from irradiated DNA-PKcs mutant M059J glioma cells or M059K glioma cells that have inhibited DNA-PKcs kinase activity due to the specific inhibitor NU7026 or siRNA knockdown. Moreover, IR-elevated vascular endothelial growth factor (VEGF) secretion was abrogated by DNA-PKcs suppression. Supplemental VEGF antibody to irradiated-conditioned media was negated enhanced cell motility with a concomitant decrease in phosphorylation of the FAK Try925 and Src Try416 . Furthermore, DNA-PKcs suppression was markedly abrogated in IR-induced transcription factor hypoxia inducible factor-1α (HIF-1α) accumulation, which is related to activation of VEGF transcription. These findings, taken together, demonstrate that depletion of DNA-PKcs in glioblastoma cells at least partly suppressed IR-inflicted migration, invasion and tube formation of HMEC-1 cells, which may be associated with the reduced HIF-1α level and VEGF secretion. Inhibition of DNA-PKcs may be a promising therapeutic approach to enhance radio-therapeutic efficacy for glioblastoma by hindering its angiogenesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Stem cells have potential in the retrieval and repair of injured tissue and renovation of organ function. To date, several studies have been carried out to elucidate how differentiation of stem cells can be used in regenerative medicine applications. Adipose tissue is an abundant and accessible source of stem cell, useful for regenerative therapeutic use. Adipose stem cells (ASCs) are favorable for future translational research and can be applied in many clinical settings. Adipose tissue repair has been recently adopted in clinical trials to prove that ASCs can be successfully used in patients. Variability in cell culture procedures (isolation, characterization and differentiation) may have an influence on the experimental outcome. In this report, we consider the selection mechanisms of ASCs using flow cytometry, cell culture, freezing/thawing, cell cycle evaluation, histochemistry/immunofluorescence and differentiation of ASCs. Both researchers and regulatory institutions should consider a new policy for GMP procedures and protocols, paying special attention to stem cell bio-physiology, to facilitate more clinically oriented studies. ASCs show angiogenic properties, with prospects of repairing tissue damaged by radiotherapy, as well as possessing the ability to heal chronic wounds. They can also be useful in surgical practice. We focus on the potential clinical application of ASCs that are currently available regarding translational medicine and the methods and procedures for their isolation, differentiation and characterization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Hypoxic non-small cell lung cancer (NSCLC) is dependent on Notch-1 signaling for survival. Targeting Notch-1 by means of γ-secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post-mortem analysis of GSI-treated, NSCLC-burdened mice suggested enhanced phosphorylation of 4E-BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non-canonical 4E-BP1 phosphorylation pattern rearrangement-a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF-4F composition indicating increased recruitment of eIF-4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF-4A assembly into eIF-4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap- and IRES-dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin-1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC-1) inhibition affected 4E-BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC-1. Key phenomena described in this study were reversed by overexpression of the APP C-terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC-1 regulation of cap-dependent protein synthesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site-specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The pregnane X receptor (PXR, NR1I2) is a ligand-activated nuclear receptor which plays an essential role in organism's metabolic detoxification system by sensing the presence of xenobiotics and triggering detoxification responses. In addition to its role in xenobiotic metabolism, PXR has pleiotropic functions in regulating immune/inflammatory responses, cell proliferation, bile acid/cholesterol metabolism, glucose and lipid metabolism, steroid/endocrine homeostasis and bone metabolism. Recent research suggests that the PXR is required for maintaining healthy commensalism between microbiota and gut. Interestingly, the metabolites such as indole derivatives from commensal microbes serve as the ligands for the PXR in intestinal epithelium forming an intricate mutualistic interaction between host and microbiota. PXR-regulated gene responses are controlled at epigenetic level by chromatin modifications, DNA methylation and noncoding RNA. Developmental alterations of the epigenome by exposure to the xenobiotics or diseases may produce persistent changes in PXR-regulated physiological responses. These new areas of research promise to vastly increase our understanding of PXR-regulated responses. In this review we highlight recent results on the epigenetic mechanisms for the PXR-regulated gene expression and discuss the physiological significance of these findings. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Ketoprofen L-lysine salt (KLS), a NSAID, is widely used for its analgesic efficacy and tolerability. L-lysine salification was reported to increase the solubility and the gastric absorption and tolerance of Ketoprofen. Since the management of NSAIDs gastrotoxicity still represents a major limitation in prolonged therapies, mainly when gastric lesions are present, this study investigated the gastro-protective activity of L-lysine by using a well-established model of gastric mucosa injury, the ethanol-gastric injury model. Several evidences show that the damaging action of ethanol could be attributed to the increase of ROS, which plays a key role in the increase of lipid peroxidation products, including malonyldialdehyde and 4-hydroxy-2-nonenal. With the aim to unravel the mechanism of L-lysine gastroprotection, cellular MDA levels and 4-HNE protein adducts as markers of lipid peroxidation and a panel of key endogenous gastro-protective proteins were assayed. The data obtained indicate a gastroprotective effect of L-lysine on gastric mucosa integrity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that defends against oxidative damage due to reactive oxygen species (ROS). A new isoform of MnSOD with cytotoxic activity was recently discovered in liposarcoma cells. Here, we tested the effectiveness of a recombinant form of this isoform (rMnSOD) on leukemic T cells, Jurkat cells, and normal lymphocytes. Our results confirm that leukemic T cells can internalize rMnSOD and that rMnSOD causes apoptosis of 99% of leukemic cells without showing toxic effects on healthy cells. Using light and electron microscopy, we determined that an rMnSOD concentration of 0.067 μM most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 μM rMnSOD resulted in high expression of the pro-apoptotic protein Bax and low expression of the anti-apoptotic protein Bcl-2 in leukemia cells. Concerning signal transduction pathway no influence was observed after treatment except for Jurkat cells showing a slightly decreased expression of ERK phosphorylation. These results suggest that rMnSOD may be an effective and non-toxic treatment option for T-cell leukemia. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: We hypothesized that O 2 tension influences the redox state and the immunomodulatory responses of inflammatory cells to dimethyl fumarate (DMF), an activator of the nuclear factor Nrf2 that controls antioxidant genes expression. This concept was investigated in macrophages permanently cultured at either physiological (5% O 2 ) or atmospheric (20% O 2 ) oxygen levels and then treated with DMF or challenged with lipopolysaccharide (LPS) to induce inflammation. RAW 264.7 macrophages cultured at 20% O 2 exhibited a pro-oxidant phenotype, reflected by a lower content of reduced glutathione, higher oxidized glutathione and increased production of reactive oxygen species when compared to macrophages continuously grown at 5% O 2 . At 20% O 2 , DMF induced a stronger antioxidant response compared to 5% O 2 as evidenced by a higher expression of heme oxygenase-1, NAD(P)H:quinone oxydoreductase-1 and superoxide dismutase-2. After challenge of macrophages with LPS, several pro-inflammatory (iNOS, TNF-α, MMP-2, MMP-9), anti-inflammatory (arginase-1, IL-10) and pro-angiogenic (VEGF-A) mediators were evaluated in the presence or absence of DMF. All markers, with few interesting exceptions, were significantly reduced at 5% O 2 . This study brings new insights on the effects of O 2 in the cellular adaptation to oxidative and inflammatory stimuli and highlights the importance of characterizing the effects of chemicals and drugs at physiologically relevant O 2 tension. Our results demonstrate that the common practice of culturing cells at atmospheric O 2 drives the endogenous cellular environment towards an oxidative stress phenotype, affecting inflammation and the expression of antioxidant pathways by exogenous modulators. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members [ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (B-Raf), MEK]. Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Following the elucidation of the human genome and components of the epigenome, it is timely to revisit what is known of vitamin D receptor (VDR) function. Early transcriptomic studies using microarray approaches focused on the protein coding mRNA that were regulated by the VDR, usually following treatment with ligand. These studies quickly established the approximate size and surprising diversity of the VDR transcriptome, revealing it to be highly heterogenous and cell type and time dependent. Investigators also considered VDR regulation of non-protein coding RNA and again, cell and time dependency was observed. Attempts to integrate mRNA and miRNA regulation patterns are beginning to reveal patterns of co-regulation and interaction that allow for greater control of mRNA expression, and the capacity to govern more complex cellular events. Alternative splicing in the trasncriptome has emerged as a critical process in transcriptional control and there is evidence of the VDR interacting with components of the splicesome. ChIP-Seq approaches have proved to be pivotal to reveal the diversity of the VDR binding choices across cell types and following treatment, and have revealed that the majority of these are non-canonical in nature. The underlying causes driving the diversity of VDR binding choices remain enigmatic. Finally, genetic variation has emerged as important to impact the transcription factor affinity towards genomic binding sites, and recently the impact of this on VDR function has begun to be considered. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Cell culture conditions can vary between laboratories and have been optimised for 2D cell culture. In this study, engineered muscle was cultured in 5.5 mM low glucose (LG) or 25 mM high glucose (HG) and in the absence or presence (+S) of streptomycin and the effect on C2C12 tissue-engineered muscle function and metabolism was determined. Following 2 weeks differentiation, streptomycin (3-fold) and LG (0.5-fold) significantly decreased force generation. LG and/or streptomycin resulted in upward and leftward shifts in the force-frequency curve and slowed time-to-peak tension and half-relaxation time. Despite changes in contractile dynamics, no change in myosin isoform was detected. Instead, changes in troponin isoform, calcium sequestering proteins (CSQ and parvalbumin) and the calcium uptake protein SERCA predicted the changes in contractile dynamics. Culturing in LG and/or streptomycin resulted in increased fatigue resistance despite no change in the mitochondrial enzymes SDH, ATPsynthase and cytochrome C. However, LG resulted in increases in the β-oxidation enzymes LCAD and VLCAD and the fatty acid transporter CPT-1, indicative of a greater capacity for fat oxidation. In contrast, HG resulted in increased GLUT4 content and the glycolytic enzyme PFK, indicative of a more glycolytic phenotype. These data suggest that streptomycin has negative effects on force generation and that glucose can be used to shift engineered muscle phenotype via changes in calcium-handling and metabolic proteins. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. Mechanisms by which PEDF regulates matrix mineralization remain unknown. We examined effect of exogenous PEDF on expression of osteoblastic and osteocytic related genes and proteins in mineralizing osteoblast culture. Mineralizing human osteoblasts supplemented with exogenous PEDF for 14 days deposited 47% more mineral than cells cultured without PEDF. Analysis of selected gene expression by cells in mineralizing cultures supplemented with exogenous PEDF showed reduction in expression of Sclerostin (Sost) by 70%, matrix extracellular phosphoglycoprotein (MEPE) by 75% and dentin matrix protein (DMP-1) by 20% at day 14 of culture. Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) expression was not affected. Western blotting and immunoprecipitation showed that sclerostin and MEPE synthesis by osteocytes were reduced by 50% and 60% respectively in mineralizing osteoblasts containing exogenous PEDF. Primary osteocytes exposed to PEDF also reduced synthesis of Sost/sclerostin by 50% within 24h. For osteoblastic genes, Bone sialoprotein (BSP) was expressed at 75% higher by day 7 in cultures containing exogenous PEDF while Col1A1 expression remained high at all-time points. Total beta-catenin was increased in mineralizing osteoblastic cells suggesting increased Wnt activity. Taken together, the data indicate that PEDF suppressed expression of factors that inhibit mineralization while enhancing those that promote mineralization. The findings also suggest that PEDF may regulate Sost expression by osteocytes leading to enhanced osteoblastic differentiation and increased matrix mineralization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The ability of a cell to undergo malignant transformation is both associated with and dependent on a concomitant increase in protein synthesis due to increased cell division rates and biosynthetic activities. Protein synthesis, in turn, depends upon the synthesis of ribosomes and thus ultimately on the transcription of ribosomal RNA by RNA polymerase I that occurs in the nucleolus. Enlargement of nucleoli has long been considered a hallmark of the malignant cell, but it is only recently that the rate of synthesis of rRNA in the nucleolus has been recognized as both a critical regulator of cellular proliferation and a potential target for therapeutic intervention. As might be expected, the factors regulating rRNA synthesis are both numerous and complex. It is the objective of this review to highlight recent advances in understanding how rRNA synthesis is perturbed in transformed mammalian cells and to consider the impact of these findings on the development of new approaches to the treatment of malignancies. In-depth analysis of the process of rRNA transcription