ALBERT

Description: Bone remodeling is a natural process that enables growth and maintenance of the skeleton. It involves the deposition of mineralized matrix by osteoblasts and resorption by osteoclasts. Several cancers that metastasize to bone negatively perturb the remodeling process through a series of interactions with osteoclasts, and osteoblasts. These interactions have been described as the “vicious cycle” of cancer metastasis in bone. Due to the inaccessibility of the skeletal tissue it is difficult to study this system in vivo . In contrast, standard tissue culture lacks sufficient complexity. We have developed a specialized three-dimensional culture system that permits growth of a non-vascularized, multiple-cell-layer of mineralized osteoblastic tissue from pre-osteoblasts. In this study, the essential properties of bone remodeling were created in vitro by co-culturing the mineralized collagenous osteoblastic tissue with actively resorbing osteoclasts followed by reinfusion with proliferating pre-osteoblasts. Cell-cell and cell-matrix interactions were determined by confocal microscopy as well as by assays for cell specific cytokines and growth factors. Osteoclasts, differentiated in the presence of osteoblasts, led to degradation of the collagen-rich extracellular matrix. Further addition of metastatic breast cancer cells to the co-culture mimicked the vicious cycle; i.e. there was a further reduction in osteoblastic tissue thickness, an increase in osteoclastogenesis, chemotaxis of cancer cells to osteoclasts and formation of cancer cells into large colonies. The resulting model system permits detailed study of fundamental osteobiological and osteopathological processes in a manner that will enhance development of therapeutic interventions to skeletal diseases. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Cervical carcinoma represents the paradigm of virus-induced cancers, where virtually all cervical cancers come from previous “high-risk” HPV infection. The persistent expression of the HPV viral oncoproteins E6 and E7 is responsible for the reprogramming of fundamental cellular functions in the host cell, thus generating a noticeable, yet only partially explored, imbalance in protein molecular networks and cell signaling pathways. Eighty-eight cellular factors, identified as HPV direct or surrogate targets, were chosen and monitored in a retrospective analysis for their mRNA expression in HPV-induced cervical lesions, from dysplasia to cancer. Real-time quantitative PCR (qPCR) was performed by using formalin-fixed, paraffin embedded archival samples. Gene expression analysis identified 40 genes significantly modulated in LSIL, HSIL and squamous cervical carcinoma. Interestingly, among these, the expression level of a panel of four genes, TOP2A, CTNNB1, PFKM and GSN, was able to distinguish between normal tissues and cervical carcinomas. Immunohistochemistry was also done to assess protein expression of two genes among those up-regulated during the transition between dysplasia and carcinoma, namely E2F1 and CDC25A, and their correlation with clinical parameters. Besides the possibility of significantly enhancing the use of some of these factors in diagnostic or prognostic procedures, these data clearly outline specific pathways, and thus key biological processes, altered in cervical dysplasia and carcinoma. Deeper insight on how these molecular mechanisms work may help widen the spectrum of novel innovative approaches to these virus-induced cell pathologies. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Cellular pathways are numerous and are highly integrated in function in the control of cellular systems. They collectively regulate cell division, proliferation, survival and apoptosis of cells and mutagenesis of key genes that control these pathways can initiate neoplastic transformations. Understanding these pathways is crucial to future therapeutic and preventive strategies of the disease. Ovarian cancers are of three major types; epithelial, germ-cell and stromal. However, ovarian cancers of epithelial origin, arising from the mesothelium, are the predominant form. Of the subtypes of ovarian cancer, the high-grade serous tumors are fatal, with low survival rate due to late detection and poor response to treatments. Close examination of preserved ovarian tissues and in vitro studies have provided insights into the mechanistic changes occurring in cells mediated by a few key genes. This review will focus on pathways and key genes of the pathways that are mutated or have aberrant functions in the pathology of ovarian cancer. Non-genetic mechanisms that are gaining prominence in the pathology of ovarian cancer, miRNAs and epigenetics, will also be discussed in the review. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Normal pregnancy is associated with systemic vasodilation and decreased vascular contraction, partly due to increased release of endothelium-derived vasodilator substances. Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor acting via endothelin receptor type A (ET A R) and possibly type B (ET B R) in vascular smooth muscle cells (VSMCs), with additional vasodilator effects via endothelial ET B R. However, the role of ET-1 receptor subtypes in the regulation of vascular function during pregnancy is unclear. We investigated whether the decreased vascular contraction during pregnancy reflects changes in the expression/activity of ET A R and ET B R. Contraction was measured in single aortic VSMCs isolated from virgin, mid-pregnant (mid-Preg, day 12) and late-Preg (day 19) Sprague-Dawley rats, and the mRNA expression, protein amount, tissue and cellular distribution of ET A R and ET B R were examined using RT-PCR, Western blots, immunohistochemistry and immunofluorescence. Phenylephrine (Phe, 10 −5  M), KCl (51 mM) and ET-1 (10 −6  M) caused VSMC contraction that was in late-Preg 〈 mid-Preg and virgin rats. In VSMCs treated with ET B R antagonist BQ788, ET-1 caused significant contraction that was still in late-Preg 〈 mid-Preg and virgin rats. In VSMCs treated with the ET A R antagonist BQ123, ET-1 caused a small contraction; and the ET B R agonists IRL-1620 and sarafotoxin 6c (S6c) caused similar contraction that was in late-Preg 〈 mid-Preg and virgin rats. RT-PCR revealed similar ET A R, but greater ET B R mRNA expression in pregnant vs. virgin rats. Western blots revealed similar ET A R, and greater protein amount of ET B R in endothelium-intact vessels, but reduced ET B R in endothelium-denuded vessels of pregnant vs. virgin rats. Immunohistochemistry revealed prominent ET B R staining in the intima, but reduced ET A R and ET B R in the aortic media of pregnant rats. Immunofluorescence signal for ET A R and ET B R was less in VSMCs of pregnant vs. virgin rats. The pregnancy-associated decrease in ET A R- and ET B R-mediated VSMC contraction appears to involve downregulation of ET A R and ET B R expression/activity in VSM, and may play a role in the adaptive vasodilation during pregnancy. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Neuroregenerative medicine is an ever-growing field in which regeneration of lost cells/tissues due to a neurodegenerative disease is the ultimate goal. With the scarcity of available replacement alternatives, stem cells provide an attractive source for regenerating neural tissue. While many stem cell sources exist, including: mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), the limited cellular potency, technical difficulties, and ethical considerations associated with these make finding alternate sources a desirable goal. Periodontal ligament stem cells (PDLSCs) derived from the neural crest were induced into neural-like cells using a combination of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Morphological changes were evident in our treated group, seen under both light microscopy and scanning electron microscopy (SEM). A statistically significant increase in the expression of neuron-specific β-tubulin III and the neural stem/progenitor cell marker nestin, along with positive immunohistochemical staining for glial fibrillary acidic protein (GFAP), demonstrated the success of our treatment in inducing both neuronal and glial phenotypes. Positive staining for synaptophysin demonstrated neural connections and electrophysiological recordings indicated that when subjected to whole cell patch clamping, our treated cells displayed inward currents conducted through voltage-gated sodium (Na + ) channels. Taken together, our results indicate the success of our treatment in inducing PDLSCs to neural-like cells. The ease of sourcing and expansion, their embryologic neural crest origin, and the lack of ethical implications in their use make PDLSCs an attractive source for use in neuroregenerative medicine. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Urotensin II (UII), a vasoactive peptide modulates renal hemodynamics. However, the physiological functions of UII in glomerular cells are unclear. In particular, whether UII alters mesangial tone remains largely unknown. The present study investigates the physiological effects of UII on intracellular Ca 2+ ([Ca 2+ ] i ) and contraction in glomerular mesangial cells (GMCs). This study also tested the hypothesis that the regulator of G-protein signaling (RGS) controls UII receptor (UTR) activity in GMCs. RT-PCR, Western immunoblotting, and immunofluorescence revealed UTR expression and localization in cultured murine GMCs. Mouse UII (mUII) stimulated [Ca 2+ ] i elevation in GMCs in the absence and presence of extracellular Ca 2+ . mUII also caused a reduction in planar GMC surface area. mUII-induced [Ca 2+ ] i elevation and contraction in GMCs were attenuated by SB 657510, a UTR antagonist, araguspongin B, an inositol 1,4,5-trisphosphate receptor antagonist, thapsigargin, a sarco/endoplasmic reticulum Ca 2+ -ATPase inhibitor, and La 3+ , a store-operated Ca 2+ channel blocker, but not nimodipine, an L-type Ca 2+ channel blocker. In situ proximity ligation assay indicated molecular proximity between endogenous RGS2 and UTR in the cells. Treatment of GMCs with mUII increased plasma membrane association of RGS2 by ∼ 2-fold. mUII also increased the interaction between RGS2 and UTR in the cells. siRNA-mediated knockdown of RGS2 in murine GMCs increased mUII-induced [Ca 2+ ] i elevation and contraction by ∼ 35 and 31%, respectively. These findings indicate that mUII induces [Ca 2+ ] i elevation and contraction in murine GMCs. Data also suggest that UTR activation stimulates RGS2 recruitment to GMC plasma membrane as a negative feedback mechanism to regulate UTR signaling. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Poly-N-acetyllactosamine (PLN) is a unique glycan composed of repeating units of the common disaccharide (Galβ1,4-GlcNAcβ1,3) n . The expression of PLN on glycoprotein core structures minimally requires enzyme activities for β1,4-galactosyltransferase (β4GalT) and β1,3-N-acetylglucosminyltransferase (β3GnT). Because β4GalTs are ubiquitous in most cells, PLN expression is generally ascribed to the tissue-specific transcription of 8 known β3GnT genes in mice. In the olfactory epithelium (OE), β3GnT2 regulates expression of extended PLN chains that are essential for axon guidance and neuronal survival. N-glycan branching and core composition, however, can also modulate the extent of PLN modification. Here we show for the first time that the β1,6-branching glycosyltransferase GCNT2 (formerly known as IGnT) is expressed at high levels specifically in the OE and other sensory ganglia. Postnatally, GCNT2 is maintained in mature olfactory neurons that coexpress β3GnT2 and PLN. This highly specific coexpression suggests that GCNT2 and β3GnT2 function cooperatively in PLN synthesis. In support of this, β3GnT2 and GCNT2 cotransfection in HEK293T cells results in high levels of PLN expression on the cell surface and on adenylyl cyclase 3, a major carrier of PLN glycans in the OE. These data clearly suggest that GCNT2 functions in vivo together with β3GnT2 to determine PLN levels in olfactory neurons by regulating β1,6-branches that promote PLN extension. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Spermatogenesis is a special process by which spermatogonial stem cells (SSCs) divide and differentiate to male gametes called mature spermatozoa. SSCs are the unique cells because they are adult stem cells that transmit genetic information to subsequent generations. Accumulating evidence has demonstrated that SSCs can be reprogrammed to acquire pluripotency to become embryonic stem-like cells that differentiate into all cell lineages of the three germ layers, highlighting potential important applications of SSCs for regenerative medicine. Recent studies from peers and us have made great achievements on the characterization, isolation and culture of mouse and human SSCs, which could lead to better understanding the biology of SSCs and the applications of SSCs in both reproductive and regenerative medicine. In this review, we first compared the cell identity and biochemical phenotypes between mouse SSCs and human SSCs. Notably, the cell types of mouse and human SSCs are distinct, and human SSCs share some but not all phenotypes with mouse SSCs. The approaches for isolating SSCs as well as short- and long- term culture of mouse SSCs and short-period culture of human SSCs were also discussed. We further addressed the new advances on the self-renewal of SSCs with an aim to establish the long-term culture of human SSCs which has not yet been achieved. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Angiogenin (ANG) undergoes nuclear translocation and promotes ribosomal RNA (rRNA) transcription thereby enhancing cell growth and proliferation. However, the mode of action of ANG in stimulating rRNA transcription is unclear. Here, we show that ANG enhances the formation of RNA polymerase I (Pol I) pre-initiation complex at the ribosomal DNA (rDNA) promoter. ANG binds at the upstream control element (UCE) of the promoter and enhances promoter occupancy of RNA Pol I as well as the selectivity factor SL1 components TAF I 48 and TAF I 110. We also show that ANG increases the number of actively transcribing rDNA by epigenetic activation through promoter methylation and histone modification. ANG binds to histone H3, inhibits H3K9 methylation, and activates H3K4 methylation as well as H4 acetylation at the rDNA promoter. These data suggest that one of the mechanisms by which ANG stimulates rRNA transcription is through an epigenetic activation of rDNA promoter. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Constitutive androstane receptor (CAR) was originally identified as xenobiotic sensor that regulates the expression of cytochrome P450 genes. However, recent studies suggest that this nuclear receptor is also involved in the regulation of energy metabolism including glucose and lipid homeostasis. This study investigated the role of CAR in the regulation of bone mass in vivo using CAR -/- mice. Endogenous mRNA expression of CAR was observed in both primary osteoblasts and osteoclast precursors. CAR -/- mice have exhibited significant increase in whole body bone mineral density (BMD) by 9.5% ( p  〈 0.01) and 5.5% (p 〈 0.05) at 10 and 15 weeks of age, respectively, compared with WT mice in males. Microcomputed tomography analysis of proximal tibia demonstrated a significant increase in trabecular bone volume (62.7%), trabecular number (54.1%) in male CAR -/- mice compared with WT mice. However, primary culture of calvarial cells exhibited no significant changes in osteogenic differentiation potential between CAR -/- and WT. In addition, the number of tartrate-resistant acid-phosphatase positive osteoclasts in the femur and serum level of CTx was not different between CAR -/- and WT mice. The higher BMD and microstructural parameters were not observed in female mice. Interestingly, serum level of testosterone in male CAR -/- mice was 2.5-fold higher compared with WT mice and the mRNA expressions of Cyp2b9 and 2b10 in the liver, which regulate testosterone metabolism, were significantly down-regulated in male CAR -/- mice. Furthermore, the difference in BMD between CAR -/- and WT mice disappeared at 8 weeks after performing orchiectomy. CAR -/- mice also exhibited significant increase in serum1,25(OH) 2 D 3 levels but Cyp 27B1 which converts 25(OH)D 3 to 1,25(OH) 2 D 3 was significantly down-regulated compared to WT mice. These results suggest that in vivo deletion of CAR resulted in higher bone mass, which appears to be a result from reduced metabolism of testosterone due to down-regulation of Cyp2b. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Testosterone exerts important effects in the heart. Cardiomyocytes are target cells for androgens, and testosterone induces rapid effects via Ca 2+ release and protein kinase activation and long-term effects via cardiomyocyte differentiation and hypertrophy. Furthermore, it stimulates metabolic effects such as increasing glucose uptake in different tissues. Cardiomyocytes preferentially consume fatty acids for ATP production, but under particular circumstances, glucose uptake is increased to optimize energy production. We studied the effects of testosterone on glucose uptake in cardiomyocytes. We found that testosterone increased uptake of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose and [ 3 H]2-deoxyglucose, which was blocked by the glucose transporter 4 (GLUT4) inhibitor indinavir. Testosterone stimulation in the presence of cyproterone or albumin-bound testosterone induced glucose uptake, which suggests an effect that is independent of the intracellular androgen receptor. To determine the degree of GLUT4 cell surface exposure, cardiomyocytes were transfected with the plasmid GLUT4 myc -eGFP. Subsequently, testosterone increased GLUT4 myc- GFP exposure at the plasma membrane. Inhibition of Akt by the Akt-inhibitor-VIII had no effect. However, inhibition of Ca 2+ /calmodulin protein kinase (CaMKII) (KN-93 and autocamtide-2 related inhibitory peptide II) and AMP-activated protein kinase (AMPK) (compound C and siRNA for AMPK) prevented glucose uptake induced by testosterone. Moreover, GLUT4 myc- eGFP exposure at the cell surface caused by testosterone was also abolished after CaMKII and AMPK inhibition. These results suggest that testosterone increases GLUT4-dependent glucose uptake, which is mediated by CaMKII and AMPK in cultured cardiomyocytes. Glucose uptake could represent a mechanism by which testosterone increases energy production and protein synthesis in cardiomyocytes. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Although the role of Cbl-family proteins as key cellular regulators has been established, phenomena regulated in a Cbl-dependent fashion are multiple and the mechanisms mediating the effects of Cbl proteins are diverse. This finding makes it important to consider different phenomena affected by functions of Cbl proteins individually. Among effects of Cbl on various biological functions there are many cases of regulation of cellular phenomena related to cytoskeletal rearrangements, such as cell adhesion, motility and invasion. Some of these regulatory functions are mediated by adaptor-type interactions of Cbl, especially by Cbl-dependent modulation of phosphatidyl-inositol-3' kinase (PI3K), while others are caused by Cbl-dependent ubiquitylation of various cytoskeletal and regulatory proteins, identified and unidentified. The role of Cbl in regulation of cytoskeleton-dependent cellular functions is discussed in this review. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Widespread changes in gene expression underlie B cell development and activation, yet our knowledge of which chromatin-remodeling factors are essential is limited. Here, we demonstrate that the BRG1 catalytic subunit of SWI/SNF complexes was dispensable for murine B cell development but played an important, albeit selective, role during activation. Although BRG1 was dispensable for CD69 induction and differentiation into plasma cells based on the ability of mutant B cells to undergo hypertrophy and secrete IgM antibodies, it was required for robust cell proliferation in response to activation. Accordingly, BRG1 was required for only ∼100 genes to be expressed at normal levels in naïve B cells but 〉1,000 genes during their activation. BRG1 upregulated 5-fold more genes than it downregulated, and the toll-like receptor pathway and JAK/STAT cytokine-signaling pathways were particularly dependent on BRG1. The importance of BRG1 in B cell activation was underscored by the occurrence of opportunistic Pasteurella infections in conditionally mutant mice. B cell activation has long served as a model of inducible gene expression, and the results presented here identify BRG1 as a chromatin-remodeling factor that upregulates the transcriptome of B cells during their activation to promote rapid cell proliferation and to mount an effective immune response. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Optical tweezers were used to scan individual Chronic Myelogenous Leukemia cells to determine if the cell death depends on the scanning conditions. Although increasing the scanning frequency or amplitude means greater force applied to the cells, their effects on cell death are not a simple increasing trend, as observed in the optical microscopy. Indeed, cell death sharply increased at particular screening frequencies and amplitudes, whereas other frequencies or amplitudes were less detrimental. These results suggest that cell damage was more sensitive to certain scanning conditions, rather than simply high applied forces. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of odontoblasts. We have recently found that Krüppel-like factor 4 ( Klf4 ) was expressed in the polarizing and elongating odontoblasts, but the function of Klf4 in the differentiation of odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of odontoblasts by up-regulating some odontoblast-related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4 significantly up-regulated odontoblast-related genes, such as Dmp1 , Dspp , and Alp , and promoted the accumulation of mineral nodules. Knock-down of Klf4 down-regulated expression of Dmp1 , Dspp , and Alp , and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4 — Dmp1 . Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of odontoblasts via the up-regulation of Dmp1 . © 2013 Wiley Periodicals, Inc.

Description: Nociception is the sensory mechanism used to detect cues that can harm an organism. The understanding of the neural networks and molecular controls of the reception of pain remains an ongoing challenge for biologists. While we have made significant progress in identifying a number of molecules and pathways that are involved in transduction of noxious stimuli, from the skin through the sensory receptor cell and from this to the spinal cord on into the central nervous system, we still lack a clear understanding of the perceptual processes, the responses to pain and the regulation of pain perception. Mice and rat animal models have been extensively used for nociception studies. However, the study of pain and noiception in these organisms can be rather laborious, costly and time consuming. Conversely the use of Drosophila and C. elegans may be affected by the large evolutionary distance between these animals and humans. We outline here the reasons why zebrafish presents a new and attractive model for studying pain reception and responses and the most interesting findings in the study of nociception that have been obtained using the zebrafish model. © 2013 Wiley Periodicals, Inc.

Description: Transforming growth factor (TGF)-β is a multifunctional cytokine acting during development, tissue homeostasis, regeneration processes and disease progression. Due to its pleiotropic effects, tight regulation of the induced signaling cascades is mandatory. Caveolin proteins regulate a specific endocytic pathway and modulate diverse signaling pathways and thus have been related to severe disorders, e.g. cancer and fibrosis. Caveolin affects TGF-β/-Smad and non-Smad signaling in many ways and thus can determine the cellular outcome upon TGF-β challenge. Reciprocal regulation of caveolin and TGF-β is also evident, ranging from gene expression to miRNA regulation. Finally, there is in vivo evidence that this crosstalk influences disease development and progression. This review gives an overview about the multifaceted relations of caveolin and TGF-β. © 2013 Wiley Periodicals, Inc.

Description: Transforming growth factor (TGF)-β1 regulates diverse cellular functions. Particularly, TGF-β1 induces monocyte migration to sites of injury or inflammation in early period, whereas TGF-β1 inhibits cell migration in late phase. In this study, we attempted to understand how TGF-β1 suppresses cell migration in late phase. We found that TGF-β1 of short exposure induces the production of chemokines, such as macrophage inflammatory protein (MIP)-1α, by Raw 264.7 cells. However, knock-down of small GTPase RhoA by sh-RhoA inhibited the production of MIP-1α and macrophage migration, suggesting that RhoA is essential for expression of this chemokine. An activator of Epac ( e xchange p roteins directly a ctivated by c AMP; a guanine nucleotide exchange factor of Rap1), 8CPT-2Me-cAMP which leads to Rap1 activation abrogated MIP-1α expression and macrophage migration. Indeed, GTP-RhoA and GTP-Rap1 levels were reciprocally regulated in a time-dependent manner following TGF-β1 stimulation. 8CPT-2Me-cAMP suppressed GTP-RhoA levels, whereas si-Rap1 augmented GTP-RhoA levels and cell migration. TGF-β1 produced cAMP in late period and si-RNAs of Epac1 (exchange protein directly activated by cAMP 1) and Epac2 reduced GTP-Rap1 levels leading to promotion of GTP-RhoA levels. Furthermore, si-RNA of ARAP3 (Rap-dependent RhoGAP) increased GTP-RhoA level and cell migration. Therefore, we propose the mechanism that prolonged TGF-β1 treatment produce cAMP, which activates sequentially Epac, Rap1 and ARAP3, resulting in suppression of RhoA, chemokine expression, and macrophage migration. Contrary to the general concept that Rap1 stimulates cell migration, we demonstrated in this study that Rap1 inhibits cell migration by suppression of RhoA activity in response to TGF-β1. © 2013 Wiley Periodicals, Inc.

Description: Polycyclic Aromatic Hydrocarbons (PAHs) are the products of incomplete combustion of organic materials, which are present in cigarette smoke, deep-fried food, and in natural crude oil. Since PAH-metabolites form DNA adducts and cause oxidative DNA damage, we asked if these environmental carcinogens could affect transforming potential of the human Polyomavirus JC oncoprotein, T-antigen (JCV T-antigen). We extracted DMSO soluble PAHs from Deepwater Horizon oil spill in the Gulf of Mexico (oil-PAHs), and detected several carcinogenic PAHs. The oil-PAHs were tested in exponentially growing cultures on normal mouse fibroblasts (R508), and in R508 stably expressing JCV T-antigen (R508/T). The oil-PAHs were cytotoxic only at relatively high doses (1:50–1:100 dilution), and at 1:500 dilution the growth and cell survival rates were practically unaffected. This non-toxic dose triggered however, a significant accumulation of reactive oxygen species (ROS), caused oxidative DNA damage and the formation of DNA double strand breaks (DSBs). Although oil-PAHs induced similar levels of DNA damage in R508 and R508/T cells, only T-antigen expressing cells demonstrated inhibition of high fidelity DNA repair by homologous recombination (HRR). In contrast, low-fidelity repair by non-homologous end joining (NHEJ) was unaffected. This potential mutagenic shift between DNA repair mechanisms was accompanied by a significant increase in clonal growth of R508/T cells chronically exposed to low doses of the oil-PAHs. Our results indicate for the first time carcinogenic synergy in which oil-PAHs trigger oxidative DNA damage and JCV T-antigen compromises DNA repair fidelity. © 2013 Wiley Periodicals, Inc.

Description: The intracellular pH is regulated by a delicate balance of ion distribution across the plasma membrane and the physico-chemical properties of intra- and extracellular components. We analysed the effects of glycosaminoglycans on the intracellular pH of fibroblasts by using the fluorescent pH indicator BCECF-AM. Addition of hyaluronan, hyaluronan oligosaccharides, chondroitin sulfate or heparin to the culture medium of fibroblasts caused intracellular acidification from pH 7.2 to pH 6.7 in a concentration dependent manner. High molecular weight hyaluronan acidified more than hyaluronan oligosaccharides at the same concentrations. Hyaluronidase treatment or inhibition of hyaluronan export with xanthohumol led to intracellular alkalization. These observations indicated that extracellular glycosaminoglycans participate in intracellular pH regulation. The mechanism was explained by Donnan effects and molecular crowding. © 2013 Wiley Periodicals, Inc.

