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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  [1598 journals]
  • SHP‐2 Phosphatase Prevents Colonic Inflammation by Controlling
           Secretory Cell Differentiation and Maintaining Host‐Microbiota
           Homeostasis
    • Abstract: Polymorphisms in the PTPN11 gene encoding for the tyrosine phosphatase SHP‐2 were described in patients with ulcerative colitis. We have recently demonstrated that mice with an intestinal epithelial cell‐specific deletion of SHP‐2 (SHP‐2IEC‐KO) develop severe colitis one month after birth. However, the mechanisms by which SHP‐2 deletion induces colonic inflammation remain to be elucidated. We generated SHP‐2IEC‐KO mice lacking Myd88 exclusively in the intestinal epithelium. The colonic phenotype was histologically analyzed and cell differentiation was determined by electron microscopy and lysozyme or Alcian blue staining. Microbiota composition was analyzed by 16S sequencing. Results show that innate defense genes including those specific to Paneth cells were strongly up‐regulated in SHP‐2‐deficient colons. Expansion of intermediate cells (common progenitors of the Goblet and Paneth cell lineages) was found in the colon of SHP‐2IEC‐KO mice while Goblet cell number was clearly diminished. These alterations in Goblet/intermediate cell ratio were noticed two weeks after birth, before the onset of inflammation and were associated with significant alterations in microbiota composition. Indeed, an increase in Enterobacteriaceae and a decrease in Firmicutes were observed in the colon of these mice, indicating that dysbiosis also occurred prior to inflammation. Importantly, loss of epithelial Myd88 expression inhibited colitis development in SHP‐2IEC‐KO mice, rescued Goblet/intermediate cell ratio and prevented NFκB hyperactivation and inflammation. These data indicate that SHP‐2 is functionally important for the maintenance of appropriate barrier function and host‐microbiota homeostasis in the large intestine. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-21T14:15:45.654798-05:
      DOI: 10.1002/jcp.25407
       
  • Cover Image, Volume 231, Number 8, August 2016
    • Authors: Rahul Mittal; Brandon Chan, M'hamed Grati, Jeenu Mittal, Kunal Patel, Luca H. Debs, Amit P. Patel, Denise Yan, Prem Chapagain, Xue Zhong Liu
      Abstract: Cover: The cover image, by Xue Liu et al., is based on the Review Article Molecular Structure and Regulation of P2X Receptors With a Special Emphasis on the Role of P2X2 in the Auditory System,
      DOI : 10.1002/jcp.25274.
      PubDate: 2016-04-18T08:25:11.16477-05:0
       
  • Runx2/DICER/miRNA Pathway in Regulating Osteogenesis
    • Authors: Leilei Zheng; Qisheng Tu, Shu Meng, Lan Zhang, Liming Yu, Jinlin Song, Yun Hu, Lei Sui, Jin Zhang, Michel Dard, Jessica Cheng, Dana Murray, Jane Lian, Gary Stein, Jake Chen
      Abstract: DICER is the central enzyme that cleaves precursor microRNAs (miRNAs) into 21‐25 nucleotide duplex in cell lineage differentiation, identity and survival. In the current study, we characterized the specific bone metabolism genes and corresponding miRNAs and found that DICER and Runt‐related transcription factor 2 (Runx2) expressions increased simultaneously during osteogenic differentiation. Luciferase assay showed that Runx2 significantly increased the expression levels of DICER luciferase promoter reporter. Our analysis also revealed weaker DICER expression in embryos of Runx2 knock out mice (Runx2 ‐/‐) compared with that of Runx2 +/− and Runx2 +/+ mice. We further established the calvarial bone critical‐size defect (CSD) mouse model. The bone marrow stromal cells (BMSCs) transfected with siRNA targeting DICER were combined with silk scaffolds and transplanted into calvarial bone CSDs. Five weeks post‐surgery, micro‐CT analysis revealed impaired bone formation and repairing in calvarial defects with the siRNA targeting DICER group. In conclusion, our results suggest that DICER is specifically regulated by osteogenic master gene Runx2 that binds to the DICER promoter. Consequently, DICER cleaves precursors of miR‐335‐5p and miR‐17‐92 cluster to form mature miRNAs, which target and decrease the Dickkopf‐related protein 1 (DKK1) and proapoptotic factor BIM levels, respectively, leading to an enhanced Wnt/β‐catenin signaling pathway. These intriguing results reveal a central mechanism underlying lineage‐specific regulation by a Runx2/DICER/miRNAs cascade during osteogenic differentiation and bone development. Our study also suggests a potential application of modulating DICER expression for bone tissue repair and regeneration. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-11T12:31:05.364501-05:
      DOI: 10.1002/jcp.25406
       
  • Antibodies Against Mimotopes of Simian Virus 40 Large T Antigen, the
           Oncoprotein, in Serum Samples from Elderly Healthy Subjects
    • Authors: Elisa Mazzoni; Giovanni Guerra, Maria Vittoria Casali, Silvia Pietrobon, Ilaria Bononi, Andrea Puozzo, Andrea Tagliapietra, Pier Francesco Nocini, Mauro Tognon, Fernanda Martini
      Abstract: Simian Virus 40 (SV40), a monkey polyomavirus, was administered to human populations by early anti‐poliomylitis vaccines contaminated by this small DNA tumor virus. Data on SV40 infection in humans remain controversial. Elderly subjects represent an interesting cohort to investigate, because they were not immunized with SV40‐contaminated vaccines. Taking advantage of the Italian population, the second oldest worldwide, elderly subjects (n = 237) up to 100 years old were enrolled in this study. Their sera were analyzed, by ELISA tests with synthetic peptides mimicking the viral epitopes, for IgG antibodies reacting with SV40 large Tumor antigen (Tag), the viral oncoprotein. An overall seroprevalence of 22% was revealed in subjects aged 66‐100 years, ranging from 19% in individuals 66‐74 years old, to 24% in subjects 82‐100 years old, with a lower SV40 titer detected in the oldest group. Our data show that: (i) SV40 infection is not frequent in old individuals; (ii) the infection rate increases in elderly with the age; (iii) the antibody titer of SV40 Tag decreases with the age. In conclusion, SV40 infection seems to spread in old subjects independently from SV40‐contaminated vaccines. This study seems to confirm that SV40 is also a human virus. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-11T12:30:53.004404-05:
      DOI: 10.1002/jcp.25405
       
  • Properties of Adult Lung Stem and Progenitor Cells
    • Authors: Ivan Bertoncello
      Abstract: The last decade has seen significant progress in understanding the organisation of regenerative cells in the adult lung. Cell lineage tracing and in vitro clonogenic assays have enabled the identification and characterisation of endogenous lung epithelial stem and progenitor cells. Selective lung injury models, and genetically engineered mice have revealed highly conserved gene networks, factors, signalling pathways, and cellular interactions important in maintaining lung homeostasis and regulating lung regeneration and repair following injury. This review describes the current models of lung epithelial stem and progenitor cell organisation in adult mice, and the impediments encountered in translational studies aiming to identify and characterise their human homologs. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-07T16:26:52.308325-05:
      DOI: 10.1002/jcp.25404
       
  • Metabolic Adaptation of the Small Intestine to Short‐ and
           Medium‐Term High‐Fat Diet Exposure
    • Abstract: The small intestine is the main organ involved in the digestion and absorption of nutrients, i.e., it is in an ideal position to sense the availability of energy in the lumen in addition to its absorptive function. Consumption of a high‐fat diet (HFD) influences the metabolic characteristics of the small intestine. Therefore, to better understand the metabolic features of the small intestine and their changes in response to dietary fat, we characterized the metabolism of duodenal, jejunal and hepatic cell lines and assessed the metabolic changes in the enterocytes and the liver after short‐term (3 days) or medium‐term (14 days) HFD feeding in mice. Experiments with immortalized enterocytes indicated a higher glycolytic capacity in the duodenal cell line compared to the other two cell lines, whereas the jejunal cell line exhibited a high oxidative metabolism. Short‐term HFD feeding induced changes in the expression of glucose and lipid metabolism‐related genes in the duodenum and the jejunum of mice, but not in the liver. When focusing on fatty acid oxidation both, short‐ and medium‐term HFD feeding induced an upregulation of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A, the key enzyme of ketogenesis, at the protein level in the intestinal epithelial cells, but not in the liver. These results suggest that HFD feeding induces an early adaptation of the small intestine rather than the liver in response to a substantial fat load. This highlights the importance of the small intestine in the adaptation of the body to the metabolic changes induced by HFD exposure. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-07T16:26:19.058388-05:
      DOI: 10.1002/jcp.25402
       
  • Epigenetics in Intestinal Epithelial Cell Renewal
    • Abstract: A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt‐villus axis. One important check‐point in this process occurs in the transit amplifying zone of the intestinal crypts where different signalling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development and tissue renewal. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-07T16:25:48.709253-05:
      DOI: 10.1002/jcp.25401
       
  • Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of
           Osteoblast Growth and Differentiation
    • Abstract: Inhibition of RUNX2 is one of many mechanisms that suppress bone formation in glucocorticoid (GC)‐induced osteoporosis (GIO). We profiled mRNA expression in ST2/Rx2dox cells after treatment with doxycycline (dox; to induce Runx2) and/or the synthetic GC dexamethasone (dex). As expected, dex typically antagonized Runx2‐driven transcription. Select genes, however, were synergistic stimulated and this was confirmed by RT‐qPCR. Among the genes synergistically stimulated by GCs and Runx2 was Wnt inhibitory Factor 1 (Wif1), and Wif1 protein was readily detectable in medium conditioned by cultures co‐treated with dox and dex, but neither alone. Cooperation between Runx2 and GCs in stimulating Wif1 was also observed in primary preosteoblast cultures. GCs strongly inhibited dox‐driven alkaline phosphatase (ALP) activity in control ST2/Rx2dox cells, but not in cells in which Wif1 was silenced. Unlike its anti‐mitogenic activity in committed osteoblasts, induction of Runx2 transiently increased the percentage of cells in S‐phase and accelerated proliferation in the ST2 mesenchymal pluripotent cell culture model. Furthermore, like the inhibition of Runx2‐driven ALP activity, dex antagonized the transient mitogenic effect of Runx2 in ST2/Rx2dox cultures, and this inhibition eased upon Wif1 silencing. Plausibly, homeostatic feedback loops that rely on Runx2 activation to compensate for bone loss in GIO are thwarted, exacerbating disease progression through stimulation of Wif1. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-06T17:02:53.67624-05:0
      DOI: 10.1002/jcp.25399
       
  • Differentiation of Keratinocytes Modulates Expression of Epidermal
           Elements of the Cutaneous Analog of
           Hypothalamus‐Pituitary‐Adrenal Axis
    • Abstract: It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus‐pituitary‐adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1‐3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from Day 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-06T17:02:14.046921-05:
      DOI: 10.1002/jcp.25400
       
  • SRC Family Kinase Inhibition in Ewing Sarcoma Cells Induces p38 MAP
           Kinase‐Mediated Cytotoxicity and Reduces Cell Migration
    • Authors: Paola Indovina; Nadia Casini, Iris Maria Forte, Tiziana Garofano, Daniele Cesari, Carmelina Antonella Iannuzzi, Leonardo Del Porro, Francesca Pentimalli, Luca Napoliello, Silvia Boffo, Silvia Schenone, Maurizio Botta, Antonio Giordano
      Abstract: Ewing sarcoma (ES) is a highly aggressive bone and soft tissue cancer, representing the second most common primary malignant bone tumor in children and adolescents. Although the development of a multimodal therapy, including both local control (surgery and/or radiation) and systemic multidrug chemotherapy, has determined a significant improvement in survival, patients with metastatic and recurrent disease still face a poor prognosis. Moreover, considering that ES primarily affects young patients, there are concerns about long‐term adverse effects of the therapy. Therefore, more rational strategies, targeting specific molecular alterations underlying ES, are required. Recent studies suggest that SRC family kinases (SFKs), which are aberrantly activated in most cancer types, could represent key therapeutic targets also for ES. Here, we challenged ES cell lines with a recently developed selective SFK inhibitor (a pyrazolo[3,4‐d]pyrimidine derivative, called SI221), which was previously shown to be a valuable proapoptotic agent in other tumor types while not affecting normal cells. We observed that SI221 significantly reduced ES cell viability and proved to be more effective than the well‐known SFK inhibitor PP2. SI221 was able to induce apoptosis in ES cells and also reduced ES cell clonogenic potential. Furthermore, SI221 was also able to reduce ES cell migration. At the molecular level, our data suggest that SFK inhibition through SI221 could reduce ES cell viability at least in part by hindering an SFK‐NOTCH1 receptor‐p38 mitogen‐activated protein kinase (MAPK) axis. Overall, our study suggests a potential application of specific SFK inhibition in ES therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-02T06:40:50.273009-05:
      DOI: 10.1002/jcp.25397
       
  • Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and
           Epithelial Factors
    • Abstract: Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long‐term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre‐culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co‐culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-02T06:35:50.209015-05:
      DOI: 10.1002/jcp.25398
       
  • MG132 Induces Expression of Monocyte Chemotactic Protein‐ Induced
           Protein 1 in Vascular Smooth Muscle Cells
    • Abstract: Monocyte chemoattractant protein‐1 (MCP‐1) has been reported to induce the expression of monocyte chemotactic protein‐induced protein 1 (MCPIP1), which undergoes ubiquitination degradation. Therefore, we predict that in vascular smooth muscle (VSMCs), MCPIP1 may be induced by MCP‐1 and undergo degradation, which can be inhibited by the proteasome inhibitor, MG132. Our results showed that treatment of human VSMCs with MCP‐1 did not increase the expression of MCPIP1. Treatment with MG132, however, elevated MCPIP1 protein levels through stimulation of the gene transcription, but not thorugh increasing protein stability. MCPIP1 expression induced by MG132 was inhibited by α‐amanitin inhibition of gene transcription or cycloheximide inhibition of protein synthesis. Our further studies showed that MCPIP1 expression induced by MG132 was inhibited by the inhibitors of AKT and p38 kinase, suggesting a role of the AKT‐p38 pathway in MG132 effects. We also found that treatment with MG132 induces apoptosis, but overexpression of MCPIP1 inhibited bromodeoxyuridine (BrdU) incorporation of human VSMCs without induction of significant apoptosis. In summary, MCPIP1 expression is induced by MG132 likely through activation of the AKT‐p38 pathway. MCPIP1 inhibits SMC proliferation without induction of apoptosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-31T09:15:51.140802-05:
      DOI: 10.1002/jcp.25396
       
  • CRISPR‐Cas Assisted Multiplexing (CAM): Simple Same‐Day
           Multi‐Locus Engineering in Yeast
    • Authors: Jessica M. Walter; Sunil S. Chandran, Andrew A. Horwitz
      Abstract: Demands on the industrial and academic yeast strain engineer have increased significantly in the era of synthetic biology. Installing complex biosynthetic pathways and combining point mutations are tedious and time‐consuming using traditional methods. With multiplex engineering tools, these tasks can be completed in a single step, typically achieving up to six‐fold compression in strain engineering timelines. To capitalize on this potential, a variety of yeast CRISPR‐Cas methods have been developed, differing largely in how the guide RNA (gRNA) reagents that direct the Cas9 nuclease are delivered. However, in nearly all reported protocols, the time savings of multiplexing is offset by multiple days of cloning to prepare the required reagents. Here, we discuss the advantages and opportunities of CRISPR‐Cas Assisted Multiplexing (CAM), a same‐day, cloning‐free method for multi‐locus engineering in yeast. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-31T03:07:05.233542-05:
      DOI: 10.1002/jcp.25375
       
  • Cannabinoid Receptor 1 Mediates Homing of Bone Marrow‐Derived
           Mesenchymal Stem Cells Triggered by Chronic Liver Injury
    • Authors: Lin Wang; Le Yang, Lei Tian, Ping Mai, Shuangshuang Jia, Lin Yang, Liying Li
      Abstract: Cannabinoid receptors (CBs) have been implicated in the pathogenesis of various liver diseases, including liver fibrosis. Our previous studies have demonstrated that after liver injury, mouse bone marrow‐derived mesenchymal stem cells (BMSCs) can migrate to the injured liver and differentiate to myofibroblasts, contributing to hepatic fibrogenesis. However, the role of CBs in the homing of BMSCs in liver injury is yet unclear. In this study, we found that both CB1 and CB2 were expressed in BMSCs. Migration assays were performed by transwell chambers. CB1 agonist ACEA promoted the migration of BMSCs, but CB2 agonist JWH133 had no effect. Pharmacological or genetic ablation of CB1 reduced ACEA‐induced migration, whereas CB2 did not. Moreover, activation of CB1 increased active GTP‐bound Rac1, RhoA and Cdc42 protein levels. The elevated GTP‐bound Rac1 and RhoA protein levels were decreased by CB1 antagonist AM281 treatment, but not Cdc42. In addition, ACEA‐induced migration was suppressed by NSC23766 (Rac1 inhibitor) or C3 transferase (RhoA inhibitor), whereas MLS‐573151 (Cdc42 inhibitor) had no effect. Consistent with these data, Rac1 or RhoA knock‐down significantly blocked CB1‐mediated migration. Meanwhile, CB1‐mediated migration was associated with cytoskeletal remodeling. In vivo, administration of CB1 antagonist AM281 markedly inhibited the recruitment of BMSCs to the injured liver using fluorescence‐activated cell sorting. Furthermore, blockade of CB1 significantly attenuated liver fibrosis. In conclusion, our results suggest that CB1 plays a crucial role in liver fibrosis through mediating the homing of BMSCs to damaged liver, which may provide new insight into the pathogenesis and treatment of liver fibrosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-30T06:22:16.134871-05:
      DOI: 10.1002/jcp.25395
       
  • Priming Endothelial Cells with a Melanoma‐Derived Extracellular
           Matrix Triggers the Activation of Alpha v Beta3/VEGFR2 Axis
    • Abstract: The unique composition of tumor‐produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM‐ECM), ECM produced by a melanoma cell line (MV3‐ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin‐C and laminin and a lower expression of fibronectin. When cultured directly on MV3‐ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation and tubulogenesis. Interaction of endothelial cells with MV3‐ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK‐Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma‐ECM triggers integrin‐dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up‐regulate angiogenesis. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-30T05:21:31.793361-05:
      DOI: 10.1002/jcp.25358
       
  • Stimulatory Effects of Coumestrol on Embryonic and Fetal Development
           through AKT and ERK1/2 MAPK Signal Transduction
    • Authors: Whasun Lim; Gwonhwa Song
      Abstract: Successful establishment of pregnancy is required for fetal‐maternal interactions regulating implantation, embryonic development and placentation. A uterine environment with insufficient growth factors and nutrients increases the incidence of intrauterine growth restriction (IUGR) leading to an impaired uterine environment. In the present study, we demonstrated the effects of the phytoestrogen coumestrol on conceptus development in the pig that is regarded as an excellent biomedical animal model for research on IUGR. Results of this study indicated that coumestrol induced migration of porcine trophectoderm (pTr) cells in a concentration‐dependent manner. In response to coumestrol, the phosphorylation of AKT, P70S6K, S6, ERK1/2 MAPK and P90RSK proteins were activated in pTr cells and ERK1/2 MAPK and P90RSK phosphorylation was prolonged for a longer period than for the other proteins. To identify the signal transduction pathway induced by coumestrol, pharmacological inhibitors U0126 (an ERK1/2 inhibitor) and LY294002 (a PI3K inhibitor) were used to pretreat pTr cells. The results showed that coumestrol‐induced phosphorylation of ERK1/2 MAPK and P90RSK was blocked by U0126. In addition, the increased phosphorylation in response to coumestrol was completely inhibited following pre‐treatment incubation of pTr cells in the presence of LY294002 and U0126. Furthermore, these two inhibitors suppressed the ability of coumestrol to induce migration of pTr cells. Collectively, these findings suggest that coumestrol affects embryonic development through activation of the PI3K/AKT and ERK1/2 MAPK cell signal transduction pathways and improvement in the uterine environment through coumestrol supplementation may provide beneficial effects of enhancing embryonic and fetal survival and development. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-30T05:21:07.958247-05:
      DOI: 10.1002/jcp.25381
       
  • TNF‐α Induces Caspase‐1 Activation Independently of
           Simultaneously Induced NLRP3 in 3T3‐L1 Cells
    • Abstract: The intracellular cysteine protease caspase‐1 is critically involved in obesity‐induced inflammation in adipose tissue. A substantial body of evidence from immune cells, such as macrophages, has shown that caspase‐1 activation depends largely on a protein complex, called the NLRP3 inflammasome, which consists of the NOD‐like receptor (NLR) family protein NLRP3, the adaptor protein ASC, and caspase‐1 itself. However, it is not fully understood how caspase‐1 activation is regulated within adipocytes upon inflammatory stimuli. In this study, we show that TNF‐α‐induced activation of caspase‐1 is accompanied by robust induction of NLRP3 in 3T3‐L1 adipocytes but that caspase‐1 activation may not depend on the NLRP3 inflammasome. Treatment of 3T3‐L1 cells with TNF‐α induced mRNA expression and activation of caspase‐1. Although the basal expression of NLRP3 and ASC was undetectable in unstimulated cells, TNF‐α strongly induced NLRP3 expression but did not induce ASC expression. Interestingly, inhibitors of the ERK MAP kinase pathway strongly suppressed NLRP3 expression but did not suppress the expression and activation of caspase‐1 induced by TNF‐α, suggesting that NLRP3 is dispensable for TNF‐α‐induced caspase‐1 activation. Moreover, we did not detect the basal and TNF‐α‐induced expression of other NLR proteins (NLRP1a, NLRP1b, and NLRC4), which do not necessarily require ASC for caspase‐1 activation. These results suggest that TNF‐α induces caspase‐1 activation in an inflammasome‐independent manner in 3T3‐L1 cells and that the ERK‐dependent expression of NLRP3 may play a role independently of its canonical role as a component of inflammasomes. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-30T05:20:43.933647-05:
      DOI: 10.1002/jcp.25385
       
  • Quercetin Partially Preserves Development of Osteoblast Phenotype in Fetal
           Rat Calvaria Cells in an Oxidative Stress Environment
    • Authors: Jonathan G. Messer; Stephanie La, Robin G. Hopkins, Deborah E. Kipp
      Abstract: Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up‐regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide‐induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for four days (D5‐9) with 300 µM hydrogen peroxide resulted in fewer alkaline phosphatase‐positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression (p 
      PubDate: 2016-03-29T03:41:44.132435-05:
      DOI: 10.1002/jcp.25392
       
  • Enhancement of Lytic Activity by Leptin Is Independent from Lipid Rafts in
           Murine Primary Splenocytes
    • Abstract: Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation and survival. Leptin receptor is integrated inside cholesterol‐rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl‐β‐cyclodextrin (β‐MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/mL). Unlike β‐MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-29T03:36:47.50878-05:0
      DOI: 10.1002/jcp.25394
       
  • Hypoxia Modulates the Swelling‐Activated Cl Current in Human
           Glioblastoma Cells: Role in Volume Regulation and Cell Survival
    • Authors: Luigi Sforna; Marta Cenciarini, Silvia Belia, Antonio Michelucci, Mauro Pessia, Fabio Franciolini, Luigi Catacuzzeno
      Abstract: The malignancy of glioblastoma multiform (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non‐tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling‐activated Cl current (ICl,swell). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia‐activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably the hypoxia‐induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell. Since a hypoxia‐induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia‐activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia‐induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-29T03:36:23.324328-05:
      DOI: 10.1002/jcp.25393
       
  • MMP‐8 Is Critical for Dexamethasone Therapy in Alkali‐Burned
           Corneas Under Dry Eye Conditions
    • Abstract: Our previous studies have shown that Dexamethasone (Dex) reduced the expression of matrix‐metalloproteinases (MMPs‐1,‐3,‐9,‐13), IL‐1β and IL‐6, while it significantly increased MMP‐8 mRNA transcripts in a concomitant dry eye and corneal alkali burn murine model (CM). To investigate if MMP‐8 induction is responsible for some of the protective effects of Dex in CM, MMP‐8 knock out mice (MMP‐8KO) were subjected to the CM for 2 or 5 days and topically treated either with 2µL of 0.1% Dexamethasone (Dex), or saline QID. A separate group of C57BL/6 mice were topically treated with Dex or BSS and received either 100nM CAM12 (MMP‐8 inhibitor) or vehicle IP, QD. Here we demonstrate that topical Dex treated MMP‐8KO mice subjected to CM showed reduced corneal clarity, increased expression of inflammatory mediators (IL‐6, CXCL1 and MMP‐1 mRNA) and increased neutrophil infiltration at 2D and 5D compared to Dex treated WT mice. C57BL/6 mice topically treated with Dex and CAM12 recapitulated findings seen with MMP‐8KO mice. These results suggest that some of the anti‐inflammatory effects of Dex are mediated through increased MMP‐8 expression. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-28T02:41:14.282199-05:
      DOI: 10.1002/jcp.25364
       
