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Journal Cover   Journal of Cellular Physiology
  [SJR: 1.948]   [H-I: 125]   [5 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
   Published by John Wiley and Sons Homepage  [1597 journals]
  • The IP3R Binding Protein Released with Inositol 1,4,5‐Trisphosphate
           Is Expressed in Rodent Reproductive Tissue and Spermatozoa
    • Authors: Heike Borth; Nele Weber, Dorke Meyer, Andrea Wartenberg, Elisabeth Arlt, Susanna Zierler, Andreas Breit, Gunther Wennemuth, Thomas Gudermann, Ingrid Boekhoff
      Abstract: Besides its capacity to inhibit the 1,4,5‐trisphosphate (IP3) receptor, the regulatory protein IRBIT (IP3 receptor‐binding protein released with IP3) is also able to control the activity of numerous ion channels and electrolyte transporters and thereby creates an optimal electrolyte composition of various biological fluids. Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co‐localize with the proton pumping V‐ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co‐localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturations steps after ejaculation and/or the final fertilization process. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-06T06:35:29.112421-05:
      DOI: 10.1002/jcp.25209
  • Reduced dentin matrix protein expression in Camurati‐Engelmann
           disease transgenic mouse model
    • Authors: Angela Gullard; Christina M. Croney, Xiangwei Wu, Olga Mamaeva, Philip Sohn, Xu Cao, Mary MacDougall
      Abstract: Overexpression of transforming growth factor‐beta 1 (TGF‐β1) has been shown to lead to mineralization defects in both the enamel and dentin layers of teeth. A TGFB1 point mutation (H222D), derived from published cases of Camurati‐Engelmann disease (CED), has been shown to constitutively activate TGF‐β1, leading to excess bone matrix production. Although CED has been well documented in clinical case reports, there are no published studies on the effect of CED on the dentition. The objective of this study was to determine the dental manifestations of hyperactivated TGF‐β1 signaling using an established mouse model of CED‐derived TGF‐β1 mutation. Murine dental tissues were studied via radiography, micro‐CT, immunohistochemistry, and qRT‐PCR. Results showed that initial decreased dental mineralized tissue density is resolved Proliferation assays of incisor pulp and alveolar bone cell cultures revealed that cells from transgenic animals displayed a reduced rate of growth compared to alveolar bone cultures from wild‐type mice. TGF‐β family gene expression analysis indicated significant fold changes in the expression of Alpl, Bmp2‐5, Col‐1, ‐2,‐4 and ‐6, Fgf, Mmp, Runx2, Tgfb3, Tfgbr3, and Vdr genes. Assessment of SIBLINGs reveals downregulation of Ibsp, Dmp1, Dspp, Mepe, and Spp1, as well as reduced staining for BMP‐2 and VDR in mesenchymal‐derived pulp tissue in CED animals. Treatment of dental pulp cells with recombinant human TGF‐β1 resulted in increased SIBLING gene expression. Conclusions: Our results provide in vivo evidence suggesting that TFG‐β1 mediates expression of important dentin extracellular matrix components secreted by dental pulp, and when unbalanced, may contribute to abnormal dentin disorders. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-01T11:13:46.627626-05:
      DOI: 10.1002/jcp.25207
  • Smooth muscle cell differentiation: Model systems, regulatory mechanisms,
           and vascular diseases
    • Abstract: Smooth muscle cell (SMC) differentiation is an important process during vascular development. The highly differentiated mature SMCs play critical roles in maintaining structural and functional integrity of blood vessels. However, SMCs are not terminally differentiated, and their phenotype can be modulated between contractile and proliferative states in response to various environmental conditions. Alterations in SMC phenotype contribute to a number of major cardiovascular diseases such as atherosclerosis, hypertension and restenosis following angioplasty, etc. The goal of this review is to provide a brief overview of the recent advancements in our understanding of SMC differentiation and the development of in vitro SMC differentiation models, with a particular emphasis on examination of molecular mechanisms involved in the regulation of SMC differentiation and phenotypic modulation. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-01T11:13:24.410441-05:
      DOI: 10.1002/jcp.25208
  • Involvement of Notch‐1 in Resistance to Regorafenib in Colon Cancer
    • Authors: Giovanna Mirone; Stefania Perna, Arvind Shukla, Gabriella Marfe
      Abstract: Regorafenib, an oral small‐molecule small‐molecule multi kinase inhibitor, is able to block Vascular Endothelial Growth Factor Receptors (VEGFR‐1, 2 and 3), Platelet‐Derived Growth Factor Receptors (PDGF), Fibroblast Growth Factor (FGF) receptor 1, Raf, TIE‐2, and the kinases KIT, RET, and BRAF. Different studies have displayed its antitumor activity in several cancer models (both in vitro and in vivo), particularly in colorectal and gastrointestinal stromal cancers. The mechanism of resistance to regorafenib is largely unknown. In our investigation, we have generated regorafenib‐resistant SW480 cells (Reg‐R‐SW480 cells) by culturing such cells with increasing concentration of regorafenib. Examination of intracellular signaling found that Akt signaling was activated in Reg‐R‐SW480 cells but not in wild‐type SW480 cells, after regorafenib treatment as measured byWestern Blot. The Notch pathway is a fundamental signaling system in the development and homeostasis of tissues since it regulates different cellular process such as proliferation, differentiation, and apoptosis and it can be a potential driver of resistance to a wide array of targeted therapies. In this study, we found that Notch‐1 was significantly up‐regulated in resistant tumor cells as well as HES1 and HEY. Addionally, inhibition of Notch‐1 in resistant cells partially restored sensitivity to regorafenib treatment in vitro. Collectively, these data suggest a key role of Notch‐1 in mediating the resistant effects of regorafenib in colorectal cancer cells, and also provide a rationale to improve the therapeutic efficacy of regorafenib. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-30T05:48:18.10011-05:0
      DOI: 10.1002/jcp.25206
  • Genes in the GABA Pathway Increase in the Lateral Thalamus of Sprague
           Dawley Rats During the Proestrus/Estrus Phase
    • Authors: Mikhail Umorin; Crystal Stinson, Larry L. Bellinger, Phillip R. Kramer
      Abstract: Pain can vary over the estrous cycle as a result of changes in estradiol concentration but the mechanism causing this variation is unclear. Because the thalamus is important in pain control, gene expression in the lateral thalamus (ventral posteromedial, ventral posterolateral, reticular thalamic nuclei) was screened at different phases of the estrous cycle. Gene expression changes in Sprague‐Dawley rats were further analyzed by real‐time PCR and ELISA and plasma estradiol levels were measured by RIAs at different phases of the estrous cycle. Our results indicated that both the RNA and protein expression of glutamate decarboxylase 1 and 2 (GAD1, GAD2), GABA(A) receptor‐associated protein like 1 (GABARAPL1) and vesicular GABA transporter (VGAT) significantly increased in the lateral thalamus when plasma estradiol levels were elevated. Estradiol levels were elevated during the proestrus and estrus phases of the estrous cycle. Estrogen receptor α (ERα) was observed to be co‐localized in thalamic cells and thalamic infusion of an ERα antagonist significantly reduced GAD1 and VGAT transcript. GAD1, GAD2 GABARAPL1 and VGAT have been shown to effect neuronal responses suggesting that modulation of pain during the estrous cycle can be dependent, in part, through estradiol induced changes in thalamic gene expression. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-30T02:18:29.573669-05:
      DOI: 10.1002/jcp.25198
  • Evidences of Biological Functions of Biliverdin Reductase A in the Bovine
    • Abstract: Epididymal sperm binding protein 1 (ELSPBP1) is secreted by the epididymal epithelium via epididymosomes and is specifically transferred to dead spermatozoa during epididymal transit. We identified biliverdin reductase A (BLVRA) as a partner of ELSPBP1 by immunoprecipitation followed by tandem mass spectrometry. Pull down assays showed that these two proteins interact in the presence of zinc ions. The BLVRA enzyme is known to convert biliverdin to bilirubin, both of which possess antioxidant activity. Assessment by real‐time RT‐PCR showed that BLVRA is highly expressed in the caput and the corpus epididymis, but is expressed at lower levels in the testis and the cauda epididymis. It is primarily found in the soluble fraction of the caput epididymal fluid, is barely detectable in the cauda fluid, and is detectable to a lesser extent in the epididymosome fraction of both caput and cauda fluids. Immunocytometry on epididymal sperm showed that BLVRA is found on all sperm recovered from the caput region, whereas it is undetectable on cauda sperm. biliverdin and bilirubin are found in higher concentrations in the caput epididymal fluid, as measured by mass spectrometry. Lipid peroxidation was limited by 1 μM of biliverdin, but not bilirubin when caput spermatozoa were challenged with 500 μM H2O2. Since immature spermatozoa are a source of reactive oxygen species, BLVRA may be involved in the protection of maturing spermatozoa. It is also plausible that BLVRA is implicated in haemic protein catabolism in the epididymal luminal environment. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-30T02:18:00.222198-05:
      DOI: 10.1002/jcp.25200
  • Table of Contents: Volume 231, Number 1
    • PubDate: 2015-09-28T16:08:41.616187-05:
      DOI: 10.1002/jcp.25194
  • Highlights: Volume 231, Number 1
    • PubDate: 2015-09-28T16:08:30.6357-05:00
      DOI: 10.1002/jcp.25195
  • Editor's Choice
    • PubDate: 2015-09-28T16:08:24.598244-05:
      DOI: 10.1002/jcp.25193
  • Epigenetic Modulation in Periodontitis: Interaction of Adiponectin and
           JMJD3‐IRF4 Axis in Macrophages
    • Authors: Dongying Xuan; Qianqian Han, Qisheng Tu, Lan Zhang, Liming Yu, Dana Murry, Tianchi Tu, Jane Lian, Gary S. Stein, Jincai Zhang, Jake Chen
      Abstract: Emerging evidence suggests an important role for epigenetic mechanisms in modulating signals during macrophage polarization and inflammation. JMJD3, a JmjC family histone demethylase necessary for M2 polarization is also required for effective induction of multiple M1 genes by lipopolysaccharide (LPS). However, the effects of JMJD3 to inflammation in the context of obesity remains unknown. To address this deficiency, we firstly examined the expression of JMJD3 in macrophage isolated from bone marrow and adipose tissue of diet induced obesity (DIO) mice. The results indicated that JMJD3 was down‐regulated in obesity. Adiponectin (APN), a factor secreted by adipose tissue which is down‐regulated in obesity, functions to switch macrophage polarization from M1 to M2 (Jenke et al., 2014), thereby attenuating chronic inflammation. Intriguingly, our results indicated that APN contributed to JMJD3 up‐regulation, reduced macrophage infiltration in obese adipose tissue, and abolished the up‐regulation of JMJD3 in peritoneal macrophages isolated from DIO mice when challenged with Porphyromonas gingivalis LPS (pg.lps). To elucidate the interaction of APN and JMJD3 involved in macrophage transformation in the context of inflammation, we designed the loss and gain‐function experiments of APN in vivo with APN‐/‐ mice with experimental periodontitis and in vitro with macrophage isolated from APN ‐/‐ mice. For the first time, we found that APN can help to reduce periodontitis‐related bone loss, modulate JMJD3 and IRF4 expression and macrophage infiltration. Therefore, it can be inferred that APN may contribute to anti‐inflammation macrophage polarization by regulating JMJD3 expression, which provides a basis for macrophage‐centered epigenetic therapeutic strategies. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-24T06:51:20.993116-05:
      DOI: 10.1002/jcp.25201
  • Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently
           Generated from Hemogenic Endothelial Cells in Human Pluripotent Stem Cells
    • Authors: Hao Bai; Yanfeng Liu, Yinliang Xie, Dixie L. Hoyle, Robert A. Brodsky, Linzhao Cheng, Tao Cheng, Zack Z. Wang
      Abstract: Generation of fully functional hematopoietic multipotent progenitor (MPP) cells from human pluripotent stem cells (hPSCs) has a great therapeutic potential to provide an unlimited cell source for treatment of hematological disorders. We previously demonstrated that CD34+CD31+CD144+ population derived from hPSCs contain hemato‐endothelial progenitors (HEPs) that give rise to hematopoietic and endothelial cells. Here, we report a differentiation system to generate definitive hematopoietic MPP cells from HEPs via endothelial monolayer. In the presence of angiogenic factors, HEPs formed an endothelial monolayer, from which hematopoietic clusters emerged through the process of endothelial‐to‐hematopoietic transition (EHT). EHT was significantly enhanced by hematopoietic growth factors. The definitive MPP cells generated from endothelial monolayer were capable of forming multilineage hematopoietic colonies, giving rise to T lymphoid cells, and differentiating into enucleated erythrocytes. Emergence of hematopoietic cells from endothelial monolayer occurred transiently. Hematopoietic potential was lost during prolonged culture of HEPs in endothelial growth conditions. Our study demonstrated that CD34+CD31+CD144+ HEPs gave rise to hematopoietic MPP cells via hemogenic endothelial cells that exist transiently. The established differentiation system provides a platform for future investigation of regulatory factors involved in de novo generation of hematopoietic MPP cells and their applications in transplantation. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-23T04:29:09.590376-05:
      DOI: 10.1002/jcp.25199
  • Downhill Running Excessive Training Inhibits Hypertrophy in Mice Skeletal
           Muscles with Different Fiber Type Composition
    • Abstract: The aim of this study was to verify the effects of running overtraining protocols performed in downhill, uphill and without inclination on the proteins related to hypertrophy signaling pathway in extensor digitorum longus (EDL) and soleus of C57BL/6 mice. We also performed histological and stereological analyses. Rodents were divided into control (CT; sedentary mice), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR). The incremental load, exhaustive and grip force tests were used as performance evaluation parameters. 36h after the grip force test, EDL and soleus were removed and immediately used for immunoblotting analysis or stored at ‐80°C for histological and stereological analyses. For EDL, OTR/down decreased the protein kinase B (Akt) and tuberous sclerosis protein 2 (TSC2) phosphorylation (p), and increased myostatin, receptor‐activated Smads (pSMAD2‐3) and insulin receptor substrate‐1 (pIRS‐1; Ser307/636). OTR/down also presented low and high relative proportions of cytoplasm and connective tissue, respectively. OTR/up increased the mammalian target of rapamycin (pmTOR), 70‐kDa ribosomal protein S6 kinase 1 (pS6K1) and pSMAD2‐3, and decreased pTSC2. OTR decreased pTSC2 and increased pIRS‐1 (Ser636). For soleus, OTR/down increased S6 ribosomal protein (pS6RP) and pSMAD2‐3, and decreased pIRS‐1 (Ser639). OTR/up decreased pS6K1, pS6RP and pIRS‐1 (Ser639), and increased pTSC2 (Ser939) and pSMAD2‐3. OTR increased pS6RP, 4E‐binding protein‐1 (p4E‐BP1), pTSC2 (Ser939), and pSMAD2‐3, and decreased pIRS‐1 (Ser639). In summary, OTR/down inhibited the skeletal muscle hypertrophy with concomitant signs of atrophy in EDL. The effects of OTR/up and OTR depended on the analyzed skeletal muscle type. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T06:49:06.822849-05:
      DOI: 10.1002/jcp.25197
  • Mechanical Loading Stimulates Expression of Collagen Cross‐Linking
           Associated Enzymes in Periodontal Ligament
    • Authors: Masaru Kaku; Juan Marcelo Rosales Rocabado, Megumi Kitami, Takako Ida, Yosuke Akiba, Mitsuo Yamauchi, Katsumi Uoshima
      Abstract: Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post‐translationally modified by a series of specific enzymes. Among the collagen‐modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross‐linking and lysyl hydroxylases (LHs) to regulate the cross‐linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen‐modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intraperitoneal injection of a LOX inhibitor, β‐aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL‐derived cells. The mechanical loading also elevated formation of collagen α‐chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2‐positive cells was observed only at the bone‐side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up‐regulating collagen‐modifying enzymes and subsequent collagen cross‐linking which are important for PDL tissue maintenance. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T04:03:40.772205-05:
      DOI: 10.1002/jcp.25184
  • Mitotic Inheritance of mRNA Facilitates Translational Activation of the
           Osteogenic‐Lineage Commitment Factor Runx2 in Progeny of
           Osteoblastic Cells
    • Authors: Nelson Varela; Alejandra Aranguiz, Carlos Lizama, Hugo Sepulveda, Marcelo Antonelli, Roman Thaler, Ricardo D. Moreno, Martin Montecino, Gary S. Stein, Andre J. van Wijnen, Mario Galindo
      Abstract: Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype‐committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post‐mitotic re‐activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone‐related runt‐related transcription factor Runx2. Here, we characterized a non‐genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self‐renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non‐chromosomal tubulin‐containing compartment. Subsequently, transcription‐independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast‐specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T04:02:34.052494-05:
      DOI: 10.1002/jcp.25188
  • TRPV4 Channels in Human White Adipocytes: Electrophysiological
           Characterization and Regulation by Insulin
    • Abstract: Intracellular calcium homeostasis in adipocytes is important for the regulation of several functions and is involved in pathological changes in obesity and other associated diseases. TRPV4 (Transient Receptor Potential Vanilloid 4) channels are an important route for calcium entry that operates in a variety of cells and intervenes in a number of functions. In this study, the expression and operation of TRPV4 channels in human cultured adipocytes was evaluated using RT‐PCR, Western blotting, the whole‐cell patch‐clamp technique and fluorescence measurements to characterize these channels and determine intracellular calcium responses. Both the hypoosmolarity and 4αPDD (4alpha‐phorbol‐didecanoate), a specific TRPV4 agonist, induced a similar HC‐067047‐sensitive current, which was predominantly inward, and an intracellular Ca2+ concentration increase, which was exclusively dependent on extracellular calcium, and membrane depolarization. The current had a reverse potential of +31 ± 6 mV and exhibited preferential permeability to Ca2+. Insulin, which regulates metabolic homeostasis in adipocytes, attenuated the TRPV4‐mediated effects. These results confirm the function of TRPV4 in human cultured adipocytes and its regulation by insulin. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T03:56:29.75762-05:0
      DOI: 10.1002/jcp.25187
  • Prolyl Endopeptidase (PREP) is Associated with Male Reproductive Functions
           and Gamete Physiology in Mice
    • Authors: Raffaele Dotolo; Jung Dae Kim, Paolo Pariante, Sergio Minucci, Sabrina Diano
      Abstract: Prolyl endopeptidase (PREP) is a serine protease which has been implicated in many biological processes, such as the maturation and degradation of peptide hormones and neuropeptides, learning and memory, cell proliferation and differentiation, and glucose metabolism. A small number of reports have also suggested PREP participation in both male and female reproduction‐associated processes. In the present work we examined PREP distribution in male germ cells and studied the effects of its knockdown (Prepgt/gt) on testis and sperm in adult mice. The protein is expressed and localized in elongating spermatids and luminal spermatozoa of wild type (wt) mice, as well as Sertoli, Leydig and peritubular cells. PREP is also expressed in the head and midpiece of epididymal spermatozoa, whereas the remaining tail region shows a weaker signal. Furthermore, testis weight, histology of seminiferous tubules, and epididymal sperm parameters were assessed in wt and Prepgt/gt mice: wild type testes have larger average tubule and lumen diameter; in addition, lumenal composition of seminiferous tubules is dissimilar between wt and Prepgt/gt, as the percentage of spermiated tubules is much higher in wt. Finally, total sperm count, sperm motility and normal morphology are also higher in wt than in Prepgt/gt. These results show for the first time that the expression of PREP could be necessary for a correct reproductive function, and suggest that the enzyme may play a role in mouse spermatogenesis and sperm physiology. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T02:47:20.832753-05:
      DOI: 10.1002/jcp.25178
  • Alterations of the Plasma Peptidome Profiling in Colorectal Cancer
    • Authors: Chiara Bedin; Sara Crotti, Eugenio Ragazzi, Salvatore Pucciarelli, Lisa Agatea, Ennio Tasciotti, Mauro Ferrari, Pietro Traldi, Flavio Rizzolio, Antonio Giordano, Donato Nitti, Marco Agostini
      Abstract: Early detection of colorectal cancer (CRC) remains a challenge. It has been highlighted that the pathological alterations within an organ and tissues might be reflected in serum or plasma proteomic/peptidic patterns. The aim of the study was to follow the changes in the plasma peptides associated to colorectal cancer progression by mass spectrometry. This study included 27 adenoma, 67 CRC (n = 33 I‐II stage and n = 34 III‐IV stage), 23 liver metastasis from CRC patients and 34 subjects disease‐free as controls. For plasma peptides analysis, samples purification was performed on the Nanoporous Silica Chips technology followed by matrix‐assisted laser desorption/ionisation‐time of flight analysis. Since the high complexity of the obtained dataset, multivariate statistical analysis and discriminant pattern recognition were performed for study groups classification. Forty‐four of 88 ionic species were successfully identified as fragments of peptides and proteins physiologically circulating in the blood and belonging to immune and coagulation systems and inflammatory mediators. Many peptides clustered into sets of overlapping sequences with ladder‐like truncation clearly associated to proteolytic processes of both endo‐ and exoproteases activity. Comparing to controls, a different median ion intensity of the group‐type fragments distribution was observed. Moreover, the degradation pattern obtained by proteolytic cleavage was different into study groups. This pattern was specific and characteristic of each group: controls, colon tumour disease (including adenoma and CRC) and liver metastasis, revealing a role as biomarker in early diagnosis and prognosis. Our findings highlighted peculiar changes in protease activity characteristic of CRC progression from pre‐cancer lesion to metastatic disease. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-17T07:24:37.275826-05:
      DOI: 10.1002/jcp.25196
  • Alpha‐Catulin Co‐Localizes with Vimentin Intermediate
           Filaments and Functions in Pulmonary Vascular Endothelial Cell Migration
           via ROCK
    • Authors: Michael D. Bear; Tiegang Liu, Shereen Abualkhair, Maher A. Ghamloush, Nicholas S. Hill, Ioana Preston, Barry L. Fanburg, Usamah S. Kayyali, Deniz Toksoz
      Abstract: The ubiquitous α‐catulin acts as a scaffold for distinct signalosomes including RhoA/ROCK; however, its function is not well understood. While α‐catulin has homology to the cytoskeletal linkers α‐catenin and vinculin, it appears to be functionally divergent. Here we further investigated α‐catulin function in pulmonary vascular endothelial cells (VEC) on the premise that α‐catulin has a unique cytoskeletal role. Examination of endogenous α‐catulin intracellular localization by immunofluorescence revealed a highly organized cytosolic filamentous network suggestive of a cytoskeletal system in a variety of cultured VEC. Double‐immunofluorescence analyses of VEC showed endogenous α‐catulin co‐localization with vimentin intermediate filaments. Similar to vimentin, α‐catulin was found to distribute into detergent‐soluble and ‐insoluble fractions. Treatment of VEC with withaferinA, an agent that targets vimentin filaments, disrupted the α‐catulin network distribution and altered α‐catulin solubility. Vimentin participates in cell migration, and withaferinA was found to inhibit VEC migration in vitro; similarly, α‐catulin knock‐down reduced VEC migration. Based on previous reports showing that ROCK modulates vimentin, we found that ROCK depletion attenuated VEC migration; furthermore, α‐catulin depletion was shown to reduce ROCK‐induced signaling. These findings indicate that α‐catulin has a unique function in co‐localization with vimentin filaments that contributes to VEC migration via a pathway that may involve ROCK signaling. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-17T04:29:20.726411-05:
      DOI: 10.1002/jcp.25185
  • HhAntag, a Hedgehog Signaling Antagonist, Suppresses Chondrogenesis and
           Modulates Canonical and Non‐Canonical BMP Signaling
    • Authors: Christina Mundy; Adebayo Bello, Federica Sgariglia, Eiki Koyama, Maurizio Pacifici
      Abstract: Chondrogenesis subtends the development of most skeletal elements and involves mesenchymal cell condensations differentiating into growth plate chondrocytes that proliferate, undergo hypertrophy and are replaced by bone. In the pediatric disorder Hereditary Multiple Exostoses, however, chondrogenesis occurs also at ectopic sites and causes formation of benign cartilaginous tumors –exostoses‐ near the growth plates. No treatment is currently available to prevent or reverse exostosis formation. Here, we asked whether chondrogenesis could be stopped by targeting the hedgehog pathway, one of its major regulators. Micromass cultures of limb mesenchymal cells were treated with increasing amounts of the hedgehog inhibitor HhAntag or vehicle. The drug effectively blocked chondrogenesis and did so in a dose‐dependent manner as monitored by: alcian blue‐positive cartilage nodule formation; gene expression of cartilage marker genes; and reporter activity in Gli1‐LacZ cell cultures. HhAntag blocked chondrogenesis even when the cultures were co‐treated with bone morphogenetic protein 2 (rhBMP‐2), a strong pro‐chondrogenic factor. Immunoblots showed that HhAntag action included modulation of canonical (pSmad1/5/8) and non‐canonical (pp38) BMP signaling. In cultures co‐treated with HhAntag plus rhBMP‐2, there was a surprising strong up‐regulation of pp38 levels. Implantation of rhBMP‐2‐coated beads near metacarpal elements in cultured forelimb explants induced formation of ectopic cartilage that however, was counteracted by HhAntag co‐treatment. Collectively, our data indicate that HhAntag inhibits not only hedgehog signaling, but also modulates canonical and non‐canonical BMP signaling and blocks basal and rhBMP2‐stimulated chondrogenesis, thus representing a potentially powerful drug‐based strategy to counter ectopic cartilage growth or induce its involution. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-12T09:39:16.573423-05:
      DOI: 10.1002/jcp.25192
  • Lipidomics of Mesenchymal Stromal Cells: Understanding the Adaptation of
           Phospholipid Profile in Response to Pro‐Inflammatory Cytokines
    • Abstract: Mesenchymal stromal cells (MSCs) present anti‐inflammatory properties and are being used with great success as treatment for inflammatory and autoimmune diseases. In clinical applications MSCs are subjected to a strong pro‐inflammatory environment, essential to their immunosuppressive action. Despite the wide clinical use of these cells, how MSCs exert their effect remains unclear. Several lipids are known to be involved in cell's signaling and modulation of cellular functions. The aim of this paper is to examine the variation in lipid profile of MSCs under pro‐inflammatory environment, induced by the presence of tumor necrosis factor alpha (TNF‐α) and interferon gamma (IFN‐γ), using the most modern lipidomic approach. Major changes in lipid molecular profile of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysoPC (LPC) and sphingomyelin (SM) classes were found. No changes were observed in the phosphatidylinositol (PI) profile. The levels of PC species with shorter fatty acids (FAs) acyl chains, mainly C16:0, decreased under pro‐inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of LPC(18:0), which is known to be an anti‐inflammatory LPC, observed in MSCs subjected to TNF‐α and IFN‐γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF‐α and IFN‐γ also enhanced the levels of PE(40:6) and decreased the levels of PE(O‐38:6). Higher expression of PS(36:1) and SM(34:0) along with a decrease in PS(38:6) levels were observed. These results indicate that lipid metabolism and signaling are modulated during MSCs activation, which suggests that lipids may be involved in MSCs functional and anti‐inflammatory activities. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-12T09:38:24.556302-05:
      DOI: 10.1002/jcp.25191
  • Review: Tumor Microenvironment Versus Cancer Stem Cells in
           Cholangiocarcinoma: Synergistic Effects'
    • Authors: Maurizio Romano; Francesco De Francesco, Enrico Gringeri, Antonio Giordano, Giuseppe A. Ferraro, Marina Di Domenico, Umberto Cillo
      Abstract: Cholangiocarcinoma (CCAs) may be defined as tumors that derived from the biliary tree with the differentiation in the biliary epithelial cells. This tumor is malignant, extremely aggressive with a poor prognosis. It can be treated surgically and its pathogenesis is poorly understood. The tumor microenvironment (TME) is a very important factor in the regulation of tumor angiogenesis, invasion, and metastasis. Besides cancer stem cells (CSCs) can modulate tumor growth, stroma formation and migratory capability. The initial stage of tumorigenesis is characterized by genetic mutations and epigenetic alterations due to intrinsic factors which lead to the generation of oncogenes thus inducing tumorigenesis. CSCs may result from precancerous stem cells, cell de‐differentiation, normal stem cells or an epithelial‐mesenchymal transition (EMT). CSCs have been found in the cancer niche, and EMT may occur early within the tumor microenvironment. Previous studies have demonstrated evidence of cholangiocarcinoma stem cells (CD133, CD24, EpCAM, CD44, and others) and the presence of these markers has been associated with malignant potential. The interaction between TME and cholangiocarcinoma stem cells via signaling mediators may create an environment that accommodates tumor growth, yielding resistance to cytotoxic insults (chemotherarapeutic). While progress has been made in the understanding of the mechanisms, the interactions in the tumorigenic process still remain a major challenge. Our review, addresses recent concepts of TME‐CSCs interaction and will emphasize the importance of early detection with the use of novel diagnostic mechanisms such as CCA‐CSC biomarkers and the importance of tumor stroma to define new treatments. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-11T04:31:25.258196-05:
      DOI: 10.1002/jcp.25190
  • Glucose and Inflammatory Cells Decrease Adiponectin in Epicardial Adipose
           Tissue Cells: Paracrine Consequences on Vascular Endothelium
    • Abstract: Epicardial adipose tissue (EAT) is a source of energy for heart that expresses the insulin‐sensitizer, anti‐inflammatory and anti‐atherogenic protein, adiponectin. But, in coronary artery disease, adiponectin production declines. Our objective was to determine its regulation by glucose and inflammation in stromal cells from EAT and subcutaneous adipose tissue (SAT) and its paracrine effect on endothelial cells. Stromal cells of EAT and SAT were obtained from patients who underwent cardiac surgery. Adipogenesis was induced at 117, 200 or 295 mg/dL glucose, with or without macrophage‐conditioned medium (MCM). Expression of adiponectin, GLUT‐4 and the insulin receptor was analyzed by real‐time PCR. The paracrine effect of stromal cells was determined in co‐cultures with endothelial cells, by exposing them to high glucose and/or MCM, and, additionally, to leukocyte‐conditioned medium from patients with myocardial infarction. The endothelial response was determined by analyzing vascular adhesion molecule expression. Our results showed a U‐shaped dose‐response curve of glucose on adiponectin in EAT, but not in SAT stromal cells. Conversely, MCM reduced the adipogenesis‐induced adiponectin expression of EAT stromal cells. The presence of EAT stromal increased the inflammatory molecules of endothelial cells. This deleterious effect was emphasized in the presence of inflammatory cell‐conditioned medium from patients with myocardial infarction. Thus, high glucose and inflammatory cells reduced adipogenesis‐induced adiponectin expression of EAT stromal cells, which induced an inflammatory paracrine process in endothelial cells. This inflammatory effect was lower in presence of mature adipocytes, producers of adiponectin. These results contribute to understanding the role of EAT dysfunction on coronary atherosclerosis progression. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-10T06:38:31.719517-05:
      DOI: 10.1002/jcp.25189
  • Neurotransmitters and Neuropeptides in the Endocrine Pancreas: New Players
           in the Control of Islet Cells Mass and Function
    • Authors: Eliana S. Di Cairano; Stefania Moretti, Paola Marciani, Vellea Franca Sacchi, Michela Castagna, Alberto Davalli, Franco Folli, Carla Perego
      Abstract: Islets of Langerhans control whole body glucose homeostasis, as they respond, releasing hormones, to changes in nutrient concentrations in the blood stream. The regulation of hormone secretion has been the focus of attention for a long time because it is related to many metabolic disorders, including diabetes mellitus. Endocrine cells of the islet use a sophisticate system of paracrine and autocrine signals to synchronize their activities. These signals provide a fast and accurate control not only for hormone release but also for cell differentiation and survival, key aspects in islet physiology and pathology. Among the different categories of paracrine/autocrine signals, this review highlights the role of neurotransmitters and neuropeptides. In a manner similar to neurons, endocrine cells synthesize, accumulate, release neurotransmitters in the islet milieu, and possess receptors able to decode these signals. In this review, we provide a comprehensive description of neurotransmitter/neuropetide signaling pathways present within the islet. Then, we focus on evidence supporting the concept that neurotransmitters/neuropeptides and their receptors are interesting new targets to preserve β‐cell function and mass. A greater understanding of how this network of signals works in physiological and pathological conditions would advance our knowledge of islet biology and physiology and uncover potentially useful areas of pharmacological intervention. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-10T02:41:38.974466-05:
      DOI: 10.1002/jcp.25176
  • Osteosarcoma Stem Cells Have Active Wnt/β‐Catenin and
           Overexpress SOX2 and KLF4
    • Abstract: Osteosarcoma is a bone tumor displaying significant cellular and histological heterogeneity and a complex genetic phenotype. Although multiple studies strongly suggest the presence of cancer stem cells in osteosarcoma a consensus on their characterization is still missing. We used a combination of functional assays (sphere‐forming, Aldefluor and side‐population) for identification of cancer stem cell populations in osteosarcoma cell lines. Expression of stemness‐related transcription factors, quiescent nature, in vivo tumorigenicity and Wnt/β‐catenin activation were evaluated. We show that different cancer stem cell populations may co‐exist in osteosarcoma cell lines exhibiting distinct functional properties. Osteosarcoma spheres are slowly‐proliferating populations, overexpress SOX2 and KLF4 stemness‐related genes and have enhanced tumorigenic potential. Additionally, spheres show specific activation of Wnt/β‐catenin signaling as evidenced by increased nuclear β‐catenin, TCF/LEF activity and AXIN2 expression, in a subset of the cell lines. Aldefluor‐positive populations were detected in all osteosarcoma cell lines and overexpress SOX2, but not KLF4. The side‐population phenotype is correlated with ABCG2 drug‐efflux transporter expression. Distinct functional methods seem to identify cancer stem cells with dissimilar characteristics. Intrinsic heterogeneity may exist within osteosarcoma cancer stem cells and can have implications on the design of targeted therapies aiming to eradicate these cells within tumors. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T04:44:02.134755-05:
      DOI: 10.1002/jcp.25179
  • Kidney Injury Molecule‐1 Enhances Endocytosis of Albumin in Renal
           Proximal Tubular Cells
    • Authors: Xueying Zhao; Chen Jiang, Rebecca Olufade, Dong Liu, Nerimiah Emmett
      Abstract: Receptor‐mediated endocytosis plays an important role in albumin reabsorption by renal proximal tubule epithelial cells. Kidney injury molecule‐1 (KIM‐1) is a scavenger receptor that is upregulated on the apical membrane of proximal tubules in proteinuric kidney disease. In this study, we examined the cellular localization and functional role of KIM‐1 in cultured renal tubule epithelial cells (TECs). Confocal immunofluorescence microscopy reveals intracellular and cell surface localization of KIM‐1 in primary renal TECs. Albumin stimulation resulted in a redistribution of KIM‐1 and tight junction protein zonula occludens‐1 in primary TEC monolayer. An increase in albumin internalization was observed in both primary TECs expressing endogenous KIM‐1 and rat kidney cell line (NRK‐52E) overexpressing exogenous KIM‐1. KIM‐1‐induced albumin accumulation was abolished by its specific antibody. Moreover, endocytosed KIM‐1 and its cargo proteins were delivered from endosomes to lysosomes for degradation in a clathrin‐dependent pathway. Supportive evidence includes a) detection of KIM‐1 in Rab5‐positive early endosomes, Rab7‐positive late endosomes/multivesicular bodies, and LAMP1‐positive lysosomes, b) colocalization of KIM‐1 and clathrin in the intracellular vesicles, and c) blockade of KIM‐1‐mediated albumin internalization by chlorpromazine, an inhibitor of clathrin‐dependent endocytosis. KIM‐1 expression was upregulated by albumin but downregulated by transforming growth factor‐β1. Taken together, our data indicate that KIM‐1 increases albumin endocytosis in renal tubule epithelial cells, at least partially via a clathrin‐dependent mechanism. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T04:39:37.965251-05:
      DOI: 10.1002/jcp.25181
  • Modulating Intrafollicular Hormonal Milieu in Controlled Ovarian
           Stimulation: Insights from PPAR Expression in Human Granulosa Cells
    • Abstract: Controlled ovarian stimulation (COS) leading to ovulation of multiple follicles is a crucial aspect of biomedical infertility care. Nevertheless, biomarkers useful for COS management are still lacking. Peroxisome proliferator‐activated receptors (PPARs) are nuclear hormone receptors relevant to steroid metabolism in granulosa cells (GCs). We investigated whether PPARs and their steroidogenic targets were differentially expressed in GCs differentiated under different recombinant or urinary gonadotropin preparations. GCs from women subjected to COS with r‐hFSH, r‐hFSH/r‐hLH or hMG‐HP were processed to assess expression of PPARα, PPARβ/δ, PPARγ and steroidogenic enzymes under PPAR modulation. As an evidence of their activation, all PPAR isotypes with their coactivators, the retinoic‐X‐receptors (RXRs), localized in the nucleus. When GCs from r‐hFSH/r‐hLH group were compared with r‐hFSH, a significant reduction of PPARα protein was observed. By contrast, an increase of PPARβ/δ at both protein and mRNA levels along with that of PPARγ protein were detected. The steroidogenic enzymes 17βHSD IV, 3βHSD II and HMG‐CoA red were downregulated in the r‐hFSH/r‐hLH group in comparison to r‐hFSH unlike CYP19A1 that remained unchanged. In GCs from urinary FSH‐LH stimulation (hMG‐HP), PPARα was more expressed in comparison with r‐hFSH/r‐hLH group. Likewise, 3βHSD II and 17βHSD IV were increased suggesting that hMG‐HP partially mimicked r‐hFSH/r‐hLH effects. In summary, transcript analysis associated to protein investigation revealed differential effects of COS protocols on PPARs and their steroidogenic targets in relation to LH and gonadotropin source. These observations candidate PPARs as new biomarkers of follicle competence opening new hypotheses on COS effects on ovarian physiology. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T04:35:04.594288-05:
      DOI: 10.1002/jcp.25182
  • Downregulation of Long Noncoding RNA Meg3 Affects Insulin Synthesis and
           Secretion in Mouse Pancreatic Beta Cells
    • Authors: LiangHui You; Ning Wang, DanDan Yin, LinTao Wang, FeiYan Jin, YaNan Zhu, QingXin Yuan, Wei De
      Abstract: Increasing evidence indicates that long noncoding RNAs (lncRNAs) are involved in diverse biological process. Mouse maternal expressed gene 3 (Meg3), is an imprinted gene and essential for development. Here, we explored the relationship between Meg3 and the function of mouse beta cells in vitro and vivo. Real‐time PCR analyses revealed Meg3 was more abundantly expressed in Balb/c mouse islets than exocrine glands. Moreover, the expression of Meg3 in islets was decreased in T1DM (NOD female mice) and T2DM (db/db mice) models. Meg3 expression was modulated dynamically by glucose in Min6 cells and isolated mouse islets. The function role of Meg3 was investigated in Min6 cells and normal mouse by knockdown of Meg3 using small interfering RNA. After suppression of Meg3 expression in vitro, insulin synthesis and secretion were impaired and the rate of beta cells apoptosis was increased. Moreover, knockdown of Meg3 in vivo led to the impaired glucose tolerance and decreased insulin secretion, consisted with the reduction of insulin positive cells areas by immunochemistry assays. Notably, islets from Meg3 interference groups showed significant decrease of Pdx‐1 and MafA expression in mRNA and protein levels. These results indicate that Meg3 may function as a new regulator of maintaining beta cells identity via affecting insulin production and cell apoptosis. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T04:04:54.515521-05:
      DOI: 10.1002/jcp.25175
  • Circulating MicroRNA and Long Noncoding RNA as Biomarkers of
           Cardiovascular Diseases
    • Authors: Qiang Shi; Xi Yang
      Abstract: Although >90 of the human genome is transcribed, only  200 nucleotides). It is now clear that these RNAs fulfil critical roles as transcriptional and post‐transcriptional regulators and as guides of chromatin‐modifying complexes. Although not translated into protein, noncoding RNAs can regulate cardiac function through diverse mechanisms and their dysregulation is increasingly linked with cardiovascular pathophysiology. Furthermore, a series of recent studies have discovered that noncoding RNAs can be found in the bloodstream and some species are remarkably stable. This has raised the possibility that such noncoding RNAs may be measured in body fluids and serve as novel diagnostic biomarkers. Here, we summarize the current knowledge of noncoding RNAs' function and biomarker potential in cardiac diseases, concentrating mainly on circulating miRNAs and lncRNAs. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T03:49:20.492542-05:
      DOI: 10.1002/jcp.25174
  • Beta Adrenergic Receptor Stimulation Suppresses Cell Migration in
           Association With Cell Cycle Transition in Osteoblasts – Live Imaging
           Analyses Based on FUCCI System
    • Authors: Sakie Katsumura; Yoichi Ezura, Yayoi Izu, Jumpei Shirakawa, Atsushi Miyawaki, Kiyoshi Harada, Masaki Noda
      Abstract: Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed‐ridden conditions in patients suffering from aging‐associated diseases including cardiovascular, neurological and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed‐ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis is still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppresses bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, FUCCI (Fluorescent Ubiquitination‐based Cell Cycle Indicator), in osteoblast biology and examined isoproterenol regulation of cell cycle transition cell migration in osteoblasts. Isoproterenol treatment suppresses the first entry peak of quiescent osteoblastic cells into cell cycle phase from G1/G0 to S/G2/M and also suppresses second major peak population size that enters into S/G2/M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1/S nor in G2/M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T03:42:30.977253-05:
      DOI: 10.1002/jcp.25096
  • Systematic Review and Meta‐Analysis: Circulating miRNAs for