Description: Purpose To test whether the use of a striatum weighted image may improve registration accuracy and diagnostic outcome in patients with parkinsonian syndromes (PS). Methods Weighted images were generated by increasing signal intensity of striatal voxels and used as intermediate dataset for co-registering the brain image onto template. Experimental validation was performed using an anthropomorphic striatal phantom. 123 I-FP-CIT SPECT binding ratios were manually determined in 67 PS subjects an and compared to those obtained using unsupervised standard (UWR) and weighted registered (WR) approach. Normalized cost function was used to evaluate the accuracy of phantom and subjects registered images to the template. Reproducibility between unsupervised and manual ratios was assessed by using intra-class correlation coefficient (ICC) and Bland and Altman analysis. Correlation coefficient was used to assess the dependence of semi-quantitative ratios on clinical findings. Results Weighted method improves accuracy of brain registration onto template as determined by cost function in phantom (0.86 ± 0.06vs.0.98 ± 0.02; Student's t test, P = 0.04) and in subject scans (0.69 ± 0.06vs.0.53 ± 0.06; Student's t test, P 〈 0.0001). Agreement between manual and unsupervised derived binding ratios as measured by ICC was significantly higher on WR as compared to UWR images (0.91vs.0.76). Motor UPDRS score was significantly correlated with manual and unsupervised derived binding potential. In phantom as well as in subjects studies, correlations were more significant using the WR method (BPm: R 2  = 0.36,p = 0.0001; BPwr: R 2  = 0.368, P  = 0.0001; BPuwr: R 2  = 0.300, P  = 0.0008). Conclusion Weighted registration improves accuracy of binding potential estimates and may be a promising approach to enhance the diagnostic outcome of SPECT imaging, correlation with disease severity, and for monitoring disease progression in parkinsonian syndromes. © 2013 Wiley Periodicals, Inc.

Description: G-protein coupled receptors (GPCRs) are a large family of proteins that coordinate extracellular signals to produce physiologic outcomes. Adenosine receptors (AR) are one class of GPCRs that have been shown to regulate functions as diverse as inflammation, blood flow, and cellular differentiation. Adenosine signals through four GPCRs that either inhibit (A1AR and A3AR) or activate (A2aAR and A2bAR) adenylyl cyclase. This review will focus on the role of GPCRs, and in particular, adenosine receptors, in adipogenesis. Preadipocytes differentiate to mature adipocytes as the adipose tissue expands to compensate for the consumption of excess nutrients. These newly generated adipocytes contribute to maintaining metabolic homeostasis. Understanding the key drivers of this differentiation process can aid the development of therapeutics to combat the growing obesity epidemic and associated metabolic consequences. Although much literature has covered the transcriptional events that culminate in the formation of an adipocyte, less focus has been on receptor-mediated extracellular signals that direct this process. This review will highlight GPCRs and their downstream messengers as significant players controlling adipocyte differentiation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: The Toll and Toll-Like Receptor signaling pathways are evolutionarily conserved pathways that regulate innate immunity in insects and mammals. While efforts have been made to clarify the signal transduction events that occur during infection, much less is known about the components that maintain immune quiescence. Here we show that retromer, an intracellular protein complex known for regulating vesicle trafficking, functions in modulating the Toll pathway in Drosophila melanogaster . In mutant animals lacking retromer function, the Toll pathway but not JAK-STAT or IMD pathway is activated, triggering both cellular and humoral responses. Genetic epistasis and clonal analysis suggest that retromer regulates a component that acts upstream of Toll. Our data further show that in the mutant the Toll ligand Spätzle has a processing pattern similar to that of after infection. Together, the results suggest a novel function of retromer in regulating Toll pathway and innate immunity at a step that modulates ligand processing or activity. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

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: Beta cell apoptosis is a hallmark of diabetes. Since we have previously shown that galectin-3 deficient (LGALS3 –/– ) mice are relatively resistant to diabetes induction, the aim of this study was to examine whether beta cell apoptosis depends on the presence of galectin-3 and to delineate the underlying mechanism. Deficiency of galectin-3, either hereditary or induced through application of chemical inhibitors, β-lactose or TD139, supported survival and function of islet beta cells compromised by TNF-α+IFN-γ+IL-1β stimulus. Similarly, inhibition of galectin-3 by β-lactose or TD139 reduced cytokine-triggered apoptosis of beta cells, leading to conclusion that endogenous galectin-3 propagates beta apoptosis in the presence of an inflammatory milieu . Exploring apoptosis-related molecules expression in primary islet cells before and after treatment with cytokines we found that galectin-3 ablation affected the expression of major components of mitochondrial apoptotic pathway, such as BAX, caspase-9, Apaf, SMAC, caspase-3, and AIF. In contrast, anti-apoptotic molecules Bcl-2 and Bcl-XL were up-regulated in LGALS3 –/– islet cells when compared to wild type (WT) counterparts (C57BL/6), resulting in increased ratio of anti-apoptotic versus pro-apoptotic molecules. However, Fas-triggered apoptotic pathway as well as extracellular signal-regulated kinase 1/2 (ERK1/2) was not influenced by LGALS-3 deletion. All together, these results point to an important role of endogenous galectin-3 in beta cell apoptosis in the inflammatory milieu that occurs during diabetes pathogenesis and implicates impairment of mitochondrial apoptotic pathway as a key event in protection from beta cell apoptosis in the absence of galectin-3. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Pin1 is a unique enzyme that can isomerize specific phospho-Ser/Thr-Pro peptide bonds, inducing a conformational change in the target protein. Such activity represents a novel and tightly controlled signaling mechanism regulating a spectrum of protein functions during the normal physiology of the cell and in pathological conditions. Our last study demonstrated that Pin1 interacts with the androgen receptor protein and that this interaction is important for its transcriptional activity. Here, we consider the activity that Pin1 plays on the N-terminal domain of different nuclear receptors and provide an interpretation of this phenomenon. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Mechanical stress is an important signal to determine the levels of bone mass. Unloading-induced osteoporosis is a critical issue in bed- ridden patients and astronauts. Many molecules have been suggested to be involved in sensing mechanical stress in bone, though the mechanisms involved in this phenomenon are not fully understood. Nck1is an adaptor protein known to mediate signaling from plasma membrane-activated receptors to cytosolic effectors regulating actin cytoskeleton remodeling. Nck1 has also been implicated in cellular responses to endoplasmic reticulum stress. In vitro, in case of cell stress the actin cytoskeleton is disrupted and in such cases Nck1 has been reported to enter the nucleus of the cells to mediate the nuclear actin polymerization. However, the role of Nck1 in vivo during the bone response to mechanical stimuli is unknown. The purpose of this study is to examine the role of Nck1 in unloading- induced bone loss in vivo. Sciatic and femoral nerve resection was conducted. Neurectomy- based unloading enhanced Nck1 gene expression in bone about two fold. Using the Nck1 deficient mice and control Nck1+/+, effects of neurectomy-based unloading on bone structure were examined. Unloading reduced bone volume in wild type mice by 30% whereas the levels in bone loss were exacerbated to 50% in Nck1 deficient mice due to neurectomy after four weeks. These data demonstrate that Nck1 gene deficiency accelerates the mechanical unloading-induced bone loss suggesting Nck1 to be a crucial molecule in mechanical stress mediated regulation in bone metabolism. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: During mitosis, histone deacetylase 2 (HDAC2) becomes highly phosphorylated through the action of CK2, and HDAC1 and 2 are displaced from mitotic chromosomes. HDAC1 and 2 are components of corepressor complexes, which function with lysine acetyltransferases to catalyze dynamic protein acetylation and regulate gene expression. In this study, we show that HDAC1 and 2 associate with F-actin in mitotic cells. Inhibition of Aurora B or protein kinase CK2 did not prevent the displacement of HDAC1 and 2 from mitotic chromosomes in HeLa cells. Further, proteins of the HDAC1 and 2 corepressor complexes and transcription factors recruiting these corepressors to chromatin were dissociated from mitotic chromosomes independent of Aurora B activity. HDAC1 and 2 returned to the nuclei of daughter cells during lamin A/C reassembly and before Sp1, Sp3 and RNA polymerase II. Our results show that HDAC1 and 2 corepressor complexes are removed from the mitotic chromosomes and are available early in the events leading to the re-establishment of the gene expression program in daughter cells. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Recent studies have suggested that changes in serum phosphate levels influence pathological states associated with aging such as cancer, bone metabolism, and cardiovascular function, even in individuals with normal renal function. The causes are only beginning to be elucidated but are likely a combination of endocrine, paracrine, autocrine, and cell autonomous effects. We have used an integrated quantitative biology approach, combining transcriptomics and proteomics to define a multi-phase, extracellular phosphate-induced, signaling network in pre-osteoblasts as well as primary human and mouse mesenchymal stromal cells. We identified a rapid mitogenic response stimulated by elevated phosphate that results in the induction of immediate early genes including c-fos . The mechanism of activation requires FGF receptor signaling followed by stimulation of N-ras and activation of AP-1 and serum response elements. A distinct long-term response also requires FGF receptor signaling and results in N-ras activation and expression of genes and secretion of proteins involved in matrix regulation, calcification, and angiogenesis. The late response is synergistically enhanced by addition of FGF23 peptide. The intermediate phase results in increased oxidative phosphorylation and ATP production and is necessary for the late response providing a functional link between the phases. Collectively, the results define elevated phosphate, as a mitogen and define specific mechanisms by which phosphate stimulates proliferation and matrix regulation. Our approach provides a comprehensive understanding of the cellular response to elevated extracellular phosphate, functionally connecting temporally coordinated signaling, transcriptional, and metabolic events with changes in long-term cell behavior. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Osteocalcin (OC) is a non-collagenous, Vitamin K-dependent protein secreted in the late stage of osteoblasts differentiation. The presence of the three residues of γ-carbossiglutamatic acid, specific of the active form of OC protein, allows the protein to bind calcium and consequently hydroxyapatite. The osteoblastic OC protein is encoded by the bone γ-carbossiglutamate gene whose transcription is principally regulated by the Runx2/Cbfa1 regulatory element and stimulated by Vitamin D 3 through a steroid-responsive enhancer sequence. Even if data obtained in literature are controversial, the dual role of OC in bone can be presumed as follows: firstly, OC acts as a regulator of bone mineralization; secondly, OC regulates osteoblast and osteoclast activity. Recently the metabolic activity of OC, restricted to the un-carboxylated form has been demonstrated in osteoblast-specific knockout mice. This effect is mediated by the regulation of pancreatic β-cell proliferation and insulin secretion and adiponectin production by adipose tissue and leads to the regulation of glucose metabolism and fat mass. Nevertheless clinical human studies only demonstrated the correlation between OC levels and factors related to energy metabolism. Thus further investigations in humans are required to demonstrate the role of OC in the regulation of human energy metabolism. Moreover it is presumable that OC also acts on blood vessels by inducing angiogenesis and pathological mineralization. This review highlights the recent studies concerning skeletal and extra-skeletal effects of OC. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Invasive ductal adenocarcinoma (IDA) of the pancreas manifests poor prognosis due to the early invasion and distant metastasis. In contrast, intraductal papillary mucinous adenoma or carcinoma (IPMA or IPMC) reveals better clinical outcomes. Various molecular mechanisms contribute to these differences but entire picture is still unclear. Recent researches emphasized the important role of miRNA in biological processes including cancer invasion and metastasis. We previously described that miR-126 is down-regulated in IDA compared with IPMA or IPMC, and miR-126 regulates the expression of invasion related molecule disintegrin and metalloproteinase domain-containing protein 9 (ADAM9). Assessing the difference of miRNA expression profiles of IDA, IPMA and IPMC, we newly identified miR-197 as an up-regulated miRNA specifically in IDA. Expression of miR-197 in pancreatic cancer cells resulted in the induction of epithelial-mesenchymal transition (EMT) along with the down-regulation of p120 catenin which is a putative target of miR-197. Direct interaction between miR-197 and p120 catenin mRNA sequence was confirmed by 3'UTR assay, and knockdown of p120 catenin recapitulated EMT induction in pancreatic cancer cells. In situ hybridization of miR-197 and immunohistochemistry of p120 catenin showed mutually exclusive patterns suggesting pivotal role of miR-197 in the regulation of p120 catenin. This miR-197 / p120 catenin axis could be a novel therapeutic target. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Endometriosis is a common benign pathology, characterised by the presence of endometrial tissue outside the endometrial cavity with a prevalence of 10–15% in reproductive-aged women. The pathogenesis is not completely understood, and several theories have been proposed to explain the etiology. Our group has recently described the presence of ectopic endometrium in a consistent number of human female foetuses analysed by autopsy, reinforcing the hypothesis that endometriosis may be generated by defects during the organogenesis of the female reproductive trait. Herein, in order to identify, at molecular level, changes involved in the disease, we compared the transcriptional profiling of ectopic endometrium with the corresponding eutopic one. Statistical analyses lead us to identify some genes specifically deregulated in the ectopic endometrium, that are involved in gonad developmental process or in wound healing process. Among them, we identified BMP4 and GREM1. BMP4 was never associated before to endometriosis and is involved in the mesoderm-Müllerian duct differentiation. GREM1 is needed for the initial step of the ureter growth and perhaps could possibly be involved in Müller ducts differentiation. These molecules might be related to the endometriosis etiology since we showed that their expression is not related to the menstrual cycle phase both at RNA and at protein levels. These data support the theory that embryological defects could be responsible of the endometriosis generation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Cancerous stroma coevolves alongside tumour progression, thereby promoting the malignant conversion of epithelial carcinoma cells. To date, an abundance of data have supported crucial roles of the tumour microenvironment (TME) in providing cancer cells with proliferative, migratory, survival and invasive propensities favouring the processes of tumourigenesis. The cancerous reactive stroma is frequently populated by a large number of myofibroblasts (MFs), which are activated, non-transformed fibroblasts expressing α-smooth muscle actin (α-SMA). MFs together with non-MF cells present in the tumour-associated stroma are collectively referred to as carcinoma-associated fibroblasts (CAFs), one of the major stromal cell types recognised in various human carcinomas. Recruitment of fibroblasts and/or their progenitors to a tumour mass and their subsequent transdifferentiation into MFs, as well as ongoing maintenance of their activated state, are believed to be essential processes facilitating tumour progression. However, the complex networks of signalling pathways mediating the phenotypic conversion into CAFs, as well as those underlying their tumour-promoting interactions with other tumour-constituting cells, have yet to be fully explored. Histopathological confirmation of the presence of large numbers of CAF MFs within TME and their altered gene expression profiles are known to be associated with disease progression and to serve as independent negative prognostic factors for a wide range of tumour types. In this review, we examine the current evidence shedding light on the emerging roles of tumour-promoting CAFs, cells that are pivotal for epithelial cancer development and progression, and discuss the therapeutic potential of targeting these cells. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Although the expression of p27 has been regarded as a prognostic parameter in human liver cancer since the implication of decreased p27 expression levels in the genesis and progression of hepatocellular carcinoma (HCC), the molecular mechanism linking p27 deficiency and HCC development is still unclear. Here, we report an increase in tumorigenesis and progression as well as an enhanced inflammatory response in p27 deficient mice (p27 -/- ) and hypothesize the possible mechanism. We show that p27 -/- mice display increased proliferation and decreased apoptosis of tumor cells, accompanied by an increase in the serum inflammatory cytokines IL-6 and TNF-α. Furthermore, our data indicated that the increased number and signal transducers and activator of transcription 3 (STAT3) phosphorylation status of infiltrated inflammatory cells was accompanied by increased IL-6 and TNF-α mRNA levels in tumor and normal liver tissue in the p27 -/- mice. Moreover, using tumor cell and splenocytes co-culture and tumor homologous transplantation, we validated our hypothesis in vitro and in vivo. Collectively, these data demonstrate that the loss of p27 promotes carcinogens-induced HCC genesis and progression via the elevation of inflammatory cytokines and the augmented activation of STAT3 signaling in tumor cells and infiltrated inflammatory cells. Altogether, the loss of the cyclin kinase inhibitor p27, traditionally regarded as a consequence of DNA damage, can in turn promote HCC progression through enhancing the inflammatory response, potentially representing a promising therapeutic target in the prevention of HCC genesis and progression. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 minutes, returning to baseline at 60 min. No increase in ATP was observed in tissue incubated without stretch, or tissue stretched in the presence of the Rho kinase inhibitor Y27632. The increase in fibroblast cross sectional area in response to tissue stretch was blocked by both suramin (a purinergic receptor blocker) and apyrase (an enzyme that selectively degrades extracellular ATP). Furthermore, connexin channel blockers (octanol and carbenoxolone), but not VRAC (fluoxetine) or pannexin (probenecid) channel blockers, inhibited fibroblast expansion. Together, these results support a mechanism in which extracellular ATP signaling via connexin hemichannels mediated the active change in fibroblast shape that occurs in response to a static increase in tissue length. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: The communication between the tumour cells and the surrounding cells helps drive the process of tumour progression. Since the microenvironment of breast cancer includes CCL20 chemokine, the purpose of this study was to determine whether CCL20 modulates the physiology of healthy breast epithelial cells in areas adjacent to the tumour. Therefore, primary cultures of mammary cells taken from normal peritumoral areas were used. We assessed that breast cells expressed CCR6 CCL20 receptor. Using molecular (siRNA) and pharmacological (inhibitors) techniques we found multiple signalling kinases to be activated by CCR6 and involved in CCL20-induced breast cell proliferation and migration. The binding of 10 ng/ml CCL20 to CCR6 induced cell migration whilst higher concentrations (from 15 to 25 ng/ml) led to cell proliferation. CCL20 controlled cell migration and MMP-9 expression by PKC-alpha that activated Src, which caused the activation of downstream Akt, JNK and NF-kB pathways. Furthermore, higher CCL20 concentrations increased cycE and decreased p27Kip expression ending in enhanced cell proliferation. Cell proliferation occurred through PKC-epsilon activation that transactivated EGFR and ERK1/2/MAPK pathway. Although activated by different CCL20 concentrations, these pathways function in parallel and crosstalk to some extent, inasmuch as Akt activation was responsible for ERK1/2 nuclear translocation and enhanced the transcription of of c-fos and c-myc, involved in cell proliferation. In summary, tumour cells exchange signals with the surrounding healthy cells modifying the extracellular matrix through enzyme secretion; thus, CCL20 might be a factor involved in the ontogeny of breast carcinoma. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: We report here that C6orf89, which encodes a protein that interacts with bombesin receptor subtype-3 and accelerates cell cycle progression and wound repair in human bronchial epithelial cells (Liu et al. PLoS One 2011), encodes one soluble and two type II membrane proteins that function as histone deacetylases (HDAC) enhancers. Soluble 34/64sp is selectively targeted to the nucleolus and is retained in nucleolar organiser regions (NORs) in mitotic cells. Nucleolar 34/64sp is integrated into the ribosomal gene transcription machinery, colocalises and coimmunoprecipitates with the Pol I transcription factor UBF, and undergoes a dramatic relocalisation to the nucleolus upon the arrest of rDNA transcription, protein synthesis and PI3K/mTORC2 signalling. Membrane 42/116mp localises to the Golgi and the midbody, and its controlled ectopic expression provokes the disruption of the Golgi cisternae and hinders the separation of daughter cells and the completion of mitosis. The latter effect is also produced by the microinjection of an affinity-purified amfion antibody. The identification of C60rf89 as a gene that encodes three distinct proteins with the capacity to enhance the activity of histone deacetylases (HDACs) in the nucleolus, the Golgi and the midbody provides new information regarding the components of the acetylome and their capacity to interact with different functional groups in the cell. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress-induced autophagy-associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over-expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi-based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1-binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: The rodent heart contains a population of nestin (+) cells derived from the embryonic neural crest and migrate to the scar after myocardial infarction. The present study tested the hypothesis that intron 2 of the nestin gene drives expression and a subpopulation of nestin (+) cells participate in reparative vascularisation. The directed expression of the green fluorescent protein (GFP) by the second intron of the nestin gene identified GFP/nestin (+) cells intercalated among ventricular myocytes in the heart of normal transgenic mice. Ischemic injury led to the migration of GFP (+) cells to the scar and a subpopulation was detected in CD31/nestin (+) endothelial cells of newly formed blood vessels. The direct contribution to reparative vascularisation provided the impetus to test the hypothesis that increasing the population of nestin (+) cells in the infarcted heart will improve scar healing. Skin-derived cells isolated from E18 Sprague-Dawley rats grew as spheres, expressed nestin, sox2, neural crest-related transcriptional genes and a panel of peptide growth factors. Skin-derived cells transplanted in the non-infarcted left ventricle of 3-day post-MI rats migrated to the peri-infarct/infarct region and remained engrafted for 21 days. A significantly smaller infarct, increased number of small calibre blood vessels and improved ventricular function were observed in engrafted infarcted rat hearts. Thus, the second intron of the nestin gene drives expression in the mouse heart and a subpopulation of GFP/nestin (+) cells directly participate in reparative vascularisation. Increasing the population of nestin (+) cells via the transplantation of skin-derived cells represents a potential approach to limit ischemic damage to the heart. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: MicroRNAs (miRNAs) can efficiently regulate gene expression by targeting mRNA to cause mRNA cleavage or translational repression. Growing evidence indicates that miRNAs exist not only in cells but also in a variety of body fluids, which stimulates substantial interest in the transport mechanism and regulating process of extracellular miRNAs. This article reviews the basic biogenesis of miRNAs in detail to explore the origin of extracellular miRNAs. Different miRNA transporters have been summarized (e.g. exosomes, microvesicles, apoptosis bodies and RNA-binding proteins). In addition, we discuss the regulators affecting miRNA transport (e.g. ATP and ceramide) and the selection mechanism for different miRNA transporters. Studies about miRNA transporters and the transport mechanism are new and developing. With the progress of the research, new functions of extracellular miRNAs may be uncovered in the future. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.

Description: We enthusiastically read the recent manuscript by Intapad et al. from the Catravas group in the August 227 issue of Journal of Cellular Physiology entitled “ Regulation of Asthmatic Airway Relaxation by Estrogen and Heat Shock Protein 90 ” [1]. The authors show impaired relaxation of murine tracheal rings sensitized with serum from human asthmatics to nitric oxide (NO) donors, and potentiation of such relaxation upon treatment with estradiol (E 2 ), or the ERa- or ERb-selective ligands PPT and DPN, respectively [1]. They conclude that estrogens can potentiate NO-mediated bronchodilation in normal and asthmatic airways. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Many squamous cell carcinomas (SCCs) are characterized by high levels of EGFR and by overexpression of the ΔNp63α isoform. Here, we investigated the regulation of ΔNp63α expression upon EGFR activation and the role of the EGFR-ΔNp63α axis in proliferation of SCC tumor-initiating cells (TICs). SCC cell lines A-431, Cal-27 and SCC-25 treated with EGF showed a time-dependent increase in ΔNp63α expression at the protein and mRNA levels, which was blocked by the tyrosine kinase inhibitor (TKI) Lapatinib. RNA interference experiments suggested the role of STAT3 in regulating ΔNp63α expression downstream of EGFR. Inactivation of EGFR by the monoclonal antibody Cetuximab and RNA interference against STAT3 or ΔNp63α impaired the TICs ability to grow under non-differentiating conditions. Radiation treatment, which triggers EGFR activation, induced ΔNp63α accumulation without affecting TICs proliferation, whereas the combination Cetuximab plus radiation significantly reduced TICs growth under non-differentiating conditions. Together, our findings provide evidence that ΔNp63α expression is regulated by EGFR activation through STAT3 and that the EGFR-ΔNp63α axis is crucial for proliferation of TICs present in SCCs. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The archetype driving the drug targeting approach to cancer therapy is the success of imatinib against chronic phase chronic myeloid leukemia (CML-CP). Molecular targeting success of this magnitude has yet to be repeated for most solid tumors. To answer why imatinib remains an exception of cancer research, we summarize key features and patterns of evolution that contrast CML-CP from prostate cancer, an example of a solid tumor that also shares a signature fusion gene. Distinctive properties of CML-CP include: a large cell population size that is not geographically constrained, a highly penetrant dominant oncogene that sweeps the entire cell population, subsequent progressive and ordered clonal genetic changes, and the effectiveness of molecular targeting within the chronic phase, which is comparable to the benign phase of solid tumors. CML-CP progression resembles a clonal, stepwise model of evolution, whereas the pattern of solid tumor evolution is highly dynamic and stochastic. The distinguishing features and evolutionary pattern of CML-CP support why the success of imatinib does not carry over to most solid tumors. Changing the focus of cancer research from a gene-based view to a genome-based concept will provide insight into solid tumor evolutionary dynamics. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Epithelial plasticity characterizes embryonic development and diseases such as cancer. Epithelial-mesenchymal transition (EMT) is a reversible and guided process of plasticity whereby embryonic or adult epithelia acquire mesenchymal properties. Multiple signaling pathways control EMT, and the transforming growth factor β (TGFβ) pathway plays a central role as its inducer. Here, we analyzed the role of the tumor suppressor protein p53 in TGFβ-induced EMT in a well-established mammary epithelial cell model. We found that diploid NMuMG mammary cells express bi-allelically a wild type and a missense mutant (R277C) form of p53. Global reduction of both forms of p53 led to an enhanced EMT response to TGFβ. Conversely, stabilization of wild type p53 using the compound nutlin had a negative impact on EMT. After silencing both p53 forms, rescue experiments using either wild type or R277C mutant p53 revealed that wild type p53 inhibited, whereas the R277C mutant did not significantly affect, the TGFβ-driven EMT response. Under serum-free culture conditions, silencing of total p53 levels led to higher numbers of mammospheres characterized by larger size. Rescue of the silenced endogenous p53 with R277C mutant p53, in contrast, suppressed both size and numbers of the mammospheres. This work proposes that wild type p53 controls the efficiency by which mammary epithelial cells undergo EMT in response to TGFβ. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: We have previously determined that integrin α11β1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of α11 -/- mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed α11 -/- mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in α11-deficient iPDL cells, including matrix metalloproteinase-13 (MMP-13) and cathepsin K. α11 -/- iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP-13 protein expression levels. The MMP-13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the α11 integrin-mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by α11β1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of α11β1-dependent matrix remodeling, involving MMP-13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Fat accumulation in obese individuals worsens the clinical outcomes of cardiovascular disease (CVD). Paradoxically, increased circulating adipocytokines secreted from visceral fat may confer cardioprotective effects. Visfatin, a novel adipocytokine, has anti-diabetic, anti-tumor, and pro-inflammatory properties. However, its effects on cardiomyocytes and the underlying mechanisms remain unknown. This paper demonstrated that visfatin counteracted H 2 O 2 -induced apoptotic damage in H9c2 cardiomyocytes in a time-dependent manner. Qualitative immunofluorescence approaches demonstrated that visfatin pretreatment attenuated H 2 O 2 -induced DNA fragmentation (TdT-mediated dUTP-biotin nick end-labeling), phosphatidyl serine exposure (Annexin V/PI staining), and mitochondrial membrane potential (ΔΨm) depolarization (JC-1 staining). Biochemical studies on cardiomyoctes showed improved cell viability and reduced caspase-3 activation caused by visfatin pretreatment. Visfatin did not inhibit the death receptor-dependent apoptotic pathways, as characterized by its absence in both Fas and TNFR1 down-regulation. Instead, visfatin specifically suppressed the mitochondria-dependent apoptotic pathways, as characterized by changed levels of p53 and its downstream Bcl-2 family genes. Visfatin also up-regulated the protein levels of phosphorylated AMPK, and the anti-apoptotic action of visfatin was attenuated by the AMPK-specific inhibitor compound C. These results suggested that visfatin plays a critical role in cardioprotection by suppressing myocardial apoptosis via AMPK activation. These findings may be the missing link between obesity and CVD. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Mollugin is a naphthohydroquine found in the roots of Rubia cordifolia , and has been reported to have a variety of biological activities, including anti-inflammatory and apoptotic effects. In the present study, we investigated the molecular mechanisms by which mollugin exerts anti-tumor effect in HER2-overexpressing cancer cells. Our results showed that mollugin exhibited potent inhibitory effects on cancer cell proliferation, especially in HER2-overexpressing SK-BR-3 human breast cancer cells and SK-OV-3 human ovarian cancer cell lines in a dose- and time-dependent manner without affecting immortalized normal mammary epithelial cell line MCF-10A. Furthermore, we found that a blockade of Akt/SREBP-1c signaling through mollugin treatment significantly reduced FAS expression and subsequently suppressed cell proliferation and induced apoptosis in HER2-overexpressing cancer cells. Mollugin treatment caused a dose-dependent inhibition of HER2 gene expression at the transcriptional level, potentially in part through suppression of NF-κB activation. The combination of mollugin with a MEK1/2 inhibitor may be required in order to achieve optimal efficacy in HER2-overexpressing cancers. These data provide evidence that mollugin inhibits proliferation and induces apoptosis in HER2-overexpressing cancer cells by blocking expression of the FAS gene through modulation of a HER2/Akt/SREBP-1c signaling pathway. Our findings suggest that mollugin is a novel modulator of the HER2 pathway in HER2-overexpressing cancer cells with a potential role in the treatment and prevention of human breast and ovarian cancer with HER2 overexpression. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Azithromycin is an antibiotic with anti-inflammatory properties used as an adjunct to treat periodontitis, a common inflammatory mediated condition featuring pathologic alveolar bone resorption. This study aimed to determine the effect of azithromycin on human osteoclast formation and resorptive activity in vitro . Osteoclasts were generated from peripheral blood mononuclear cells stimulated with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B (RANK) ligand. The effects of azithromycin at concentrations ranging from 0.5µg/mL to 40µg/mL were tested. Osteoclast formation and activity, acidification, actin ring formation and expression of mRNA and protein encoding for key osteoclast genes were assessed. The results demonstrated that azithromycin reduced osteoclast resorptive activity at all concentrations tested with osteoclast formation being significantly reduced at the higher concentrations (20µg/mL and 40µg/mL). mRNA and protein expression of key osteoclast transcription factor Nuclear Factor of Activated T cells (NFATc1) was significantly reduced by azithromycin at later stages of osteoclast development (day 17). Azithromycin also reduced tumour necrosis factor receptor associated factor-6 (TRAF6) mRNA expression at day 14, and cathepsin K mRNA expression at day 14 and 17. Integrin β3 and MMP-9 mRNA expression was reduced by azithromycin at day 17 in osteoclasts cultured on dentine. The osteoclast proton pump did not appear to be affected by azithromycin, however formation of the actin ring cytoskeleton was inhibited. This study demonstrates that azithromycin inhibits human osteoclast function in vitro, which may account for at least some of the beneficial clinical effects observed with azithromycin treatment in periodontitis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Tumor-associated macrophages (TAMs) are a key component of the tumor microenvironment and orchestrate various aspects of cancer. Diversity and plasticity are hallmarks of cells of the monocyte-macrophage lineage. In response to distinct signals macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a spectrum of activation states. Metabolic adaptation is a key component of macrophage plasticity and polarization, instrumental to their function in homeostasis, immunity and inflammation. Generally, TAMs acquire a M2-like phenotype that plays important roles in many aspects of tumor growth and progression. There is now evidence that also neutrophils can be driven towards distinct phenotypes in response to microenvironmental signals. The identification of mechanisms and molecules associated with macrophage and neutrophil plasticity and polarized activation provides a basis for new diagnostic and therapeutic strategies. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Low-power laser irradiation (LPLI) has been shown to exert promotive effects on cell survival and proliferation through activation of various signaling pathways. Estrogen receptors (ERs, ERa and ERβ) are ligand-activated transcription factors, which regulate target gene expression, promote cell proliferation and resist apoptosis. However, it is unclear whether LPLI could induce ligand-independent activation of ERs. In the present study, we investigated the subcellular pools, nuclear redistribution and transcriptional activity of ERs under LPLI (1.2 J/cm 2 , 633 nm) treatment using single-molecule fluorescence imaging and dual-luciferase reporter assay. We found that ERs were not only localized to nucleus, but also existed in mitochondria. Moreover, we found that LPLI induced nuclear redistribution and transcriptional activity of ERs in a ligand-independent manner. Our further investigation showed that PI3-K/Akt signaling cascade was involved in LPLI-induced activation of ERs. Wortmannin, a PI3-K inhibitor, or triciribine (API-2), a specific Akt inhibitor, potently suppressed the nuclear redistribution and transcriptional activity of ERs induced by LPLI, revealing that PI3-K/Akt signaling cascade was required for the activation of ERs induced by LPLI. Collectively, we demonstrated the first time that LPLI induced the ligand-independent nuclear redistribution and transcriptional activity of ERs, which were dependent on the activity of PI3-K/Akt. Our findings provide direct evidence for the molecular mechanisms of LPLI-induced transcription factor activation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias . In this study we investigated the effects, structure-activity relationships, and mechanism of action of pregnane glycosides on steroidogenesis in human adrenocortical H295R cells. Administration of pregnane glycosides for 24 h suppressed the basal and forskolin-stimulated release of androstenedione, corticosterone, and cortisone from H295R cells. The conversion of progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to either androstenedione or 11-deoxycortisol was most strongly affected, with 12-cinnamoyl-, benzoyl-, and tigloyl-containing pregnanes showing the highest activity. Incubation of pregnane glycosides for 24 h had no effect on mRNA transcripts of CYP11A1, CYP21A1, CYP11B1 cytochrome enzymes and steroidogenic acute regulatory protein (StaR) protein, yet resulted in 2-fold decrease in HSD3B1 mRNA levels. At the same time, pregnane glycosides had no effect on the CYP1, 2, or 3 drug and steroid metabolism enzymes and showed weak Na + /K + ATPase and glucocorticoid receptor binding. Taken together, these data suggest that pregnane glycoside specifically suppress steroidogenesis through strong inhibition of 11β-hydroxylase and steroid 17-alpha-monooxygenase, and weak inhibition of cytochrome P450 side chain cleavage enzyme and 21β-hydroxylase, but not 3β-hydroxysteroid dehydrogenase/isomerase. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: We used vertical growth phase (VGP) human VMM5 melanoma cells to ask whether the tumor microenvironment could induce Matrix Metalloproteinase-1 (MMP-1) in vivo , and whether this induction correlated with metastasis. We isolated two clones from parental VMM5 cells: a low MMP-1 producing clone (C4) and high producing clone (C9). When these clones were injected orthotopically (intradermally) into nude mice, both were equally tumorigenic and produced equivalent and abundant amounts of MMP-1. However, the tumors from the C4 clones displayed different growth kinetics and distinct profiles of gene expression from the C9 population. The C4 tumors, which had low MMP-1 levels in vitro , appeared to rely on growth factors and cytokines in the microenvironment to increase MMP-1 expression in vivo , while MMP-1 levels remained constant in the C9 tumors. C9 cells, but not C4 cells, grew as spheres in culture and expressed higher levels of Jarid 1b, a marker associated with melanoma initiating cells. We conclude that VMM5 melanoma cells exhibit striking intra-tumor heterogeneity, and that the tumorigenicity of these clones is driven by different molecular pathways. Our data suggest that there are multiple mechanisms for melanoma progression within a tumor, which may require different therapeutic strategies. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Numerous studies have reported that Vpr alters NF-κB signaling in various cell types, however, the findings have been largely conflicting with reports of both stimulatory and inhibitory effects of Vpr. Our aim was to investigate the role of Vpr signaling in myeloid cells using an adenovirus based expression and indicator system. Our results show that Vpr is inhibitory to NF-κB, however, this effect is dependant on the particular manner of NF-κB stimulation. Consistent with this notion, we report that Vpr has inhibitory effects that are specific to the TNF-α pathway, but not affecting the LPS pathway, suggesting that differential targets of Vpr may exist for NF-κB regulation. Further, we identify VprBP as one possible cellular component of Vpr's regulation of IκBα in response to TNF-α stimulation. We did not identify such a role for HSP27, which instead seems to inhibit Vpr functions. Chronically HIV-1 infected U1 cells with knockdown constructs for Vpr were unexpectedly less responsive to TNF-α mediated viral replication, perhaps suggesting that other HIV-1 components may antagonize these anti-NF-κB effects in infected cells. We hypothesize that Vpr may serve an important role in the context of viral infection and immune function in vivo, through its selective inhibition of NF-κB pathways. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Colorectal cancer is ranked among the top leading causes of cancer death in industrialized populations. Polycomb group proteins (PcGs), including Suz12 and Ezh2, are epigenetic regulatory proteins that act as transcriptional repressors of many differentiation-associated genes and are overexpressed in a large subset of colorectal cancers. Retinoic acid (RA) acts as a negative regulator of PcG actions in stem cells, but has shown limited therapeutic potential in some solid tumors, including colorectal cancer, in part because of RARβ silencing. Through treatment with RA, Suz12 shRNA knockdown, or Ezh2 pharmacological inhibition with 3-deazaneplanocin A (DZNep), we increased TRAIL-mediated apoptosis in human colorectal cancer cell lines. This increased apoptosis in human colon cancer cells after RA or DZNep treatment was associated with a ∼2.5-fold increase in TNFRSF10B (DR5) transcript levels and a 42% reduction in the H3K27me3 epigenetic mark at the TNFRSF10B promoter after DZNep addition. Taken together, our findings indicate that pharmacological inhibition of PRC2 histone methyltransferase activity may constitute a new epigenetic therapeutic strategy to overcome RA non-responsiveness in a subset of colorectal tumors by increasing TRAIL-mediated apoptosis sensitivity. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The Pur proteins are an ancient family of sequence-specific single-stranded nucleic acid-binding proteins. They bind a G-rich element in either single- or double-stranded nucleic acids and are capable of displacing the complementary C-rich strand. Recently several reports have described Pur family member knockouts, mutations and disease aberrations. Together with a recent crystal structure of Purα, these data reveal conserved structural features of these proteins that have been adapted to serve functions unique to higher eukaryotes. In humans Pur proteins are critical for myeloid cell development, muscle development, and brain development, including trafficking of mRNA to neuronal dendrites. Pur family members have been implicated in diseases as diverse as cancer, premature aging and fragile-X mental retardation syndrome. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Malignant mesothelioma (MM) is an intractable tumor of the peritoneal and pleural cavities primarily linked to exposure to asbestos. Recently we described an interplay between mitochondrial-derived oxidants and expression of FOXM1, a redox-responsive transcription factor that has emerged as a promising therapeutic target in solid malignancies. Here have investigated the effects of nitroxides targeted to mitochondria via triphenylphosphonium (TPP) moieties on mitochondrial oxidant production, expression of FOXM1 and peroxiredoxin 3 (PRX3), and cell viability in MM cells in culture. Both Mito-carboxy-proxyl (MCP) and Mito-TEMPOL (MT) caused dose-dependent increases in mitochondrial oxidant production that was accompanied by inhibition of expression of FOXM1 and PRX3 and loss of cell viability. At equivalent concentrations TPP, CP and TEMPOL had no effect on these endpoints. Live cell ratiometric imaging with a redox-responsive green fluorescent protein targeted to mitochondria (mito-roGFP) showed that MCP and MT, but not CP, TEMPOL, or TPP, rapidly induced mitochondrial fragmentation and swelling, morphological transitions that were associated with diminished ATP levels and increased production of mitochondrial oxidants. Mdivi-1, an inhibitor of mitochondrial fission, did not rescue mitochondria from fragmentation by MCP. Immunofluorescence microscopy experiments indicate a fraction of FOXM1 coexists in the cytoplasm with mitochondrial PRX3. Our results indicate that MCP and MT inhibit FOXM1 expression and MM tumor cell viability via perturbations in redox homeostasis caused by marked disruption of mitochondrial architecture, and suggest that both compounds, either alone or in combination with thiostrepton or other agents, may provide credible therapeutic options for the management of MM. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Adipose-derived stem cells (ASCs) have been discovered for more than a decade. Due to the large numbers of cells that can be harvested with relatively little donor morbidity, they are considered to be an attractive alternative to bone marrow derived mesenchymal stem cells. Consequently, isolation and differentiation of ASCs draw great attention in the research of tissue engineering and regenerative medicine. Cartilage defects cause big therapeutic problems because of their low self-repair capacity. Application of ASCs in cartilage regeneration gives hope to treat cartilage defects with autologous stem cells. In recent years, a lot of studies have been performed to test the possibility of using ASCs to re-construct damaged cartilage tissue. In this paper, we have reviewed the most up-to-date articles utilizing ASCs for cartilage regeneration in basic and translational research. Our topic covers differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes, increased cartilage formation by co-culture of ASCs with chondrocytes and enhancing chondrogenic differentiation of ASCs by gene manipulation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: OncostatinM (OSM) belongs to IL-6 subfamily and is mostly produced by T lymphocytes. High levels of OSM are detected in the pannus of rheumatoid arthritis (RA) patients and it may arouse the inflammation responses in joints and eventually leads to bone erosion. Placenta growth factor (PLGF) is an angiogenic factor and highly homologous with vascular endothelial growth factor (VEGF). It has been recently reported that PLGF is highly expressed in synovial tissue and enhances the production of proinflammatory cytokines including TNF-α and IL-6. Here we demonstrated that OSM increased mRNA and protein levels of PLGF in a time- and concentration-dependent manner in RA synovial fibroblasts. Inhibitors of JAK3 and PI3K antagonized OSM-induced production of PLGF. OSM enhanced the phosphorylation of Tyr705-STAT3, Ser727-STAT3, Ser473-Akt, and increased the nuclear translocation of phosphorylated STAT3 time-dependently. Transfection of dominant negative Akt or application of PI3K inhibitor LY294002 significantly inhibited p-Tyr705-STAT3, p-Ser727-STAT3 and PLGF expression, indicating that Akt is involved in JAK3/STAT3/PLGF signaling cascade. To further examine whether STAT3 binds to the promoter region of PLGF, Chip assay was used and it was found that OSM could bind with PLGF promoter, which was inhibited by JAK3 and PI3K inhibitors. Accumulation of PLGF in the pannus may contribute to the inflammation, angiogenesis and joints destruction in RA patients. These findings demonstrated the important role of OSM in the pathology network of RA and provided novel therapeutic drug targets for RA treatment. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The contribution of caveolae in Bone Morphogenetic Protein 2 (BMP2) activated Smad signaling was quantified using a system biology approach. BMP2 plays crucial roles during processes such as hematopoiesis, embryogenesis, and skeletal development. BMP2 signaling is tightly regulated on the plasma membrane by its receptors. The localization of BMP receptors in caveolae and endocytosis through clathrin coated pits are thought to regulate the signaling; however the conclusions in the current literature are inconsistent. Therefore published literature was used to establish a mathematical model that was validated using confocal AFM (atomic force microscopy), confocal microscopy, sucrose density centrifugation followed by western blots, and reporter gene assays. The model and experiments confirmed that both caveolae and CCPs regulate the Smad-dependent signaling pathway, however caveolae are centers at the plasma membrane where receptor - ligand interaction is crucial, Smad phosphorylation occurs, and a high degree of Smad signaling is regulated. This demonstrates a role for caveolae that needs to be considered and further studied. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: MicroRNAs are short non-coding RNAs that modulate gene expression by translational repression. Because of their high stability in intracellular as well as extracellular environments, miRNAs have recently emerged as important biomarkers in several human diseases. However, they have not been tested in the cerebrospinal fluid (CSF) of HIV-1 positive individuals. Here, we present results of a study aimed at determining the feasibility of detecting miRNAs in the CSF of HIV-infected individuals with and without encephalitis (HIVE). We also evaluated similarities and differences between CSF and brain tissue miRNAs in the same clinical setting. We utilized a high throughput approach of miRNA detection arrays and identified differentially expressed miRNAs in the frontal cortex of three cases each of HIV+, HIVE, and HIV- controls, and CSF of ten HIV-positive and ten HIV-negative individuals. For the CSF samples, the group of HIV+ individuals contained nine cases of HIV-Associated Neurological Disorders (HAND) and, among those, four had HIVE. All the HIV-negative samples had non-viral acute disseminate encephalomyelitis. A total of 66 miRNAs were found differentially regulated in HIV+ compared to HIV- groups. The greatest difference in miRNA expression was observed when four cases of HIVE were compared to five non-HIVE cases, previously normalized with the HIV-negative group. After statistical analyses, eleven miRNAs were fund significantly up-regulated in HIVE. Although more clinical samples should be examined, this work represents the first report of CSF miRNAs in HIV-infection and offers the basis for future investigation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The majority of hematopoietic stem/progenitor cells (HSPCs) reside in bone marrow (BM) surrounded by a specialized environment, which governs HSPC function. Here we investigated the potential role of bone remodeling cells (osteoblasts and osteoclasts) in homeostasis and stress-induced HSPC mobilization. Peripheral blood (PB) and BM in steady/mobilized state were collected from healthy donors undergoing allogeneic transplantation and from mice treated with granulocyte colony stimulating factor (G-CSF), parathyroid hormone (PTH), or receptor activator of nuclear factor kappa-B ligand (RANKL). The number and the functional markers of osteoblasts and osteoclasts were checked by a series of experiments. Our data showed that the number of CD45 - Ter119 - osteopontin (OPN) + osteoblasts was significantly reduced from 4085 ± 135 cells/femur on day 0 to 1032 ± 55 cells/femur on day 5 in mice (P = 0.02) and from 21.38 ± 0.66 on day 0 to 14.78 ± 0.65 on day 5 in healthy donors (P 〈 0.01). Decrease of osteoblast number leads to reduced level of HSPC mobilization regulators stromal cell-derived factor-1 (SDF-1), stem cell factor (SCF) and OPN. The osteoclast number at bone surface (OC.N/B.s) was significantly increased from 1.53 ± 0.12 on day 0 to 4.42 ± 0.46 on day 5 ( P  〈 0.01) in G-CSF-treated mice and from 0.88 ± 0.20 on day 0 to 3.24 ± 0.31 on day 5 ( P  〈 0.01) in human. Serum TRACP-5b level showed a biphasic trend during G-CSF treatment. The ratio of osteoblasts number per bone surface (OB.N/B.s) to OC.N/B.s was changed after adding PTH plus RANKL during G-CSF treatment. In conclusion, short term G-CSF treatment leads to reduction of osteoblasts and stimulation of osteoclasts, and interrupting bone remodeling balance may contribute to HSPC mobilization. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Thyroid cancer is not very common, accounting for 1-2% of all cancers, with a population incidence of about 0.004%. Currently, the ability to discriminate between follicular adenoma and carcinoma represents the major challenge in preclinical diagnosis of thyroid proliferative lesions. Better discrimination between the two would help avoid unnecessary thyroidectomy and save valuable resources. Over the years, Galectin-3 has been proposed as a diagnostic marker with varied success. In this paper we used Environmental Scanning Electron Microscopy Immunogold Labelling (ESEM-IGL) to investigate the expression of galectin-3 on thin-prep fine needle cytology (FNAC). We optimized the ESEM-IGL method on thyroid cell lines (RO-82 and FTC-133) comparing our membrane Galectin-3 (Gal-3) labelling data with Western Blot. We evaluated 183 thyroid FNAC from Italian patients with a uncertain pre-surgical diagnosis. ESEM-IGL method marker sensitivity is 71.2%, while specificity is 53.3% and diagnostic efficacy is 61.2%. Our results confirmed that Galectin-3 expression is associated with situations of hypertrophy and/or cellular hyperproliferation, pathophysiological situations common both to adenomas and to thyroid carcinomas. The innovation of thyroid FNAC Thin-Prep ESEM-IGL shows the levels of Gal-3 immunolabeling clearly, even through the individual cells of a thyroid nodule. However, galectin-3 alone, as a molecular marker of thyroid cancer, can still have a limited application in pre-surgery diagnosis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Obesity has been shown to be associated with the risk of colorectal cancer (CRC). Adipokines produced by the adipose tissue are linked to some malignancies, including CRC. Visfatin is an adipokine shown to be a biomarker of CRC malignant potential. In addition, the stromal cell-derived factor-1 (SDF-1) has been reported to play a role in CRC progression. Although the relationship between visfatin and CRC has been established, the underlying mechanism has not been clarified. We investigated the molecular mechanism governing the interaction between visfatin stimulation and SDF-1 expression in human CRC cell lines. We found that visfatin stimulation led to an increase in the expression and secretion of SDF-1 in CRC DLD-1 and SW48 cells. Experiments involving specific inhibitors and small interfering RNA demonstrated that the activation of ERK and p38 mitogen-activated protein kinase (MAPK) pathways are critical for visfatin-induced SDF-1 expression. Analysis of transcription factor binding using ELISA and luciferase reporter assays revealed that visfatin increased NF-κB- and AP-1-DNA-binding activities in DLD-1 cells. Inhibition of NF-κB and AP-1 activation blocked the visfatin-induced expression and activity of the SDF-1 promoter. The effect of visfatin on DLD-1 signaling and SDF-1 expression was mediated by β1 integrin. In summary, these findings provide novel insights pertaining to the pathophysiological role of visfatin in CRC. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative-RT-PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord-derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Previous studies revealed that gap junction intercellular communication (GJIC) between uterine stromal cells plays critical roles in modulating decidualization, neovasularization and embryo implantation. Connexin (Cx) proteins are the major component of gap junctions and Cx43 is the most widely expressed connexin in endometrium. Phosphorylation of Cx43 was found to impair gap junction communication in this tissue. Using primary human endometrial stromal cells (ESC) and a stable high telomerase-expressing ESC transfectant (T-HESC), we found that retinoic acid (RA) altered the phosphorylation status of Cx43 protein such that there was a decrease in the phosphorylated (P1 and P2) species accompanied by an increase in the non-phosphorylated (P0) form. This process is dependent on protein phosphatase 2A (PP2A) activity since selective PP2A inhibitors prevented the ability of RA to dephosphorylate Cx43. Although RA had no effect on total PP2A expression or activity, it significantly increased the intracellular association of Cx43 and PP2A. Inhibition of transcription and protein synthesis by actinomycin D and cycloheximide, respectively, had no effect on the RA-induced changes in the Cx43 phosphorylation pattern. Furthermore, BMS493, a potent antagonist of the classical RA-mediated transcriptional pathway, did not inhibit RA-induced Cx43 dephosphorylation. Our data indicate that RA stimulates physical association of PP2A with Cx43, resulting in the dephosphorylation of Cx43 and, as a consequence, up-regulation of GJIC in endometrial stromal cells. This process is independent of new mRNA and protein synthesis and suggests a novel mechanism by which aberrant retinoid metabolism can explain certain reproductive disorders manifested by dysfunctional endometrial cell GJIC. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Tamoxifen is a triphenylethylene non-steroidal antiestrogen anticancer agent. It also shows inhibitory effects on metastasis of estrogen receptor (EsR)-independent tumors, but the underlying mechanism is unclear. It was demonstrated in this study that, in EsR-negative and highly metastatic human hepatocellular carcinoma MHCC97H cells, tamoxifen inhibited cell migration, volume-activated Cl - currents ( I cl,vol ) and regulatory volume decrease (RVD) in a concentration–dependent manner with a similar IC 50 . Analysis of the relationships between migration, I cl,vol and RVD showed that cell migration was positively correlated with I cl,vol and RVD. Knockdown of the expression of ClC-3 Cl - channel proteins by ClC-3 shRNA or siRNA inhibited I cl,vol , and cell migration, and these inhibitory effects could not be increased further by addition of tamoxifen in the medium. The results suggest that knockdown of ClC-3 expression may deplete the effects of tamoxifen; tamoxifen may inhibit cell migration by modulating I cl,vol and cell volume. Moreover, tamoxifen decreased the activity of Protein Kinase C (PKC) and the effects were reversed by the PKC activator PMA. Activation of PKC by PMA could competitively downregulate the inhibitory effects of tamoxifen on I Cl,vol . PMA promoted cell migration, and knockdown of ClC-3 expression by ClC-3 siRNA abolished the PMA effect on cell migration. The results suggest that tamoxifen may inhibit I Cl,vol by suppressing PKC activation; I cl,vol may be an EsR-independent target for tamoxifen in the anti-metastatic action on cancers, especially on EsR-negative cancers. The finding may have an implication in the clinical use of tamoxifen in the treatments of both EsR-positive and EsR-negative cancers. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Angiopoietin 1 (Ang1) plays an important role in various endothelial functions, such as vascular integrity and angiogenesis; however, less is known about its function outside of the endothelium. In this study, we examined whether Ang1 has direct effects on skeletal muscle cells. We found that Ang1 exhibited myogenic potential, as it promoted the proliferation, migration, and differentiation of mouse primary skeletal myoblasts. The positive effect of Ang1 on myoblast proliferation could have been mediated by the α7 and β1 integrins. We also found that Ang1 potentiated cellular Ca 2+ movements in differentiated myotubes in response to stimuli, possibly through the increased expression of two Ca 2+ -related proteins, namely, Orai1 and calmodulin. Ang1 also increased Orai1 and calmodulin expression in mouse hearts in vivo . These results provide an insight into the molecular mechanisms by which Ang1 directly affects the myogenesis of striated muscle. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: High mobility group box 1 (HMGB1) protein is a crucial cytokine that mediates response to infection, injury, and inflammation. Rosmarinic acid (RA) is an important component of the leaves of Perilla frutescens and has neuroprotective, anti-microbial, anti-oxidant, and anti-cancer effects but little is known of its effects on HMGB1-mediated inflammatory response. Here, we investigated this issue by monitoring the effects of RA on the lipopolysaccharide (LPS) or cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated modulation of inflammatory responses. RA potently inhibited the release of HMGB1 and down-regulated HMGB1-dependent inflammatory responses in human endothelial cells. RA also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. Furthermore, RA reduced CLP-induced HMGB1 release and sepsis-related mortality. Given these results, RA should be viewed as a candidate therapeutic agent for the treatment of various inflammatory diseases via inhibition of the HMGB1 signaling pathway. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Serine/threonine protein phosphatase 2A (PP2A) participates in regulating many important physiological processes such as cell cycle, growth, apoptosis, and signal transduction. Osterix is a zinc-finger-containing transcription factor that is essential for osteoblast differentiation and regulation of many bone-related genes. We have recently reported that decrease in α-isoform of PP2A catalytic subunit (PP2A Cα) accelerates osteoblast differentiation through the expression of bone-related genes. In this study, we further examined the role of PP2A Cα in osteoblast differentiation by establishing the stable cell lines that overexpress PP2A Cα. Overexpression of PP2A Cα reduced alkaline phosphatase (ALP) activity. Osteoblast differentiation and mineralization were also decreased in PP2A Cα-overexpressing cells, with reduction of bone-related genes including Osterix, Bone sialoprotein (Bsp), and Osteocalcin (OCN). Luciferase assay showed that the transcriptional activity of the Osterix promoter region was decreased in PP2A Cα-overexpressing cells. Introduction of ectopic Osterix rescued the expression of Bsp and OCN in PP2A Cα-overexpressing cells. These results indicate that PP2A Cα and its activity play a negative role in osteoblast differentiation and Osterix is a key factor responsible for regulating the expressions of Bsp and OCN during PP2A Cα-mediated osteoblast differentiation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: We have recently shown that many mediators of the JAK/STAT signaling pathway are present in ejaculated human spermatozoa. Among them, STAT3 is detected mainly in membranes and flagellar cytoskeletal fractions. In order to determine the importance of STAT3-mediated signaling, sperm were incubated with Stattic V, a specific inhibitor. Effects on motility were evaluated by CASA, sperm acrosomal integrity was evaluated by FITC conjugated lectin (PSA or PNA) staining, and protein phosphotyrosine content was assessed by western blot using a monoclonal anti-phosphotyrosine antibody. INDO1-AM and JC-1 were used to measure sperm intracellular calcium and mitochondrial membrane potential, respectively, by flow cytometry, and reactive oxygen species (ROS) production was investigated by luminol-based assay. Percentages of motility and motility parameters were significantly affected by Stattic V. This later also significantly increased intracellular Ca 2+ levels, progesterone- and calcium ionophore (A23187)-induced acrosome reaction. On the other hand, a significant decrease in ATP content was measured when sperm were treated with Stattic V, associated with depolarization of mitochondrial membrane and elevated ROS production. These results suggest that STAT3 is involved in sperm functions, at least through regulation of mitochondrial activity. This further emphasizes that STAT3 mediates cellular activities in a manner different than strictly the activation of gene transcription. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Regardless of the original causes and etiology, the progression to renal function declines follows a final common pathway associated with tubulointerstitial injury, in which the proximal tubular epithelial cells (PTEC) are instrumental. Kidney injury molecule-1 (KIM-1) is an emerging biomarker, and its expression and release are induced in PTEC upon injury. KIM-1 plays the role as a double-edged sword and implicates in the process of kidney injury and healing. Expression of KIM-1 is also associated with tubulointerstitial inflammation and fibrosis. More importantly, KIM-1 expressing PTEC play the role as the residential phagocytes, contribute to the removal of apoptotic cells and facilitate the regeneration of injured tubules. The precise mechanism of KIM-1 and its shedded ectodomain on restoration of tubular integrity after injury is not fully understood. Other than PTEC, macrophages (Mø) also implicate in tubular repair. Understanding the crosstalk between Mø and the injured PTEC, is essential for designing appropriate methods for controlling the sophisticated machinery in tubular regeneration and healing. This paper will review the current findings of KIM-1, beginning with its basic structure, utility as a biomarker, and possible functions, with focus on the role of KIM-1 in regeneration and healing of injured PTEC. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The metabolome is a data-rich source of information concerning all the low-molecular-weight metabolites in a biofluid, which can indicate early biological changes to the host due to perturbations in metabolic pathways. Major changes can be seen after minor stimuli, which make it a valuable target for analysis. Due to the diverse and sensitive nature of the metabolome, studies must be designed in a manner to maintain consistency, reduce variation between subjects, and optimize information recovery. Technological advancements in experimental design, mouse models and instrumentation have aided in this effort. Metabolomics has the ultimate potential to be valuable in a clinical setting where it could be used for early diagnosis of a disease and as a predictor of treatment response and survival. During drug treatment, the metabolic status of an individual could be monitored and used to indicate possible toxic effects. Metabolomics therefore has great potential for improving diagnosis, treatment and aftercare of disease. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Transforming growth factor (TGF) β1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K) is one of the signaling pathways implicated in TGFβ1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD) or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFβ1 induced pro-inflammatory cytokine and contractile proteins in ASM cells. Results: A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFβ1 increased 110d mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 μM), the p110β inhibitor TGX221 (0.1, 1, 10 μM) nor the PI3K pan inhibitor LY294002 (3, 10 μM) had any effect on basal IL-6, calponin or smooth muscle α-actin (α-SMA) expression. However, TGFβ1 increased calponin and α-SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221 and LY294002 reduced TGFβ1 induced IL-6 release in a dose related manner in all groups of ASM cells. Conclusion: PI3K p110δ is important for TGFβ1 induced production of the contractile proteins calponin and α-SMA and the proinflammatory cytokine IL-6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: We tested the hypothesis that asthmatic mouse airways exhibit impaired relaxation to NO donors. Mouse tracheal rings were incubated overnight in serum from asthmatic human subjects or from non-asthmatic controls. The next day, cumulative concentration-response curves (CCRC) to sodium nitroprusside (SNP) and nitroglycerine (NTG) were obtained. Both SNP and NTG relaxed the pre-constricted normal tracheal rings. Tracheal rings exposed to serum from asthmatic patients exhibited a more than a 3-fold increase in the EC50 of SNP and NTG. Pre-incubation of tracheal rings with heat shock protein 90 inhibitors decreased the relaxation of both normal and asthmatic tracheal rings to SNP and NTG. Pre-incubation with estradiol did not affect normal tracheal ring relaxation but exhibited an increase in asthmatic tracheal ring relaxation, which was abolished by an estrogen receptor (ER) antagonist. ER subtype-selective agonists, but not GPR30 agonists, mimicked the action of estradiol on tracheal ring relaxation. Co-incubation of rings with radicicol and estradiol produced an ER-dependent increase in the relaxation response to SNP of both normal and asthmatic ASM. Estrogen-induced relaxation of ASM was abolished by overnight incubation with radicicol and this was associated with reduced expression of ERβ. These data suggest that asthmatic ASM is considerably less responsive to NO-donors and that both estrogen and hsp90 play important roles in ASM relaxation. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: An excessive food supply has resulted in an increasing prevalence of overweight and obesity, conditions accompanied by serious health problems. Several studies have confirmed the significant inverse correlation between testosterone and obesity. Indeed after decades of intense controversy, a consensus has emerged that androgens are important regulators of fat mass and distribution in mammals and that androgen status affects cellularity in vivo . The high correlation of testosterone levels with body composition and its contribution to the balance of lipid metabolism are also suggested by the fact that testosterone lowering is associated with important clinical disorders such as dyslipidemia, atherosclerosis, cardiovascular diseases, metabolic syndrome and diabetes. In contrast, testosterone supplementation therapy in hypogonadic men has been shown to improve the lipid profile by lowering cholesterol, blood sugar and insulin resistance. Leptin, ghrelin and adiponectin are some of the substances related to feeding as well as androgen regulation. Thus, complex and delicate mechanisms appear to link androgens with various tissues (liver, adipose tissue, muscles, coronary arteries, heart) and the subtle alteration of some of these interactions might be the cause of correlated diseases. This review underlines some aspects regarding the high correlations between testosterone physiology and body fat composition. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Endothelium is a highly dynamic tissue that controls vascular homeostasis. This requires constant rearrangements of the shape or function of endothelial cells that cannot set aside the role of the cytoskeleton. The aim of this study was to determine the mechanisms by means of which cytoskeletal alterations induce cyclooxygenase-2 expression in human endothelial cells using compounds that interfere with microtubule or actin architecture. Microtubule disruption by nocodazole markedly increased cyclooxygenase-2 expression and activity, and provoked paracellular gap formation, a cardinal feature of endothelial barrier dysfunction. The cyclooxygenase-2 metabolite prostacyclin down-regulated cyclooxygenase-2 through an autocrine receptor-mediated mechanism, and partially prevented the disassembly of endothelial monolayers. There was also an interaction between microtubules and actin filaments in nocodazole-induced cyclooxygenase-2 expression. Nocodazole provoked the dissolution of the F-actin cortical ring and stress fibre formation, increased actin glutathionylation and concomitantly lowered intracellular levels of reduced glutathione. The restoration of glutathione levels by N-acetylcysteine opposed cyclooxygenase-2 expression and preserved the integrity of endothelial monolayers. Among the signalling pathways connecting microtubule disruption with cyclooxygenase-2 up-regulation, crucial roles are played by Src family kinase activation, serine/threonine phosphatase 2A inhibition, and the phosphorylation of mitogen activated protein kinase p38. Our findings provide a mechanistic insight into the observation that cyclooxygenase-2 is induced in endothelial cells under cytoskeleton-perturbing conditions such as those occurring in the presence of atherogenic/inflammatory stimuli and oxidative stress. In this scenario, cyclooxygenase-2 up-regulation by endothelia exposed to noxious conditions can be considered protective of the vasodilatory and anti-thrombotic properties of the vessel wall. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data presents ether à go-go (hEag1) K + channels as relevant player in controlling cell cycle and proliferation of non-invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA-MB-231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA-MB-231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca 2+ entry and MDA-MB-231 cell migration without affecting cell proliferation. Recent studies have reported that Ca 2+ entry through Orai1 channels is required for MDA-MB-231 cell migration. Down-regulation of hEag1 or Orai1 reduced Ca 2+ influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca 2+ influx or cell migration were observed in cells co-transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM + ). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum-induced migration of BC cell by controlling the Ca 2+ entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: The size of human cervical intraepithelial neoplasia biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small cervical intraepithelial neoplasia tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue-derived fibroblasts and keratinocytes were co-cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17 and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Annexin A1 (AnxA1) originating from mature neutrophils and their microparticles (MPs) plays an important anti-inflammatory role during the resolution phase of inflammation. However, the role of AnxA1 during the process of granulocytic differentiation is still unknown. All-trans retinoic acid (ATRA) can induce acute promyelocytic leukemic (APL) cells to differentiate along the granulocytic lineage and has been used successfully in treating APL patients. In this study we investigated whether or not AnxA1 contributed to the anti-inflammatory properties of ATRA-treated APL (NB4; ATRA-NB) cells using the transmigratory and adhesive assays. We found that ATRA was able to enhance the surface expression of AnxA1 and its receptor (FPR2/ALX) and the release of AnxA1-containing MPs from ATRA-NB4 cells, while the expression of annexin V was not elevated on the latter cells. Further studies demonstrated that exogenous AnxA1 could inhibit ATRA-NB4 cells in their transmigratory activity and adhesion to endothelial cells. In addition, the transmigratory activity of ATRA-NB4 cells can be significantly enhanced by pretreatment with a FPR2/ALX neutralizing antibody, suggesting that endogenous AnxA1 may contribute to the anti-migratory effects. Finally, ATRA-NB4-derived MPs could also inhibit recipient cells in their transmigratory and adhesive activities and these anti-inflammatory effects could be inhibited by pretreatment of MPs with a specific anti-AnxA1 antibody. Flowcytometry studies further demonstrated that FITC-labeled AnxA1 could be transport from MPs to the membrane of recipient ATRA-NB4 cells. We conclude that biologically active AnxA1 may play a role in the anti-inflammatory properties of ATRA-treated APL cells during the process of granulocytic differentiation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.