  • The Integrin‐Mediated ILK‐Parvin‐αPix Signalling
           Axis Controls Differentiation in Mammary Epithelial Cells
    • Authors: Nicholas Rooney; Pengbo Wang, Keith Brennan, Andrew P Gilmore, Charles H Streuli
      Abstract: Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behaviour and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1‐integrin (β1‐itg) to establish apico‐basal polarity and to differentiate in response to prolactin. Downstream of β1‐itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico‐basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue‐specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA‐mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin‐binding guanine nucleotide exchange factor αPix prevented prolactin‐induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation‐specific bifurcation point in β1‐itg‐ILK adhesive signalling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin‐ILK signalling axis for MEC differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:41:06.985668-05:
      DOI: 10.1002/jcp.25390
       
  • Atp2c2 Is Transcribed from a Unique Transcriptional Start Site in Mouse
           Pancreatic Acinar Cells
    • Authors: Melissa Fenech; Caitlin M. Sullivan, Lucimar T. Ferreira, Rashid Mehmood, William A. MacDonald, Peter B. Stathopulos, Christopher L. Pin
      Abstract: Regulation of cytosolic Ca2+ is critical for pancreatic acinar cell function. Disruptions in normal Ca2+ concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca2+ homeostasis by promoting rapid Ca2+ movement. Determining how expression of Ca2+ modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas‐specific isoform for Secretory pathway Ca2+ ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA‐seq), chromatin immunoprecipitation for epigenetic modifications and promoter‐reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last 4 exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca2+. RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca2+ levels. Combined, these results suggest SPCA2C affects Ca2+ homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca2+ATPases. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:40:45.708336-05:
      DOI: 10.1002/jcp.25391
       
  • iPSCs, a Future Tool for Therapeutic Intervention in Mitochondrial
           Disorders: Pros and Cons
    • Abstract: Mitochondrial disorders, although individually are rare, taken together constitute a big group of diseases that share a defect in the oxidative phosphorylation system. Up to now, the development of therapies for these diseases is very slow and ineffective due in part to the lack of appropriate disease models. Therefore, there is an urgent need for the discovery of new therapeutic interventions. Regarding this, the generation of induced pluripotent stem cells (iPSCs) has opened new expectations in the regenerative medicine field. However, special cares and considerations must be taken into account previous to a replacement therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:37:01.892538-05:
      DOI: 10.1002/jcp.25386
       
  • DC‐STAMP: A Key Regulator in Osteoclast Differentiation
    • Abstract: Bone is a dynamic tissue that continuously undergoes remodeling with diverse functions including locomotion, hemopoiesis, and protection of internal organs. Osteoimmunology has recently become an emerging field of musculoskeletal research, which connects the bone and immune systems. Results from osteoimmunology studies provide a new concept that bone is not only an essential component of the musculoskeletal system, but it also participates in immune regulation. Many important factors involved in osteoimmunology regulation also participate in bone homeostasis. Bone homeostasis is achieved by a coordinated action between bone‐synthesizing osteoblasts and bone‐degrading osteoclasts. An imbalanced action between osteoblasts and osteoclasts often results in pathological bone diseases: osteoporosis is caused by an excessive osteoclast activity over that of osteoblasts, whereas osteopetrosis results from an increased osteoblast activity. This review focus on Dendritic Cell‐Specific Transmembrane Protein (DCSTAMP), an important protein currently considered as the master regulator of osteoclastogenesis. DC‐STAMP is required for cell‐cell fusion during osteoclast differentiation. Intriguingly, the frequency of circulating DCSTAMP+ cells is elevated during the pathogenesis of psoriatic diseases. Current data collectively suggest that DC‐STAMP also plays an imperative role in bone homeostasis by regulating the differentiation of both osteoclasts and osteoblasts. This article summarizes our current knowledge on DC‐STAMP by focusing on its interacting proteins, its regulation on osteoclastogenesis‐related genes, its possible involvement in immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐mediated signaling cascade, and its potential of developing therapeutics for clinical applications. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:36:35.866617-05:
      DOI: 10.1002/jcp.25389
       
  • Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,
           Differentiation and Myotube Hypertrophy
    • Authors: K. van der Meijden; N. Bravenboer, N.F. Dirks, A.C. Heijboer, M. den Heijer, G.M.J. de Wit, C. Offringa, P. Lips, R.T. Jaspers
      Abstract: An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2D3 and 25(OH)D3 affect myoblast proliferation, differentiation and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2D3. We showed that myoblasts not only respond to 1,25(OH)2D3, but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2D3 as well as 25(OH)D3 stimulate VDR mRNA expression and in myotubes 1,25(OH)2D3 also stimulates MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes express CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2D3. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:36:11.821671-05:
      DOI: 10.1002/jcp.25388
       
  • Quantification of Exosomes
    • Authors: E. H. Koritzinsky; J. M. Street, R. A. Star, P.S.T. Yuen
      Abstract: Exosomes are released by cells as self‐contained vesicles with an intact lipid bilayer that encapsulates a small portion of the parent cell. Exosomes have been studied widely as information‐rich sources of potential biomarkers that can reveal cellular physiology. We suggest that quantification is essential to understand basic biological relationships between exosomes and their parent cells and hence the underlying interpretation of exosome signals. The number of methods for quantifying exosomes has expanded as interest in exosomes has increased. However, a consensus on proper quantification has not developed, making each study difficult to compare to another. Overcoming this ad hoc approach will require widely available standards that have been adequately characterized, and multiple comparative studies across platforms. We outline the current status of these technical approaches and our view of how they can become more coherent. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:35:45.891505-05:
      DOI: 10.1002/jcp.25387
       
  • Role of PTHrP(1‐34) Pulse Frequency versus Pulse Duration to Enhance
           Mesenchymal Stromal Cell Chondrogenesis
    • Authors: Jennifer Fischer; Marlen Ortel, Sebastien Hagmann, Andreas Hoeflich, Wiltrud Richter
      Abstract: Generation of phenotypically stable, articular chondrocytes from mesenchymal stromal cells (MSCs) is still an unaccomplished task, with formation of abundant, hyaline extracellular matrix and avoidance of hypertrophy being prime challenges. We recently demonstrated that parathyroid hormone‐related protein (PTHrP) is a promising factor to direct chondrogenesis of MSCs towards an articular phenotype, since intermittent PTHrP application stimulated cartilage matrix production and reduced undesired hypertrophy. We here investigated the role of frequency, pulse duration, total exposure time and underlying mechanisms in order to unlock the full potential of PTHrP actions. Human MSC subjected to in vitro chondrogenesis for six weeks were exposed to 2.5 nM PTHrP(1‐34) pulses from day 7‐42. Application frequency was increased from three times weekly (3 × 6h/week) to daily maintaining either the duration of individual pulses (6 hours/day) or total exposure time (18 hours/week; 2.6 hours/day). Daily PTHrP treatment significantly increased extracellular matrix deposition regardless of pulse duration and suppressed alkaline‐phosphatase activity by 87%. High total exposure time significantly reduced cell proliferation at day 14. Pulse duration was critically important to significantly reduce IHH expression, but irrelevant for PTHrP‐induced suppression of the hypertrophic markers MEF2C and IBSP. COL10A1, RUNX2 and MMP13 expression remained unaltered. Decreased IGFBP‐2, ‐3, and ‐6 expression suggested modulated IGF‐I availability in PTHrP groups, while drop of SOX9 protein levels during the PTHrP‐pulse may delay chondroblast formation and hypertrophy. Overall, the significantly optimized timing of PTHrP‐pulses demonstrated a vast potential to enhance chondrogenesis of MSC and suppress hypertrophy possibly via superior balancing of IGF‐related and SOX9‐related mechanisms. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-21T04:00:51.980611-05:
      DOI: 10.1002/jcp.25369
       
  • Dissection of Individual Prostate Lobes in Mouse Models of Prostate Cancer
           to Obtain High Quality RNA
    • Authors: Areg Zingiryan; Nicholas H Farina, Kristiaan H Finstad, Janet L Stein, Jane B Lian, Gary S Stein
      Abstract: Genetically engineered mouse models of prostate cancer allow for study of disease progression from localized tumor formation through distal metastasis. The anatomy of the mouse prostate differs dramatically from the human prostate, being composed of 4 lobe pairs (anterior, dorsal, lateral, and ventral), making the identification and dissection technically challenging. While the entire murine prostate and surrounding tissue, including urethra, bladder, seminal vesicles, and associated adipose tissue, can be quickly dissected for en bloc analysis, it is necessary to isolate individual prostate lobes for gene expression studies elucidating the molecular mechanisms of prostate cancer. The procedure as described here includes full color images, allowing the researcher to appreciate the unique prostate morphology and tissue manipulation required to harvest individual prostate lobes. Along with removing all extraneous tissue, the procedure allows for direct comparison of the different prostate lobes by established downstream techniques. Importantly, high quality RNA required for next‐generation gene expression analysis can only consistently be obtained from ventral and lateral lobes. Finally, preclinical studies using prostate targeted therapies can be monitored specifically in individual prostate lobes on the histological and gene expression levels. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-18T08:27:02.29893-05:0
      DOI: 10.1002/jcp.25384
       
  • Stiffness of Intact Endothelial Cells from Fresh Aortic Bifurcations of
           Atherosclerotic Rabbits ‐ Atomic Force Microscopic Study
    • Authors: Kozaburo Hayashi; Michitaka Higaki
      Abstract: Stiffness of intact endothelial cells (ECs) in the abdominal aorta (AA) and in the medial and lateral wall of the common iliac artery (CIA(Medial) and CIA(Lateral), respectively), which were freshly obtained from cholesterol‐fed rabbits, were measured with an atomic force microscopic indentation method. In the areas away from atherosclerotic plaques (Off‐plaque), ECs were significantly stiffer in CIA(Medial) than in the other two locations; this result was similar to that from normal diet‐fed animals. On the other hand, there were no significant differences in the stiffness of ECs located on atherosclerotic plaques (On‐plaque) among the three sites; the stiffness was equal to those in “Off‐plaque” wall of CIA(Lateral) and AA. Moreover, the stiffness of ECs covering plaques decreased with the progression of atherosclerosis. The precise quantification of the stiffness of vascular ECs would provide a better understanding of cellular remodeling and adaptation in atherosclerosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:43:15.558698-05:
      DOI: 10.1002/jcp.25379
       
  • L‐glutamine Improves Skeletal Muscle Cell Differentiation and
           Prevents Myotube Atrophy after Cytokine (TNF‐α) Stress via
           Reduced p38 MAPK Signal Transduction
    • Authors: Matthew Girven; Hannah F. Dugdale, Daniel J. Owens, David C. Hughes, Claire E. Stewart, Adam P. Sharples
      Abstract: Tumour Necrosis Factor‐ Alpha (TNF‐α) is chronically elevated in conditions where skeletal muscle loss occurs. As L‐glutamine can dampen the effects of inflamed environments, we investigated the role of L‐glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF‐α (20 ng.ml−1) ± L‐glutamine (20 mM).TNF‐α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf‐I and Igfbp5. Furthermore, when administered to mature myotubes, TNF‐α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf‐I, Igfbp2 and glutamine synthetase and parallel increases in Fox03, Cfos, p53 and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non‐significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF‐α. Importantly, 20 mM L‐glutamine reduced p38 MAPK activity in TNF‐α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. L‐glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf‐Ir, Myhc2 & 7, Tnfsfr1b, Adra1d and restored atrophic gene expression of Fox03 back to baseline in TNF‐α conditions. In conclusion, L‐glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. L‐glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:42:28.208974-05:
      DOI: 10.1002/jcp.25380
       
  • XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of
           Telomeres in Tumor Cells
    • Abstract: Previous work has shown that the three‐dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT‐185, KPT‐330/selinexor, and KPT‐8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment‐naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non‐lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:42:03.802445-05:
      DOI: 10.1002/jcp.25378
       