           Diagnosis of Hepatocellular Carcinoma
    • Abstract: Because early‐stage hepatocellular carcinoma (HCC) is difficult to diagnose using the existing techniques, identifying better biomarkers would likely improve the patients' prognoses. We performed a systematic review and meta‐analysis of published studies to appraise the utility of microRNAs (miRNAs) for the early diagnosis of HCC. Pertinent literature was collected from the Medline, Embase, and Chinese National Knowledge Infrastructure databases. We analyzed 50 studies that included 3423 cases of HCC, 2403 chronic hepatic disease (CH) patients, and 1887 healthy controls in 16 articles. Summary receiver operating characteristic analyses of all miRNAs showed an area under the curve (AUC) of 0.82, with 75.8% sensitivity and 75.0% specificity in discriminating patients with HCC from healthy controls. miR‐21 and miR‐122 individually distinguished patients with HCC from healthy controls, with an AUC of 0.88 for miR‐21 and 0.77 for miR‐122. The sensitivity and specificity for miR‐21 were 86.6% and 79.5%, respectively; those for miR‐122 were 68.0% and 73.3%. We conclude that circulating miRNAs, particularly miR‐21 and miR‐122, are promising biomarkers for the early diagnosis of HCC. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T03:39:08.751175-05:
      DOI: 10.1002/jcp.25135
  • Peripheral Blood Mononuclear Cells Spontaneous Osteoclastogenesis:
    • Abstract: In vitro peripheral blood mononuclear cells differentiate into osteoclasts under the influence of osteoclast‐stimulating factors. However, accumulating evidence suggests spontaneous osteoclasts formation and activity in patients affected by local or systemic bone remodeling diseases in comparison with healthy controls. Therefore, within this review, we summarize the studies where spontaneous osteoclastogenesis of peripheral blood mononuclear cells was observed in pathological conditions of the skeletal system. We indicate a linkage between immunoregulation by T cells and spontaneous osteoclasts formation with increased levels of tumor necrosis factors‐α, receptor activator of nuclear factor kappa‐B ligand and inteleukin‐7 production. In the light of these results, it would be of crucial importance to deepen the correlation between systemic bone remodeling diseases and spontaneous osteoclastogenesis as well as to investigate in detail the mechanisms underlying this phenomenon and the clinical relevance in bone remodeling disease diagnosis and monitoring. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T03:32:50.397314-05:
      DOI: 10.1002/jcp.25134
  • Stress‐Induced Activation of Apoptosis Signal‐Regulating
           Kinase 1 Promotes Osteoarthritis
    • Abstract: Apoptosis signal‐regulated kinase 1 (ASK1) has been shown to affect a wide range of cellular processes including stress‐related responses, cytokine and growth factor signaling, cell cycle and cell death. Recently, we reported that lack of ASK1 slowed chondrocyte hypertrophy, terminal differentiation and apoptosis resulting in an increase in trabecular bone formation. Herein, we investigated the role of ASK1 in the pathogenesis of osteoarthritis (OA). Immunohistochemistry performed on articular cartilage samples from patients with OA showed ASK1 expression increased with OA severity. In vitro analysis of chondrocyte hypertrophy, maturation and ASK1 signaling in embryonic fibroblasts from ASK1 knockout (KO) and wild type (WT) mice was examined. Western analysis demonstrated an increase in ASK1 signaling commensurate with chondrogenic maturation during differentiation or in response to stress by the cytokines, tumor necrosis factor alpha or interleukin 1 beta in WT, but not in ASK1 KO embryonic fibroblasts. Surgically induced moderate or severe OA or OA due to natural aging in WT and ASK1 KO mice was assessed by microCT of subchondral bone, immunohistochemistry, histology and OARSI scoring. Immunohistochemistry, microCT and OARSI scoring all indicated that the lack of ASK1 protected against OA joint degeneration, both in surgically induced OA and in aging mice. We propose that the ASK1 MAP kinase signaling cascade is an important regulator of chondrocyte terminal differentiation and inhibitors of this pathway could be useful for slowing chondrocyte maturation and cell death observed with OA progression. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-07T05:44:12.455325-05:
      DOI: 10.1002/jcp.25186
  • Epigenetic Priming Confers Direct Cell Trans‐Differentiation from
           Adipocyte to Osteoblast in a Transgene‐Free State
    • Abstract: The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans‐differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5′‐aza‐dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3‐L1 adipocytes with 5′‐aza‐dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5′‐aza‐dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue‐engineering applications. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-03T17:22:44.577997-05:
      DOI: 10.1002/jcp.25183
  • BMP‐2 Enhances Lgr4 Gene Expression in Osteoblastic Cells
    • Authors: Chantida Pawaputanon Na Mahasarakham; Yoichi Ezura, Makiri Kawasaki, Arayal Smriti, Shuichi Moriya, Takayuki Yamada, Yayoi Izu, Katsuhiko Nishimori, Yuichi Izumi, Masaki Noda
      Abstract: Osteoporosis is one of the most prevalent diseases and the number of patients suffering from this disease is soaring due to the increase in the aged population in the world. The severity of bone loss in osteoporosis is based on the levels of impairment in the balance between bone formation and bone resorption, two arms of the bone metabolism and bone remodeling. However, determination of bone formation levels is under many layers of control that are as yet fully defined. Bone morphogenetic protein (BMP) plays a key role in regulation of bone formation while its downstream targets are still incompletely understood. Lgr4 gene encodes an orphan receptor and has been identified as a genetic determinant for bone mass in osteoporotic patients. Here, we examine the effects of BMP on the expression of Lgr4 in osteoblastic cells. Lgr4 gene is expressed in an osteoblastic cell line, MC3T3E1 in a time dependent manner during the culture. BMP treatment enhances Lgr4 mRNA expression at least in part via transcriptional event. When Lgr4 mRNA is knocked down, the levels of BMP‐induced increase in alkaline phosphatase (Alp) activity and Alp mRNA are suppressed. BMP enhancement of Lgr4 gene expression is suppressed by FGF and reversed by dexamethasone. BMP also enhances Lgr4 expression in primary cultures of calvarial osteoblasts. These data indicate that Lgr4 gene is regulated by BMP and is required for BMP effects on osteoblastic differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-02T17:08:43.507094-05:
      DOI: 10.1002/jcp.25180
  • Mesenchymal Stromal Cells Differentiating to Adipocytes Accumulate
           Autophagic Vesicles Instead of Functional Lipid Droplets
    • Abstract: Adult bone marrow mesenchymal stromal cells (BMSCs) can easily be differentiated into a variety of cells. In vivo transplantation of BMSCs‐differentiated cells has had limited success, suggesting that these cells may not be fully compatible with the cells they are intended to replace in vivo. We investigated the structural and functional features of BMSCs‐derived adipocytes as compared with adipocytes from adipose tissue, and the structure and functionality of lipid vesicles formed during BMSCs differentiation to adipocytes. Gas chromatography‐mass spectrometry showed fatty acid composition of BMSCs‐derived adipocytes and adipocytes from the adipose tissue to be very different, as is the lipid rafts composition, caveolin‐1 expression, caveolae distribution in their membranes, and the pattern of expression of fatty acid elongases. Confocal microscopy confirmed the absence from BMSCs‐derived adipocytes of markers of lipid droplets. BMSCs‐derived adipocytes cannot convert deuterated glucose into deuterated species of fatty acids and cannot uptake the deuterated fatty acid‐bovine serum albumin complexes from the culture medium, suggesting that intra‐cellular accumulation of lipids does not occur by lipogenesis. We noted that BMSCs differentiation to adipocytes is accompanied by an increase in autophagy. Autophagic vesicles accumulate in the cytoplasm of BMSCs‐derived adipocytes and their size and distribution resembles that of Nile Red‐stained lipid vesicles. Stimulation of autophagy in BMSCs triggers the intra‐cellular accumulation of lipids while inhibition of autophagy prevents this accumulation. In conclusion, differentiation of BMSCs‐derived adipocytes leads to intra‐cellular accumulation of autophagic vesicles rather than functional lipid droplets, suggesting that these cells are not authentic adipocytes. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T18:31:06.575644-05:
      DOI: 10.1002/jcp.25177
  • Toll‐Like Receptors and Tissue Remodeling: The Pro/Cons Recent
    • Authors: Alessandra Micera; Bijorn Omar Balzamino, Antonio Di Zazzo, Filippo Biamonte, Gigliola Sica, Stefano Bonini
      Abstract: The Toll‐like Receptor (TLR) family ensures prompt response towards pathogens, protecting the host against infections, and guarantees a realistic balance between protective and detrimental activities. Multiple regulating mechanisms characterize TLR activity that is not limited to innate and adaptive antimicrobial immune responses, as observed in the inflammatory (either infective, allergic or autoimmune) responses associated with tissue remodeling. Following the insult and the arise of inflammatory response, tissue remodeling takes place and might develop in fibrosis, depending on microenvironment as a result of imbalanced fibroblasts (FBs) and myofibroblasts (myoFBs) activation/survival. The process is driven by an epithelial‐fibroblast‐immune cell cross‐talk. While the main FB function is the matrix metabolism for tissue homeostasis or repair, the myoFB differentiation represents a crucial step in attempting repair of injury. FBs/myoFBs provide more than structural support at site of injury, synthesizing and/or reacting to different cytokines, growth factors, neuromediators and soluble/lipid mediators. TLR‐bearing FBs/myoFBs might contribute at the innate immune level, providing a second line of protection/defense as well as being a target/effector cell of tissue remodeling. TLRs might also interfere with acute inflammation as well as with established fibrosis, triggering structural/functional changes in agreement with the genetic background, the site of lesion, the entity of associated infection, the poor blood circulation or the pharmacological treatments, all together strictly influencing tissue repair/remodeling process. This review will focus on the recent findings on TLRs at launch and long‐lasting tissue remodeling process, that strongly suggest TLRs as optional targets for future therapies. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T03:50:56.588812-05:
      DOI: 10.1002/jcp.25124
  • G Protein‐Coupled Receptor 120 Signaling Negatively Regulates
           Osteoclast Differentiation, Survival, and Function
    • Abstract: G protein‐coupled receptor 120 (GPR120) plays an important role in the regulation of inflammation and lipid metabolism. In this study, we investigated the role of GPR120 in osteoclast development and found that GPR120 regulates osteoclast differentiation, survival and function. We observed that GPR120 was highly expressed in osteoclasts compared to their precursors, bone marrow‐derived macrophages (BMMs). Activation of GPR120 by its ligand GW9508 suppressed receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast differentiation and the expression of nuclear factor of activated T cells c1 (NFATc1), a key modulator of osteoclastogenesis. GPR120 activation further inhibited the RANKL‐stimulated phosphorylation of IκBα and JNK. In addition to osteoclast differentiation, GPR120 activation increased the apoptosis of mature osteoclasts by inducing caspase‐3 and Bim expression. Activation of GPR120 also interfered with cell spreading and actin cytoskeletal organization mediated by M‐CSF but not by RANKL. Coincident with the impaired cytoskeletal organization, GPR120 activation blocked osteoclast bone resorbing activity. Furthermore, knockdown of GPR120 using small hairpin RNA abrogated all these inhibitory effects on osteoclast differentiation, survival, and function. Together, our findings identify GPR120 as a negative modulator of osteoclast development that may be an attractive therapeutic target for bone‐destructive diseases. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T02:47:54.011233-05:
      DOI: 10.1002/jcp.25133
  • Vitamin D Induces Cyclooxygenase 2 Dependent Prostaglandin E2 Synthesis in
           HaCaT keratinocytes
    • Abstract: The active metabolite of vitamin D calcitriol and its analogs are well‐known for their anti‐inflammatory action in the skin, while their main side effect associated with topical treatment of inflammatory disorders is irritant contact dermatitis. Prostaglandin E2 (PGE2) is pro‐inflammatory at the onset of inflammation and anti‐inflammatory at its resolution. We hypothesized that induction of PGE2 synthesis by calcitriol in epidermal keratinocytes may contribute both to its pro‐inflammatory and anti‐inflammatory effects on the skin. Treatment of human immortalized HaCaT keratinocytes with calcitriol (3–100 nM, 2–24 h) increased PGE2 production due to increased mRNA and protein expression of COX‐2, but not to increase of COX‐1 or release of arachidonic acid. The effect of calcitriol on COX‐2 mRNA was observed also in primary human keratinocytes. The increase in COX‐2 mRNA is associated with COX‐2 transcript stabilization. Calcitriol exerts this effect by a rapid (2 h) and protein synthesis independent mode of action that is dependent on PKC and Src kinase activities. Treatment with a COX‐2 inhibitor partially prevented the attenuation of the keratinocyte inflammatory response by calcitriol. We conclude that up‐regulation of COX‐2 expression with the consequent increase in PGE2 synthesis may be one of the mechanisms explaining the Janus face of calcitriol as both a promoter and attenuator of cutaneous inflammation. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T01:48:46.324798-05:
      DOI: 10.1002/jcp.25132
  • Comparison of Dasatinib, Nilotinib and Imatinib in the Treatment of
           Chronic Myeloid Leukemia
    • Authors: Roberto Ciarcia; Sara Damiano, Maria Valeria Puzio, Serena Montagnaro, Francesco Pagnini, Carmen Pacilio, Giuseppe Caparrotti, Cristiana Bellan, Tiziana Garofano, Maria Sole Polito, Antonio Giordano, Salvatore Florio
      Abstract: To overcome the drug resistance phenomenon induced by Imatibib (IM), in clinical practice, are often used second generation of tyrosine kinase inhibitors as Nilotinib (NIL) a such potent inhibitor of the BCR/ABL kinase and Dasatinib (DAS) a inhibitor of BCR/ABL kinase and inhibitor SrC family kinase. In this study we evaluated the in vivo effect of DAS, NIL and IM on intracellular calcium concentration, oxidative stress and apoptosis in peripheral blood leukocytes of 45 newly diagnosed patients with chronic myeloid leukaemia (CML‐PBM). Our data demonstrated that treatment with DAS and NIL showed an higher modulating potential than IM on intracellular calcium concentration by inhibiting the thapsigargin, a sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor and Lithium (Li) an inositol 1,4,5‐triphosphate (InsP3) receptor inhibitor activities. Moreover our data demonstrated that NIL and DAS have significantly increased apoptosis more than IM by involving both intracellular calcium signaling as well as oxidative stress. The acquisition of the oxidative stress and calcium channels receptors values data could help the hematologist to modulate and improve the treatment of chronic myeloid leukaemia (CML) pathology. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T01:42:17.468069-05:
      DOI: 10.1002/jcp.25118
  • Selective β2‐AR Blockage Suppresses Colorectal Cancer Growth
           through Regulation of EGFR‐Akt/ERK1/2 Signaling, G1‐phase
           Arrest, and Apoptosis
    • Abstract: The stress‐upregulated catecholamines‐activated β1‐ and β2‐adrenergic receptors (β1/2‐ARs) have been shown to accelerate the progression of cancers such as colorectal cancer (CRC). We investigated the underlying mechanism of the inhibition of β1/2‐ARs signaling for the treatment of CRC and elucidated the significance of β2‐AR expression in CRC in vitro and in clinical samples. The impacts of β1/2‐AR antagonists in CRC in vitro and CRC‐xenograft in vivo were examined. We found that repression of β2‐AR but not β1‐AR signaling selectively suppressed cell viability, induced G1‐phase cell cycle arrest, caused both intrinsic and extrinsic pathways‐mediated apoptosis of specific CRC cells and inhibited CRC‐xenograft growth in vivo. Moreover, the expression of β2‐AR was not consistent with the progression of CRC in vitro or in clinical samples. Our data evidences that the expression profiles, signaling, and blockage of β2‐AR have a unique pattern in CRC comparing to other cancers. β2‐AR antagonism selectively suppresses the growth of CRC accompanying active β2‐AR signaling, which potentially carries wild‐type KRAS, in vitro and in vivo via the inhibition of β2‐AR transactivated EFGR‐Akt/ERK1/2 signaling pathway. Thus, β2‐AR blockage might be a potential therapeutic strategy for combating the progressions of β2‐AR‐dependent CRC. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T01:40:01.495255-05:
      DOI: 10.1002/jcp.25092
  • Cell Surface Human Airway Trypsin‐Like Protease Is Lost During
           Squamous Cell Carcinogenesis
    • Authors: Michael J. Duhaime; Khaliph O. Page, Fausto A. Varela, Andrew S. Murray, Michael E. Silverman, Gina L. Zoratti, Karin List
      Abstract: Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro‐cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin‐like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non‐proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-22T17:23:43.72242-05:0
      DOI: 10.1002/jcp.25173
  • MDA‐9/Syntenin Control
    • Authors: Julie Philley; Anbarasu Kannan, Santanu Dasgupta
      Abstract: MDA‐9/Syntenin is a small PDZ domain containing scaffolding protein with diverse array of function regulating membrane trafficking, cell adhesion, neural and synaptic development, ubiquitination and exosome biogenesis. An appreciable number of studies also established a pivotal role of MDA‐9/Syntenin in cancer development and progression. In this review, we will discuss the dynamic role of MDA‐9/Syntenin in regulating normal and abnormal fate of various cellular processes. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-20T17:45:36.201263-05:
      DOI: 10.1002/jcp.25136
  • Defining Plasma MicroRNAs Associated With Cognitive Impairment in
           Hiv‐Infected Patients
    • Abstract: Human Immunodeficiency Virus (HIV)‐infected individuals are at increased risk for developing neurocognitive disorders and depression. These conditions collectively affect more than 50% of people living with HIV/AIDS and adversely impact adherence to HIV therapy. Thus, identification of early markers of neurocognitive impairment could lead to interventions that improve psychosocial functioning and slow or reverse disease progression through improved treatment adherence. Evidence has accumulated for the role and function of microRNAs in normal and pathological conditions. We have optimized a protocol to profile microRNAs in body fluids. Using this methodology, we have profiled plasma microRNA expression for 30 age‐matched, HIV‐infected (HIV+) patients and identified highly sensitive and specific microRNA signatures distinguishing HIV+ patients with cognitive impairment from those without cognitive impairment. These results justify follow‐on studies to determine whether plasma microRNA signatures can be used as a screening or prognostic tool for HIV+ patients with neurocognitive impairment. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-18T14:15:44.419205-05:
      DOI: 10.1002/jcp.25131
  • Autophagy‐Induced Apoptosis in Lung Cancer Cells By a Novel
           Digitoxin Analog
    • Authors: Yogesh M. Kulkarni; Vivek Kaushik, Neelam Azad, Clayton Wright, Yon Rojanasakul, George O'Doherty, Anand Krishnan V. Iyer
      Abstract: We have synthesized a novel derivative of Digitoxin, termed “MonoD”, which demonstrates cytotoxic effects in lung cancer cells with much higher potency as compared to Digitoxin. Our data show that within one hour of MonoD treatment, H460 cells showed increased oxidative stress, increased formation of autophagic vacuoles and increased expression of pro‐autophagic markers Beclin‐1 and LC3‐II. Cells pretreated with MnTBAP, a superoxide scavenger not only lowered superoxide production, but also had lower levels of LC3‐II and Beclin‐1. Prolonged treatment with MonoD induced apoptosis in lung cancer cells. We investigated MonoD‐dependent regulation of Akt and Bcl2, proteins that are known regulators of both autophagy and apoptosis. Molecular and pharmacologic inhibitors of Bcl2 and Akt, when combined with MonoD, led to higher expression of LC3‐II and Beclin‐1 as compared to MonoD alone, suggesting a repressive effect for these proteins in MonoD‐dependent autophagy. Pretreatment of cells with an autophagy inhibitor repressed the apoptotic potential of MonoD, confirming that early autophagic flux is important to drive apoptosis. Therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis may prove advantageous over current therapies that have unimodal basis for action and may drive sustained tumor regression, which is highly desirable. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-11T17:32:01.320926-05:
      DOI: 10.1002/jcp.25129
  • The Histone‐Deacetylase‐Inhibitor Suberoylanilide Hydroxamic
           Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix
           Metalloproteinase‐13 Activity
    • Authors: Henry F. Duncan; Anthony J. Smith, Garry J. P. Fleming, Nicola C. Partridge, Emi Shimizu, Gary P. Moran, Paul R. Cooper
      Abstract: Direct application of histone‐deacetylase‐inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular‐reparative events. We have previously demonstrated that HDACis (Valproic acid, Trichostatin A) increase mineralization in dental papillae‐derived cell‐lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 μM); while low concentrations (1 μM) SAHA did not increase apoptosis. HDACi‐exposure for 24 h induced mineralization‐per‐cell dose‐dependently after 2 weeks; however, constant 14d SAHA‐exposure inhibited mineralization. Microarray analysis (24 h and 14d) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0‐fold change at 24 h, which reduced to 36 genes at 14d. 59% of genes were down‐regulated at 24 h and 36% at 14d, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA‐supplementation increased MMP‐13 protein expression (7d, 14 d) and enzyme activity (48 h, 14d). Selective MMP‐13‐inhibition (MMP‐13i) dose‐dependently accelerated mineralization in both SAHA‐treated and non‐treated cultures. MMP‐13i‐supplementation promoted expression of several mineralization‐associated markers, however, HDACi‐induced cell migration and wound healing were impaired. Data demonstrate that short‐term low‐dose SAHA‐exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP‐13i further increased mineralization‐associated events, but decreased HDACi cell migration indicating a specific role for MMP‐13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-11T17:31:15.884875-05:
      DOI: 10.1002/jcp.25128
  • Nuclear Translocation of p65 is Controlled by Sec6 Via the Degradation of
    • Authors: Toshiaki Tanaka; Mitsuyoshi Iino
      Abstract: Nuclear factor‐κB (NF‐κB) is an inducible transcription factor that mediates immune and inflammatory responses. NF‐κB pathways are also involved in cell adhesion, differentiation, proliferation, autophagy, senescence, and protection against apoptosis. The deregulation of NF‐κB activity is found in a number of disease states, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease. The 90 kDa ribosomal S6 kinase (p90RSK) family, which is serine/threonine kinases, is phosphorylated by extracellular signal‐regulated kinase1/2 (ERK1/2) and is related to NF‐κB pathways. Our previous studies revealed that Sec6, a component of the exocyst complex, plays specific roles in cell‐cell adhesion and cell cycle arrest. However, the mechanism by which Sec6 regulates the NF‐κB signaling pathway is unknown. We demonstrated that Sec6 knockdown inhibited the degradation of IκBα and delayed the nucleus‐cytoplasm translocation of p65 in HeLa cells transfected with Sec6 siRNAs after treatment with tumor necrosis factor alpha (TNF‐α). Furthermore, the binding of p65 and cAMP response element binding protein (CREB) binding protein (CBP) or p300 decreased and NF‐κB related genes which were inhibitors of NF‐κB alpha (IκBα), A20, B cell lymphoma protein 2 (Bcl‐2), and monocyte chemoattractant protein‐1 (MCP‐1) were low in cells transfected with Sec6 siRNAs in response to TNF‐α stimulation. Sec6 knockdown decreased the expression of p90RSKs and the phosphorylation of ERK or p90RSK1 at Ser380 or IκBα at Ser32. The present study suggests that Sec6 regulates NF‐κB transcriptional activity via the control of the phosphorylation of IκBα, p90RSK1, and ERK. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-06T07:01:22.740635-05:
      DOI: 10.1002/jcp.25122
  • CD44 Influences Fibroblast Behaviors Via Modulation of Cell‐Cell and
           Cell‐Matrix Interactions, Affecting Survivin and Hippo Pathways
    • Authors: Masayuki Tsuneki; Joseph A. Madri
      Abstract: CD44 has been studied in a wide variety of cell types, in a diverse array of cell behaviors and in a diverse range of signaling pathways. We now document a role for CD44 in mediating fibroblast behaviors via regulation of N‐cadherin, extracellular matrix expression, Survivin and the Hippo pathway. Here we report our findings on the roles of CD44 in modulating proliferation, apoptosis, migration and invasion of murine wild‐type (WT‐FB) and CD44 knockout dermal fibroblasts (CD44KO‐FB). As we have documented in microvascular endothelial cells lacking CD44, we found persistent increased proliferation, reduced activation of cleaved caspase 3, increased initial attachment, but decreased strength of cell attachment in high cell density, post confluent CD44KO‐FB cultures. Additionally, we found that siRNA knock‐down of CD44 mimicked the behaviors of CD44KO‐FB, restoring the decreases in N‐cadherin, collagen type I, fibronectin, Survivin, nuclear fractions of YAP and phospho‐YAP and decreased levels of cleaved caspase 3 to the levels observed in CD44KO‐FB. Interestingly, plating CD44KO‐FB on collagen type I or fibronectin resulted in significant decreases in secondary proliferation rates compared to plating cells on non‐coated dishes, consistent with increased cell adhesion compared to their effects on WT‐FB. Lastly, siRNA knockdown of CD44 in WT‐FB resulted in increased fibroblast migration compared to WT‐FB, albeit at reduced rates compared to CD44KO‐FB. These results are consistent with CD44's pivotal role in modulating several diverse behaviors important for adhesion, proliferation, apoptosis, migration and invasion during development, growth, repair, maintenance and regression of a wide variety of mesenchymal tissues. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-06T07:00:50.701007-05:
      DOI: 10.1002/jcp.25123
  • Ca2+ Signalling in Endothelial Progenitor Cells: Friend or Foe'
    • Authors: Francesco Moccia; Germano Guerra
      Abstract: Endothelial progenitor cells (EPCs) are mobilized either from the bone marrow and/or the arterial to replace dysfunctional endothelial cells and rescue blood perfusion in ischemic tissues. In addition, they may contribute to the angiogenic switch, thereby sustaining tumour growth and metastatization. Understanding the molecular mechanisms utilized by vascular endothelial growth factor (VEGF) to stimulate EPCs might unveil novel targets to enhance their clinical outcome in regenerative medicine and to adverse tumour vascularisation. VEGF stimulates peripheral blood‐derived EPCs to undergo repetitive Ca2+ oscillations shaped by the interaction between inositol‐1,4,5‐trisphosphate (InsP3)‐dependent Ca2+ release and store‐operated Ca2+ entry (SOCE). However, the Ca2+ machinery underlying VEGF‐induced Ca2+ spikes changes in umbilical cord blood‐derived EPCs, which require TRPC3‐mediated Ca2+ entry to trigger the interplay between InsP3 and SOCE. Surprisingly, VEGF fails to elicit pro‐angiogenic Ca2+ signals when EPCs derive from renal cellular carcinoma patients, thus questioning the suitability of VEGFR‐2 as a target for anti‐angiogenic treatments in these individuals. The lack of response to VEGF is likely due to the dramatic rearrangement of the Ca2+ toolkit occurring in RCC‐derived EPCs. Finally, primary myelofibrosis‐derived EPCs display a further pattern of reorganization of the Ca2+ machinery and proliferate independently of SOCE. Thus, the Ca2+ machinery in human ECFCs is extremely plastic and may change depending on the physio‐pathological background of the donor. As a consequence, the Ca2+ toolkit could properly be used to enhance the regenerative outcome of cell‐based therapy or adverse tumor vascularisation. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-06T07:00:23.572265-05:
      DOI: 10.1002/jcp.25126
  • MicroRNAs in Control of Plant Development
    • Authors: Chao Li; Baohong Zhang
      Abstract: In the long evolutionary history, plant has evolved elaborate regulatory network to control functional gene expression for surviving and thriving, such as transcription factor‐regulated transcriptional programming. However, plenty of evidences from the past decade studies demonstrate that the 21‐24 nucleotides small RNA molecules, majorly microRNAs (miRNAs) play dominant roles in post‐transcriptional gene regulation through base pairing with their complementary mRNA targets, especially prefer to target transcription factors in plants. Here, we review current progresses on miRNA‐controlled plant development, from miRNA biogenesis dysregulation‐caused pleiotropic developmental defects to specific developmental processes, such as SAM regulation, leaf and root system regulation, plant floral transition. We also summarize some miRNAs that are experimentally proved to greatly affect crop plant productivity and quality. In addition, recent reports show that a single miRNA usually display multiple regulatory roles, such as organ development, phase transition and stresses responses. Thus, we infer that miRNA may act as a node molecule to coordinate the balance between plant development and environmental clues, which may shed the light on finding key regulator or regulatory pathway for uncovering the mysterious molecular network. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-06T06:59:32.765442-05:
      DOI: 10.1002/jcp.25125
  • Retinoblastoma Protein Knockdown Favors Oxidative Metabolism and Glucose
           and Fatty Acid Disposal in Muscle Cells
    • Abstract: Deficiency in the retinoblastoma protein (Rb) favors leanness and a healthy metabolic profile in mice largely attributed to activation of oxidative metabolism in white and brown adipose tissues. Less is known about Rb modulation of skeletal muscle metabolism. This was studied here by transiently knocking down Rb expression in differentiated C2C12 myotubes using small interfering RNAs. Compared with control cells transfected with non‐targeting RNAs, myotubes silenced for Rb (by 80–90%) had increased expression of genes related to fatty acid uptake and oxidation such as Cd36 and Cpt1b (by 61% and 42%, respectively), increased Mitofusin 2 protein content (∼2.5‐fold increase), increased mitochondrial to nuclear DNA ratio (by 48%), increased oxygen consumption (by 65%) and decreased intracellular lipid accumulation. Rb silenced myotubes also displayed up‐regulated levels of glucose transporter type 4 expression (∼5‐fold increase), increased basal glucose uptake, and enhanced insulin‐induced Akt phosphorylation. Interestingly, exercise in mice led to increased Rb phosphorylation (inactivation) in skeletal muscle as evidenced by immunohistochemistry analysis. In conclusion, the silencing of Rb enhances mitochondrial oxidative metabolism and fatty acid and glucose disposal in skeletal myotubes, and changes in Rb status may contribute to muscle physiological adaptation to exercise. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-04T06:58:00.065611-05:
      DOI: 10.1002/jcp.25121
  • Virtual Reality in Health System: Beyond Entertainment. A
           Mini‐Review on the Efficacy of VR During Cancer Treatment
    • Authors: Andrea Chirico; Fabio Lucidi, Michele De Laurentiis, Carla Milanese, Alessandro Napoli, Antonio Giordano
      Abstract: Virtual Reality (VR), a computer‐generated virtual environment, has been increasingly used in the entertainment world becoming a very new evolving field, but VR technology has also found a variety of applications in the biomedical field. VR can offer to subjects a safe environment within which to carry on different interventions ranging from the rehabilitation of discharged patients directly at home, to the support of hospitalized patients during different procedures and also of oncological inpatient subjects. VR appears as a promising tool for support and monitoring treatments in cancer patients influencing psychological and physiological functions. The aim of this systematic review is to provide an overview of all the studies that used VR intervention on cancer patients and analyze their main findings. Nineteen studies across nearly a thousand articles were identified that explored effects of VR interventions on cancer patients. Although these studies varied greatly in setting and design, this review identified some overarching themes. Results found that VR improved patients' emotional well‐being, and diminished cancer related psychological symptoms. The studies explored various relevant variables including different types of settings (i.e. during chemotherapy, during pain procedures, during hospitalization). Here, we point to the need of a global and multi‐disciplinary approach aimed at analyzing the effects of VR taking advantage of the new technology systems like bio sensors as well as electroencephalogram monitoring pre‐during and after intervention. Devoting more attention to bio physiological variables, standardized procedures, extending duration to longitudinal studies and adjusting for motion sickness related to VR treatment need to become standard of this research field. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-03T21:58:46.057434-05:
      DOI: 10.1002/jcp.25117
  • Impaired Hematopoiesis and Disrupted Monocyte/Macrophage Homeostasis in
           Mucopolysaccharidosis Type I Mice
    • Abstract: Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disease caused by alpha‐L‐iduronidase deficiency in which heparan and dermatan sulfate degradation is compromised. Besides primary lysosomal glycosaminoglycan accumulation, further changes in cellular functions have also been described in several murine MPS models. Herein, we evaluated alterations in hematopoiesis and its implications on the production of mature progeny in a MPS I murine model. Despite the significant increase in hematopoietic stem cells, a reduction in common myeloid progenitors and granulocyte‐macrophage progenitor cells was observed in Idua ‐/‐ mice bone marrow. Furthermore, no alterations in number, viability nor activation of cell death mechanisms were observed in Idua ‐/‐ mice mature macrophages but they presented higher sensitivity to apoptotic induction after staurosporine treatment. In addition, changes in Ca2+ signaling and a reduction in phagocytosis ability were also found. In summary, our results revealed significant intracellular changes in mature Idua ‐/‐ macrophages related to alterations in Idua ‐/‐ mice hematopoiesis, revealing a disruption in cell homeostasis. These results provide new insights into physiopathology of MPS I. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-03T06:59:30.534856-05:
      DOI: 10.1002/jcp.25120
  • Fucoidan Suppresses the Growth of Human Acute Promyelocytic Leukemia Cells
           In Vitro and In Vivo
    • Authors: Farzaneh Atashrazm; Ray M Lowenthal, Gregory M Woods, Adele F Holloway, Samuel S Karpiniec, Joanne L Dickinson
      Abstract: Fucoidan, a natural component of seaweeds, is reported to have immunomodulatory and anti‐tumor effects. The mechanisms underpinning these activities remain poorly understood. In this study, the cytotoxicity and anti‐tumor activities of fucoidan were investigated in acute myeloid leukemia (AML) cells. The human AML cell lines NB4, KG1a, HL60 and K562 were treated with fucoidan and cell cycle, cell proliferation and expression of apoptotic pathways molecules were analyzed. Fucoidan suppressed the proliferation and induced apoptosis through the intrinsic and extrinsic pathways in the acute promyelocytic leukemia (APL) cell lines NB4 and HL60, but not in KG1a and K562 cells. In NB4 cells, apoptosis was caspase‐dependent as it was significantly attenuated by pre‐treatment with a pan‐caspase inhibitor. P21/WAF1/CIP1 was significantly up‐regulated leading to cell cycle arrest. Fucoidan decreased the activation of ERK1/2 and down‐regulated the activation of AKT through hypo‐phosphorylation of Thr(308) residue but not Ser(473). In vivo, a xenograft model using the NB4 cells was employed. Mice were fed with fucoidan and tumor growth was measured following inoculation with NB4 cells. Subsequently, splenic natural killer (NK) cell cytotoxic activity was also examined. Oral doses of fucoidan significantly delayed tumor growth in the xenograft model and increased cytolytic activity of NK cells. Taken together, these data suggest that the selective inhibitory effect of fucoidan on APL cells and its protective effect against APL development in mice warrant further investigation of fucoidan as a useful agent in treatment of certain types of leukemia. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-03T06:58:16.202878-05:
      DOI: 10.1002/jcp.25119
  • IPSCs, a Promising Tool to Restore Muscle Atrophy
    • Abstract: Induced pluripotent stem cells (iPSCs) are a promising tool for regenerative medicine in chronic conditions associated with muscle atrophy since iPSCs are easier to obtain, pose less ethical limitations and can better capture human genetic diversity compared with human embryonic stem cells. We highlight the potentiality of iPSCs for treating muscle‐affecting conditions for which no effective cure is yet available, notably aging sarcopenia and inherited neurometabolic conditions. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-30T05:15:28.036587-05:
      DOI: 10.1002/jcp.25114
  • Hypoxia Promotes the Inflammatory Response and Stemness Features in
           Visceral Fat Stem Cells from Obese Subjects
    • Authors: Elisa Petrangeli; Giuseppe Coroniti, Anna T. Brini, Laura de Girolamo, Deborah Stanco, Stefania Niada, Gianfranco Silecchia, Emanuela Morgante, Carla Lubrano, Matteo A. Russo, Luisa Salvatori
      Abstract: Low‐grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of 2 subpopulations of adipose‐derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of 2 subpopulations of ASC from the same isolation sites of non‐obese individuals. In particular, the behavior of ASC of obese vs non‐obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV‐ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation‐related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue‐specific NF‐kB activation and an NF‐kB‐mediated increase in inflammatory and fibrogenic responses. Moreover, obV‐ASC, which showed a less stem‐like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV‐ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF‐kB inhibitors. These novel findings suggest that the deregulated hyper‐responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low‐grade chronic inflammation, which has been implicated in obesity‐related morbidity. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-30T05:15:07.745025-05:
      DOI: 10.1002/jcp.25113
  • Regulation of Spermatogenic Cell T‐Type Ca2+ Currents by Zn2+:
           Implications in Male Reproductive Physiology
    • Abstract: Zn2+ is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis and prostate contain high Zn2+ concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express CaV3.2‐encoded T‐type Ca2+ currents (ICaT) which are positively or negatively modulated by Zn2+ in other tissues. To explore whether ICaT could be regulated by Zn2+ and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell ICaT in the absence or presence of different Zn2+ concentrations. Zn2+ decreased ICaT in a concentration‐dependent manner (IC50= 2 μM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, ICaT did not show a tonic inhibition by Zn2+. Further analysis showed that Zn2+ did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady‐state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong ICaT reduction. Using non‐stationary fluctuation analysis, we found that Zn2+ mainly reduced the number of available Ca2+ channels without changing the single channel current amplitude. ICaT modulation by Zn2+ could be relevant for spontaneous Ca2+ oscillations during spermatogenesis and in pathophysiological conditions such as diabetes. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-29T09:24:14.760195-05:
      DOI: 10.1002/jcp.25112
  • BMP‐2 Induced Expression of PLCβ1 That Is a Positive Regulator
           of Osteoblast Differentiation
    • Authors: Giulia Ramazzotti; Alberto Bavelloni, William Blalock, Manuela Piazzi, Lucio Cocco, Irene Faenza
      Abstract: Bone morphogenetic protein 2 (BMP‐2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide‐phospholipase Cβ 1 (PLCβ1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCβ1. Here, the function of PLCβ1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP‐2 we assist to a remarkable increase in PLCβ1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCβ1 promotes osteogenic differentiation by up‐regulating alkaline phosphatase (ALP). Moreover, PLCβ1 is present in the nuclear compartment of these cells and overexpression of a cytosolic‐PLCβ1mutant (cyt‐PLCβ1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR‐214 is a regulator of Osterix (Osx) which is an osteoblast‐specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCβ1 could be a potential target of miR‐214 in the control of osteogenic differentiation by gain‐ and loss‐ of function experiment. The results indicated that inhibition of miR‐214 in C2C12 cells significantly enhances the protein level of PLCβ1 and promotes C2C12 BMP‐2‐induced osteogenesis by targeting PLCβ1. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:43:25.727826-05:
      DOI: 10.1002/jcp.25107
  • Leptin Effect on Acetylation and Phosphorylation of PGC1α in Muscle
           Cells Associated With AMPK and Akt Activation in High‐Glucose Medium
    • Abstract: Leptin is crucial in energy metabolism, including muscle regulation. Peroxisome proliferator activated receptor gamma co‐activator 1α (PGC1α) orchestrates energy metabolism and is tightly controlled by post‐translational covalent modifications such as phosphorylation and acetylation. We aimed to further the knowledge of PGC1α control by leptin (at physiological levels) in muscle cells by time‐sequentially analysing the activation of AMP activated protein kinase (AMPK), p38 mitogen‐activated protein kinase (p38 MAPK) and Akt (Protein kinase B) – all known to phosphorylate PGC1α and to be involved in the regulation of its acetylation status – in C2C12 myotubes placed in a high‐glucose serum‐free medium. We also studied the protein levels of PGC1α, Sirtuin 1, adiponectin, COX IV, mitofusin 2 (Mfn2) and pyruvate dehydrogenase kinase 4 (PDK4). Our main findings suggest an important role of leptin regulating AMPK and Akt phosphorylation, mitofusin 2 induction and PGC1α acetylation status, with the novelty that the latter in transitorily increased in response to leptin, an effect dependent, at least in part, on AMPK regulation. These post‐translational reversible changes in PGC1α in response to leptin, especially the increase in acetylation status, may be related to the physiological role of the hormone in modulating muscle cell response to the physiological/nutritional status. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:41:43.604498-05:
      DOI: 10.1002/jcp.25109
  • Sustained Inhibition of Proliferative Response After Transient FGF
           Stimulation is Mediated by Interleukin 1 Signaling
    • Authors: Ashleigh Poole; Doreen Kacer, Emily Cooper, Francesca Tarantini, Igor Prudovsky
      Abstract: Transient FGF stimulation of various cell types results in FGF memory – a sustained blockage of efficient proliferative response to FGF and other growth factors. FGF memory establishment requires HDAC activity, indicating its epigenetic character. FGF treatment stimulates proinflammatory NFκB signaling, which is also critical for FGF memory formation. The search for FGF‐induced mediators of FGF memory revealed that FGF stimulates HDAC‐dependent expression of the inflammatory cytokine IL1α. Similarly to FGF, transient cell treatment with recombinant IL1α inhibits the proliferative response to further FGF and EGF stimulation, but does not prevent FGF receptor‐mediated signaling. Interestingly, like cells pretreated with FGF1, cells pretreated with IL1α exhibit enhanced restructuring of actin cytoskeleton and increased migration in response to FGF stimulation. IRAP, a specific inhibitor of IL1 receptor, and a neutralizing anti‐IL1α antibody prevent the formation of FGF memory and rescue an efficient proliferative response to FGF restimulation. A similar effect results following treatment with the anti‐inflammatory agents aspirin and dexamethasone. Thus, FGF memory is mediated by proinflammatory IL1 signaling. It may play a role in the limitation of proliferative response to tissue damage and prevention of wound‐induced hyperplasia. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:41:20.046371-05:
      DOI: 10.1002/jcp.25111
  • Failure to Target RANKL Signaling Through p38‐MAPK Results in
           Defective Osteoclastogenesis in the Microphthalmia Cloudy‐eyed
    • Abstract: The microphthalmia‐associated transcription factor (MITF) is a basic helix‐loop‐helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of receptor activator of NFκB ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C‐terminal end. Here we show that the Mitfce allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitfce/ce mice. The loss of the leucine zipper domain in Mitfce/ce mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitfce. This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site‐directed mutations in the docking site interfered with the interaction between MITF and its co‐factors FUS and BRG1. MITF‐ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/ p38 MAPK interactions in osteoclast differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:40:54.04586-05:0
      DOI: 10.1002/jcp.25108
  • IL‐6 Activates PI3K and PKCζ Signaling and Determines Cardiac
           Differentiation in Rat Embryonic H9c2 Cells
    • Authors: Maria Angela D'amico; Barbara Ghinassi, Pascal Izzicupo, Annalisa Di Ruscio, Angela Di Baldassarre
      Abstract: Introduction IL‐6 influences several biological processes, including cardiac stem cell and cardiomyocyte physiology. Although JAK‐STAT3 activation is the defining feature of IL‐6 signaling, signaling molecules such as PI3K, PKCs and ERK1/2 are also activated and elicit different responses. Moreover, most studies on the specific role of these signaling molecules focus on the adult heart, and few studies are available on the biological effects evoked by IL‐6 in embryonic cardiomyocytes. Aim The aim of this study was to clarify the biological response of embryonic heart derived cells to IL‐6 by analyzing the morphological modifications and the signaling cascades evoked by the cytokine in H9c2 cells. Results IL‐6 stimulation determined the terminal differentiation of H9c2 cells, as evidenced by the increased expression of cardiac transcription factors (NKX2.5 and GATA4), structural proteins (α‐myosin heavy chain and cardiac Troponin T) and the gap junction protein Connexin 43. This process was mediated by the rapid modulation of PI3K, Akt, PTEN and PKCζ phosphorylation levels. PI3K recruitment was an upstream event in the signaling cascade and when PI3K was inhibited, IL‐6 failed to modify PKCζ, PTEN and Akt phosphorylation. Blocking PKCζ activity affected only PTEN and Akt. Finally, the overexpression of a constitutively active form of PKCζ in H9c2 cells largely mimicked the morphological and molecular effects evoked by IL‐6. Conclusion This study demonstrated that IL‐6 induces the cardiac differentiation of H9c2 embryonic cells though a signaling cascade that involves PI3K, PTEN and PKCζ activities. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:37:46.759391-05:
      DOI: 10.1002/jcp.25101
  • RhoA‐Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate
    • Abstract: Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho‐family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M) and nanotopography (N) to evaluate the effect of surface topography on RhoA‐mediated functions (cell spreading, adhesion, migration and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S< M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness‐dependent (S 
      PubDate: 2015-07-23T18:37:20.245806-05:
      DOI: 10.1002/jcp.25100
  • The PPARβ/δ Agonist GW0742 Induces Early Neuronal Maturation of
           Cortical Post‐Mitotic Neurons: Role of PPARβ/δ in Neuronal
    • Authors: E. Benedetti; S. Di Loreto, B. D'Angelo, L. Cristiano, M. d'Angelo, A. Antonosante, A. Fidoamore, Golini Raffaella, B. Cinque, MG Cifone, R Ippoliti, A. Giordano, A. Cimini
      Abstract: Increasing evidences support that signalling lipids participate in synaptic plasticity and cell survival, and that the lipid signalling is closely associated with neuronal differentiation, learning and memory and with pathologic events such as epilepsy and Alzheimer's disease. The Peroxisome Proliferator‐Activated Receptors (PPAR) are strongly involved in the fatty acid cell signalling, as many of the natural lypophylic compounds are PPAR ligands. We have previously shown that PPARβ/δ is the main isotype present in cortical neuron primary cultures and that during neuronal maturation, PPARβ/δ is gradually increased and activated. To get more insight into the molecular mechanism by which PPARβ/δ may be involved in neuronal maturation processes, in this work a specific PPARβ/δ agonist, GW0742 was used administered alone or in association with a specific PPARβ/δ antagonist, the GSK0660 and the parameters involved in neuronal differentiation and maturation were assayed. The data obtained demonstrated the strong involvement of PPARβ/δ in neuronal maturation, triggering the agonist an anticipation of neuronal differentiation and the antagonist abolishing the observed effects. These effects appear to be mediated by the activation of both BDNF canonical (BDNF7TrkB) pathway. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:36:55.606691-05:
      DOI: 10.1002/jcp.25103
  • Cellular and Molecular Mechanisms of Phenotypic Switch in Gastrointestinal
           Smooth Muscle
    • Authors: Annunziata Scirocco; Paola Matarrese, Marilia Carabotti, Barbara Ascione, Walter Malorni, Carola Severi
      Abstract: As a general rule, smooth muscle cells (SMC) are able to switch from a contractile phenotype to a less mature synthetic phenotype. This switch is accompanied by a loss of differentiation with decreased expression of contractile markers, increased proliferation as well as the synthesis and the release of several signaling molecules such as pro‐inflammatory cytokines, chemotaxis‐associated molecules and growth factors. This SMC phenotypic plasticity has extensively been investigated in vascular diseases but interest is also emerging in the field of gastroenterology. It has in fact been postulated that altered micro‐environmental conditions, including the composition of microbiota, could trigger the remodeling of the enteric SMC, with phenotype changes and consequent alterations of contraction and impairment of gut motility. Several molecular actors participate in this phenotype remodeling. These include extracellular molecules such as cytokines and extracellular matrix proteins, as well as intracellular proteins, e.g. transcription factors. Epigenetic control mechanisms and miRNA have also been suggested to participate. In this review key roles and actors of smooth muscle phenotypic switch, mainly in GI tissue, are described and discussed in the light of literature data available so far. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:36:30.672067-05:
      DOI: 10.1002/jcp.25105
  • CyclinD1 Down Regulation and Increased Apoptosis Are Common Features of
    • Abstract: Genetic variants within components of the cohesin complex (NIPBL, SMC1A, SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a spectrum of human syndromes known as “cohesinopathies” that includes Cornelia de Lange Syndrome (CdLS). CdLS is a multiple malformation syndrome affecting almost any organ and causing severe developmental delay. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in cohesin components. However, it is still unclear how mutations in different components of the cohesin complex affect the output of gene regulation. In this study, zebrafish embryos and SMC1A‐mutated patient‐derived fibroblasts were used to analyze abnormalities induced by SMC1A loss of function. We show that the knockdown of smc1a in zebrafish impairs neural development, increases apoptosis and specifically down‐regulates Ccnd1 levels. The same down‐regulation of cohesin targets is observed in SMC1A‐mutated patient fibroblasts. Previously, we have demonstrated that haploinsufficiency of NIPBL produces similar effects in zebrafish and in patients fibroblasts indicating a possible common feature for neurological defects and mental retardation in cohesinopathies. Interestingly, expression analysis of Smc1a and Nipbl in developing mouse embryos reveals a specific pattern in the hindbrain, suggesting a role for cohesin in neural development in vertebrates. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:36:04.714796-05:
      DOI: 10.1002/jcp.25106
  • Osteogenic Potential of Human Oral‐Periosteal Cells (PCs) Isolated
           from Different Oral Origin: An In Vitro Study
    • Authors: Gabriele Ceccarelli; Antonio Graziano, Laura Benedetti, Marcello Imbriani, Federica Romano, Francesco Ferrarotti, Mario Aimetti, Gabriella M. Cusella De Angelis
      Abstract: The periosteum is a specialized connective tissue containing multipotent stem cells capable of bone formation. In this study, we aimed at demonstrating that human oral periosteal cells derived from three different oral sites (upper vestibule, lower vestibule and hard palate) represent an innovative cell source for maxillo‐facial tissue engineering applications in terms of accessibility and self‐commitment towards osteogenic lineage. Periosteal cells (PCs) were isolated from patients with different ages (20‐30 yy, 40‐50 yy, 50‐60 yy); we then analyzed the in vitro proliferation capacity and the bone self‐commitment of cell clones culturing them without any osteogenic supplement to support their differentiation. We found that oral PCs, independently of their origin and age of patients, are mesenchymal stem cells with stem cell characteristics (clonogenical and proliferative activity) and that, even in absence of any osteogenic induction, they undertake the osteoblast lineage after 45 days of culture. These results suggest that oral periosteal cells could replace mesenchymal cells from bone marrow in oral tissue‐engineering applications. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:35:38.582213-05:
      DOI: 10.1002/jcp.25104
  • Reciprocal Control of Osteogenic and Adipogenic Differentiation by ERK/MAP
           Kinase Phosphorylation of Runx2 and PPARγ Transcription Factors
    • Authors: Chunxi Ge; William P. Cawthorn, Yan Li, Guisheng Zhao, Ormond A. MacDougald, Renny T. Franceschi
      Abstract: In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPARγ, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPARγ. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPARγ phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPARγ‐dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant‐negative suppression of activity had the opposite effect. In rescue experiments using Runx2‐/‐ mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation‐deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation‐resistant S112A mutant PPARγ strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg ‐/‐ MEFs, while S112E mutant PPARγ was less active. Competition between RUNX2 and PPARγ was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPARγ phosphorylation in the control of osteoblast and adipocyte lineages. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:35:14.838053-05:
      DOI: 10.1002/jcp.25102
  • Selective Life‐Long Skeletal Myofiber – Targeted VEGF Gene
           Ablation Impairs Exercise Capacity in Adult Mice
    • Authors: K. Tang; Y. Gu, N.D. Dalton, H. Wagner, K. L. Peterson, P. D. Wagner, E. C. Breen
      Abstract: Exercise is dependent on adequate oxygen supply for mitochondrial respiration in both cardiac and locomotor muscle. To determine whether skeletal myofiber VEGF is critical for regulating exercise capacity, independent of VEGF function in the heart, ablation of the VEGF gene was targeted to skeletal myofibers (skmVEGF‐/‐) during embryogenesis (∼ E9.5), leaving intact VEGF expression by all other cells in muscle. In adult mice, VEGF levels were decreased in the soleus (by 65%), plantaris (94%), gastrocnemius (74%), EDL (99%) and diaphragm (64%) (p 
      PubDate: 2015-07-22T18:12:32.284024-05:
      DOI: 10.1002/jcp.25097
  • Endoplasmic Reticulum Stress Interacts with Inflammation in Human Diseases
    • Authors: Stewart Siyan Cao; Katherine L. Luo, Lynn Shi
      Abstract: The endoplasmic reticulum (ER) is a critical organelle for normal cell function and homeostasis. Disturbance in the protein folding process in the ER, termed ER stress, leads to the activation of unfolded protein response (UPR) that encompasses a complex network of intracellular signaling pathways. The UPR can either restore ER homeostasis or activate pro‐apoptotic pathways depending on the type of insults, intensity and duration of the stress, and cell types. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune, infectious, neurodegenerative, and metabolic disorders. In the cell, ER stress and inflammatory signaling share extensive regulators and effectors in a broad spectrum of biological processes. In spite of different etiologies, the two signaling pathways have been shown to form a vicious cycle in exacerbating cellular dysfunction and causing apoptosis in many cells and tissues. However, the interaction between ER stress and inflammation in many of these diseases remains poorly understood. Further understanding of the biochemistry, cell biology, and physiology may enable the development of novel therapies that spontaneously target these pathogenic pathways. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-22T18:12:07.728989-05:
      DOI: 10.1002/jcp.25098
  • Androgen Receptor Coactivators in Regulation of Growth and Differentiation
           in Prostate Cancer
    • Authors: Zoran Culig
      Abstract: Androgen receptor (AR) is a key factor in regulation of growth and differentiation in normal and malignant prostate. Endocrine therapies for prostate cancer include inhibition of androgen production either by analogues of luteinizing hormone releasing hormone or abiraterone acetate and/or use of anti‐androgens such as hydroxyflutamide, bicalutamide, and enzalutamide. Castration therapy‐resistant cancer develops inevitably in patients who undergo treatment. AR coactivators are proteins which interact with one or more regions of the AR thus enhancing its function. Although several functions of AR coactivators may be redundant, specific functions have been identified and analyzed. The p160 group of coactivators, SRC‐1, ‐2, and ‐3 not only potentiate the activation of the AR, but are also implicated in potentiation of function of insulin‐like growth factor‐I and activation of the Akt pathway. Transcriptional integrators p300 and CBP are up‐regulated by androgen ablation and may influence antagonist/agonist balance of non‐steroidal anti‐androgens. A therapy approach designed to target p300 in prostate cancer revealed its role in regulation of proliferation of migration of androgen‐sensitive and –insensitive prostate cancer cells. Coactivators p300 and SRC‐1 are required for AR activation by interleukin‐6 (IL‐6), a cytokine that is overexpressed in castration‐therapy resistant prostate cancer. Some coactivators, such as Vav3 are involved in regulation of transcriptional activity of truncated AR, which emerge during endocrine thrapy. Stimulation of cellular migration and invasion by AR coactivators has also been described. Translational studies with aim to introduce anti‐AR coactivator therapy have not been successfuly implemented in the clinic so far. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-22T18:10:57.542696-05:
      DOI: 10.1002/jcp.25099
  • Nitric Oxide Up‐Regulates RUNX2 in LNCaP Prostate Tumours:
           Implications for Tumour Growth In Vitro and In Vivo
    • Authors: H Nesbitt; G Browne, KM O'Donovan, NM Byrne, J Worthington, SR McKeown, DJ McKenna
      Abstract: Background Aberrant expression of the transcription factor RUNX2 in prostate cancer has a number of important consequences including increased resistance to apoptosis, invasion and metastasis to bone. We previously demonstrated that hypoxia up‐regulated RUNX2 in tumour cells, which in turn up‐regulated the anti‐apoptotic factor Bcl‐2. Here, we investigate the impact of nitric oxide (NO) on RUNX2 and Bcl‐2 expression in prostate cancer and further, how RUNX2 over‐expression can impact tumour growth, angiogenesis and oxygenation in vivo. Methods The effect of NO levels on RUNX2 and thus Bcl‐2 expression was examined in prostate cancer cells in vitro using methods including gene and protein expression analyses, nitrite quantitation, protein‐DNA interaction assays (ChIP) and viability assays (XTT). The effect of RUNX2 over‐expression on tumour physiology (growth, oxygenation and angiogenesis) was also assessed in vivo using LNCaP xenografts. Results A low (but not high) concentration of NO (10µM) induced expression of RUNX2 and Bcl‐2, conferring resistance to docetaxel. These effects were induced via the ERK and PI3K pathways and were dependent on intact AP‐1 binding sites in the RUNX2 promoter. RUNX2 over‐expression in LNCaP tumours in vivo decreased the time to tumour presentation and increased tumour growth. Moreover, these tumours exhibited improved tumour angiogenesis and oxygenation. Conclusion Low levels of NO increase expression of RUNX2 and Bcl‐2 in LNCaP prostate tumour cells, and in vivo up‐regulation of RUNX2 created tumours with a more malignant phenotype. Collectively, our data reveals the importance of NO‐regulation of key factors in prostate cancer disease progression. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-19T22:28:37.075832-05:
      DOI: 10.1002/jcp.25093
  • D‐Aspartate Induces Proliferative Pathways in Spermatogonial
           GC‐1 Cells
    • Authors: Alessandra Santillo; Sara Falvo, Paolo Chieffi, Maria Maddalena Di Fiore, Rosalba Senese, Gabriella Chieffi Baccari
      Abstract: D‐aspartate (D‐Asp) is an endogenous amino acid present in vertebrate tissues, with particularly high levels in the testis. In vivo studies indicate that D‐Asp indirectly stimulates spermatogenesis through the hypothalamic‐pituitary‐gonadal axis. Moreover, in vitro studies have demonstrated that D‐Asp up‐regulates testosterone production in Leydig cells by enhancing expression of the steroidogenic acute regulatory protein. In this study, a cell line derived from immortalized type‐B mousespermatogonia retaining markers of mitotic germ cells (GC‐1) was employed to explore more direct involvement of D‐Asp in spermatogenesis.Activity and protein expression of markers of cell proliferation were determined at intervals during incubation in D‐Asp‐containing medium. D‐Asp induced phosphorylation of ERK and Akt proteins, stimulated expression of PCNA and Aurora B, and enhanced mRNA synthesis and protein expression of P450 aromatase and protein expressionof Estrogen Receptor β (ERβ). These results are the first demonstration of a direct effect of D‐Asp on spermatogonial mitotic activity. Considering thatspermatogonia express the NR1 subunit of the N‐Methyl‐D‐Aspartic Acid receptor (NMDAR), we suggest that their response to D‐Asp depends on NMDAR‐mediated activation of the ERK and Akt pathways and is further enhanced by activation of the P450 aromatase/ERβ pathway. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-19T22:27:42.589855-05:
      DOI: 10.1002/jcp.25095
  • Hydroxytyrosol Inhibits Cannabinoid CB1 Receptor Gene Expression in
           3T3‐L1 Preadipocyte Cell Line
    • Authors: Valeria Tutino; Antonella Orlando, Francesco Russo, Maria Notarnicola
      Abstract: The 3T3‐L1 preadipocyte cell line is a well characterized cell model for studying the adipocyte status and the molecular mechanisms involved in differentiation of these cells. 3T3‐L1 preadipocytes have the ability to synthesize and degrade endocannabinoid anandamide (AEA) and their differentiation into adipocytes increases the expression of cannabinoid (CB1) and PPAR‐γ receptors. Clinically, the blocking stimulation of the endocannabinoid pathway has been one of the first approaches proposed to counteract the obesity and obesity‐associated diseases (such as diabetes, metabolic syndrome and cancer). In this connection, here we studied in cultured 3T3‐L1 pre‐adipocytes the effects of n‐3‐PUFA, α‐Linolenic acid (OM‐3), n‐6‐PUFA, Linoleic acid (OM‐6) and hydroxytyrosol (HT) on the expression of CB1 receptor gene and the adipogenesis‐related genes PPAR‐γ, Fatty Acid Synthase (FAS) and Lipoprotein Lipase (LPL). HT was able to inhibit 3T3‐L1 cell differentiation by down‐regulating cell proliferation and CB1 receptor gene expression. HT exhibited anti‐adipogenic effects, whereas OM‐3 and OM‐6 exerted an inhibitory action on cell proliferation associated with an induction of the preadipocytes differentiation and CB1 receptor gene expression. Moreover, the expression of FAS and LPL genes resulted increased after treatment with both HT and OM‐3 and OM‐6. The present study points out that the intake of molecules such as HT, contained in extra virgin olive oil, may be considered also in view of antiobesity and antineoplastic properties by acting directly on the adipose tissue and modulating CB1 receptor gene transcription. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-19T22:27:19.213701-05:
      DOI: 10.1002/jcp.25094
  • Intravenous Immunoglobulin (IVIG) Attenuates TNF‐Induced Pathologic
           Bone Resorption and Suppresses Osteoclastogenesis by Inducing A20
    • Abstract: Investigations on the therapeutic effects of intravenous immunoglobulin (IVIG) have focused on the suppression of autoantibody‐ and immune complex‐mediated inflammatory pathogenesis. Inflammatory diseases such as rheumatoid arthritis are often accompanied by excessive bone erosion but the effect of IVIG on osteoclasts, bone‐resorbing cells, has not been studied. Here, we investigate whether IVIG directly regulates osteoclast differentiation and has therapeutic potential for suppressing osteoclast‐mediated pathologic bone resorption. IVIG or cross‐linking of Fcγ receptors with plate‐bound IgG suppressed receptor activator of nuclear factor‐kappa B ligand (RANKL)‐induced osteoclastogenesis and expression of osteoclast‐related genes such as integrin β3 and cathepsin K in a dose‐dependent manner. Mechanistically, IVIG or plate‐bound IgG suppressed osteoclastogenesis by downregulating RANKL‐induced expression of NFATC1, the master regulator of osteoclastogenesis. IVIG suppressed NFATC1 expression by attenuating RANKL‐induced NF‐κB signaling, explained in part by induction of the inflammatory signaling inhibitor A20. IVIG administration attenuated in vivo osteoclastogenesis and suppressed bone resorption in the tumor necrosis factor (TNF)‐induced calvarial osteolysis model. Our findings show that, in addition to suppressing inflammation, IVIG directly inhibits osteoclastogenesis through a mechanism involving suppression of RANK signaling. Direct suppression of osteoclast differentiation may provide beneficial effects on preserving bone mass when IVIG is used to treat rheumatic disorders. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-17T08:49:03.091744-05:
      DOI: 10.1002/jcp.25091
  • Distinct Roles for Intestinal Epithelial Cell‐Specific Hdac1 and
           Hdac2 in the Regulation of Murine Intestinal Homeostasis
    • Abstract: The intestinal epithelium responds to and transmits signals from the microbiota and the mucosal immune system to insure intestinal homeostasis. These interactions are in part conveyed by epigenetic modifications, which respond to environmental changes. Protein acetylation is an epigenetic signal regulated by histone deacetylases, including Hdac1 and Hdac2. We have previously shown that villin‐Cre‐inducible intestinal epithelial cell (IEC)‐specific Hdac1 and Hdac2 deletions disturb intestinal homeostasis. To determine the role of Hdac1 and Hdac2 in the regulation of IEC function and the establishment of the dual knockout phenotype, we have generated villin‐Cre murine models expressing one Hdac1 allele without Hdac2, or one Hdac2 allele without Hdac1. We have also investigated the effect of short‐term deletion of both genes in naphtoflavone‐inducible Ah‐Cre and tamoxifen‐inducible villin‐CreER mice. Mice with one Hdac1 allele displayed normal tissue architecture, but increased sensitivity to DSS‐induced colitis. In contrast, mice with one Hdac2 allele displayed intestinal architecture defects, increased proliferation, decreased goblet cell numbers as opposed to Paneth cells, increased immune cell infiltration associated with fibrosis, and increased sensitivity to DSS‐induced colitis. In comparison to dual knockout mice, intermediary activation of Notch, mTOR and Stat3 signaling pathways was observed. While villin‐CreER Hdac1 and Hdac2 deletions led to an impaired epithelium and differentiation defects, Ah‐Cre‐mediated deletion resulted in blunted proliferation associated with the induction of a DNA damage response. Our results suggest that IEC determination and intestinal homeostasis are highly dependent on Hdac1 and Hdac2 activity levels, and that changes in the IEC acetylome may alter the mucosal environment. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-14T09:19:09.198588-05:
      DOI: 10.1002/jcp.25090
  • S‐Adenosylmethionine Affects ERK1/2 and STAT3 Pathways and Induces
           Apoptosis in Osteosarcoma Cells
    • Authors: Concetta Paola Ilisso; Luigi Sapio, Donatella Delle Cave, Michela Illiano, Annamaria Spina, Giovanna Cacciapuoti, Silvio Naviglio, Marina Porcelli
      Abstract: Osteosarcoma is a very aggressive bone tumor. Its clinical outcome remains discouraging despite intensive surgery, radiotherapy and chemotherapy. Thus, novel therapeutic approaches are demanded. S‐Adenosylmethionine (AdoMet) is a naturally occurring molecule that is synthesized in our body by methionine adenosyltransferase isoenzymes and is also available as a nutritional supplement. AdoMet is the principal methyl donor in numerous methylation reactions and is involved in many biological functions. Interestingly, AdoMet has been shown to exert antiproliferative action in various cancer cells. However, the underlying molecular mechanisms are just starting to be studied. Here, we investigated the effects of AdoMet on the proliferation of osteosarcoma U2OS cells and the underlying mechanisms. We carried out direct cell number counting, MTT and flow cytometry‐based assays, and immunoblotting experiments in response to AdoMet treatment. We found that AdoMet strongly inhibits proliferation of U2OS cells by slowing‐down cell cycle progression and by inducing apoptosis. We also report that AdoMet consistently causes an increase of p53 and p21 cell‐cycle inhibitor, a decrease of cyclin A and cyclin E protein levels, and a marked increase of pro‐apoptotic Bax/Bcl‐2 ratio, with caspase‐3 activation and PARP cleavage. Moreover, the AdoMet‐induced antiproliferative effects were dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels. Altogether, our data enforce the evidence of AdoMet acting as a biomolecule with antiproliferative action in osteosarcoma cells, capable of down‐regulating ERK1/2 and STAT3 pathways leading to cell cycle inhibition and apoptosis, and provide a rationale for the possible use of AdoMet in osteosarcoma therapy. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-14T09:19:04.107771-05:
      DOI: 10.1002/jcp.25089
  • Grp78 is Critical for Amelogenin‐Induced Cell Migration in a
           Multipotent Clonal Human Periodontal Ligament Cell Line
    • Authors: Kyosuke Toyoda; Takao Fukuda, Terukazu Sanui, Urara Tanaka, Kensuke Yamamichi, Ryo Atomura, Hidefumi Maeda, Atsushi Tomokiyo, Takaharu Taketomi, Takeshi Uchiumi, Fusanori Nishimura
      Abstract: Periodontal ligament stem cells (PDLSCs) are known to play a pivotal role in regenerating the periodontium. Amelogenin, which belongs to a family of extracellular matrix (ECM) proteins, is a potential bioactive molecule for periodontal regenerative therapy. However, its downstream target molecules and/or signaling patterns are still unknown. Our recent proteomic study identified glucose‐regulated protein 78 (Grp78) as a new amelogenin‐binding protein. In this study, we demonstrate, for the first time, the cellular responses induced by the biological interaction between amelogenin and Grp78 in the human undifferentiated PDL cell line 1‐17, which possesses the most typical characteristics of PDLSCs. Confocal co‐localization experiments revealed the internalization of recombinant amelogenin (rM180) via binding to cell surface Grp78, and the endocytosis was inhibited by the silencing of Grp78 in 1‐17 cells. Microarray analysis indicated that rM180 and Grp78 regulate the expression profiles of cell migration‐associated genes in 1‐17 cells. Moreover, Grp78 overexpression enhanced rM180‐induced cell migration and adhesion without affecting cell proliferation, while silencing of Grp78 diminished these activities. Finally, binding of rM180 to Grp78 promoted the formation of lamellipodia, and the simultaneous activation of Rac1 was also demonstrated by NSC23766, a widely accepted Rac1 inhibitor. These results suggest that Grp78 is essential for enhancing amelogenin‐induced migration in 1‐17 cells. The biological interaction of amelogenin with Grp78 offers significant therapeutic potential for understanding the biological components and specific functions involved in the signal transduction of amelogenin‐induced periodontal tissue regeneration. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-03T17:49:10.870409-05:
      DOI: 10.1002/jcp.25087
  • Alterations in Skeletal Muscle Oxidative Phenotype in Mice Exposed to
           Three Weeks of Normobaric Hypoxia
    • Authors: Ilse G.M. Slot; Annemie M.W.J. Schols, Chiel C. de Theije, Frank J.M. Snepvangers, Harry R. Gosker
      Abstract: Skeletal muscle of patients with chronic respiratory failure is prone to loss of muscle mass and oxidative phenotype. Tissue hypoxia has been associated with cachexia and emphysema in humans. Experimental research on the role of hypoxia in loss of muscle oxidative phenotype however has yielded inconsistent results. Animal studies are frequently performed in young animals, which may hinder translation to generally older aged patients. Therefore in this study we tested the hypothesis that hypoxia induces loss of skeletal muscle oxidative phenotype in a model of aged (52 weeks) mice exposed to three weeks of hypoxia. Additional groups of young (4 weeks) and adult (12 weeks) mice were included to examine age effects. To verify hypoxia‐induced cachexia, fat pad and muscle weights as well as muscle fiber cross‐sectional areas were determined. Muscle oxidative phenotype was assessed by expression and activity of markers of mitochondrial metabolism and fiber‐type distribution. A profound loss of muscle and fat was indeed accompanied by a slightly lower expression of markers of muscle oxidative capacity in the aged hypoxic mice. In contrast, hypoxia‐associated changes of fiber‐type composition were more prominent in the young mice. The differential response of the muscle of young, adult, and aged mice to hypoxia suggests that age matters and that the aged mouse is a better model for translation of findings to elderly patients with chronic respiratory disease. Furthermore, the findings warrant further mechanistic research into putative accelerating effects of hypoxia‐induced loss of oxidative phenotype on the cachexia process in chronic respiratory disease. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:54:30.354054-05:
      DOI: 10.1002/jcp.25083
  • TRPV3 Channel Negatively Regulates Cell Cycle Progression and Safeguards
           the Pluripotency of Embryonic Stem Cells
    • Authors: Iek Chi Lo; Hing Chung Chan, Zenghua Qi, Kwun Lam Ng, Chun So, Suk Ying Tsang
      Abstract: Embryonic stem cells (ESCs) have tremendous potential for research and future therapeutic purposes. However, the calcium handling mechanism in ESCs is not fully elucidated. Aims of this study are 1) to investigate if transient receptor potential vanilloid‐3 (TRPV3) channels are present in mouse ESCs (mESCs) and their subcellular localization; 2) to investigate the role of TRPV3 in maintaining the characteristics of mESCs. Western blot and immunocytochemistry showed that TRPV3 was present at the endoplasmic reticulum (ER) of mESCs. Calcium imaging showed that, in the absence of extracellular calcium, TRPV3 activators camphor and 6‐tert‐butyl‐m‐cresol increased the cytosolic calcium. However, depleting the ER store in advance of activator addition abolished the calcium increase, suggesting that TRPV3 released calcium from the ER. To dissect the functional role of TRPV3, TRPV3 was activated and mESC proliferation was measured by trypan blue exclusion and MTT assays. The results showed that TRPV3 activation led to a decrease in mESC proliferation. Cell cycle analysis revealed that TRPV3 activation increased the percentage of cells in G2/M phase; consistently, Western blot also revealed a concomitant increase in the expression of inactive form of cyclin‐dependent kinase 1, suggesting that TRPV3 activation arrested mESCs at G2/M phase. TRPV3 activation did not alter the expression of pluripotency markers Oct‐4, Klf4 and c‐Myc, suggesting that the pluripotency was preserved. Our study is the first study to show the presence of TRPV3 at ER. Our study also reveals the novel role of TRPV3 in controlling the cell cycle and preserving the pluripotency of ESCs. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:54:08.77255-05:0
      DOI: 10.1002/jcp.25086
  • Potential Role of Glycogen Synthase Kinase‐3β in Regulation of
           Myocardin Activity in Human Vascular Smooth Muscle Cells
    • Abstract: Glycogen synthase kinase (GSK)‐3β, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK‐3β results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co‐activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study therefore aimed to examine whether and how inhibition of GSK‐3β regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK‐3β inhibitors AR‐A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22) and ACTA2 (SM α‐actin). In human SMCs overexpressing myocardin through the T‐REx system, treatment with either GSK‐3β inhibitor also inhibited the expression of CNN1, TAGLN1 and ACTA2. These effects of GSK‐3β inhibitors were mimicked by transfection with GSK‐3β siRNA. Notably, both AR‐A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR‐A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant‐negative GSK‐3β mutant in myocardin‐overexpressing SMCs reduced the expression of calponin, SM22 and SM α‐actin. As expected, overexpression of constitutively‐active or wild‐type GSK‐3β in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK‐3β reduces myocardin transcriptional activity, suggesting a role for GSK‐3β in myocardin transcriptional activity and smooth muscle differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:53:47.238546-05:
      DOI: 10.1002/jcp.25084
  • Agents Which Inhibit NF‐κB Signaling Block Spontaneous
           Contractile Activity and Negatively Influence Survival of Developing
    • Authors: C. George Carlson; Lauren Stein, Elizabeth Dole, Ross M. Potter, David Bayless
      Abstract: Inhibiting the NF‐κB signaling pathway provides morphological and functional benefits for the mdx mouse, a model for Duchenne muscular dystrophy characterized by chronic elevations in the nuclear expression of p65, the transactivating component of the NF‐κB complex. The purpose of this study was to examine p65 expression in nondystrophic and mdx myotubes using confocal immunofluorescence, and determine whether inhibitors of the NF‐κB pathway alter myotube development. Primary cultures of nondystrophic and mdx myotubes had identical levels of nuclear and cytosolic p65 expression and exhibited equivalent responses to TNF‐α, thus excluding the hypothesis that the lack of dystrophin is sufficient to induce increases in NF‐κB signaling. The NF‐κB inhibitors pyrrolidine dithiocarbamate (PDTC) and sulfasalazine decreased spontaneous contractile activity and reduced myotube viability in a dose‐ and time‐dependent manner. Similarly, a vivo‐morpholino designed to block translation of murine p65 (m‐p65tb‐vivomorph1) rapidly abolished spontaneous contractile activity, reduced p65 expression measured by confocal immunofluorescence, and induced cell death in primary cultures of nondystrophic and mdx myotubes. Similar effects on p65 immunofluorescence and cell viability were observed following m‐p65tb‐vivomorph1 exposure to spontaneously inactive C2C12 myotubes, while exposure to a control scrambled vivo morpholino had no effect. These results indicate a direct role of the NF‐κB pathway in myotube development and identify a potential therapeutic limitation to the use of NF‐κB inhibitors in treating Duchenne and related muscular dystrophies. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:53:25.163707-05:
      DOI: 10.1002/jcp.25085
  • Pathophysiology of Corneal Dystrophies: From Cellular Genetic Alteration
           to Clinical Findings
    • Abstract: Corneal dystrophies are a heterogeneous group of bilateral, inherited, rare diseases characterized by slowly progressive corneal opacities, that lead to visual impairment. Most of them have an autosomal dominant pattern of inheritance with variable expressivity, but new mutations have been described. Many corneal dystrophies have been genetically characterized and the specific gene mutations identified, such as for the epithelial–stromal TGFBI dystrophies. Current classification systems identified four main groups of corneal dystrophies based on clinical, histologic and genetic information. Diagnosis is performed during a routine ophthalmic examination that shows typical cellular abnormalities of the corneal epithelium, stroma or endothelium. Disease progression should be carefully monitored to decide the proper clinical management. The treatment of corneal dystrophies is variable, depending on symptoms, clinical course, severity and type of dystrophy. Management aimed to reduce symptoms and to improve vision, includes different surgical approaches. Novel cellular and genetic therapeutic approaches are under evaluation. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-23T18:46:57.198342-05:
      DOI: 10.1002/jcp.25082
  • Critical Role of AMPK/FoxO3A Axis in Globular Adiponectin‐Induced
           Cell Cycle Arrest and Apoptosis in Cancer Cells
    • Abstract: Adiponectin predominantly secreted from adipose tissue has exhibited potent anti‐proliferative properties in cancer cells via modulating cell cycle and apoptosis. FoxO3A, a Forkhead box O member of the transcription factor, plays a critical role in modulating expression of genes involved in cell death and/or survival. In this study, we investigated the role of FoxO3A signaling in anti‐cancer activities of adiponectin. Herein, we have shown that treatment with globular adiponectin (gAcrp) increases p27 but decreases cyclinD1 expression in human hepatoma (HepG2) and breast (MCF‐7) cancer cells. Gene ablation of FoxO3A prevented gAcrp‐induced increase in p27 and decreased in cyclin D1 expression, and further ameliorated cell cycle arrest by gAcrp, indicating a critical role of FoxO3A in gAcrp‐induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase‐3/7 activation and increased Fas ligand (FasL) expression in both HepG2 and MCF‐7 cells. Transfection with FoxO3A siRNA inhibited gAcrp‐induced caspase‐3/7 activation and FasL expression, suggesting that FoxO3A signaling also plays an important role in gAcrp‐induced apoptosis of cancer cells. We also found that gene silencing of AMPK prevented gAcrp‐induced nuclear translocation of FoxO3A in HepG2 and MCF‐7 cells. In addition, suppression of AMPK also blocked gAcrp‐induced cell cycle arrest and further attenuated gAcrp‐induced caspase‐3/7 activation, indicating that AMPK signaling plays a pivotal role in both gAcrp‐induced cell cycle arrest and apoptosis via acting as an upstream signaling of FoxO3A. Taken together, our findings demonstrated that AMPK/FoxO3A axis plays a cardinal role in anti‐proliferative effect of adiponectin in cancer cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-18T18:54:36.875656-05:
      DOI: 10.1002/jcp.25080
  • Platelet‐Derived CCL5 Regulates CXC Chemokine Formation and
           Neutrophil Recruitment in Acute Experimental Colitis
    • Authors: Changhui Yu; Songen Zhang, Yongzhi Wang, Su Zhang, Lingtao Luo, Henrik Thorlacius
      Abstract: Accumulating data suggest that platelets not only regulate thrombosis and haemostasis but also inflammatory processes. Platelets contain numerous potent pro‐inflammatory compounds, including the chemokines CCL5 and CXCL4 although their role in acute colitis remains elusive. The aim of this study was to examine the role of platelets and platelet‐derived chemokines in acute colitis. Acute colitis was induced in female Balb/c mice by administration of 5% dextran sodium sulphate (DSS) for five days. Animals received a platelet‐depleting, anti‐CCL5, anti‐CXCL4 or a control antibody prior to DSS challenge. Colonic tissue was collected for quantification of myeloperoxidase (MPO) activity, CXCL5, CXCL2, interleukin‐6 (IL‐6) and CCL5 levels as well as morphological analyses. Platelet depletion reduced tissue damage and clinical disease activity index in DSS‐exposed animals. Platelet depletion not only reduced levels of CXCL2 and CXCL5 but also levels of CCL5 in the inflamed colon. Immunoneutralization of CCL5 but not CXCL4 reduced tissue damage, CXC chemokine expression and neutrophil recruitment in DSS‐treated animals. These findings show that platelets play a key role in acute colitis by regulating CXC chemokine generation, neutrophil infiltration and tissue damage in the colon. Moreover, our results suggest that platelet‐derived CCL5 is an important link between platelet activation and neutrophil recruitment in acute colitis. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-18T18:54:10.466808-05:
      DOI: 10.1002/jcp.25081
  • Genetic and Functional Analysis of Polymorphisms in the Human Dopamine
           Receptor and Transporter Genes in Small Cell Lung Cancer
    • Authors: Emanuela Cherubini; Arianna Di Napoli, Alessia Noto, Giorgia Amira Osman, Maria Cristina Esposito, Salvatore Mariotta, Rossella Sellitri, Luigi Ruco, Giuseppe Cardillo, Gennaro Ciliberto, Rita Mancini, Alberto Ricci
      Abstract: The regulatory role of dopamine (DA) in endocrine, cardiovascular and renal functions has been extensively studied and used for clinical purposes. More recently DA has been indicated as a regulatory molecule for immune cells and malignant cell proliferation. We assessed the expression and the functional role DA, DA receptors and transporters in primary small cell lung cancer (SCLC). By HPLC DA plasma levels were more elevated in SCLC patients in comparison with NSCLC patients and healthy controls. SCLC cell expressed DA D1‐ and D2‐like receptors and membrane and vesicular transporters at protein and mRNA levels. We also investigated the effects of independent D1‐ or D2‐like receptor stimulation on SCLC cell cultures. DA D1 receptor agonist SKF38393 induced the increase of cAMP levels and DARPP‐32 protein expression without affecting SCLC growth rate. Cell treatment with the DA D1 receptor antagonist SCH23390 inhibited SKF38393 effects. In contrast, the DA D2 receptor agonist quinpirole (10μM) counteracted, in a dose and time dependent way, SCLC cell proliferation, it did not affect cAMP levels and decreased phosphorylated AKT that was induced by DA D2 receptor antagonist sulpiride. However, in only one SCLC line, stimulation of DA D2 receptor failed to inhibit cell proliferation in vitro. This effect was associated to the existence of rs6275 and rs6277 polymorphisms in the D2 gene. These results gave more insight into DA control of lung cancer cell behavior and suggested the existence of different SCLC phenotypes. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-16T17:46:14.085232-05:
      DOI: 10.1002/jcp.25079
  • Aquaporin‐1 Plays Important Role in Proliferation by Affecting Cell
           Cycle Progression
    • Abstract: Aquaporin‐1 (AQP1) has been associated with tumor development. Here, we investigated how AQP1 may affect cell proliferation. The proliferative rate of adult carotid body (CB) cells, known to proliferate under chronic hypoxia, was analyzed in wild‐type (AQP1 +/ +) and knock out (AQP1 ‐/‐) mice, maintained in normoxia or exposed to hypoxia while BrdU was administered. Fewer numbers of total BrdU+ and TH‐BrdU+ cells were observed in AQP1 ‐/‐ mice, indicating a role for AQP1 in CB proliferation. Then, by flow cytometry, cell cycle state and proliferation of cells overexpressing AQP1 were compared to those of wild‐type cells. In the AQP1‐overexpressing cells, we observed higher cell proliferation and percentages of cells in phases S and G2/M and fewer apoptotic cells after nocodazole treatment were detected by annexin V staining. Also in these cells, proteomic assays showed higher expression of cyclin D1 and E1 and microarray analysis revealed changes in many cell proliferation‐related molecules, including, Zeb 2, Jun, NF‐kβ, Cxcl9, Cxcl10, TNF, and the TNF receptor. Overall, our results indicate that the presence of AQP1 modifies the expression of key cell cycle proteins apparently related to increases in cell proliferation. This contributes to explaining the presence of AQP1 in many different tumors. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-16T17:43:07.293339-05:
      DOI: 10.1002/jcp.25078
  • Dexamethasone Downregulates SLC7A5 Expression and Promotes Cell Cycle
           Arrest, Autophagy and Apoptosis in BeWo Cells
    • Authors: Bin He; Nana Zhang, Ruqian Zhao
      Abstract: Synthetic glucocorticoids (GCs) such as dexamethasone (Dex) are widely given to pregnant women to induce maturation and improve viability of preterm infants. Despite the beneficial effects, synthetic GCs have adverse effects on placental growth and nutrient transport system. However, the molecular mechanisms involved in these events remain unknown. Here we use a human placental choriocarcinoma cell line (BeWo) as model to explore the pathway linking amino acids transport with cell viability under Dex challenge. BeWo cells treated with Dex (100 nM) for 24 h demonstrated G1/S cell cycle arrest together with enhanced autophagy and apoptosis. Concurrently, the amino acid carrier SLC7A5 was down‐regulated in association with impaired cellular amino acids uptake and inhibition of mammalian target of rapamycin (mTOR) signaling. Similar cellular responses were observed in BeWo cells treated with BCH, a classical System L inhibitor which inactivates SLC7A5. The glucocorticoid receptor (GR) antagonist RU486 was able to diminish Dex‐induced translocation of GR into nucleus and to abolish these effects. Furthermore, Dex treatment significantly promoted the binding of GR to the proximal promoter sequence of SLC7A5 gene. Taken together, our results show that Dex downregulates SLC7A5 expression via GR‐mediated transrepression. The impaired amino acids uptake leads to inhibition of mTOR signaling which in turn causes inhibited proliferation and enhanced autophagy and apoptosis in BeWo cells. These findings indicate that SLC7A5 mediates the effect of Dex on cell viability, thus providing a novel molecular target for the prevention and treatment of Dex‐induced cell cycle arrest and apoptosis in placental cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-10T18:47:13.684676-05:
      DOI: 10.1002/jcp.25076
  • Electrospun Type 1 Collagen Matrices Using a Novel Benign Solvent for
           Cardiac Tissue Engineering
    • Authors: Alan M. Punnoose; Anuradha Elamparithi, Sarah Kuruvilla
      Abstract: Electrospinning is a well‐established technique that uses a high electric field to fabricate ultra fine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers due to its biocompatibility and widespread occurrence in nature. Electrospinning of Collagen alone has been reported with fluoroalcohols such as Hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE), which are toxic to the environment. In this study we describe the use of a novel benign binary solvent to generate nanofibers of Collagen type 1, which is non‐toxic and economical. Transmission electron microscopy (TEM) analysis revealed the characteristic feature of native collagen namely the 67 nm banding pattern, confirming that the triple helical structure was maintained. Scanning Electron Microscopy (SEM) analysis showed the fiber diameters to be in the 200‐800 nm range. Biocompatibility of the three dimensional (3D) scaffolds was established by MTT assays using skeletal myoblasts and Confocal Microscopic analysis of immunofluorescent stained sections for muscle specific markers such as Desmin and Actin. Primary neonatal rat ventricular cardiomyocytes seeded onto the scaffolds were able to maintain their contractile function for a period of 17 days. Our work provides evidence that Collagen 1 can be electrospun without combining with other polymers using a novel benign solvent and we are currently exploring the potential of this approach for cardiac and skeletal muscle tissue engineering. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:48:51.922438-05:
      DOI: 10.1002/jcp.25035
  • Protein Kinase C Is Involved in the Induction of ATP‐Binding
           Cassette Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor
           Agonist in Human Macrophages
    • Authors: Etimad A. Huwait; Nishi N. Singh, Daryn R. Michael, Thomas S. Davies, Joe W. E. Moss, Dipak P. Ramji
      Abstract: The transcription of the ATP‐binding cassette transporter A1 (ABCA1) gene, which plays a key anti‐atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c‐Jun N‐terminal kinase (JNK) and phosphoinositide 3‐kinase (PI3K) in the LXRs‐mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have therefore investigated a potential role for PKC in the action of LXR/RXR agonist 22‐(R)‐hydroxycholesterol (22‐(R)‐HC)/9‐cis‐retinoic acid (9cRA) in THP‐1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein‐1 (AP‐1) DNA binding activity in macrophages treated with 22‐(R)‐HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype‐specific inhibitors. These studies therefore reveal a potentially important role for PKC in the action of 22‐(R)‐HC and 9cRA in human macrophages. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-07T18:01:38.140641-05:
      DOI: 10.1002/jcp.25157
  • p14ARF Prevents Proliferation of Aneuploid Cells by Inducing
           p53‐Dependent Apoptosis
    • Authors: Lorena Veneziano; Viviana Barra, Laura Lentini, Sergio Spatafora, Aldo Di Leonardo
      Abstract: Weakening the Spindle Assembly Checkpoint by reduced expression of its components induces chromosome instability and aneuploidy that are hallmarks of cancer cells. The tumor suppressor p14ARF is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14ARF is involved in the maintenance of aneuploid cells in primary human cells suggesting that it could be part of a pathway controlling their proliferation. To investigate further this aspect, p14ARF was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. p14ARF re‐expression reduced the number of aneuploid cells in MAD2 post‐transcriptionally silenced cells. Also aberrant mitoses, frequently displayed in MAD2‐depleted cells, were decreased when p14ARF was expressed at the same time. In addition p14ARF ectopic expression in MAD2‐depleted cells induced apoptosis associated with increased p53 protein levels. Conversely, p14ARF ectopic expression did not induce apoptosis in HCT116 p53KO cells. Collectively, our results suggest that the tumor suppressor p14ARF may have an important role in counteracting proliferation of aneuploid cells by activating p53‐dependent apoptosis. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-08T22:21:10.033109-05:
      DOI: 10.1002/jcp.24976
  • Is the Reg3α (HIP/PAP) Protein Really an Obesogenic Factor'
    • First page: 1
      Abstract: We would like to point out an internal inconsistency in the data presented by V. Secq et al. in the article entitled “PAP/HIP protein is an obesogenic factor” published in the February 2014 issue of “Journal of Cellular Physiology”, which cast serious doubt on the validity of the statement serving as the title of the article. We, and others, including J. Iovanna and his collaborators, have long shown that the C‐type lectin PAP/HIP (also known as HIP/PAP or Reg3α) has anti‐inflammatory and antioxidant activities in a number of cell types, among which pancreatic cells, hepatocytes and neurons (Gironella et al., 2005; Closa et al., 2007; Moniaux et al., 2011; Kapur et al., 2012; Haldipur et al., 2014) and that, therefore, it might help cure, or prevent, inflammatory and metabolic diseases. Given its envisaged clinical uses, the question of whether PAP/HIP significantly modifies metabolism toward weight increase is essential and must be subjected to rigorous scrutiny. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-19T17:57:14.333248-05:
      DOI: 10.1002/jcp.25046
  • Response to “Is the Reg3α (HIP/PAP) Protein Really an
           Obesogenic Factor'”
    • Authors: Veronique Secq; Cecilia Mallmann, Meritxell Gironella, Belen Lopez, Daniel Closa, Stephane Garcia, Laurence Christa, Giuseppe Montalto, Nelson Dusetti, Juan L. Iovanna
      First page: 2
      Abstract: We are grateful to Gonzalez and colleagues for their comments and remarks, which give us the opportunity to clarify a few points that were probably not sufficiently detailed in the manuscript. In our work we demonstrate that TgPAP/HIP mice, which express the PAP/HIP transgene in their liver and have a high level of circulating PAP/HIP, are obese. This statement is based on the augmented body weight but not only: circulating leptin, which is known to correlate with the amount of adipose tissue, is significantly elevated in these mice as presented in Figure 2. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T03:52:49.224005-05:
      DOI: 10.1002/jcp.25130
  • Mechanisms and Consequences of Double‐Strand DNA Break Formation in
    • Authors: Wendy J. Cannan; David S. Pederson
      Pages: 3 - 14
      Abstract: All organisms suffer double‐strand breaks (DSBs) in their DNA as a result of exposure to ionizing radiation. DSBs can also form when replication forks encounter DNA lesions or repair intermediates. The processing and repair of DSBs can lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. The most common pathway used to repair DSBs in metazoans (non‐homologous DNA end joining) is more commonly mutagenic than the alternative pathway (homologous recombination mediated repair). Thus, factors that influence the choice of pathways used DSB repair can affect an individual's mutation burden and risk of cancer. This review describes radiological, chemical, and biological mechanisms that generate DSBs, and discusses the impact of such variables as DSB etiology, cell type, cell cycle, and chromatin structure on the yield, distribution, and processing of DSBs. The final section focuses on nucleosome‐specific mechanisms that influence DSB production, and the possible relationship between higher order chromosome coiling and chromosome shattering (chromothripsis). J. Cell. Physiol. 230: 3–14, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:22.910291-05:
      DOI: 10.1002/jcp.25048
  • Error‐Prone Repair of DNA Double‐Strand Breaks
    • Authors: Kasey Rodgers; Mitch McVey
      Pages: 15 - 24
      Abstract: Preserving the integrity of the DNA double helix is crucial for the maintenance of genomic stability. Therefore, DNA double‐strand breaks represent a serious threat to cells. In this review, we describe the two major strategies used to repair double strand breaks: non‐homologous end joining and homologous recombination, emphasizing the mutagenic aspects of each. We focus on emerging evidence that homologous recombination, long thought to be an error‐free repair process, can in fact be highly mutagenic, particularly in contexts requiring large amounts of DNA synthesis. Recent investigations have begun to illuminate the molecular mechanisms by which error‐prone double‐strand break repair can create major genomic changes, such as translocations and complex chromosome rearrangements. We highlight these studies and discuss proposed models that may explain some of the more extreme genetic changes observed in human cancers and congenital disorders. J. Cell. Physiol. 230: 15–24, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:26.720925-05:
      DOI: 10.1002/jcp.25053
  • MicroRNA as Biomarkers and Diagnostics
    • Authors: Jin Wang; Jinyun Chen, Subrata Sen
      Pages: 25 - 30
      Abstract: MicroRNAs (miRNAs) are a group of small non‐coding RNAs that are involved in regulating a range of developmental and physiological processes; their dysregulation has been associated with development of diseases including cancer. Circulating miRNAs and exosomal miRNAs have also been proposed as being useful in diagnostics as biomarkers for diseases and different types of cancer. In this review, miRNAs are discussed as biomarkers for cancer and other diseases, including viral infections, nervous system disorders, cardiovascular disorders, and diabetes. We summarize some of the clinical evidence for the use of miRNAs as biomarkers in diagnostics and provide some general perspectives on their use in clinical situations. The analytical challenges in using miRNAs in cancer and disease diagnostics are evaluated and discussed. Validation of specific miRNA signatures as biomarkers is a critical milestone in diagnostics. J. Cell. Physiol. 230: 25–30, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:38.503525-05:
      DOI: 10.1002/jcp.25056
  • C‐ing the Genome: A Compendium of Chromosome Conformation Capture
           Methods to Study Higher‐Order Chromatin Organization
    • Pages: 31 - 35
      Abstract: Three‐dimensional organization of the chromatin has important roles in transcription, replication, DNA repair, and pathologic events such as translocations. There are two fundamental ways to study higher‐order chromatin organization: microscopic and molecular approaches. In this review, we briefly introduce the molecular approaches, focusing on chromosome conformation capture or “3C” technology and its derivatives, which can be used to probe chromatin folding at resolutions beyond that provided by microscopy techniques. We further discuss the different types of data generated by the 3C‐based methods and how they can be used to answer distinct biological questions. J. Cell. Physiol. 230: 31–35, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:30.697378-05:
      DOI: 10.1002/jcp.25062
  • Translating Regenerative Biomaterials Into Clinical Practice
    • Pages: 36 - 49
      Abstract: Globally health care spending is increasing unsustainably. This is especially true of the treatment of musculoskeletal (MSK) disease where in the United States the MSK disease burden has doubled over the last 15 years. With an aging and increasingly obese population, the surge in MSK related spending is only set to worsen. Despite increased funding, research and attention to this pressing health need, little progress has been made toward novel therapies. Tissue engineering and regenerative medicine (TERM) strategies could provide the solutions required to mitigate this mounting burden. Biomaterial‐based treatments in particular present a promising field of potentially cost‐effective therapies. However, the translation of a scientific development to a successful treatment is fraught with difficulties. These barriers have so far limited translation of TERM science into clinical treatments. It is crucial for primary researchers to be aware of the barriers currently restricting the progression of science to treatments. Researchers need to act prospectively to ensure the clinical, financial, and regulatory hurdles which seem so far removed from laboratory science do not stall or prevent the subsequent translation of their idea into a treatment. The aim of this review is to explore the development and translation of new treatments. Increasing the understanding of these complexities and barriers among primary researchers could enhance the efficiency of biomaterial translation. J. Cell. Physiol. 230: 36–49, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:39.091914-05:
      DOI: 10.1002/jcp.25071
  • Potential for Stem Cell‐Based Periodontal Therapy
    • Authors: Seyed Hossein Bassir; Wichaya Wisitrasameewong, Justin Raanan, Sasan Ghaffarigarakani, Jamie Chung, Marcelo Freire, Luciano C. Andrada, Giuseppe Intini
      Pages: 50 - 61
      Abstract: Periodontal diseases are highly prevalent and are linked to several systemic diseases. The goal of periodontal treatment is to halt the progression of the disease and regenerate the damaged tissue. However, achieving complete and functional periodontal regeneration is challenging because the periodontium is a complex apparatus composed of different tissues, including bone, cementum, and periodontal ligament. Stem cells may represent an effective therapeutic tool for periodontal regeneration due to their plasticity and their ability to regenerate different tissues. This review presents and critically analyzes the available information on stem cell‐based therapy for the regeneration of periodontal tissues and suggests new avenues for the development of more effective therapeutic protocols. J. Cell. Physiol. 230: 50–61, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:41.730577-05:
      DOI: 10.1002/jcp.25067
  • Correction of the FSHD Myoblast Differentiation Defect by Fusion With
           Healthy Myoblasts
    • First page: 62
      Abstract: Facioscapulohumeral dystrophy (FSHD) is a neuromuscular disease with a prevalence that could reach 1 in 8,000 characterized by progressive asymmetric muscle weakness. Myoblasts isolated from FSHD muscles exhibit morphological differentiation defects and show a distinct transcription profile. These abnormalities may be linked to the muscle weakness in FSHD patients. We have tested whether fusion of FSHD myoblasts with primary myoblasts isolated from healthy individuals could correct the differentiation defects. Our results show that the number of hybrid myotubes with normal phenotype increased with the percentage of normal myoblasts initially cultured. We demonstrated that a minimum of 50% of normal nuclei is required for a phenotypic correction of the FSHD phenotype. Moreover, transcriptomic profiles of phenotypically corrected hybrid myotubes showed that the expression of deregulated genes in FSHD myotubes became almost normal. The number of deregulated pathways also decreased from 39 in FSHD myotubes to one in hybrid myotubes formed with 40% FSHD and 60% normal myoblasts. We thus propose that while phenotypical and functional correction of FSHD is feasible, it requires more than 50% of normal myoblasts, it creates limitations for cell therapy in the FSHD context. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:42:08.69064-05:0
      DOI: 10.1002/jcp.25110
  • Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In
           Vivo and Osteoprogenitor Differentiation Ex Vivo
    • Authors: Nagat Frara; Samir M. Abdelmagid, Gregory R. Sondag, Fouad M. Moussa, Vanessa R. Yingling, Thomas A. Owen, Steven N. Popoff, Mary F. Barbe, Fayez F. Safadi
      Pages: 72 - 83
      Abstract: Initial identification of osteoactivin (OA)/glycoprotein non‐melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post‐fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV‐promoter (OA‐Tg). Western blot analysis showed increased OA/gpnmb in OA‐Tg osteoblasts, compared to wild‐type (WT). In OA‐Tg mouse femurs versus WT littermates, micro‐CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA‐Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over‐expressed in osteoclasts in OA‐Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX‐1 and RANK‐L, and decreased osteoclast numbers in OA‐Tg, compared to WT mice, indicating decreased bone remodeling in OA‐Tg mice. The proliferation rate of OA‐Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA‐Tg osteoprogenitors. Quantitative RT‐PCR analysis showed increased TGF‐β1 and TGF‐β receptors I and II expression in OA‐Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. J. Cell. Physiol. 230: 72–83, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:32.488614-05:
      DOI: 10.1002/jcp.25020
  • Cell‐penetrating and endoplasmic reticulum‐locating
           TAT‐IL‐24‐KDEL fusion protein induces tumor apoptosis
    • Authors: Jian Zhang; Aiyou Sun, Rui Xu, Xinyi Tao, Yuguo Dong, Xinxin Lv, Dongzhi Wei
      Pages: 84 - 93
      Abstract: Interleukin‐24 (IL‐24) is a unique IL‐10 family cytokine that could selectively induce apoptosis in cancer cells without harming normal cells. Previous research demonstrated that intracellular IL‐24 protein induces an endoplasmic reticulum (ER) stress response only in cancer cells, culminating in apoptosis. In this study, we developed a novel recombinant fusion protein to penetrate into cancer cells and locate on ER. It is composed of three distinct functional domains, IL‐24, and the targeting domain of transactivator of transcription (TAT) and an ER retention four‐peptide sequence KDEL (Lys‐Asp‐Glu‐Leu) that link at its NH2 and COOH terminal, respectively. The in vitro results indicated that TAT‐IL‐24‐KDEL inhibited growth in bladder cancer cells, as well as in non‐small cell lung cancer cell line and breast cancer cell line, but the normal human lung fibroblast cell line was not affected, indicating the cancer specificity of TAT‐IL‐24‐KDEL. Western blot analysis showed that apoptosis activation was induced by TAT‐IL‐24‐KDEL through the ER stress‐mediated cell death pathway. Treatment with TAT‐IL‐24‐KDEL significantly inhibited the growth of human H460 xenografts in nude mice, and the tumor growth inhibition was correlated with increased hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) staining. These findings suggest that the artificially designed recombinant fusion protein TAT‐IL‐24‐KDEL may be highly effective in cancer therapy and worthy of further evaluation and development. J. Cell. Physiol. 230: 84–93, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:26.09135-05:0
      DOI: 10.1002/jcp.25054
  • Endotoxin‐stimulated Rat Hepatic Stellate Cells Induce Autophagy in
           Hepatocytes as a Survival Mechanism
    • Authors: Anil Dangi; Chao Huang, Ashish Tandon, Donna Stolz, Tong Wu, Chandrashekhar R. Gandhi
      Pages: 94 - 105
      Abstract: Bacterial lipopolysaccharide (LPS)‐stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα, and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/mL) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS‐stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6 h. In vitro, HSCs were treated with 100 ng/mL LPS till 24 h. The medium was transferred to hepatocytes, and determinations were made at 0–12 h. Controls were HSC‐conditioned medium or medium‐containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c‐Jun NH2 terminal kinase (JNK) and apoptosis of ∼4–5% of hepatocytes. In vitro, LPS‐conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ∼10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS‐stimulated HSCs produced IFNβ, and LPS/HSC‐induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti‐IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS‐stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia. J. Cell. Physiol. 230: 94–105, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:27.636444-05:
      DOI: 10.1002/jcp.25055
  • Enhanced Chemotherapeutic Behavior of Open‐Caged DNA@Doxorubicin
           Nanostructures for Cancer Cells
    • Authors: Vinit Kumar; Samer Bayda, Mohamad Hadla, Isabella Caligiuri, Concetta Russo Spena, Stefano Palazzolo, Susanne Kempter, Giuseppe Corona, Giuseppe Toffoli, Flavio Rizzolio
      Pages: 106 - 110