itself may be found in several recently published reviews (Drygin et al., 2010, Annu Rev Pharmacol Toxicol 50:131–156; Bywater et al., 2013,Cancer Cell 22: 51–65; Hein et al., 2013,Trends Mol Med 19:643–654). J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The Notch pathway is a highly conserved cell–cell communication pathway in metazoan involved in numerous processes during embryogenesis, development, and adult organisms. Ligand-receptor interaction of Notch components on adjacent cells facilitates controlled sequential proteolytic cleavage resulting in the nuclear translocation of the intracellular domain of Notch (NICD). There it binds to the Notch effector protein RBP-J, displaces a corepressor complex and enables the induction of target genes by recruitment of coactivators in a cell-context dependent manner. Both, the gene-specific repression and the context dependent activation require an intense communication with the underlying chromatin of the regulatory regions. Since the epigenetic landscape determines the function of the genome, processes like cell fate decision, differentiation, and self-renewal depend on chromatin structure and its remodeling during development. In this review, structural features enabling the Notch pathway to read these epigenetic marks by proteins interacting with RBP-J/Notch will be discussed. Furthermore, mechanisms of the Notch pathway to write and erase chromatin marks like histone acetylation and methylation are depicted as well as ATP-dependent chromatin remodeling during the activation of target genes. An additional fine-tuning of transcriptional regulation upon Notch activation seems to be controlled by the commitment of miRNAs. Since cells within an organism have to react to environmental changes, and developmental and differentiation cues in a proper manner, different signaling pathways have to crosstalk to each other. The chromatin status may represent one major platform to integrate these different pathways including the canonical Notch signaling. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Satellite cells derived from fast and slow muscles have been shown to adopt contractile and metabolic properties of their parent muscle. Mouse muscle shows less distinctive fiber-type profiles than rat or rabbit muscle. Therefore, in this study we sought to determine whether three-dimensional muscle constructs engineered from slow soleus (SOL) and fast tibialis anterior (TA) from mice would adopt the contractile and metabolic properties of their parent muscle. Time-to-peak tension (TPT) and half-relaxation time (1/2RT) was significantly slower in SOL constructs. In agreement with TPT, TA constructs contained significantly higher levels of fast myosin heavy chain (MHC) and fast troponin C, I, and T isoforms. Fast SERCA protein, both slow and fast calsequestrin isoforms and parvalbumin were found at higher levels in TA constructs. SOL constructs were more fatigue resistant and contained higher levels of the mitochondrial proteins SDH and ATP synthase and the fatty acid transporter CPT-1. SOL constructs contained lower levels of the glycolytic enzyme phosphofructokinase but higher levels of the β-oxidation enzymes LCAD and VLCAD suggesting greater fat oxidation. Despite no changes in PGC-1α protein, SOL constructs contained higher levels of SIRT1 and PRC. TA constructs contained higher levels of the slow-fiber program repressor SOX6 and the six transcriptional complex (STC) proteins Eya1and Six4 which may underlie the higher in fast-fiber and lower slow-fiber program proteins. Overall, we have found that muscles engineered from predominantly slow and fast mouse muscle retain contractile and metabolic properties of their native muscle. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above-mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Recently, we discovered that Aflatoxin G 1 (AFG 1 ) induces chronic lung inflammatory responses, which may contribute to lung tumorigenesis in Balb/C mice. The cancer cells originate from alveolar type II cells (AT-II cells). The activated AT-II cells express high levels of MHC-II and COX-2, may exhibit altered phenotypes, and likely inhibit antitumor immunity by triggering regulatory T cells (Tregs). However, the mechanism underlying phenotypic alterations of AT-II cells caused by AFG 1 -induced inflammation remains unknown. In this study, increased MHC-II expression in alveolar epithelium was observed and associated with enhanced Treg infiltration in mouse lung tissues with AFG 1 -induced inflammation. This provides a link between phenotypically altered AT-II cells and Treg activity in the AFG 1 -induced inflammatory microenvironment. AFG 1 -activated AT-II cells underwent phenotypic maturation since AFG 1 upregulated MHC-II expression on A549 cells and primary human AT-II cells in vitro . However, mature AT-II cells may exhibit insufficient antigen presentation, which is necessary to activate effector T cells, due to the absence of CD80 and CD86. Furthermore, we treated A549 cells with AFG 1 and TNF-α together to mimic an AFG 1 -induced inflammatory response in vitro , and we found that TNF-α and AFG 1 coordinately enhanced MHC-II, CD54, COX-2, IL-10, and TGF-β expression levels in A549 cells compared to AFG 1 alone. The phenotypic alterations of A549 cells in response to the combination of TNF-α and AFG 1 were mainly regulated by TNF-α-mediated induction of the NF-κB pathway. Thus, enhanced phenotypic alterations of AT-II cells were induced in response to AFG 1 -induced inflammation. Thus, AT-II cells are likely to suppress anti-tumor immunity by triggering Treg activity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Wg/Wnts are paracrine and autocrine ligands that activate distinct signaling pathways while being internalized through surface receptors. In the field, contrasting views exist on whether, where, and how endocytosis may modulate Wnt signaling. We gather considerable amount of evidences to elaborate the point that signal-receiving cells utilize distinct, flexible, and sophisticated vesicular trafficking mechanisms to keep Wnt signaling activity in check. Same molecules in a highly context-dependent fashion serve as regulatory hub for various signaling purposes: amplification, maintenance, inhibition, and termination. Updates are provided for the regulatory mechanisms related to the three relevant cell surface complexes, Wnt-Fzd-LRP6, Dkk1-Kremen-LRP6, and R-spondin-LGR5-RNF43, which potently influence Wnt signaling. We pay particular attentions to how cells achieve sustained and delicate control of Wnt signaling strength by employing comprehensive aspects of vesicular trafficking. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Aims The dysfunction of endothelial progenitor cells (EPCs) limits their potential for the treatment of ischemia and atherosclerosis. Therefore, we investigated the effect of tripterine on EPC function and examined the underlying mechanisms. Methods and Results The effect of tripterine, an active component of Tripterygium wilfordii Hook, on the enhancement of EPC function and the efficiency of EPC transplantation was investigated in vitro and in vivo. Treatment of EPCs with tripterine at 2.5 µM for 4 h inhibited oxidized low-density lipoprotein (ox-LDL) induced ROS production, cell apoptosis, and cell senescence and improved the migration and tube formation capacities of EPCs treated with ox-LDL (200 µg/ml). In vivo studies showed that tripterine conditioning of EPCs administered to ischemic foci improved blood perfusion and microvascular density in a mouse hindlimb ischemia model. Examination of the underlying mechanisms indicated that the effect of tripterine is mediated by the induction of heat shock protein 32 expression and the inhibition of JNK activation. Conclusion(s) The present results are of clinical significance because they suggest the potential of tripterine as a therapeutic agent to improve the efficacy of EPC transplantation for the treatment of ischemic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor β3 (TGFβ3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (-/-) TGFβ3, interferon regulatory factor 6 (IRF6) (-/-), and truncated p63 (ΔNp63) (-/-) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFβ3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFβ3 (-/-), ΔNp63 (-/-), and wild-type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFβ3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFβ3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFβ3 (-/-) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (-/-) palates because of premature fusion. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: We describe a novel model for investigation of genetically normal human osteoblasts in culture. SK11 is a clonal progenitor cell line derived from human embryonic stem cells. Initially selected based on the expression of chondrogenic markers when differentiated in micromass culture, SK11 cultures display typical mRNA expression patterns of bone phenotypic genes under osteogenic conditions. These include Osterix, α1(I) collagen, Alkaline phosphatase , Osteonectin , Osteopontin and Osteocalcin . Similar to well-characterized murine osteoblast cultures, the osteoblast master regulator RUNX2 was present during the first few days after plating, but the protein disappeared during the first week of culture. Loss of RUNX2 expression is considered an important regulatory feature for osteoblast maturation. Indeed, following ∼2 weeks of differentiation, SK11 cultures exhibited robust calcium deposition, evidenced by alizarin red staining. We also introduced a lentiviral vector encoding doxycycline (dox)-inducible FLAG-tagged RUNX2 into SK11 cells. Dox-mediated enhancement of RUNX2 expression resulted in accelerated mineralization, which was further increased by co-treatment with BMP-2. Like the endogenous RUNX2, expression of the virally coded FLAG-RUNX2 was lost during the first week of culture despite persistent dox treatment. By following RUNX2 decay after dox withdrawal from day-5 versus day-3 cultures, we demonstrated a developmentally regulated decrease in RUNX2 stability. Availability of culture models for molecular investigation of genetically normal human osteoblasts is important because differences between murine and human osteoblasts, demonstrated here for the regulation of Matrix Gla Protein , may have significant biomedical implications. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 hours at 0°C. The cultured keratinocytes from the control, non-shipped skin and the 2-day shipped skin were 43.6 +/- 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta-1. However, under the same shipping conditions, the 3-day shipped tissue failed to establish colonies in the culture. Therefore, this 2-day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long-distance collaborations. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C-terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H-Ras and NRas) localize on the Golgi apparatus whereas K-Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub-organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H-Ras is distributed throughout the Golgi stacks, the singly palmitoylated N-Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants we show that the different sub-Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane but also their distribution within the Golgi stacks. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Stem and progenitor cells play important roles in organogenesis during development and in tissue homeostasis and response to injury postnatally. As the regenerative capacity of many human tissues is limited, cell replacement therapies hold great promise for human disease management. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are prime candidates for the derivation of unlimited quantities of clinically relevant cell types through development of directed differentiation protocols, i.e. the recapitulation of developmental milestones in in vitro cell culture. Tissue-specific progenitors, including progenitors of endodermal origin, are important intermediates in such protocols since they give rise to all mature parenchymal cells. In this review, we focus on the in vivo biology of embryonic endodermal progenitors in terms of key transcription factors and signaling pathways. We critically review the emerging literature aiming to apply this basic knowledge to achieve the efficient and reproducible in vitro derivation of endodermal progenitors such as pancreas, liver and lung precursor cells. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Here we report that both PLCâ1a and PLCâ1b are relevant regulators of erythropoiesis in that kinamycin F, a potent inducer of γ-globin production in K562 cells, caused a selectively reduction of both PLCâ1 isozymes even though the results point out that the effect of the drug is mainly directed toward the expression of the PLCâ1a isoform. We have identified a different role for the two isozymes as regulators of K562 differentiation process induced by kinamycin F. The overexpression of PLCâ1b induced an increase in γ-globin expression even in the absence of kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCâ1 signaling pathway. Namely the amplification of the expression of the PLCâ1a, but not of PLCâ1b, is able to maintain high levels of expression of cyclin D3 even after treatment with kinamycin F. This could be due to their different distribution in the cell compartments since the amount of PLCâ1b is mainly present in the nucleus in respect to PLCâ1a. Our data indicate that the amplification of PLCâ1a expression, following treatment with kinamycin F, confers a real advantage to K562 cells viability and protects cells themselves from apoptosis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Mast cells (MCs) are important effector cells in asthma and pulmonary inflammation, and their proliferation and maturation is maintained by stem cell factor (SCF) via its receptor, c-Kit. Cysteinyl leukotrienes (cys-LTs) are potent inflammatory mediators that signal through CysLT 1 R and CysLT 2 R located on the MC surface, and they enhance MC inflammatory responses. However, it is not known if SCF and cys-LTs cross-talk and influence MC hyperplasia and activation in inflammation. Here, we report the concerted effort of the growth factor SCF and the inflammatory mediator LTD 4 in MC activation. Stimulation of MCs by LTD 4 in the presence of SCF enhances c-Kit-mediated proliferative responses. Similarly, SCF synergistically enhances LTD 4 -induced calcium, c-fos expression and phosphorylation, as well as MIP1β generation in MCs. These findings suggest that integration of SCF and LTD 4 signals may contribute to MC hyperplasia and hyper-reactivity during airway hyper-response and inflammation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Bone marrow (BM) failure, increased risk of myelodysplastic syndrome, acute leukaemia and solid tumors, endocrinopathies and congenital abnormalities are the major clinical problems in Fanconi Anemia patients (FA). Chromosome instability and DNA repair defects are the cellular characteristics used for the clinical diagnosis. However, these