  • E‐Cadherin Facilitates Protein Kinase D1 Activation and Subcellular
           Localization
    • Authors: Zhuo Li; Chuanyou Zhang, Li Chen, Guosheng Li, Ling Qu, K.C. Balaji, Cheng Du
      Abstract: Protein kinase D 1 (PKD1) is a serine/threonine kinase implicated in the regulation of diverse cellular functions including cell growth, differentiation, adhesion and motility. The current model for PKD1 activation involves diacylglycerol (DAG) binding to the C1 domain of PKD1 which results in the translocation of PKD1 to subcellular membranes where PKD1 is phosphorylated and activated by protein kinase C (PKC). In this study, we have identified a novel regulation of PKD1 activation. The epithelial cell membrane protein E‐cadherin physically binds to PKD1 which leads to a subcellular redistribution of PKD1. Furthermore, artificial targeting of PKD1 to the membrane leads to PKD1 activation in a PKC‐independent manner, indicating that membrane attachment is sufficient enough to activate PKD1. The presence of E‐cadherin dynamically regulates PKD1 activation by Bryostatin 1, a potent activator of PKD1, and its substrate phosphorylation specificity, implying a loss of E‐cadherin during cancer metastasis could cause the re‐distribution PKD1 and re‐wiring of PKD1 signaling for distinct functions. The knocking down of PKD1 in lung epithelial cell line A549 results in an epithelial to mesenchymal transition with changes in biomarker expression, cell migration and drug resistance. These results extend our previous understanding of PKD1 regulation and E‐cadherin signaling functions and may help to explain the diversified functions of PKD1 in various cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:41:41.652766-05:
      DOI: 10.1002/jcp.25382
       
  • Kynurenine Modulates MMP‐1 and Type‐I Collagen Expression via
           Aryl Hydrocarbon Receptor Activation in Dermal Fibroblasts
    • Abstract: Dermal fibrosis is characterized by a high deposition of extracellular matrix (ECM) and tissue cellularity. Unfortunately all means of treating this condition are unsatisfactory. We have previously reported the anti‐fibrotic effects of Kynurenine (Kyn), a tryptophan metabolite, in fibrotic rabbit ear model. Here, we report the mechanism by which Kyn modulates the expression of key ECM components in dermal fibroblasts. The results showed that Kyn activates aryl hydrocarbon receptor (AHR) nuclear translocation and up‐regulates cytochrome‐P450 (CYP1A‐1) expression, the AHR target gene. A specific AHR antagonist, 6,2′,4′‐trimethoxyflavone, inhibited the Kyn‐dependent modulation of CYP1A‐1, MMP‐1 and type‐I collagen expression. Establishing the anti‐fibrogenic effect of Kyn and its mechanism of action, we then developed nano‐fibrous Kyn slow‐releasing dressings and examined their anti‐fibrotic efficacy in vitro and in a rat model. Our results showed the feasibility of incorporating Kyn into PVA/PLGA nanofibers, prolonging the Kyn release up to 4 days tested. Application of medicated‐dressings significantly improved the dermal fibrosis indicated by MMP‐1 induction, alpha‐smooth muscle actin and type‐I collagen suppression, and reduced tissue cellularity, T‐cells and myofibroblasts. This study clarifies the mechanism by which Kyn modulates ECM expression and reports the development of a new slow‐releasing anti‐fibrogenic dressing. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:40:42.51292-05:0
      DOI: 10.1002/jcp.25383
       
  • Alteration in Phospholipidome Profile of Myoblast H9c2 Cell Line in a
           Model of Myocardium Starvation and Ischemia
    • Abstract: Myocardium infarction is one of the most deathly cardiovascular diseases. It is characterized by myocardium ischemia as a result of nutrients depletion and hypoxia. The cell can respond to this injury by autophagy or apoptosis, which determines the evolution and recovery of the myocardium infarction. Lipids play an important role in cardiovascular disease. However reports stating lipidome variations in cardiovascular disease are scarce and the role that lipids play in this pathological condition is not completely understood. The aim of this work was to identify changes in lipid profile of a myoblast H9c2 cell line under starvation and ischemia, to better understand and recognize new biomarkers for myocardial infarction. Lipidomic profile was evaluated by HILIC‐LC‐MS and GC‐MS. Cardiac cells showed alterations in phosphatidylcholines PC (34:1) and PC (36:2), lysophosphatidylcholines lyso PC(16:0), lysoPC(18:1) and lysoPC(18:0), phosphatidylethanolamine PE (34:1), phosphatidylserine PS (36:1), phosphatidylinositol PI (36:2), PI (38:3) and PI (38:5), sphingomyelin SM (34:1) and cardiolipins CL(68:4), CL(72:5) and CL(74:7) in ischemia and/or starvation, in comparison with control. Specific differences observed only in starvation were decrease of SM (34:1) and FA (20:4), and increase of PS (36:1). Differences observed only in ischemia were decrease of PC (36:2), lyso PC (16:0) and FA (18:1) and simultaneous increase of FA (16:0), FA (18:0). PC (34:1) increased in ischemia and decreased in starvation. In conclusion, our work suggests that lipids are potential markers for evaluation of cell fate, either cell death or recovery, which will be useful to improve diagnosis and prognostic of cardiovascular diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-16T04:47:07.267837-05:
      DOI: 10.1002/jcp.25344
       
  • KCa3.1–/– Mice Do Not Develop CIA: Regulatory Role for KCa3.1
           in Autoimmune Arthritis
    • Authors: Smriti K. Raychaudhuri; Heike Wulff, Siba P. Raychaudhuri
      Abstract: This communication is in response to the recent publication by Friebel K, Schönherr R, Kinne RW, Kunisch E. “Functional role of the KCa3.1 potassium channel in synovial fibroblasts from rheumatoid arthritis patients” J Cell Physiol. 2015 Jul;230(7):1677‐88. Friebel et al. reported a regulatory role for the calcium‐activated potassium channel KCa3.1 on fibroblast like synovial cells (FLS) and its pathologic significance in rheumatoid arthritis (RA) (1). More specifically the authors observed that KCa3.1 regulates proliferation of FLS and several other functions of FLS relevant to pannus formation in the synovial joints such as secretion of proinflammtory cytokines and metalloproteinase. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-16T04:46:08.860573-05:
      DOI: 10.1002/jcp.25356
       
  • Are Wnts Retrogradely Transported to the ER'
    • Authors: Bor Luen Tang
      Abstract: A recent report showed that Drosophila miR‐307a initiates endoplasmic reticulum (ER) stress in wingless (wg)‐expressing cells by suppression of the evolutionarily conserve Wnt secretion factor Wntless (Wls). Interestingly, the authors noted that wg has a putative C‐terminal dilysine motif (KKVY), which is required for its apparent retrograde Golgi‐to‐ER transport. Wls suppression resulted in ER stress, which was phenocopied by several other manipulations that impaired wg secretion in flies, as well as Wnt5a secretion in mammalian cells. The authors surmised that their data “reveals a previously unknown Golgi‐to‐ER retrograde route of wg, and elucidates a correlation between Wnt secretion and ER stress”. However, there are obvious caveats to this interpretation, as ER stress resulting from Wnt secretion impairment could be readily explained by its inability to leave the ER, and not resulting from Golgi‐to‐ER retrograde transport. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-16T04:45:42.41417-05:0
      DOI: 10.1002/jcp.25360
       
  • Inheritance of Cytosine Methylation
    • Authors: Desiree Tillo; Sanjit Mukherjee, Charles Vinson
      Abstract: There are numerous examples of parental transgenerational inheritance that is epigenetic. The informational molecules include RNA, chromatin modifications, and cytosine methylation. With advances in DNA sequencing technologies, the molecular and epigenetic mechanisms mediating these effects are now starting to be uncovered. This mini‐review will highlight some of the examples of epigenetic inheritance, the establishment of cytosine methylation in sperm, and recent genomic studies linking sperm cytosine methylation to epigenetic effects on offspring. A recent paper examining changes in diet and sperm cytosine methylation from pools of eight animals each, found differences between a normal diet, a high fat diet, and a low protein diet. However, epivariation between individuals within a group was greater than the differences between groups obscuring any potential methylation changes linked to diet. Learning more about epivariation may help unravel the mechanisms that regulate cytosine methylation. In addition, other experimental and genetic systems may also produce more dramatic changes in the sperm methylome, making it easier to unravel potential transgenerational phenomena. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-15T04:05:44.400751-05:
      DOI: 10.1002/jcp.25350
       
  • Programmable Site‐Specific Nucleases for Targeted Genome Engineering
           in Higher Eukaryotes
    • Authors: Ganesan Govindan; Sivaprakash Ramalingam
      Abstract: Recent advances in the targeted genome engineering enable molecular biologists to generate sequence specific modifications with greater efficiency and higher specificity in complex eukaryotic genomes. Programmable site‐specific DNA cleavage reagents and cellular DNA repair mechanisms have made this possible. These reagents have become powerful tools for delivering a site‐specific genomic double‐strand break (DSB) at the desired chromosomal locus, which produces sequence alterations through error‐prone non‐homologous end joining (NHEJ) resulting in gene inactivations/knockouts. Alternatively, the DSB can be repaired through homology‐directed repair (HDR) using a donor DNA template, which leads to the introduction of desired sequence modifications at the predetermined site. Here we summarize the role of three classes of nucleases; zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALEN), and clustered regularly interspaced palindromic repeats (CRISPR)/(CRISPR associated protein 9) Cas9 system in achieving targeted genome modifications. Further, we discuss the progress towards the applications of programmable site‐specific nucleases (SSNs) in treating human diseases and other biological applications in economically important higher eukaryotic organisms such as plants and livestock. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-15T04:01:14.466847-05:
      DOI: 10.1002/jcp.25367
       
  • Laser Direct‐Write onto Live Tissues: A Novel Model for Studying
           Cancer Cell Migration
    • Abstract: Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. Recently, our laboratories have combined a novel tissue culture model and laser direct‐write, a form of bioprinting, to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Herein, we highlight this new model as a tool for quantifying cancer cell motility and effects on angiogenesis and lymphangiogenesis in an intact network that matches the complexity of a real tissue. Application of our proposed methodology offers an innovative ex vivo tissue perspective for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-15T04:00:44.186655-05:
      DOI: 10.1002/jcp.25363
       
  • Generation of Human Lens Epithelial‐Like Cells from
           Patient‐Specific Induced Pluripotent Stem Cells
    • Authors: Dan Li; Xiaodi Qiu, Jin Yang, Tianjin Liu, Yi Luo, Yi Lu
      Abstract: Cataractogenesis begins from the dynamic lens epithelial cells (LECs) and adjacent fiber cells. LECs derived from cell lines cannot maintain the crystalline expression as the primary LECs. The current study aimed to efficiently generate large numbers of human LECs from patient‐specific induced pluripotent stem cells (iPSCs). Anterior lens capsules were collected from cataract surgery and were used to culture primary hLECs. iPSCs were induced from these primary hLECs by lentiviral transduction of Oct4, Sox2, Klf4 and c‐Myc. Then, the generated iPSCs were re‐differentiated into hLECs by the 3‐step addition of defined factor combinations (Noggin, BMP4/7, bFGF and EGF) modified from an established method. During the re‐differentiation process, colonies of interest were isolated using a glass picking tool and cloning cylinders based on the colony morphology. After two steps of isolation, populations of LEC‐like cells (LLCs) were generated and identified by the expression of lens marker genes by qPCR, western blot and immunofluorescence staining. The study introduced a modified protocol to isolate LLCs from iPSCs by defined factors in a short time frame. This technique could be useful for mechanistic studies of lens‐related diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-15T02:56:45.612403-05:
      DOI: 10.1002/jcp.25374
       
  • Serum from Advanced Heart Failure Patients Promotes Angiogenic Sprouting
           and Affects the Notch Pathway in Human Endothelial Cells
    • Authors: M Pannella; C Caliceti, F Fortini, G Aquila, F Vieceli Dalla Sega, A Pannuti, C Fortini, MB Morelli, A Fucili, G Francolini, R Voltan, P Secchiero, G Dinelli, E Leoncini, M Ferracin, S Hrelia, L Miele, P Rizzo
      Abstract: It is unknown whether components present in heart failure (HF) patients' serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined 1) sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel and 2) protein levels of Notch receptors 1, 2, 4 and ligands Jagged1, Delta‐like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients' serum and controls. The circulating levels of NT‐proBNP (N‐terminal of the pro‐hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera‐ treated cells (r = ‐0.526 and r = ‐0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-14T08:12:10.748909-05:
      DOI: 10.1002/jcp.25373
       