      Abstract: In cancer therapy, it is imperative to increase the efficacy and reduce side effects of chemotherapeutic drugs. Nanotechnology offers the unique opportunity to overcome these barriers. In particular, in the last few years, DNA nanostructures have gained attention for their biocompatibility, easy customized synthesis and ability to deliver drugs to cancer cells. Here, an open‐caged pyramidal DNA@Doxorubicin (Py‐Doxo) nanostructure was constructed with 10 DNA sequences of 26–28 nucleotides for drug delivery to cancer cells. The synthesized DNA nanostructures are sufficiently stable in biological medium. Py‐Doxo exhibited significantly enhanced cytotoxicity of the delivered doxorubicin to breast and liver cancer cells up to twofold compared to free doxorubicin. This study demonstrates the importance of the shape and structure of the designed transporter DNA nanostructures for biomedical applications. J. Cell. Physiol. 230: 106–110, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:28.294072-05:
      DOI: 10.1002/jcp.25057
  • EGF‐Induced Connexin43 Negatively Regulates Cell Proliferation in
           Human Ovarian Cancer
    • Pages: 111 - 119
      Abstract: Connexin43 (Cx43) has been shown to regulate cell proliferation and its downregulation is correlated with poor prognosis and survival in several types of human cancer. Cx43 expression levels are frequently downregulated in human ovarian cancer, suggesting a potential role for Cx43 in regulating the progression of this disease. Epidermal growth factor (EGF) is a well‐characterized hormone that stimulates ovarian cancer cell proliferation. Although EGF is able to regulate Cx43 expression in other cell types, it is unclear whether EGF can regulate Cx43 expression in ovarian cancer cells. Additionally, it remains unknown whether Cx43 is involved in EGF‐stimulated ovarian cancer cell proliferation. In the present study, we demonstrate that treatment with EGF upregulates Cx43 expression in two ovarian cancer cell lines, SKOV3 and OVCAR4. Although treatment with EGF activates both ERK1/2 and Akt signaling pathways, pharmacological inhibition and siRNA‐mediated knockdown suggest that only the activation of Akt1 is required for EGF‐induced Cx43 upregulation. Functionally, Cx43 knockdown enhanced basal and EGF‐induced cell proliferation, whereas the proliferative effects of EGF were reduced by Cx43 overexpression. Co‐treatment with the gap junction inhibitor carbenoxolone did not alter the suppressive effects of Cx43 overexpression on EGF‐induced cell proliferation, suggesting a gap junction‐independent mechanism. This study reveals an important role for Cx43 as a negative regulator of EGF‐induced human ovarian cancer cell proliferation. J. Cell. Physiol. 230: 111–119, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:40.1841-05:00
      DOI: 10.1002/jcp.25058
  • MicroRNAs Provide Feedback Regulation of Epithelial‐Mesenchymal
           Transition Induced by Growth Factors
    • Authors: Zailong Qin; Wei He, Jingqun Tang, Qiurong Ye, Wei Dang, Yuanjun Lu, Jia Wang, Guiyuan Li, Qun Yan, Jian Ma
      Pages: 120 - 129
      Abstract: As regulators in gene expression, microRNAs take part in most biological processes including cell differentiation, apoptosis, cell cycle, and epithelial‐to‐mesenchymal transition (EMT). In order to evaluate their roles in EMT process, microRNA expression profile changes induced by EGF or TGF‐β treatment on nasopharyngeal carcinoma cell HK‐1 were analyzed, and miR‐21, miR‐148a, miR‐505, and miR‐1207‐5p were found to be upregulated in growth factors‐induced EMT process. miR‐21 is already known as an oncogenic miRNA to promote metastasis, however, the exact functions of other three miRNAs in EMT are unclear. To our surprise, we found that miR‐148a, miR‐505, and miR‐1207‐5p can suppress EMT and metastasis phenotypes in HK‐1 cells both in vitro and in vivo, which may relate to their inhibition on EMT and Wnt signaling molecules. MiRNAs confer robustness to biological processes by posttranscriptional repression of key transcriptional programs that are related to previous developmental stages or to alternative cell fates. Our findings indicate that miRNA feedback circuit is tuned to respond to growth factors‐induced EMT, and we suggested a new negative feedback loop which may be an important element of the EMT process and confer biological robustness. J. Cell. Physiol. 230: 120–129, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:37.948095-05:
      DOI: 10.1002/jcp.25060
  • The Association Between p38 MAPK‐Mediated TNF‐α/TNFR2
           up‐Regulation and
           Apoptosis in Human Leukemia U937 Cells
    • Pages: 130 - 141
      Abstract: The primary cause of treatment failures in acute myeloid leukemia is usually associated with defects in the apoptotic pathway. Several studies suggest that 2‐(4‐aminophenyl)‐7‐methoxybenzothiazole (7‐OMe‐APBT) may potentially induce apoptosis of cancer cells. Thus, the present study was conducted to explore the cytotoxic effect of 7‐OMe‐APBT on human leukemia U937 cells. The apoptosis of human leukemia U937 cells induced by 7‐OMe‐APBT was characterized by an increase in mitochondrial membrane depolarization, procaspase‐8 degradation, and tBid production. Down‐regulation of FADD blocked 7‐OMe‐APBT‐induced procaspase‐8 degradation and rescued the viability of 7‐OMe‐APBT‐treated cells, suggesting the involvement of a death receptor‐mediated pathway in 7‐OMe‐APBT‐induced cell death. Increased TNF‐α expression, TNFR2 expression, and p38 MAPK phosphorylation were noted in 7‐OMe‐APBT‐treated cells. Pretreatment with a p38 MAPK inhibitor abolished 7‐OMe‐APBT‐induced TNF‐α and TNFR2 up‐regulation. 7‐OMe‐APBT stimulated p38 MAPK/c‐Jun‐mediated transcriptional up‐regulation of TNFR2, while the increased TNF‐α mRNA stability led to TNF‐α up‐regulation in 7‐OMe‐APBT‐treated cells. Treatment with 7‐OMe‐APBT up‐regulated protein phosphatase 2A catalytic subunit α (PP2Acα) expression via the p38 MAPK/c‐Jun/ATF‐2 pathway, which, in turn, promoted tristetraprolin (TTP) degradation. Pretreatment with a protein phosphatase 2A inhibitor or TTP over‐expression abrogated TNF‐α up‐regulation in 7‐OMe‐APBT‐treated cells. Abolishment of TNF‐α up‐regulation or knock‐down of TNFR1/TNFR2 by siRNA restored the viability of 7‐OMe‐APBT‐treated cells. Taken together, our data indicate a connection between p38 MAPK‐mediated TNF‐α and TNFR2 up‐regulation and 7‐OMe‐APBT‐induced TNF‐α‐mediated death pathway activation in U937 cells. The same pathway also elucidates the mechanism underlying 7‐OMe‐APBT‐induced death of human leukemia HL‐60 cells. J. Cell. Physiol. 230: 130–141, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:31.986606-05:
      DOI: 10.1002/jcp.25064
  • Lanthanum Chloride Attenuates Osteoclast Formation and Function Via the
           Downregulation of Rankl‐Induced Nf‐κb and Nfatc1
    • Authors: Chuan Jiang; Jiangyinzi Shang, Zhe Li, An Qin, Zhengxiao Ouyang, Xinhua Qu, Haowei Li, Bo Tian, Wengang Wang, Chuanlong Wu, Jinwu Wang, Min Dai
      Pages: 142 - 151
      Abstract: The biological activities of lanthanum chloride (LaCl3) and the molecular mechanisms of action underlying its anti‐inflammatory, anti‐hyperphosphatemic, and osteoblast‐enhancing effects have been studied previously, but less is known about the effects of LaCl3 on osteoclasts. The present study used in vivo and in vitro approaches to explore the effects of LaCl3 on osteoclasts and osteolysis. The results indicated that LaCl3 concentrations that were non‐cytotoxic to mouse bone marrow‐derived monocytes attenuated receptor activator of nuclear factor‐κB ligand (RANKL)‐stimulated osteoclastogenesis, bone resorption, mRNA expression of osteoclastogenic genes in these cells, including cathepsin K, calcitonin receptor, and tartrate‐resistant acid phosphatase (TRAP). Further, LaCl3 inhibited RANKL‐mediated activation of the nuclear factor‐κB (NF‐κB) signaling pathway, and downregulated mRNA and protein levels of nuclear factor of activated T‐cells, cytoplasmic, calcineurin‐dependent 1 (NFATc1), and c‐fos. In vivo, LaCl3 attenuated titanium (Ti) particle‐induced bone loss in a murine calvarial osteolysis model. Histological analyses revealed that LaCl3 ameliorated bone destruction and decreased the number of TRAP‐positive osteoclasts in this model. These results demonstrated that LaCl3 inhibited osteoclast formation, function, and osteoclast‐specific gene expression in vitro, and attenuated Ti particle‐induced mouse calvarial osteolysis in vivo, where the inhibition of NF‐κB signaling and downregulation of NFATc1 and c‐fos played an important role. J. Cell. Physiol. 230: 142–151, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:29.16004-05:0
      DOI: 10.1002/jcp.25065
  • COMMD7 as a novel NEMO interacting protein involved in the termination of
           NF‐κB signaling
    • Authors: Elio Esposito; Gennaro Napolitano, Alessandra Pescatore, Giuseppe Calculli, Maria Rosaria Incoronato, Antonio Leonardi, Matilde Valeria Ursini
      Pages: 152 - 161
      Abstract: NEMO/IKKγ is the regulatory subunit of the IκB Kinase (IKK) complex, required for the activation of the NF‐κB pathway, which is involved in a variety of key processes, including immunity, inflammation, differentiation, and cell survival. Termination of NF‐κB activity on specific ‐κB responsive genes, which is crucial for the resolution of inflammatory responses, can be achieved by direct degradation of the chromatin‐bound NF‐κB subunit RelA/p65, a process mediated by a protein complex that contains Copper Metabolism Murr1 Domain 1 (COMMD1). In this study, we identify COMMD7, another member of the COMMDs protein family, as a novel NEMO‐interacting protein. We show that COMMD7 exerts an inhibitory effect on NF‐κB activation upon TNFα stimulation. COMMD7 interacts with COMMD1 and together they cooperate to down‐regulate NF‐κB activity. Accordingly, termination of TNFα‐induced NF‐κB activity on the ‐κB responsive gene, Icam1, is defective in cells silenced for COMMD7 expression. Furthermore, this impairment is not greatly increased when we silence the expression of both COMMD7 and COMMD1 indicating that the two proteins participate in the same pathway of termination of TNFα‐induced NF‐κB activity. Importantly, we have demonstrated that COMMD7's binding to NEMO does not interfere with the binding to the IKKs, and that the disruption of the IKK complex through the use of the NBP competitor impairs the termination of NF‐κB activity. We propose that an intact IKK complex is required for the termination of NF‐κB‐dependent transcription and that COMMD7 acts as a scaffold in the IKK‐mediated NF‐κB termination. J. Cell. Physiol. 230: 152–161, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:24.746807-05:
      DOI: 10.1002/jcp.25066
  • Core Binding Factor β Plays a Critical Role During Chondrocyte
    • Pages: 162 - 171
      Abstract: Core binding factor β (Cbfβ) is a partner protein of Runx family transcription factors with minimally characterized function in cartilage. Here we address the role of Cbfβ in cartilage by generating chondrocyte‐specific Cbfβ‐deficient mice (CbfbΔch/Δch) from Cbfb‐floxed mice crossed with mice expressing Cre from the Col2a1 promoter. CbfbΔch/Δch mice died soon after birth and exhibited delayed endochondral bone formation, shorter appendicular skeleton length with increased proliferative chondrocytes, and nearly absent hypertrophic chondrocyte zones. Immunohistochemical and quantitative real‐time PCR analyses showed that the number and size of proliferative chondrocytes increased and the expression of chondrocyte maturation markers at the growth plates, including Runx2, osterix, and osteopontin, significantly diminished in CbfbΔch/Δch mice compared to wild type mice. With regard to signaling pathways, both PTHrP‐Ihh and BMP signaling were compromised in CbfbΔch/Δch mice. Mechanistically, Cbfβ deficiency in chondrocytes caused a decrease of protein levels of Runx transcription factors by accelerating polyubiquitination‐mediated proteosomal degradation in vitro. Indeed, Runx2 and Runx3, but not Runx1, decreased in CbfbΔch/Δch mice. Collectively, these findings indicate that Cbfβ plays a critical role for chondrocyte differentiation through stabilizing Runx2 and Runx3 proteins in cartilage J. Cell. Physiol. 230: 162–171, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:33.208161-05:
      DOI: 10.1002/jcp.25068
  • Different Effects of p52SHC1 and p52SHC3 on the Cell Cycle of Neurons and
           Neural Stem Cells
    • Authors: Ning Tang; Dan Lyu, Tao Liu, Fangjin Chen, Shuqian Jing, Tianyu Hao, Shaojun Liu
      Pages: 172 - 180
      Abstract: SHC3 is exclusively expressed in postmitotic neurons, while SHC1 is found in neural stem cells and neural precursor cells but absent in mature neurons. In this study, we discovered that suppression of p52SHC1 expression by RNA interference resulted in proliferation defects in neural stem cells, along with significantly reduced protein levels of cyclin E and cyclin A. At the same time, p52SHC3 RNAi caused cell cycle re‐entry (9.54% in S phase and 5.70% in G2‐M phase) in primary neurons with significantly up‐regulated expression of cyclin D1, cyclin E, cyclin A, CDK2, and phosphorylated CDK2. When p52SHC3 was overexpressed, the cell cycle of neural stem cells was arrested with reduced protein levels of cyclin D1, cyclin E, and cyclin A, while overexpression of p52SHC1 did not result in significant changes in postmitotic neurons. Our results indicate that p52SHC3 plays an important role in maintaining the mitotic quiescence of neurons, while p52SHC1 regulates the proliferation of neural stem cells. J. Cell. Physiol. 230: 172–180, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:24.058846-05:
      DOI: 10.1002/jcp.25069
  • ATP Release and P2Y Receptor Signaling are Essential for Keratinocyte
    • Authors: Aimie Riding; Christine E. Pullar
      Pages: 181 - 191
      Abstract: Repair to damaged tissue requires directional cell migration to heal the wound. Immediately upon wounding an electrical guidance cue is created with the cathode of the electric field (EF) located at the center of the wound. Previous research has demonstrated directional migration of keratinocytes toward the cathode when an EF of physiological strength (100–150 mV/mm) is applied in vitro, but the “sensor” by which keratinocytes sense the EF remains elusive. Here we use a customized chamber design to facilitate the application of a direct current (DC) EF of physiological strength (100 mV/mm) to keratinocytes whilst pharmacologically modulating the activation of both connexin hemichannels and purinergic receptors to determine their role in EF‐mediated directional keratinocyte migration, galvanotaxis. In addition, keratinocytes were exposed to DiSCAC2(3) dye to visualize membrane potential changes within the cell upon exposure to the applied DC EF. Here we unveil ATP‐medicated mechanisms that underpin the initiation of keratinocyte galvanotaxis. The application of a DC EF of 100 mV/mm releases ATP via hemichannels activating a subset of purinergic P2Y receptors, locally, to initiate the directional migration of keratinocytes toward the cathode in vitro, the center of the wound in vivo. The delineation of the mechanisms underpinning galvanotaxis extends our understanding of this endogenous cue and will facilitate the optimization and wider use of EF devices for chronic wound treatment. J. Cell. Physiol. 230: 181–191, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:34.994296-05:
      DOI: 10.1002/jcp.25070
  • Characterization of Human Dermal Fibroblasts in Fabry Disease
    • Pages: 192 - 203
      Abstract: Fabry disease (FD) is a hereditary X‐linked metabolic lysosomal storage disorder due to insufficient amounts or a complete lack of the lysosomal enzyme α‐galactosidase A (α‐GalA). The loss of α‐GalA activity leads to an abnormal accumulation of globotriaosylcerami (Gb3) in lysosomes and other cellular components of different tissues and cell types, affecting the cell function. However, whether these biochemical alterations also modify functional processes associated to the cell mitotic ability is still unknown. The goal of the present study was to characterize lineages of human dermal fibroblasts (HDFs) of FD patients and healthy controls focusing on Gb3 accumulation, expression of chloride channels that regulate proliferation, and proliferative activity. The biochemical and functional analyses indicate the existence of quantitative differences in some but not all the parameters of cytoskeletal organization, proliferation, and differentiation processes. J. Cell. Physiol. 230: 192–203, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:36.168606-05:
      DOI: 10.1002/jcp.25072
  • Participation of TNF‐α in Inhibitory Effects of Adipocytes on
           Osteoblast Differentiation
    • Authors: Robrigo P.F. Abuna; Fabiola S. De Oliveira, Thiago De S. Santos, Thais R. Guerra, Adalberto L. Rosa, Marcio M. Beloti
      Pages: 204 - 214
      Abstract: Mesenchymal stem cells from bone marrow (BM‐MSCs) and adipose tissue (AT‐MSCs) are attractive tools for cell‐based therapies to repair bone tissue. In this study, we investigated the osteogenic and adipogenic potential of BM‐MSCs and AT‐MSCs as well as the effect of crosstalk between osteoblasts and adipocytes on cell phenotype expression. Rat BM‐MSCs and AT‐MSCs were cultured either in growth, osteogenic, or adipogenic medium to evaluate osteoblast and adipocyte differentiation. Additionally, osteoblasts and adipocytes were indirectly co‐cultured to investigate the effect of adipocytes on osteoblast differentiation and vice versa. BM‐MSCs and AT‐MSCs exhibit osteogenic and adipogenic potential under non‐differentiation‐inducing conditions. When exposed to osteogenic medium, BM‐MSCs exhibited higher expression of bone markers compared with AT‐MSCs. Conversely, under adipogenic conditions, AT‐MSCs displayed higher expression of adipose tissue markers compared with BM‐MSCs. The presence of adipocytes as indirect co‐culture repressed the expression of the osteoblast phenotype, whereas osteoblasts did not exert remarkable effect on adipocytes. The inhibitory effect of adipocytes on osteoblasts was due to the release of tumor necrosis factor alpha (TNF‐α) in culture medium by adipocytes. Indeed, the addition of exogenous TNF‐α in culture medium repressed the differentiation of BM‐MSCs into osteoblasts mimicking the indirect co‐culture effect. In conclusion, our study showed that BM‐MSCs are more osteogenic while AT‐MSCs are more adipogenic. Additionally, we demonstrated the key role of TNF‐α secreted by adipocytes on the inhibition of osteoblast differentiation. Thus, we postulate that the higher osteogenic potential of BM‐MSCs makes them the first choice for inducing bone repair in cell‐based therapies. J. Cell. Physiol. 230: 204–214, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:31.295234-05:
      DOI: 10.1002/jcp.25073
  • G‐CSF contributes at the healing of tunica media of
           arteriotomy‐injured rat carotids by promoting differentiation of
           vascular smooth muscle cells
    • Authors: Barbara Rinaldi; Mauro Finicelli, Maria Donniacuo, Giovanni Di Bernardo, Giulia Gritti, Stefania Del Gaudio, Amalia Forte, Gianfranco Peluso, Marilena Cipollaro, Francesco Rossi, Umberto Galderisi
      Pages: 215 - 223
      Abstract: Restenosis is a complex pathophysiological disease whose causative mechanisms are not fully understood. Previous studies allowed us to demonstrate the efficacy of bone marrow mesenchymal stromal cells (MSCs) transplantation in limiting the pathophysiological remodeling in a model of arteriotomy‐induced (re) stenosis. In the current research we studied the effectiveness of G‐CSF treatment on male rate rats that were subjected carotid arteriotomy in order to evaluate a potentially effective non‐invasive strategy that recapitulates the MSC‐mediated recovery of injured vessels. WKY male rats were subjected carotid arteriotomy and given a nine day treatment (3 days pre‐ to 6 days post‐arteriotomy) with G‐CSF or saline. Carotids were harvested 7 and 30 days following arteriotomy (early‐ and late‐phase, respectively). Although morphometrical analysis did not reveal differences in lumen narrowing between G‐CSF‐ and PBS‐carotids 30 days following arteriotomy, we detected a noticeable conservative effect of G‐CSF treatment on vascular wall morphology. Histological and molecular analysis revealed an increase in cellularity within the tunica media with a concomitant increase of the VSMCs differentiation markers both at early‐ and late‐phases of (re) stenotic response in G‐CSF‐treated carotids (Sm22‐alpha, Myocd, and Smtn). These findings were accompanied by the downregulation of oxidative stress‐related genes in G‐CSF‐injured rats. The effect exerted by G‐CSF in our model of arteriotomy‐induced (re) stenosis seemed support the recovery of the architecture of the tunica media of injured vessels by: (i) inducing VSMCs differentiation; and (ii) limiting the oxidative‐stress response induced by arteriotomy. J. Cell. Physiol. 230: 215–223, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:29.728595-05:
      DOI: 10.1002/jcp.25074
  • Pathways Implicated in Tadalafil Amelioration of Duchenne Muscular
    • Authors: Valeria De Arcangelis; Georgios Strimpakos, Francesca Gabanella, Nicoletta Corbi, Siro Luvisetto, Armando Magrelli, Annalisa Onori, Claudio Passananti, Cinzia Pisani, Sophie Rome, Cinzia Severini, Fabio Naro, Elisabetta Mattei, Maria Grazia Di Certo, Lucia Monaco
      Pages: 224 - 232
      Abstract: Numerous therapeutic approaches for Duchenne and Becker Muscular Dystrophy (DMD and BMD), the most common X‐linked muscle degenerative disease, have been proposed. So far, the only one showing a clear beneficial effect is the use of corticosteroids. Recent evidence indicates an improvement of dystrophic cardiac and skeletal muscles in the presence of sustained cGMP levels secondary to a blocking of their degradation by phosphodiesterase five (PDE5). Due to these data, we performed a study to investigate the effect of the specific PDE5 inhibitor, tadalafil, on dystrophic skeletal muscle function. Chronic pharmacological treatment with tadalafil has been carried out in mdx mice. Behavioral and physiological tests, as well as histological and biochemical analyses, confirmed the efficacy of the therapy. We then performed a microarray‐based genomic analysis to assess the pattern of gene expression in muscle samples obtained from the different cohorts of animals treated with tadalafil. This scrutiny allowed us to identify several classes of modulated genes. Our results show that PDE5 inhibition can ameliorate dystrophy by acting at different levels. Tadalafil can lead to (1) increased lipid metabolism; (2) a switch towards slow oxidative fibers driven by the up‐regulation of PGC‐1α; (3) an increased protein synthesis efficiency; (4) a better actin network organization at Z‐disk. J. Cell. Physiol. 230: 224–232, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-28T16:08:33.919649-05:
      DOI: 10.1002/jcp.25075
  • Erratum: Big Data Bioinformatics by C. S. Greene, J. Tan, M. Ung, J. H.
           Moore, and C. Cheng
    • Pages: 257 - 257
      PubDate: 2015-09-28T16:08:25.416724-05:
      DOI: 10.1002/jcp.25077
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