biological defects are not sufficient to explain all the clinical phenotype of FA patie nts. The known defects are structural alteration in cell cytoskeleton , altered structural organization for intermediate filaments, nuclear lamina and mitochondria. These are associated with different expression and/or maturation of the structural proteins vimentin, mitofilin and lamin A/C. suggesting the involvement of metalloproteinases (MPs). Matrix metalloproteinases (MMP) are involved in normal physiological processes such as human skeletal tissue development, maturation and hematopoietic reconstitution after bone marrow suppression. Current observations upon the eventual role of MPs in FA cells are largely inconclusive. We evaluated the overall MPs activity in FA complementation group A (FANCA) cells by exposing them to the antioxidants N-acetyl cysteine (NAC) and resveratrol (RV). This work supports the hypothesis that treatment of Fanconi patients with antioxidants may be important in FA therapy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Long suspected, recently recognized, and increasingly studied, non protein-coding RNAs (ncRNAs) are emerging as key drivers of biological control and pathology. Since their discovery in 1993, microRNAs (miRNAs) have been the subject of intense research focus and investigations have revealed striking findings, establishing that these molecules can exert a shocking level of biological control in numerous tissues. More recently, long ncRNAs (lncRNAs), the lesser-studied siblings of miRNA, have been suggested to have a similar robust role in developmental and adult tissue regulation. Mesenchymal stem cells (MSCs) are an important source of multipotent cells for normal and therapeutic tissue repair. Much is known about the critical role of miRNAs in biogenesis and differentiation of MSCs however; recent studies have suggested lncRNAs may play an equally important role in the regulation of these cells. Here we highlight the role of lncRNAs in the regulation of mesenchymal stem cell lineages including adipocytes, chondrocytes, myoblasts, and osteoblasts. In addition, the potential for these noncoding RNAs to be used as biomarkers for disease or therapeutic targets is also discussed. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The anti-mullerian hormone (AMH) is a homodimeric glycoprotein member of the transforming growth factor ƒÀ (TGF-ƒÀ) superfamily, and is secreted by Sertoli cells in the embryonic testes and is responsible of the regression of the mullerian duct. The physiological functions of this protein remain largely unknown, and its expression in human tissues has yet to be completely determined. [objective]. Firstly, we analyzed AMH expression in human tissues by immunohistochemistry. It was found that AMH was distributed in many organs, although with different tissue and cell localization and various expression levels; we also demonstrated strong AMH expression in endometriosis tissues. Secondly, we demonstrated the ability of an anti-AMH antibody, labelled with gadiolinium, to be directly detected by magnetic resonance in small endometriosis lesions (5 mm in diameter) in vivo in a mouse model. In conclusion, our data suggest that based on its expression pattern, AMH may serve to maintain physiological cellular homeostasis in different human tissues and organs. Moreover, it is strongly expressed in endometriosis lesions as a selective tissue specific contrast agent for in vivo detection of stromal endometriosis lesions. The potential significance of these findings could be further validated in a clinical setting. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Galectin-1 (Gal1), a β-galactoside-binding protein abundantly expressed in tumor microenvironments, is associated with the development of metastasis in hepatocellular carcinomas (HCC). However, the precise roles of Gal1 in HCC cell invasiveness and dissemination are uncertain. Here, we investigated whether Gal1 mediate epithelial-mesenchymal transition (EMT) in HCC cells, a key process during cancer progression. We used the well-differentiated and low invasive HepG2 cells and performed ‘gain-of-function’ and ‘loss-function’ experiments by transfecting cells with Gal1 cDNA constructs or by siRNA strategies, respectively. Epithelial and mesenchymal markers expression, changes in apico-basal polarity, independent-anchorage growth and activation of specific signaling pathways were studied using Western blot, fluorescence microscopy, soft-agar assays and FOP/TOP flash reporter system. Gal1 up-regulation in HepG2 cells induced down-regulation of the adherens junction protein E-cadherin and increased expression of the transcription factor Snail, one of the main inducers of EMT in HCC. Enhanced Gal1 expression facilitated the transition from epithelial cell morphology towards a fibroblastoid phenotype and favored up-regulation of the mesenchymal marker vimentin in HCC cells. Cells overexpressing Gal1 showed enhanced anchorage-independent growth and loss of apico-basal polarity. Remarkably, Gal1 promoted Akt activation, β-catenin nuclear translocation, TCF4/LEF1 transcriptional activity and increased cyclin D1 and c-Myc expression, suggesting activation of the Wnt pathway. Furthermore, Gal1 overexpression induced E-cadherin downregulation through a PI3K/Akt-dependent mechanism. Our results provide the first evidence of a role of Gal1 as an inducer of EMT in HCC cells, with critical implications in HCC metastasis. This article is protected by copyright. All rights reserved

Description: Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine–proline or phosphothreonine–proline motifs of numerous proteins. Previously, we reported that Pin1 deficiency resulted in defects in osteoblast differentiation during early bone development. In this study, we found that adult Pin1-deficient mice developed osteoporotic phenotypes compared to age-matched controls. Since BMP2 stored in the bone matrix plays a critical role in adult bone maintenance, we suspected that BMP R-Smads (Smad1 and Smad5) could be critical targets for Pin1 action. Pin1 specifically binds to the phosphorylated linker region of Smad1, which leads to structural modification and stabilization of the Smad1 protein. In this process, Pin1-mediated conformational modification of Smad1 directly suppresses the Smurf1 interaction with Smad1, thereby promoting sustained activation of the Smad1 molecule. Our data demonstrate that post-phosphorylational prolyl isomerization of Smad1 is a converging signal to stabilize the Smad1 molecule against the ubiquitination process mediated by Smurf1. Therefore, Pin1 is a critical molecular switch in the determination of Smad1 fate, opposing the death signal transmitted to the Smad1 linker region by phosphorylation cascades after its nuclear localization and transcriptional activation. Thus, Pin1 could be developed as a major therapeutic target in many skeletal diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Parathyroid hormone-related protein (PTHrP) is known to induce the expression of receptor activator of NF-κB ligand (RANKL) in stromal cells/osteoblasts. However, the signaling pathways involved remain controversial. In the present study, we investigated the role of cAMP/protein kinase A (PKA) and calcineurin/NFAT pathways in PTHrP-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. PTHrP-mediated induction of RANKL expression was significantly inhibited by H89 and FK506, an inhibitor of PKA and calcineurin, respectively. PTHrP upregulated CREB phosphorylation and the transcriptional activity of NFAT. Knockdown of CREB or NFATc1 