  • The Androgen Receptor Regulates PPARγ Expression and Activity in
           Human Prostate Cancer Cells
    • Authors: Emuejevoke Olokpa; Adrienne Bolden, LaMonica V. Stewart
      Abstract: The peroxisome proliferator activated receptor gamma (PPARγ) is a ligand‐activated transcription factor that regulates growth and differentiation within normal prostate and prostate cancers. However the factors that control PPARγ □within the prostate cancers have not been characterized. The goal of this study was to examine whether the androgen receptor (AR) regulates PPARγ expression and function within human prostate cancer cells. qRT‐PCR and Western blot analyses revealed nanomolar concentrations of the AR agonist dihydrotestosterone (DHT) decrease PPARγ mRNA and protein within the castration‐resistant, AR‐positive C4‐2 and VCaP human prostate cancer cell lines. The AR antagonists bicalutamide and enzalutamide blocked the ability of DHT to reduce PPARγ levels. In addition, siRNA mediated knockdown of AR increased PPARγ protein levels and ligand‐induced PPARγ transcriptional activity within the C4‐2 cell line. Furthermore, proteasome inhibitors that interfere with AR function increased the level of basal PPARγ and prevented the DHT‐mediated suppression of PPARγ. These data suggest that AR normally functions to suppress PPARγ expression within AR‐positive prostate cancer cells. To determine whether increases in AR protein would influence PPARγ expression and activity, we used lipofectamine‐based transfections to overexpress AR within the AR‐null PC‐3 cells. The addition of AR to PC‐3 cells did not significantly alter PPARγ protein levels. However, the ability of the PPARγ ligand rosiglitazone to induce activation of a PPARγ‐driven luciferase reporter and induce expression of FABP4 was suppressed in AR‐positive PC‐3 cells. Together, these data indicate AR serves as a key modulator of PPARγ expression and function within prostate tumors. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-14T07:29:46.537157-05:
      DOI: 10.1002/jcp.25368
       
  • Radiation‐Induced RhoGDIβ Cleavage Leads to Perturbation of
           Cell Polarity: A Possible Link to Cancer Spreading
    • Authors: Mamoru Fujiwara; Mayumi Okamoto, Masato Hori, Hiroshi Suga, Hiroshi Jikihara, Yuka Sugihara, Fumio Shimamoto, Toshio Mori, Koichi Nakaoji, Kazuhiko Hamada, Takahide Ota, Ralf Wiedemuth, Achim Temme, Masaaki Tatsuka
      Abstract: The equilibrium between proliferation and apoptosis is tightly balanced to maintain tissue homeostasis in normal tissues and even in tumors. Achieving and maintaining such a balance is important for cancer regrowth and spreading after cytotoxic treatments. Caspase‐3 activation and tumor cell death following anticancer therapy as well as accompanying cell death pathways are well characterized, but their association to homeostasis of cancerous tissue and tumor progression remains poorly understood. Here we proposed a novel mechanism of cancer spreading induced by caspase‐3. RhoGDIβ, known as a direct cleavage substrate of caspase‐3, is overexpressed in many epithelial cancers. The N‐terminal‐truncated RhoGDIβ (ΔN‐RhoGDIβ) is accumulated in caspase‐3‐activated cells. Stable expression of ΔN‐RhoGDIβ in HeLa cells did not induce apoptosis, but impaired directional cell migration in a wound‐healing assay accompanied by a perturbed direction of cell division at the wound edge. Subcellular protein fractionation experiments revealed that ΔN‐RhoGDIβ but not wild‐type RhoGDIβ was present in the detergent‐soluble cytoplasmic and nuclear fractions and preferentially associated with Cdc42. Furthermore, Cdc42 activity was constitutively inhibited by stable expression of ΔN‐RhoGDIβ, resulting in increased radiation‐induced compensatory proliferation linking to RhoA activation. Thus, ΔN‐RhoGDIβ dominant‐negatively regulates Cdc42 activity and contributes to loss of polarity‐related functions. The caspase‐3‐cleaved RhoGDIβ is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-14T07:29:31.108855-05:
      DOI: 10.1002/jcp.25362
       
  • Deregulated E2F5/p38/SMAD3 Circuitry Reinforces the Pro‐Tumorigenic
           Switch of TGFβ Signaling in Prostate Cancer
    • Authors: Subhadipa Majumder; Ankur Bhowal, Sanmitra Basu, Pritha Mukherjee, Urmi Chatterji, Sanghamitra Sengupta
      Abstract: Transforming growth factor‐β signaling exerts divergent effects on normal and cancer cells, although mechanism underlying this differential behavior remains unclear. In this study, expression of ninety‐four genes pertaining to the TGF‐β signaling pathway was compared between tumor and benign tissue samples from the human prostate gland to identify major discriminators driving prostate carcinogenesis. E2F5 was identified as one of the most deregulated genes in prostate cancer tissues, predominantly in samples with Gleason‐score 6. Expression of other deregulated components of TGF‐β signaling was examined by qRT‐PCR, western blot and immune‐staining. Function of E2F5 and p38 in prostate cancer was investigated using siRNA‐treatment of PC3 cell‐line followed by analyses of associated components and cell cycle. Observations revealed that E2F5 overexpression was accompanied by significantly higher phosphorylation of SMAD3 at Ser‐208 in the linker region (pSMAD3L) and p38 in tumor tissue. A striking difference in SMAD3 phosphorylation, marked by preponderance of pSMAD3L and pSMAD3C (Ser‐423 and 425) in tumor and benign tissues, respectively was noted. Co‐localization of E2F5 with pSMAD3L in the nuclei of tumor and PC3 cells indicated a functional interface between the proteins. Downregulation of E2F5 and p38 in PC3 cells resulted in marked reduction of phosphorylation of SMAD3 and perturbation of cell cycle with an arrest of cells in G1. Our findings unearthed that E2F5/p38 axis played a cardinal role in uncontrolled cellular proliferation in prostate cancer through pSMAD3L activation. It also underscores a strong potential for E2F5 to be incorporated as a tool in early detection of prostate cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-14T07:29:24.897435-05:
      DOI: 10.1002/jcp.25361
       
  • Characterization of Notch Signaling During Osteogenic Differentiation in
           Human Osteosarcoma Cell Line MG63
    • Authors: Alessia Ongaro; Agnese Pellati, Leila Bagheri, Paola Rizzo, Cristiana Caliceti, Leo Massari, Monica De Mattei
      Abstract: Osteogenic differentiation is a multi‐step process controlled by a complex molecular framework. Notch is an evolutionarily conserved intercellular signaling pathway playing a prominent role in cell fate and differentiation, although the mechanisms by which this pathway regulates osteogenesis remain controversial. This study aimed to investigate, in vitro, the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. Cells were cultured in basal condition (control) and in osteoinductive medium (OM). Notch inhibitors were also added in OM to block Notch pathway. During osteogenic differentiation, early (alkaline phosphatase activity and collagen type I) and late osteogenic markers (osteocalcin levels and matrix mineralization), as well as the gene expression of the main osteogenic transcription factors (Runx2, Osterix and Dlx5) increased. Time dependent changes in the expression of specific Notch receptors were identified in OM versus control with a significant reduction in the expression of Notch1 and Notch3 receptors in the early phase of differentiation, and an increase of Notch2 and Notch4 receptors in the late phase. Among Notch nuclear target genes, Hey1 expression was significantly higher in OM than control, whilst Hes5 expression decreased. Osteogenic markers were reduced and Hey1 was significantly inhibited by Notch inhibitors, suggesting a role for Notch through the canonical pathway. In conclusion, Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4 and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3 and Hes5, maintaining an undifferentiated status. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-11T05:18:34.276431-05:
      DOI: 10.1002/jcp.25366
       
  • Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis
           via the PI3K/AKT and ERK1/2 MAPK Pathways
    • Authors: Whasun Lim; Sunwoo Park, Fuller W. Bazer, Gwonhwa Song
      Abstract: Apigenin is a flavonoid found in parsley, onions, oranges, tea, chamomile, wheat and sprouts. It has a variety of biological properties including anti‐oxidant, anti‐mutagenic, anti‐carcinogenic, anti‐inflammatory, anti‐proliferative and anti‐spasmodic effects. Based on epidemiological and case‐control studies, apigenin is regarded as a novel chemotherapeutic agent against various cancer types. However, little is known about the effects of apigenin on choriocarcinoma cells. Therefore, we investigated the anti‐cancer effects of apigenin on choriocarcinoma cells (JAR and JEG3) in the present study. Apigenin reduced viability and migratory properties, increased apoptosis, and suppressed mitochondrial membrane potential in both the JAR and JEG3 cells. In addition, apigenin predominantly decreased phosphorylation of AKT, P70RSK and S6 whereas the phosphorylation of ERK1/2 and P90RSK was increased by apigenin treatment of JAR and JEG3 cells in a dose‐dependent manner. Moreover, treatment of JAR and JEG3 cells with both apigenin and pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126) revealed synergistic anti‐proliferative effects. Collectively, these results indicated that the apigenin is an invaluable chemopreventive agent that inhibits progression and metastasis of choriocarcinoma cells through regulation of PI3K/AKT and ERK1/2 MAPK signal transduction mechanism. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-11T02:43:28.687733-05:
      DOI: 10.1002/jcp.25372
       
  • Identification of Novel Biomarkers for Pancreatic Cancer Using Integrated
           Transcriptomics with Functional Pathways Analysis
    • Authors: Xuan Zhang; Pan Tong, Jinyun Chen, Zenglin Pei, Xiaoyan Zhang, Weiping Chen, Jianqing Xu, Jin Wang
      Abstract: Pancreatic cancer is a leading cause of cancer death largely, but the genetic alterations associated with the initiation and progression of this disease are still not well understood. To comprehensively investigate potential utility of miRNAs and protein encoding transcripts (mRNAs) in pancreatic cancer as biomarkers of the disease, we exhaustively mined genomic data from two publically available datasets: the National Center for Biotechnology Information Gene Expression Omnibus (GEO) and the Oncomine databases. We identified 26 miRNAs that were differentially expressed in pancreatic cancer tissue from five microarray datasets. Using data from five pancreatic cancer studies, we found 260 deregulated mRNAs associated with pancreatic cancer. Among these 260 deregulated transcripts, there were six transcripts encode proteins (COL1A2, CEACAM5, LAMA3, CP, ENO2, and FN1) secreted in blood, which may be developed as blood‐based biomarkers of pancreatic cancer. Further, we found that 13 abnormally expressing transcripts targeted by deregulated miRNAs were involved in the epithelial adherens junction signaling, Wnt/β‐catenin signaling, TR/RXR activation, hepatic fibrosis/hepatic stellate cell activation, and actin cytoskeleton canonical signaling pathways. Integrated bioinformatics analyses of multiple independent transcriptomic datasets revealed tumor associated deregulated miRNAs and protein encoding mRNAs involved in critical signaling pathways, which warrant further investigations to be validated as sensitive and specific biomarkers of pancreatic cancer detection and prognosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-10T04:12:01.509371-05:
      DOI: 10.1002/jcp.25353
       
  • Current Controversies in the Management of Myeloma Bone Disease
    • Authors: Rebecca Silbermann; Garson David Roodman
      Abstract: Recent significant advances in the treatment of multiple myeloma have resulted in an improvement in median overall survival from 4.6 years, for patients diagnosed between 2001 and 2005, to 6.1 years, for those diagnosed between 2006 and 2010 (Kumar et al., 2014). However, myeloma bone lesions persist in the absence of active disease and continue to be frequent and significant causes of patient morbidity and contribute to mortality. While bisphosphonate therapy in combination with anti‐myeloma therapy remains the cornerstone of skeletal disease management in myeloma, open questions regarding the optimal management of patients with myeloma bone disease remain. This article will address when to initiate and stop bone‐targeted therapy in patients with monoclonal gammopathies, duration of bisphosphonate treatment in the era of more effective anti‐myeloma treatment, the role of bone resorption markers in determining the dosing schedule for anti‐resorptive therapy, risks and benefits of long term anti‐resorptive therapy, and whether anti‐resorptive therapies should be stopped to enhance the potential anabolic effects of proteasome antagonists and other anabolic agents. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-10T04:11:25.540465-05:
      DOI: 10.1002/jcp.25351
       