Description: Human Pancreatic Cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC) and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21 , miR-221 , miR-27a , miR-27b and miR-155 , and down-regulation of miR-216a , miR-216b , miR-217 and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: The family of insulin receptor substrates (IRS) consists of four proteins (IRS-1 - IRS-4), which were initially characterized as typical cytosolic adaptor proteins involved in insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) signaling. The first cloned and characterized member of the IRS family, IRS-1, has predicted molecular weight of 132 kDa, however, as a result of its extensive serine phosphorylation it separates on a SDS gel as a band of approximately 160-185 kDa. In addition to its metabolic and growth-promoting functions, IRS-1 is also suspected to play a role in malignant transformation. The mechanism by which IRS-1 supports tumor growth is not fully understood, and the argument that IRS-1 merely amplifies the signal from the IGF-1R and/or IR requires further investigation. Almost a decade ago, we reported the presence of nuclear IRS-1 in medulloblastoma clinical samples, which express viral oncoprotein, large T-antigen of human polyomavirus JC (JCV T-antigen). This first demonstration of nuclear IRS-1 was confirmed in several other laboratories. The nuclear IRS-1 was also detected by cells expressing the SV40 T-antigen, v-Src, in immortalized fibroblasts stimulated with IGF-I, in hepatocytes, 32D cells, and in an osteosarcoma cell line. More recently, nuclear IRS-1 was detected in breast cancer cells in association with estrogen receptor alpha (ERα), and in JC virus negative medulloblastoma cells expressing ERb, further implicating nuclear IRS-1 in cellular transformation. Here, we discuss how nuclear IRS-1 acting on DNA repair fidelity, transcriptional activity, and cell growth can support tumor development and progression. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: The hallmark of acute lung injury (ALI) is the influx of proinflammatory cytokines into lung tissue and alveolar permeability that ultimately leads to pulmonary edema. However, the mechanisms involved in inflammatory cytokine production and alveolar permeability are unclear. Recent studies suggest that excessive production of ceramide has clinical relevance as a mediator of pulmonary edema and ALI. Our earlier studies indicate that the activation of inflammasome promotes the processing and secretion of proinflammatory cytokines and causes alveolar permeability in ALI. However, the role of ceramide in inflammasome activation and the underlying mechanism in relation to alveolar permeability is not known. We hypothesized that ceramide activates the inflammasome and causes inflammatory cytokine production and alveolar epithelial permeability. To test this hypothesis, we analyzed the lung ceramide levels during hyperoxic acute lung injury in mice. The effect of ceramide on activation of inflammasome and production of inflammatory cytokine was assessed in primary mouse alveolar macrophages and THP-1 cells. Alveolar transepithelial permeability was determined in alveolar epithelial type-II cells (AT-II) and THP-1 co-cultures. Our results reveal that ceramide causes inflammasome activation, induction of caspase-1, IL-1β cleavage and release of proinflammatory cytokines. In addition, ceramide further induces alveolar epithelial permeability. Short hairpin RNA silencing of inflammasome components abrogated ceramide-induced secretion of proinflammatory cytokines in vitro . Inflammasome silencing abolishes ceramide induced alveolar epithelial permeability in AT-II. Collectively, our results demonstrate for the first time that ceramide-induced secretion of proinflammatory cytokines and alveolar epithelial permeability occurs though inflammasome activation. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however the role of cyclooxygenase (COX)-2/prostaglandin (PG)E 2 on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE 2 production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE 2 production in HT-29 cells. The addition of COX activity inhibitors, including NS398, aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE 2 production in HT-29 cells. Application of PGE 2 , but not it analogues PGD 2 , PGJ 2 , or PGF 2α , protected HT-29 cells from GOS-induced DNA ladders, and the EP 1 receptor agonist, 17PT-PGE 2 , mimicked the protection induced by PGE 2 , whereas the selective EP 2 receptor agonist, butaprostol (BUT), the EP 3 receptor agonist, sulprostol (SUL), and the EP 4 receptor agonist, PGE 1 alcohol (PGE 1 ), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE 2 's protection against GOS-induced apoptosis was reversed by adding the selective EP 1 receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS398 significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to an MTT assay and DNA integrity assay. The notion of COX-2/PGE 2 activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: It remains unclear why atypical antipsychotics confer a risk for hyperglycemia compared to typical antipsychotics. Atypical antipsychotics antagonize dopamine receptors-2 (D 2 ) and serotonin (5-HT) receptors-2, while typical antipsychotics antagonize only D 2 receptors. We aimed at elucidating the mechanistic differences between the role of typical and atypical antipsychotics on prolactin levels and glucose regulation. A Medline search was conducted during 2010 using the search terms type 2 diabetes (T2D), typical/atypical antipsychotics, schizophrenia, prolactin, and serotonin. We discuss the effect of typical and atypical antipsychotics on prolactin levels and glucose regulation. Given that prolactin is under negative control by dopamine and positive control by serotonin, typical antipsychotics induce elevations in prolactin, while atypical antipsychotics do not. Research studies show protective effects of prolactin on T2D. We hypothesize that the difference in induction of T2D between typical and atypical antipsychotics is due to the antipsychotic receptor binding mediated effect in changes in prolactin levels. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Modeling breast cancer in the mouse has helped to better define the heterogeneity of human breast cancer. In the recent past, it has become evident that some limitations have restricted the potential benefits that can be achieved with this approach. In this review, we highlight some key points that should be taken into account when the mouse is used, with special emphasis on transgenic models. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Organisms frequently encounter a wide variety of proteotoxic stressors. The heat-shock response, an ancient cytoprotective mechanism, has evolved to augment organismal survival and longevity in the face of proteotoxic stress from without and within. These broadly recognized beneficial effects, ironically, contrast sharply with its emerging role as a culprit in the pathogenesis of cancers. Here, we present an overview of the normal biology of the heat-shock response and highlight its implications in oncogenic processes, including the proteotoxic stress phenotype of cancer; the function of this stress response in helping cancer survive and adapt to proteotoxic stress; and perturbation of proteome homeostasis in cancer as a potential therapeutic avenue. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: It is well established that 1a-25-dihydroxyvitamin D3 (1,25D3) regulates osteoblast function and stimulates mineralization by human osteoblasts. The aim of this study was to identify processes underlying the 1,25D3 effects on mineralization. We started with gene expression profiling analyses of differentiating human pre-osteoblast treated with 1,25D3. Bioinformatic analyses showed interferon-related and -regulated genes (ISG) to be overrepresented in the set of 1,25D3-regulated genes. 1,25D3 down-regulated ISGs predominantly during the pre-mineralization period. This pointed to an interaction between the vitamin D and IFN signaling cascades in the regulation of osteoblast function. Separately, 1,25D3 enhances while IFNβ inhibits mineralization. Treatment of human osteoblasts with 1,25D3 and IFNβ showed that 1,25D3 completely overrules the IFNβ inhibition of mineralization. This was supported by analyses of extracellular matrix gene expression, showing a dominant effect of 1,25D3 over the inhibitory effect of IFNβ. We identified processes shared by IFNβ- and 1,25D3-mediated signaling by performing gene expression profiling during early osteoblast differentiation. Bioinformatic analyses revealed that genes being correlated or anti-correlated with Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were associated with osteoblast proliferation. In conclusion, the current study demonstrates a cross-talk between 1,25D3 and IFNβ in osteoblast differentiation and bone formation/mineralization. The interaction is complex and depends on the process but importantly, 1,25D3 stimulation of mineralization is dominant over the inhibitory effect of IFNβ. These observations are of potential clinical relevance considering the impact of the immune system on bone metabolism in conditions such as rheumatoid arthritis. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of MSCs in the normal human bowel. MSCs are bone marrow multipotent cells supporting haematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from bone marrow, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146 + MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: osteogenic differentiation, hematopoietic supporting activity, immune-modulation and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29 and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon, provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Apert syndrome is characterized by craniosynostosis and syndactyly, and is predominantly caused by mutation of either S252W or P253W in the fibroblast growth factor receptor (FGFR) 2 gene. In this study, we characterized the effects of one of the mutations (S252W) using primary calvarial osteoblasts derived from transgenic mice, Ap-Tg and sAp-Tg, that expressed an Apert-type mutant FGFR2 (FGFR2IIIc-S252W; FGFR2IIIc-Ap), and the soluble form (extracellular domain only) of the mutant FGFR2 (sFGFR2IIIc-Ap), respectively. Compared to WT-derived osteoblasts, osteoblasts from Ap-Tg mouse showed a higher proliferative activity and enhanced differentiation, while those from sAp-Tg mouse exhibited reduced potential for proliferation and osteogenic differentiation. When transplanted with β-tricalcium phosphate (β-TCP) granules into immunodeficient mice, Ap-Tg-derived osteoblasts showed a higher bone forming capacity, whereas sAp-Tg-derived osteoblasts were completely deficient for this phenotype. Phosphorylation of ERK, MEK, PLCg, and p38 was increased in Ap-Tg-derived osteoblasts, whereas phosphorylation of these signaling molecules was reduced in sAp-Tg-derived osteoblasts. Interestingly, when these experiments were carried out using osteoblasts from the mice generated by crossing Ap-Tg and sAp-Tg (Ap/sAp-Tg), which co-expressed FGFR2IIIc-Ap and sFGFR2IIIc-Ap, the results were comparable to those obtained from WT-derived osteoblasts. Taken together, these results indicate that osteoblasts expressing FGFR2IIIc-Ap proliferate and differentiate via highly activated MEK, ERK and p38 pathways, while these pathways are suppressed in osteoblasts expressing sFGFR2IIIc-Ap. Our findings also suggest that altered FGFR2IIIc signaling in osteoblasts is mostly responsible for the phenotypes seen in Apert syndrome, therefore these osteoblast cell lines are useful tools for investigating the pathogenesis of Apert syndrome. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Placenta has attracted increasing attention over the past decade as a stem cell source for regenerative medicine. In particular, the amniochorionic membrane has been shown to harbour populations of mesenchymal stromal cells (MSCs). In this study, we have characterized ex vivo expanded MSCs from the human amniotic (hAMSCs) and chorionic (hCMSCs) membranes of human full term placentas and adult bone marrow (hBMSCs). Our results show that hAMSCs, hCMSCs and hBMSCs express typical mesenchymal (CD73, CD90, CD105, CD44, CD146, CD166) and pluripotent (Oct-4, Sox2, Nanog, Lin28 and Klf4) markers but not hematopoietic markers (CD45, CD34). Ex vivo expanded hAMSCs were found to be of fetal origin, while hCMSCs cultures contained only maternal cells. Cell proliferation was significantly higher in hCMSCs, compared to hAMSCs and hBMSCs. Integrin profiling revealed marked differences in the expression of α − subunits between the three cell sources. Cadherin receptors were consistently expressed on a subset of progenitors (ranging from 1% to 60%), while N-CAM (CD56) was only expressed in hAMSCs and hCMSCs but not in hBMSCs. When induced to differentiate, hAMSCs and hCMSCs displayed strong chondrogenic and osteogenic differentiation potential but very limited capacity for adipogenic conversion. In contrast, hBMSCs showed strong differentiation potential along the three lineages. These results illustrate how MSCs from different ontological sources display differential expression of cell fate mediators and mesodermal differentiation capacity. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.

Description: Heart failure is a major cause of death throughout the world. Hyperthyroidism has been shown to induce cardiac hypertrophy, which is a contributing factor to heart failure. However, the mechanism underling effect of thyroid hormone is not completely clear. The present study investigates the role of peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α) in cardiac hypertrophy induced by Triiodothyronine (T3). We investigated PGC-1α mRNA expression in rat hearts exposed to T3 in vivo and ex vivo . Surprisingly, we found that the extended periods of T3 treatment led to an increase in PGC-1α expression compared to shorter treatment times, which resulted in a reduction of PGC-1α expression. Mechanistic studies showed that suppression of PGC-1α by small interfering RNA in cardiomyocytes amplified the cellular hypertrophic response to T3 stimulation, whereas overexpression of PGC-1α was protective. Furthermore, we presented evidence to show that T3 decreased PGC-1α expression via p38 mitogen-activated protein kinases (MAPK) pathway. Our studies also revealed that overexpression of PGC-1α in cardiomyocytes inhibited basal and T3-induced p38 MAPK phosphorylation. These data indicate for the first time that PGC-1α plays protective role in T3-induced cardiac hypertrophy and that hypertrophic growth induced by T3 involves a regulatory pathway between PGC-1α and p38 MAPK. J. Cell. Physiol. © 2011 Wiley Periodicals, Inc.