blocked PTHrP-induced RANKL expression. PTHrP increased the activity of the RANKL promoter reporter that contains approximately 2 kb mouse RANKL promoter DNA sequences. Insertions of mutations in CRE-like element or in NFAT-binding element abrogated PTHrP-induced RANKL promoter activity. Chromatin immunoprecipitation assays showed that PTHrP increased the binding of CREB and NFATc1/NFATc3 to their cognate binding elements in the RANKL promoter. Inhibition of cAMP/PKA and its downstream ERK activity suppressed PTHrP-induced expression and transcriptional activity of NFATc1. CREB knockdown prevented PTHrP induction of NFATc1 expression. Furthermore, NFATc1 and CREB were co-immunoprecipitated. Mutations in CRE-like element completely blocked NFATc1-induced transactivation of the RANKL promoter reporter; however, mutations in NFAT-binding element partially suppressed CREB-induced RANKL promoter activity. Overexpression of CREB increased NFATc1 binding to the RANKL promoter and vice versa. These results suggest that PTHrP-induced RANKL expression depends on the activation of both cAMP/PKA and calcineurin/NFAT pathways, and subsequently, CREB and NFAT cooperate to transactivate the mouse RANKL gene. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Antimetabolite Methotrexate (MTX) is commonly used in childhood oncology. As a dihydrofolate reductase inhibitor it exerts its action through the reduction of cellular folate, thus its intensive use is associated with damage to soft tissues, bone marrow and bone. In the clinic, MTX is administered with folinic acid (FA) supplementation to alleviate some of this soft tissue damage. However, whether and how FA alleviates damage to the bone and bone marrow requires further investigation. As the Wnt/β-catenin signalling pathway is critical for commitment and differentiation of mesenchymal stem cells down the osteogenic or adipogenic lineage, its deregulation has been found associated with increased marrow adiposity following MTX treatment. In order to elucidate whether FA supplementation prevents MTX-induced bone marrow adiposity by regulating Wnt/β-catenin signalling, young rats were given saline or 0.75mg/kg MTX once daily for 5 days, receiving saline or 0.75mg/kg FA 6h after MTX. FA rescue alleviated the MTX-induced bone marrow adiposity, as well as well as inducing up-regulation of Wnt10b mRNA and β-catenin protein expression in the bone. Furthermore, FA blocked up-regulation of the secreted Wnt antagonist sFRP-1 mRNA expression. Moreover, secreted sFRP-1 protein in the bone marrow and its expression by osteoblasts and adipocytes was found increased following MTX treatment. This potentially indicates that sFRP-1 is a major regulator of defective Wnt/β-catenin signalling following MTX treatment. This study provides evidence that folate depletion caused by MTX chemotherapy results in increased bone marrow adiposity, and that FA rescue alleviates these defects by up-regulating Wnt/β-catenin signalling in the bone. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Therapeutic potential of mesenchymal stem cells (MSCs) demands assurance of the quality, safety, and genetic stability. Nuclear blebs (NBs) and Micronuclei (MNs) are considered as biomarkers for cancer and an increase in their numbers is associated with malignancy and other pathologic disorders. However, the status of NBs and MNs in MSCs is not known. Hence we examined the frequency of NBs and MNs in MSCs from umbilical cord (UC-MSCs) and placenta (PD-MSCs) and found a difference in the number of NBs and MNs depending on the source of the MSCs. The number of NBs and MNs was always found to be less in UC-MSCs as compared to PD-MSCs obtained from the same patient. Furthermore, we observed that the number of NBs was inversely proportional to the proliferation rate of the cells. The cryopreservation of these MSCs over 6 months also led to increase in the number of NBs and MNs thus slowing down their rate of proliferation on revival. MSCs from both the sources exhibiting high NBs and MNs showed longer S phase and G2-M arrest and increase in senescent cells without altering their CD-marker profile and differentiation potential. This feature was consistent with the upregulation of cell cycle checkpoint genes (p53, p21, Gadd45, ATM, ATR, chek2, p27, p16, and p10). In conclusion, our data demonstrates for the first time the importance of checking the occurrence of NBs and MNs in MSCs before using them for cellular therapy as a quality control measure to check their genetic stability. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Gene therapy, which involves replacement of a defective gene with a functional, healthy copy of that gene, is a potentially beneficial cancer treatment approach particularly over chemotherapy, which often lacks selectivity and can cause non-specific toxicity. Despite significant progress pre-clinically with respect to both enhanced targeting and expression in a tumor-selective manner several hurdles still prevent success in the clinic, including non-specific expression, low-efficiency delivery and biosafety. Various innovative genetic approaches are under development to reconstruct vectors/transgenes to make them safer and more effective. Utilizing cutting-edge delivery technologies, gene expression can now be targeted in a tissue- and organ-specific manner. With these advances, gene therapy is poised to become amenable for routine cancer therapy with potential to elevate this methodology as a first line therapy for neoplastic diseases. This review discusses recent advances in gene therapy and their impact on a pre-clinical and clinical level. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Glyceollins, a group of phytoalexins isolated from soybean, are known to exhibit anticancer, antiestrogenic, and antiangiogenic activities. However, whether glyceollins regulate tumor growth through regulation of hypoxia-inducible factor (HIF)-1α has not been investigated. We determined whether and how glyceollins regulate the synthesis and stability of HIF-1α. Quantitative real-time PCR revealed that glyceollins inhibited the expression of HIF-1-induced genes such as vascular endothelial growth factor ( VEGF ) in cancer cells. Enzyme-linked immunosorbent assay and reporter luciferase assay showed that glyceollins decreased VEGF secretion and its promoter activity, respectively. Treatment of various cancer cells with 0.5-100 µM glyceollins under hypoxic conditions reduced the expression of HIF-1α. Glyceollins blocked translation of HIF-1α by inhibiting the PI3K/AKT/mTOR pathway under hypoxic conditions. Glyceollins decreased the stability of HIF-1α after treatment with cycloheximide, a protein synthesis inhibitor, and increased the ubiquitination of HIF-1α after treatment with MG132, a proteasome inhibitor. Glyceollins blocked the interaction of Hsp90 with HIF-1α, as shown by immunoprecipitation assay. Chemical binding of Hsp90 with glyceollins, as confirmed by computational docking analysis, was stronger than that with geldanamycin at the HSP90 ATP-binding pocket. We found that glyceollins decreased microvessel density, as well as expression of phosphorylated AKT/mTOR and the Hsp90 client protein CDK4, in solid tumor tissues. Glyceollins potently inhibited HIF-1α synthesis and decreased its stability by blocking the PI3K/AKT/mTOR pathway and HSP90 binding activity, respectively. These results may provide new perspectives into potential therapeutic application of glyceollins for the prevention and treatment of hypervascularized diseases and into the mechanism of their anticancer activity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: pRb2/p130 phosphorylation occurs at G0/G1 transition, when cells exit from quiescence. This phenomenon leads to E2F4 and E2F5 transcription factors releasing, allowing them to activate their target genes. Hence, pRb2/p130 represents one of the most important cell cycle controller because of transcription factors binding/releasing capability. It was previously shown that the loss of expression, or function, of pRb2/p130 is involved in different neoplasms development, due to related loss of control of E2F4 and E2F5 transcription activity. Here we analyzed gastric cancer tissue samples of diffuse histotype, comparing them with the normal counterpart. Yet, we did not find any change of expression levels. Rather, we found a cytoplasmic localization of pRb2/p130 in cancer tissue samples whereas, in normal counterparts, we found the expected nuclear localization. This occurrence lead to hypothesize an almost completely uncontrolled transcriptional activity of E2F4 and E2F5 in this kind of neoplasm, actually contributing to maintenance of transformed status. Although in limited number of cases, our data support the necessity of further investigations to verify the possibility of using pRb2/p130 as diagnostic marker of diffuse gastric cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D 3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D 3 metabolite, calcitriol (1,25-dihydroxyvitamin D 3 ; 1,25(OH) 2 D 3 ), modulates responses to inflammation; however this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro-inflammatory cytokines. Cells were pre-treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP-9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ-induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: GATA-1 low/low mice have an increase in megakaryocytes (MKs) and trabecular bone. The latter is thought to result from MKs directly stimulating osteoblastic bone formation while simultaneously inhibiting osteoclastogenesis. Osteoprotegerin (OPG) is known to inhibit osteoclastogenesis and OPG -/- mice have reduced trabecular and cortical bone due to increased osteoclastogenesis. Interestingly, GATA-1 low/low mice have increased OPG levels. Here we sought to determine whether GATA-1 knockdown in OPG -/- mice could rescue the observed osteoporotic bone phenotype. GATA-1 low/low mice were bred with OPG -/- mice and bone phenotype assessed. GATA-1 low/low X OPG -/- mice have increased cortical bone porosity, similar to OPG -/- mice. Both OPG -/- and GATA-1 low/low X OPG -/- mice, were found to have increased osteoclasts localized to cortical bone, possibly producing the observed elevated porosity. Biomechanical assessment indicates that OPG -/- and GATA-1 low/low X OPG -/- femurs are weaker and less stiff than C57BL/6 or GATA-1 low/low femurs. Notably, GATA-1 low/low X OPG -/- mice had trabecular bone parameters that were not different from C57BL/6 values, suggesting that GATA-1 deficiency can partially rescue the trabecular bone loss observed with OPG deficiency. The fact that GATA-1 deficiency appears to be able to partially rescue the trabecular, but not the cortical bone phenotype suggests that MKs can locally enhance trabecular bone volume, but that MK secreted factors cannot access cortical bone sufficiently to inhibit osteoclastogenesis or that OPG itself is required to inhibit osteoclastogenesis in cortical bone. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Malignant pleural effusions (MPEs) are a common manifestation found in patients with lung cancer. After cytological and histological confirmation of malignancy, talc pleurodesis still remains the treatment of choice in patients with MPEs resistant to chemotherapy. Despite this, primary challenges include reduced quality of life and life expectancy in general. Therefore, a better understanding of the cell biology of MPEs, along with improvements in treatment is greatly needed. It has recently been demonstrated that malignant pleural effusions (MPEs) may represent an excellent source for identification of molecular mechanisms within the tumor and its environment. The present review summarizes the current understanding of MPEs cells and tumor microenvironment, and particularly focuses on dissecting the cross-talk between MPEs and epithelial to mesenchymal transition (EMT), inflammation and cancer stem cells. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large-scale gene expression profile was carried out on HPV16-CIN2, HPV16-CIN3 and normal cervical keratinocytes derived from two HPV16-CIN2, two HPV-CIN3 lesions and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16-CIN2 keratinocytes and in HPV16-CIN2 keratinocytes compared with HPV16-CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The Coiled-Coil Domain Containing 80 ( CCDC80 ) gene has been identified as strongly induced in rat thyroid PC CL3 cells immortalized by the adenoviral E1A gene. In human, CCDC80 is a potential oncosoppressor due to its down-regulation in several tumor cell lines and tissues and it is expressed in almost all tissues. CCDC80 has homologous in mouse, chicken and zebrafish. We cloned the zebrafish ccdc80 and analyzed its expression and function during embryonic development. The in silico translated zebrafish protein shares high similarity with its mammalian homologous, with nuclear localization signals and a signal peptide. Gene expression analysis demonstrates that zebrafish ccdc80 is maternally and zygotically expressed throughout the development. In particular, ccdc80 is strongly expressed in the notochord and it is under the regulation of the Hedgehog pathway. In this work we investigated the functional effects of ccdc80 -loss-of-function during embryonic development and verified its interaction with gadd45β2 in somitogenesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT Acinus-S' is a co-repressor for retinoic acid receptor (RAR)-dependent gene transcription and has been suggested to be involved in RNA processing. In this study the role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S', with the activity of Acinus-L higher than that of Acinus-S', increase the splicing of a retinoic acid (RA)-responsive minigene containing a weak 5' splice site but not a RA-responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing of the weak 5' splice site by Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus occurs to varying degrees using minigene constructs containing several different promoters while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by a RA response element (RARE)-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The RRM domain is necessary for the RA-dependent splicing activity of Acinus and the RA-independent splicing activity of Acinus is repressed by RNPS1. Importantly, measurement of the splicing of endogenous human RARβ and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA-dependent manner for RARβ and a RA-independent manner for Bcl-x. Taken together, these studies demonstrate that Acinus functions in both RAR-dependent splicing and RAR-dependent transcription. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate the stability or translation of cognate mRNAs at the posttranscriptional level. Accumulating evidence indicates that miRNAs play important roles in many aspects of muscle function, including muscle growth and development, regeneration, contractility, and muscle fiber type plasticity. In the current study, we examined the function of miR-151-3p in myoblast proliferation and differentiation. Results show that overexpression of miR-151-3p not only upregulates myoblast proliferation, but also decreases slow muscle gene expression (such as MHC-β/slow and slow muscle troponin I) in both C2C12 myotubes and in primary cultures. Alternatively, inhibition of miR-151-3p by antisense RNA was found to upregulate MHC-β/slow expression, indicating that miR-151-3p plays a role in muscle fiber type determination. Further investigation into the underlying mechanisms revealed for the first time that miR-151-3p directly targets ATP2a2, a gene encoding for a slow skeletal and cardiac muscle specific Ca 2+ ATPase, SERCA2 thus downregulating slow muscle gene expression. Mechanisms by which the alteration in SERCA2 expression induces changes in other slow muscle gene expression levels needs to be defined in future research. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: ABSTRACT The E3 ubiquitin ligase and tumor suppressor SCF Fbw7 exists as three isoforms that govern the degradation of a host of critical cell regulators, including c-Myc, cyclin E, and PGC-1α. Peroxisome proliferator activated receptor-gamma coactivator 1α (PGC-1α) is a transcriptional coactivator with broad effects on cellular energy metabolism. Cellular PGC-1α levels are tightly controlled in a dynamic state by the balance of synthesis and rapid degradation via the ubiquitin-proteasome system. Yet, isoform-specific functions of SCF Fbw7 are yet to be determined. Here, we show that the E3 ubiquitin ligase, SCF Fbw7 , regulates cellular PGC-1α levels via two independent, isoform specific, mechanisms. The cytoplasmic isoform (SCF Fbw7β ) reduces cellular PGC-1α levels via accelerated ubiquitin-proteasome degradation. In contrast, the nuclear isoform (SCF Fbw7α ) increases cellular PGC-1α levels and protein stability via inhibition of ubiquitin-proteasomal degradation. When nuclear Fbw7α proteins are redirected to the cytoplasm, cellular PGC-1α protein levels are reduced through accelerated ubiquitin-proteasomal degradation. We find that SCF Fbw7β catalyzes high molecular weight PGC-1α-ubiquitin conjugation, whereas SCF Fbw7α produces low molecular weight PGC-1α-ubiquitin conjugates that are not effective degradation signals. Thus, selective ubiquitination by specific Fbw7 isoforms represents a novel mechanism that tightly regulates cellular PGC-1α levels. Fbw7 isoforms mediate degradation of a host of regulatory proteins. The E3 ubiquitin ligase, Fbw7, mediates PGC-1α levels via selective isoform-specific ubiquitination. Fbw7β reduces cellular PGC-1α via ubiquitin-mediated degradation, whereas Fbw7α increases cellular PGC-1α via ubiquitin-mediated stabilization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The purposes of this study were to investigate the potential roles of miR-34c in regulating blood-tumor barrier (BTB) functions and its possible molecular mechanisms. The over-expression of miR-34c significantly impaired the integrity and increased the permeability of BTB, which were detected in an in vitro BTB model by transendothelial electric resistance and horseradish peroxidase flux assays respectively. Meanwhile, real-time quantitative PCR (qRT-PCR), Western blot and immunofluorescence assays successively demonstrated down-regulation of ZO-1, occludin and claudin-5. And miR-34c silencing uncovered the opposite results. Dual-luciferase reporter assays results revealed myc-associated zinc-finger protein (MAZ) is a target gene of miR-34c. Besides, mRNA and protein expressions of MAZ were reversely regulated by miR-34c. The down-expression of MAZ significantly impaired the integrity and increased the permeability of BTB as well as down-regulated the expressions of ZO-1, occludin and claudin-5. And chromatin immunoprecipitation verified that MAZ interacted with “GGGCGGG”, “CCCTCCC” and “GGGAGGG” DNA sequence of ZO-1 , occludin and claudin-5 promoter respectively. The over-expression or silencing of either miR-34c or MAZ was performed simultaneously to further explore their functional relations, and results elucidated that miR-34c and MAZ displayed reverse regulatory effects on the integrity and permeability of BTB as well as the expressions of ZO-1, occludin and claudin-5. In conclusion, our present study indicated that miR-34c regulated the permeability of BTB via MAZ-mediated expression changes of ZO-1, occludin and claudin-5. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: CRM197 is a naturally nontoxic diphtheria toxin mutant that binds and inhibits heparin-binding epidermal growth factor-like growth factor. CRM197 serves as carrier protein for vaccine and other therapeutic agents. CRM197 also inhibits the growth, migration, invasion and induces apoptosis in various tumors. Vascular cell adhesion molecule-1 (VCAM-1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we aimed to investigate the role and mechanism of CRM197 combined with shRNA interference of VCAM-1 (shRNA-VCAM-1) on the migration, invasion and apoptosis of glioblastoma cells. U87 and U251 human glioblastoma cells were treated with CRM197 (10 μg/ml) and shRNA interfering technology was employed to silence VCAM-1 expression. Cell viability, migration, invasiveness and apoptosis were assessed with CCK8, Transwell and Annexin V-PE/7-AAD staining. Activation of cleaved caspase-3, 8 and 9, activity of matrix metalloproteinase-2/9 (MMP-2/9) and expression of phosphorylated Akt (p-Akt) were also checked. Results showed that CRM197 and shRNA-VCAM-1 not only significantly inhibited the cell proliferation, migration, invasion, but also promoted the apoptosis of U87 and U251 cells. Combined treatment of both displayed enhanced inhibitory effects on the malignant biological behavior of glioma cells. The activation of cleaved caspase-3, 8, 9 was promoted, activity of MMP-2 and MMP-9 and expression of p-Akt were inhibited significantly by the treatment of CRM197 and shRNA-VCAM-1 alone or in combination, indicating that the combination of CRM197 with shRNA-VCAM-1 additively inhibited the malignant behavior of human glioblastoma cells via activating caspase-3, 8, 9 as well as inhibiting MMP-2, MMP-9 and Akt pathway. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.

Description: The CC chemokine receptor 6 (CCR6) and its ligand CCL20 are involved in human colorectal cancer (CRC) carcinogenesis and can promote the progression of CRC. In addition, interleukin-17 (IL-17), produced by a T cell subset named “Th17,” has been identified as an important player in inflammatory responses, and has emerged as a mediator in inflammation-associated cancer. However, the relevance of IL-17 in the development and progression of CRC still remains to be explored. This study aimed to investigate the effect of IL-17 on the cell migration of CRC cells. Human CRC HCT-116 cells were used to study the effect of IL-17 on CCR6 expression and cell migration in CRC cells. IL-17 treatment induced migration of HCT-116 cells across the Boyden chamber membrane and increased the expression level of the CCR6. Inhibition of CCR6 by small interfering RNA (siRNA) and neutralizing antibody inhibited IL-17-induced cell migration. By using specific inhibitors and short hairpin RNA (shRNA), we demonstrated that the activation of ERK and p38 pathways are critical for IL-17-induced CCR6 expression and cell migration. Promoter activity and transcription factor ELISA assays showed that IL-17 increased NF-κB-DNA binding activity in HCT-116 cells. Inhibition of NF-κB activation by specific inhibitors and siRNA blocked the IL-17-induced CCR6 expression. Our findings support the hypothesis that CCR6 up-regulation stimulated by IL-17 may play an active role in CRC cell migration. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.