  • Oncofetal Epigenetic Bivalency in Breast Cancer Cells: H3K4 and H3K27
           Methylation as a Biomarker for Phenotypic Plasticity†
    • Authors: Terri L. Messier; Joseph R. Boyd, Jonathan A. R. Gordon, Janet L. Stein, Jane B. Lian, Gary S. Stein
      Abstract: Alterations in the epigenetic landscape are fundamental drivers of aberrant gene expression contributing to cancer progression and pathology. Understanding specific modes of epigenetic regulation can be used to identify novel biomarkers or targets for therapeutic intervention to clinically treat solid tumors and leukemias. The bivalent marking of gene promoters by H3K4me3 and H3K27me3 is a primary mechanism to poise genes for expression in pluripotent embryonic stem cells. In this study we interrogated three well‐established mammary cell lines to model epigenetic reprogramming observed among breast cancer subtypes. Evidence is provided for a distinct bivalent signature, activating and repressive histone marks co‐residing at the same gene promoter, in the MCF7 (ESR/PGR +) luminal breast cancer cell line. We identified a subset of genes, enriched for developmental pathways that regulate cellular phenotype and signaling, and partially recapitulate the bivalent character observed in embryonic stem cells. We validated the biological relevance of this “oncofetal epigenetic” signature using data from ESR/PGR+ tumor samples from breast cancer patients. This signature of oncofetal epigenetic control is an informative biomarker and may provide novel therapeutic targets, selective for both recurring and treatment‐resistant cancers. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-10T04:02:07.887753-05:
      DOI: 10.1002/jcp.25359
       
  • Lipid‐Targeting Peptide Probes for Extracellular Vesicles
    • Authors: Aaron D. Flynn; Hang Yin
      Abstract: Extracellular vesicles released from cells are under intense investigation for their roles in cell‐cell communication and cancer progression. However, individual vesicles have been difficult to probe as their small size renders them invisible by conventional light microscopy. However, as a consequence of their small size these vesicles possess highly curved lipid membranes that offer an unconventional target for curvature‐sensing probes. In this article, we present a strategy for using peptide‐based biosensors to detect highly curved membranes and the negatively charged membrane lipid phosphatidylserine, we delineate several assays used to validate curvature‐ and lipid‐targeting mechanisms, and we explore potential applications in probing extracellular vesicles released from sources such as apoptotic cells, cancer cells, or activated platelets. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-09T12:33:48.806234-05:
      DOI: 10.1002/jcp.25354
       
  • Translocation of Endogenous Danger Signal HMGB1 from Nucleus to Membrane
           Microvesicles in Macrophages
    • Abstract: High mobility group box 1 (HMGB1) is a nuclear protein that can be released from activated or dead cells. Extracellular HMGB1 can serve as a “danger signal” and novel cytokine that mediates sterile inflammation. In addition to its soluble form, a few studies have reported that extracellular HMGB1 can also be carried by membrane microvesicles. However the cellular mechanisms responsible for nuclear HMGB1 translocation to the plasma membrane and released with membrane microvesicles have not been investigated. Tobacco smoking is a major causea of sterile inflammation in many inflammatory diseases. Smoking also increases blood levels of HMGB1. In this study, we found that exposure of macrophages to tobacco smoke extract (TSE) stimulated HMGB1 expression, redistribution, and release into the extracellular milieu both as a soluble molecule and, surprisingly, as a microvesicle‐associated form (TSE‐MV). Inhibition of chromosome region maintenance‐1 (CRM1), a nuclear exporter, attenuated TSE‐induced HMGB1 redistribution from the nucleus to the cytoplasm, as well as release with TSE‐MVs. Our study demonstrated a novel observation that nuclear HMGB1 was translocated to the plasma membrane, and then released in a microvesicle‐associated form. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-09T12:33:24.517164-05:
      DOI: 10.1002/jcp.25352
       
  • Skin Metabolite, Farnesyl Pyrophosphate, Regulates Epidermal Response to
           Inflammation, Oxidative Stress and Migration
    • Abstract: Skin produces cholesterol and a wide array of sterols and non‐sterol mevalonate metabolites, including isoprenoid derivative farnesyl pyrophosphate (FPP). To characterize FPP action in epidermis, we generated transcriptional profiles of primary human keratinocytes treated with zaragozic acid (ZGA), a squalene synthase inhibitor that blocks conversion of FPP to squalene resulting in endogenous accumulation of FPP. The elevated levels of intracellular FPP resulted in regulation of epidermal differentiation and adherens junction signaling, insulin growth factor (IGF) signaling, oxidative stress response and interferon (IFN) signaling. Immunosuppressive properties of FPP were evidenced by STAT‐1 downregulation and prominent suppression of its nuclear translocation by IFNγ. Furthermore, FPP profoundly downregulated genes involved in epidermal differentiation of keratinocytes in vitro and in human skin ex vivo. Elevated levels of FPP resulted in induction of cytoprotective transcriptional factor Nrf2 and its target genes. We have previously shown that FPP functions as ligand for the glucocorticoid receptor (GR), one of the major regulator of epidermal homeostasis. Comparative microarray analyses show significant but not complete overlap between FPP and glucocorticoid regulated genes, suggesting that FPP may have wider transcriptional impact. This was further supported by co‐transfection and chromatin immunoprecipitation experiments where we show that upon binding to GR, FPP recruits ß‐catenin and, unlike glucocorticoids, recruits co‐repressor GRIP1 to suppress keratin 6 gene. These findings have many clinical implications related to epidermal lipid metabolism, response to glucocorticoid therapy as well as pleiotropic effects of cholesterol lowering therapeutics, statins. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-09T12:31:20.088871-05:
      DOI: 10.1002/jcp.25357
       
  • The Impact of Epigenetics on Mesenchymal Stem Cell Biology
    • Authors: Yusuf Ozkul; Umberto Galderisi
      Abstract: Changes in epigenetic marks are known to be important regulatory factors in stem cell fate determination and differentiation. In the past years, the investigation of the epigenetic regulation of stem cell biology has largely focused on embryonic stem cells (ESCs). Contrarily, less is known about the epigenetic control of gene expression during differentiation of adult stem cells (AdSCs). Among AdSCs, mesenchymal stem cells (MSCs) are the most investigated stem cell population because of their enormous potential for therapeutic applications in regenerative medicine and tissue engineering. In this review we analyze the main studies addressing the epigenetic changes in MSC landscape during in vitro cultivation and replicative senescence, as well as follow osteocyte, chondrocyte, and adipocyte differentiation. In these studies, histone acetylation, DNA methylation, and miRNA expression are among the most investigated phenomena. We describe also epigenetic changes that are associated with in vitro MSC trans‐differentiation. Although at the at initial stage, the epigenetics of MSCs promise to have profound implications for stem cell basic and applied research. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-09T09:00:56.097947-05:
      DOI: 10.1002/jcp.25371
       
  • Alternative Start Codon Connects eIF5A to Mitochondria
    • Abstract: Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N‐terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N‐terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co‐purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-08T06:02:49.087117-05:
      DOI: 10.1002/jcp.25370
       
  • The Cholinergic Signaling Responsible for the Expression of a
           Memory‐Related Protein in Primary Rat Cortical Neurons
    • Abstract: Cholinergic dysfunction in the brain is closely related to cognitive impairment including memory loss. In addition to the degeneration of basal forebrain cholinergic neurons, deficits in the cholinergic receptor signaling may also play an important role. In the present study, to examine the cholinergic signaling pathways responsible for the induction of a memory‐related postsynaptic protein, a cholinergic agonist carbachol was used to induce the expression of activity‐regulated cytoskeleton associated protein (Arc) in primary rat cortical neurons. After pretreating neurons with various antagonists or inhibitors, the levels of carbachol‐induced Arc protein expression were detected by Western blot analysis. The results show that carbachol induces Arc protein expression mainly through activating M1 acetylcholine receptors and the downstream phospholipase C pathway, which may lead to the activation of the MAPK/ERK signaling pathway. Importantly, carbachol‐mediated M2 receptor activation exerts negative effects on Arc protein expression and thus counteracts the enhanced effects of M1 activation. Furthermore, it is suggested for the first time that M1‐mediated enhancement of N‐methyl‐D‐aspartate receptor (NMDAR) responses, leading to Ca2+ entry through NMDARs, contributes to carbachol‐induced Arc protein expression. These findings reveal a more complete cholinergic signaling that is responsible for carbachol‐induced Arc protein expression, and thus provide more information for developing treatments that can modulate cholinergic signaling and consequently alleviate cognitive impairment. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-08T04:59:11.493911-05:
      DOI: 10.1002/jcp.25347
       
  • Involvement of Transient Receptor Potential Cation Channel Vanilloid 1
           (TRPV1) in Myoblast Fusion
    • Authors: Mitsutoshi Kurosaka; Yuji Ogura, Toshiya Funabashi, Tatsuo Akema
      Abstract: The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)‐induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX‐induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin‐4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-08T04:58:27.133158-05:
      DOI: 10.1002/jcp.25345
       
  • Annexin A1 Is a Physiological Modulator of Neutrophil Maturation and
           Recirculation Acting on the CXCR4/CXCL12 Pathway
    • Abstract: Neutrophil production and traffic in the body compartments is finely controlled, and the strong evidences support the role of CXCL12/CXCR4 pathway on neutrophil trafficking to and from the bone marrow (BM). We recently showed that the glucocorticoid‐regulated protein, Annexin A1 (AnxA1) modulates neutrophil homeostasis and here we address the effects of AnxA1 on steady‐state neutrophil maturation and trafficking. For this purpose, AnxA1‐/‐ and Balb/C wild‐type mice (WT) were donors of BM granulocytes and mesenchymal stem cells and blood neutrophils. In vivo treatments with the pharmacological AnxA1 mimetic peptide (Ac2‐26) or the formyl peptide receptor (FPR) antagonist (Boc‐2) were used to elucidate the pathway of AnxA1 action, and with the cytosolic glucocorticoid antagonist receptor RU 38486. Accelerated maturation of BM granulocytes was detected in AnxA1‐/‐ and Boc2‐treated WT mice, and was reversed by treatment with Ac2‐26 in AnxA1‐/‐ mice. AnxA1 and FPR2 were constitutively expressed in bone marrow granulocytes, and their expressions were reduced by treatment with RU38486. Higher numbers of CXCR4+ neutrophils were detected in the circulation of AnxA1‐/‐ or Boc2‐treated WT mice, and values were rescued in Ac2‐26‐treated AnxA1‐/‐ mice. Although circulating neutrophils of AnxA1‐/‐ animals were CXCR4+, they presented reduced CXCL12‐induced chemotaxis. Moreover, levels of CXCL12 were reduced in the bone marrow perfusate and in the mesenchymal stem cell supernatant from AnxA1‐/‐ mice, and in vivo and in vitro CXCL12 expression was re‐established after Ac2‐26 treatment. Collectively, these data highlight AnxA1 as a novel determinant of neutrophil maturation and the mechanisms behind blood neutrophil homing to BM via the CXCL12/CXCR4 pathway. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-08T04:57:12.877614-05:
      DOI: 10.1002/jcp.25346
       
  • Differential Response of Human Hepatocyte Chromatin to HDAC Inhibitors as
           a Function of Microenvironmental Glucose Level
    • Authors: Marina Barreto Felisbino; Thiago Alves da Costa, Maria Silvia Viccari Gatti, Maria Luiza Silveira Mello
      Abstract: Diabetes is a complex multifactorial disorder characterized by chronic hyperglycemia due to impaired insulin secretion. Recent observations suggest that the complexity of the disease cannot be entirely accounted for genetic predisposition and a compelling argument for an epigenetic component is rapidly emerging. The use of histone deacetylase inhibitors (HDACi) in clinical setting is an emerging area of investigation. In this study, we have aimed to understand and compare the response of hepatocyte chromatin to valproic acid (VPA) and trichostatin A (TSA) treatments under normoglycemic or hyperglycemic conditions to expand our knowledge about the consequences of HDACi treatment in a diabetes cell model. Under normoglycemic conditions, these treatments promoted chromatin remodeling, as assessed by image analysis and H3K9ac and H3K9me2 abundance. Simultaneously, H3K9ac marks shifted to the nuclear periphery accompanied by HP1 dissociation from the heterochromatin and a G1 cell cycle arrest. More striking changes in the cell cycle progression and mitotic ratios required drastic treatment. Under hyperglycemic conditions, high glucose per se promoted chromatin changes similar to those promoted by VPA and TSA. Nonetheless, these results were not intensified in cells treated with HDACis under hyperglycemic conditions. Despite the absence of morphological changes being promoted, HDACi treatment seems to confer a physiological meaning, ameliorating the cellular hyperglycemic state through reduction of glucose production. These observations allow us to conclude that the glucose level to which the hepatocytes are subjected affects how chromatin responds to HDACi and their action under high‐glucose environment might not reflect on chromatin remodeling. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-06T21:56:30.869903-05:
      DOI: 10.1002/jcp.25343
       
  • Phospholipidomic Profile Variation on THP‐1 Cells Exposed to Skin or
           Respiratory Sensitizers and Respiratory Irritant
    • Abstract: Occupational exposure to low molecular weight reactive chemicals often leads to development of allergic reactions such as allergic contact dermatitis and respiratory allergies. Further insights into the interaction of these chemicals with physiopathological relevant cellular models might provide the foundations for novel non‐animal approaches to safety assessment. In this work we used the human THP‐1 cell line to determine phospholipidome changes induced by the skin sensitizer 1‐fluoro‐2,4‐dinitrobenzene (DNFB), the respiratory allergen hexamethylene diisocyanate (HDI) and the irritant methyl salicylate (MESA). We detected that these chemicals differently induce lipid peroxidation and modulate THP‐1 IL‐1β, IL‐12B, IL‐8, CD86 and HMOX1 transcription. Decreased phosphatidylethanolamine content was detected in cells exposed to MESA, while profound alterations in the relative abundance of cardiolipin species were observed in cells exposed to DNFB. All chemicals tested induced a decrease in the relative abundance of plasmanyl phosphatidylcholine species PC (O‐16:0e/18:1) and phosphatidylinositol species PI (34:1), while increasing PI (38:4). An increased abundance of oleic acid was observed in the phospholipids of cells exposed to DNFB while a decreased abundance of palmitic acid was detected in cells treated with MESA or DNFB. We conclude that both specific and common alterations at phospholipidome levels are triggered by the different chemicals, while not allowing a complete distinction between them using a Canonical Analysis of Principal Coordinates (CAP). The common effects observed at phospholipids level with all the chemicals tested might be related to unspecific cell cytotoxic mechanisms that nevertheless may contribute to the elicitation of specific immune responses. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-04T09:06:24.071788-05:
      DOI: 10.1002/jcp.25365
       
  • The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion
           and Fenestrations
    • Abstract: In eukaryotes, the nuclear envelope physically separates nuclear components and activities from rest of the cell. The nuclear envelope also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the nuclear envelope is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the nuclear envelope of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and expands asymmetrically a mitotic delay. Moreover, its nuclear envelope is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast nuclear envelope lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast nuclear envelope have uncovered interesting, and likely conserved, processes that contribute to nuclear envelope dynamics. In particular, we discuss the processes that drive and enable nuclear envelope expansion and the dramatic changes in the nuclear envelope that lead to extensions and fenestrations. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-22T17:34:06.862231-05:
      DOI: 10.1002/jcp.25355
       
  • Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer
           Therapy
    • Authors: Yuhui Yang; Svetlana Karakhanova, Werner Hartwig, Jan G. D'Haese, Pavel P. Philippov, Jens Werner, Alexandr V. Bazhin
      Abstract: Mitochondria are indispensable for energy metabolism, apoptosis regulation and cell signaling. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and participate actively in metabolic reprogramming. Mitochondria in cancer cells are characterized by reactive oxygen species (ROS) overproduction, which promotes cancer development by inducing genomic instability, modifying gene expression and participating in signaling pathways. Mitochondrial and nuclear DNA mutations caused by oxidative damage that impair the oxidative phosphorylation process will result in further mitochondrial ROS production, completing the “vicious cycle” between mitochondria, ROS, genomic instability and cancer development. The multiple essential roles of mitochondria have been utilized for designing novel mitochondria‐targeted anticancer agents. Selective drug delivery to mitochondria helps to increase specificity and reduce toxicity of these agents. In order to reduce mitochondrial ROS production, mitochondria‐targeted antioxidants can specifically accumulate in mitochondria by affiliating to a lipophilic penetrating cation and prevent mitochondria from oxidative damage. In consistence with the oncogenic role of ROS, mitochondria‐targeted antioxidants are found to be effective in cancer prevention and anticancer therapy. A better understanding of the role played by mitochondria in cancer development will help to reveal more therapeutic targets, and will help to increase the activity and selectivity of mitochondria‐targeted anticancer drugs. In this review we summarized the impact of mitochondria on cancer and gave summary about the possibilities to target mitochondria for anticancer therapies. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-19T20:43:18.622087-05:
      DOI: 10.1002/jcp.25349
       
  • A Novel Biological Role of α‐Mangostin in Modulating
           Inflammatory Response through the Activation of SIRT‐1 Signaling
           Pathway
    • Authors: Sara Franceschelli; Mirko Pesce, Alessio Ferrone, Antonia Patruno, Pasqualone Livia, Giuseppe Carlucci, Vincenzo Ferrone, Maura Carlucci, Maria Anna De Lutiis, Alfredo Grilli, Mario Felaco, Lorenza Speranza
      Abstract: Several studies have shown that xanthones obtained from Garcinia Mangostana (GM) have remarkable biological activities. α‐mangostin (α‐MG) is the main constituent of the fruit hull of the GM. Several findings have suggested that SIRT‐1, a nuclear histone deacetylase, could influence cellular function by the inhibition of NF‐kB signaling. ROS can inhibit SIRT‐1 activity by initiating oxidative modifications on its cysteine residues, and suppression of SIRT‐1 enhances the NF‐κB signaling resulting in inflammatory responses. The goals of the present study were to evaluate the quantity of α‐MG in the methanolic extract of GM (Vithagroup Spa) and to investigate the activity of this xanthone in U937 cell line and in human monocytes from responsive to inflammatory insult analyzing the possible changes on the activation of SIRT‐1 protein via NF‐Kb. Cells were treated with the methanolic extract of GM and/or LPS. The chromatographic separation of α‐MG was performed by an HPLC analysis. EX 527, a specific SIRT‐1 inhibitor, was used to determine if SIRT‐1/NfkB signaling pathway might be involved in α‐MG action on cells. Our results show that α‐MG inhibits p65 acetylation and down‐regulates the pro‐inflammatory gene products as COX‐2, iNOS via SIRT‐1 activation. Cells treated with EX 527 showed an up‐regulation of NFkB acetylation and an over expression of inducible enzymes and their product of catalysis (NO and PGE2). These results suggest that α‐MG may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-19T20:42:29.518255-05:
      DOI: 10.1002/jcp.25348
       
  • Absence of K‐Ras Reduces Proliferation and Migration But Increases
           Extracellular Matrix Synthesis in Fibroblasts
    • Abstract: The involvement of Ras‐GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H‐ and N‐Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K‐Ras in cellular processes involved in the development of fibrosis: proliferation, migration and extracellular matrix (ECM) proteins synthesis. K‐Ras knockout (KO) mouse embryonic fibroblasts (K‐ras−/−) stimulated with transforming growth factor‐β1 (TGF‐β1) exhibited reduced proliferation and impaired mobility than wild‐type fibroblasts. Moreover, an increase on ECM production was observed in K‐Ras KO fibroblasts in basal conditions. The absence of K‐Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF‐β1‐induced Smad signalling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K‐ras but reduced migration and ECM proteins expression only in wild‐type fibroblasts, while the PI3K‐AKT inhibitor LY294002 decreased cell proliferation, migration and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K‐Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K‐Ras may be a crucial mediator in TGF‐β1‐mediated effects in this cell type. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-12T20:57:45.863676-05:
      DOI: 10.1002/jcp.25340
       
  • miR‐19a, ‐19b, and ‐26b Mediate CTGF Expression and
           Pulmonary Fibroblast Differentiation
    • Abstract: Although microRNA (miRNA) dysregulation with intracellular signaling cascade disruption has been demonstrated in the pathophysiology of pulmonary fibrosis, the relationship between miRNAs and intracellular signaling cascades in pulmonary fibrosis remains unclear. Using the human embryonic lung fibroblast cell line WI‐38, we observed endothelin‐1 (ET‐1)‐ and thrombin‐induced expression of the differentiation markers α‐smooth muscle actin (α‐SMA) and vimentin along with increased connective tissue growth factor (CTGF) protein expression. Decreased CTGF protein expression by CTGF siRNA significantly blocked ET‐1‐ and thrombin‐induced α‐SMA and vimentin expression in WI‐38 cells. Activation of the mitogen‐ activated protein kinases (MAPKs) extracellular signal‐regulated kinase ERK, c‐Jun N‐ terminal kinase (JNK), and p38 contributed to ET‐1‐ and thrombin‐induced CTGF, α‐SMA, and vimentin expression in WI‐38 cells. TargetScan Human, miRanda, and PicTar prediction algorithms were used to predict miRNAs with binding sites in the 3′ untranslated region (UTR) of CTGF mRNA. miR‐19a, ‐19b, and ‐26b were candidate miRNAs of CTGF. Direct binding of the candidate miRNAs to the 3′‐UTR of CTGF mRNA was verified through luciferase assay by using SV40‐promoter‐IRES‐driven luciferase containing the 3′‐UTR of CTGF mRNA as a reporter plasmid. ET‐1 and thrombin reduced candidate miRNA levels. Candidate miRNA overexpression significantly suppressed ET‐1‐ and thrombin‐induced CTGF expression and reduced α‐SMA and vimentin expression in the WI‐38 cells. Furthermore, candidate miRNA levels were decreased in the lung tissues of mice with bleomycin‐induced pulmonary fibrosis, and intratracheal application of miR‐19a, ‐19b, and 26b reduced the pulmonary fibrotic severity induced by bleomycin. This study is the first to demonstrate crosstalk between MAPK activation and reduction in miR‐19a, ‐19b, and ‐26b expression leading to lung fibroblast differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-12T20:57:03.811817-05:
      DOI: 10.1002/jcp.25341
       
  • A New In Vitro Co‐Culture Model Using Magnetic Force‐Based
           Nanotechnology
    • Abstract: Background Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in‐vitro model of myoblast transplantation with “patterned” or “randomly‐mixed” co‐culture of SkMBs and cardiomyocytes enabling subsequent electrophysiological and arrhythmogenic evaluation. Methods SkMBs were magnetically labeled with magnetite nanoparticles and co‐cultured with neonatal rat ventricular myocytes (NRVMs) on multi‐electrode arrays. SkMBs were patterned by a magnet beneath the arrays. Excitation synchronicity was evaluated by Ca2+ imaging using a gene‐encoded Ca2+ indicator, G‐CaMP2. Results In the monoculture of NRVMs (control), conduction was well‐organized. In the randomly‐mixed co‐culture of NRVMs and SkMBs (random group), there was inhomogeneous conduction from multiple origins. In the “patterned” co‐culture where an en bloc SKMB‐layer was inserted into the NRVM‐layer, excitation homogenously propagated although conduction was distorted by the SkMB‐area. The 4‐mm distance conduction time (CT) in the random group was significantly longer (197 ± 126 ms) than in control (17 ± 3 ms). In the patterned group, CT through NRVM‐area did not change (25 ± 3 ms), although CT through the SkMB‐area was significantly longer (132 ± 77 ms). The intervals between spontaneous excitation varied beat‐to‐beat in the random group, while regular beating was recorded in the control and patterned groups. Synchronized Ca2+ transients of NRVMs were observed in the patterned group, whereas those in the random group were asynchronous. Conclusions Patterned alignment of SkMBs is feasible with magnetic nanoparticles. Using the novel in‐vitro model mimicking cell transplantation, it may become possible to predict arrhythmogenicity due to heterogenous cell transplantation. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-12T20:55:31.564092-05:
      DOI: 10.1002/jcp.25342
       
  • Lack of Dystrophin Affects Bronchial Epithelium in mdx Mice
    • Abstract: Mild exercise training may positively affect the course of Duchenne Muscular Dystrophy (DMD). Training causes mild bronchial epithelial injury in both humans and mice, but no study assessed the effects of exercise in mdx mice, a well known model of DMD. The airway epithelium was examined in mdx (C57BL/10ScSn‐Dmdmdx) mice, and in wild type (WT, C57BL/10ScSc) mice either under sedentary conditions (mdx‐SD, WT‐SD) or during mild exercise training (mdx‐EX, WT‐EX). At baseline, and after 30 and 45 days of training (5 d/wk for 6 weeks), epithelial morphology and markers of regeneration, apoptosis, and cellular stress were assessed. The number of goblet cells in bronchial epithelium was much lower in mdx than in WT mice under all conditions. At 30 days, epithelial regeneration (PCNA positive cells) was higher in EX than SD animals in both groups; however, at 45 days, epithelial regeneration decreased in mdx mice irrespective of training, and the percentage of apoptotic (TUNEL positive) cells was higher in mdx‐EX than in WT‐EX mice. Epithelial expression of HSP60 (marker of stress) progressively decreased, and inversely correlated with epithelial apoptosis (r = ‐0.66, p = 0.01) only in mdx mice. Lack of dystrophin in mdx mice appears associated with defective epithelial differentiation, and transient epithelial regeneration during mild exercise training. Hence, lack of dystrophin might impair repair in bronchial epithelium, with potential clinical consequences in DMD patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-11T21:44:42.465212-05:
      DOI: 10.1002/jcp.25339
       
  • Phosphorylation Regulates Functions of ZEB1 Transcription Factor
    • Authors: M. Candelaria Llorens; Guadalupe Lorenzatti, Natalia L. Cavallo, Maria V. Vaglienti, Ana P. Perrone, Anne L. Carenbauer, Douglas S. Darling, Ana M. Cabanillas
      Abstract: ZEB1 transcription factor is important in both development and disease, including many TGFβ‐induced responses, and the epithelial‐to‐mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA‐binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF‐1) can inhibit transcriptional repression by either a ZEB1‐ZD2 domain clone, or full‐length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA‐binding activity of ZEB1. Immunoprecipitation with anti‐ZEB1 antibodies followed by western analysis with a phospho‐Threonine‐Proline‐specific antibody indicates that the ERK consensus site at Thr‐867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA‐binding measured by EMSA, IGF‐1‐induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP‐ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF‐1. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-11T21:44:19.162798-05:
      DOI: 10.1002/jcp.25338
       
  • PERK Integrates Oncogenic Signaling and Cell Survival During Cancer
           Development
    • Authors: Yiwen Bu; J. Alan Diehl
      Abstract: Unfolded protein responses (UPR), consisting of three major transducers PERK, IRE1 and ATF6, occur in the midst of a variety of intracellular and extracellular challenges that perturb protein folding in the endoplasmic reticulum (ER). ER stress occurs and is thought to be a contributing factor to a number of human diseases, including cancer, neurodegenerative disorders and various metabolic syndromes. In the context of neoplastic growth, oncogenic stress resulting from dysregulation of oncogenes such as c‐Myc, BrafV600E and HRASG12V trigger the UPR as an adaptive strategy for cancer cell survival. PERK is an ER resident type I protein kinase harboring both pro‐apoptotic and pro‐survival capabilities. PERK, as a coordinator through its downstream substrates, reprograms cancer gene expression to facilitate survival in response to oncogenes and microenvironmental challenges, such as hypoxia, angiogenesis, and metastasis. Herein, we discuss how PERK kinase engages in tumor initiation, transformation, adaption microenvironmental stress, chemoresistance and potential opportunities and potential opportunities for PERK targeted therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-10T23:41:39.901168-05:
      DOI: 10.1002/jcp.25336
       
  • Dynamic Histone Acetylation of H3K4me3 Nucleosome Regulates MCL1
           Pre‐mRNA Splicing
    • Authors: Dilshad H. Khan; Carolina Gonzalez, Nikesh Tailor, Mohammad K. Hamedani, Etienne Leygue, James R. Davie
      Abstract: Pre‐mRNA splicing is a cotranscriptional process affected by the chromatin architecture along the body of coding genes. Recruited to the pre‐mRNA by splicing factors, histone deacetylases (HDACs) and K‐acetyltransferases (KATs) catalyze dynamic histone acetylation along the gene. In colon carcinoma HCT 116 cells, HDAC inhibition specifically increased KAT2B occupancy as well as H3 and H4 acetylation of the H3K4 trimethylated (H3K4me3) nucleosome positioned over alternative exon 2 of the MCL1 gene, an event paralleled with the exclusion of exon 2. These results were reproduced in MDA‐MB‐231, but not in MCF7 breast adenocarcinoma cells. These later cells have much higher levels of demethylase KDM5B than either HCT 116 or MDA‐MB‐231 cells. We show that H3K4me3 steady‐state levels and H3K4me3 occupancy at the end of exon 1 and over exon 2 of the MCL1 gene were lower in MCF7 than in MDA‐MB‐231 cells. Furthermore, in MCF7 cells, there was minimal effect of HDAC inhibition on H3/H4 acetylation and H3K4me3 levels along the MCL1 gene and no change in pre‐mRNA splicing choice. These results show that, upon HDAC inhibition, the H3K4me3 mark plays a critical role in the exclusion of exon 2 from the MCL1 pre‐mRNA. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-10T22:31:33.413353-05:
      DOI: 10.1002/jcp.25337
       
  • Fatty Acids and Breast Cancer: Make them on Site or have them Delivered
    • Authors: William B. Kinlaw; Paul W. Baures, Leslie E. Lupien, Wilson L. Davis, Nancy B. Kuemmerle
      Abstract: Brisk fatty acid (FA) production by cancer cells is accommodated by the Warburg effect. Most breast and other cancer cell types are addicted to fatty acids (FA), which they require for membrane phospholipid synthesis, signaling purposes, and energy production. Expression of the enzymes required for FA synthesis is closely linked to each of the major classes of signaling molecules that stimulate BC cell proliferation. This review focuses on the regulation of FA synthesis in BC cells, and the impact of FA, or the lack thereof, on the tumor cell phenotype. Given growing awareness of the impact of dietary fat and obesity on BC biology, we will also examine the less‐frequently considered notion that, in addition to de novo FA synthesis, the lipolytic uptake of preformed FA may also be an important mechanism of lipid acquisition. Indeed, it appears that cancer cells may exist at different points along a “lipogenic‐lipolytic axis”, and FA uptake could thwart attempts to exploit the strict requirement for FA focused solely on inhibition of de novo FA synthesis. Strategies for clinically targeting FA metabolism will be discussed, and the current status of the medicinal chemistry in this area will be assessed. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-04T12:41:43.136357-05:
      DOI: 10.1002/jcp.25332
       
  • Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound
           Healing Process
    • Authors: Cristina La Torre; Benedetta Cinque, Francesca Lombardi, Gianfranca Miconi, Paola Palumbo, Zoran Evtoski, Giuseppe Placidi, Donatella Fanini, Anna Maria Cimini, Elisabetta Benedetti, Maurizio Giuliani, Maria Grazia Cifone
      Abstract: An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S‐nitroso‐N‐acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analysed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0‐28 h) and the wound area was analysed through software included in Matlab®. As early as 15 minutes, SNAP induced significant cytoskeletal remodelling, as shown by immunostaining (phalloidin‐labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki‐67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP‐induced migration and proliferation were antagonized by the NO‐sensitive GC inhibitor 1H‐[1,2,4]oxadiazolo[‐4,3‐a]quinoxalin‐1‐one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP‐induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR‐beta antagonist (GSK0660) may interfere with NO‐mediated wound healing process. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-03T13:51:13.810184-05:
      DOI: 10.1002/jcp.25331
       
  • First Evidence of DAAM1 Localization during the Post‐Natal
           Development of Rat Testis and in Mammalian Sperm
    • Authors: Paolo Pariante; Raffaele Dotolo, Massimo Venditti, Diana Ferrara, Aldo Donizetti, Francesco Aniello, Sergio Minucci
      Abstract: Dishevelled‐associated activator of morphogenesis 1 (DAAM1) is a formin‐family protein involved in nucleation of unbranched actin filaments and in cytoskeletal organization through Wnt‐Dishevelled PCP pathway, which participates in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. While its role has been investigated during development and in somatic cells, its potential association with the germinal compartment and reproduction is still unexplored. In this work, we assessed the possible association of DAAM1 with the morphogenesis of rat testis. We studied its expression and profiled its localization versus actin and tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, DAAM1 shares its localization with actin in Sertoli cells, gonocytes and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, while only actin is detectable at the forming blood‐testis barrier. DAAM1, then, follows the development of the acrosome system throughout spermiogenesis, and it is finally retained inside the cytoplasmic droplet in mature gametes, as corroborated by additional immunolocalization data on both rat and human sperm. Unlike the DAAM1, actin keeps its localization in Sertoli cells, and tubulin is associated with their protruding cytoplasm during the process. Our data support, for the first time, the hypothesis of a role for DAAM1 in cytoskeletal organization during Mammalian testis morphogenesis and gamete progression, while also hinting at its possible investigation as a morphological marker of germ cell and sperm physiology. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T08:06:20.256924-05:
      DOI: 10.1002/jcp.25330
       
  • Emerging from the Unknown: Structural and Functional Features of
           Agnoprotein of Polyomaviruses
    • Abstract: Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV and SV40. In the absence of its expression, these viruses are unable to sustain their productive life cycle. It is a highly basic phosphoprotein that localizes mostly to the perinuclear area of infected cells, although a small amount of the protein is also found in nucleus. Much has been learned about the structure and function of this important regulatory protein in recent years. It forms highly stable dimers/oligomers in vitro and in vivo through its Leu/Ile/Phe‐rich domain. Structural NMR studies revealed that this domain adopts an alpha‐helix conformation and plays a critical role in the stability of the protein. It associates with cellular proteins, including YB‐1, p53, Ku70, FEZ1, HP1α, PP2A, AP‐3, PCNA and α‐SNAP; and viral proteins, including small t antigen, large T antigen, HIV‐1 Tat, and JCV VP1; and significantly contributes the viral transcription and replication. This review summarizes the recent advances in the structural and functional properties of this important regulatory protein. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T08:06:14.347529-05:
      DOI: 10.1002/jcp.25329
       
  • Corticosteroid‐Induced MKP‐1 Represses Pro‐Inflammatory
           Cytokine Secretion by Enhancing Activity of Tristetraprolin (TTP) in ASM
           Cells
    • Authors: Pavan Prabhala; Kristin Bunge, Qi Ge, Alaina J. Ammit
      Abstract: Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti‐inflammatory pharmacotherapies, including corticosteroids, are front‐line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their actions are not fully understood. The corticosteroid‐inducible gene, mitogen‐activated protein kinase (MAPK) phosphatase 1 (MKP‐1, DUSP1) has emerged as a key molecule responsible for the repressive effects of steroids. MKP‐1 is known to deactivate p38 MAPK phosphorylation and can control the expression and activity of the mRNA destabilizing protein ‐ tristetraprolin (TTP). But whether corticosteroid‐induced MKP‐1 acts via p38 MAPK‐mediated modulation of TTP function in a pivotal airway cell type, airway smooth muscle (ASM), was unknown. While pretreatment of ASM cells with the corticosteroid dexamethasone (preventative protocol) is known to reduce ASM synthetic function in vitro, the impact of adding dexamethasone after stimulation (therapeutic protocol) had not been explored. Whether dexamethasone modulates TTP in a p38 MAPK‐dependent manner in this cell type was also unknown. We address this herein and utilize an in vitro model of asthmatic inflammation where ASM cells were stimulated with the pro‐asthmatic cytokine tumor necrosis factor (TNF) and the impact of adding dexamethasone 1 hr after stimulation assessed. IL‐6 mRNA expression and protein secretion was significantly repressed by dexamethasone acting in a temporally distinct manner to increase MKP‐1, deactivate p38 MAPK and modulate TTP phosphorylation status. In this way, dexamethasone‐induced MKP‐1 acts via p38 MAPK to switch on the mRNA destabilizing function of TTP to repress pro‐inflammatory cytokine secretion from ASM cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-30T06:47:04.182305-05:
      DOI: 10.1002/jcp.25327
       
  • Bee Venom Accelerates Wound Healing in Diabetic Mice by Suppressing
           Activating Transcription Factor‐3 (ATF‐3) and Inducible Nitric
           Oxide Synthase (iNOS)‐Mediated Oxidative Stress and Recruiting Bone
           Marrow‐Derived Endothelial Progenitor Cells
    • Authors: Gamal Badr; Wael N. Hozzein, Badr M. Badr, Ahmad Al Ghamdi, Heba M. Saad Eldien, Olivier Garraud
      Abstract: Multiple mechanisms contribute to impaired diabetic wound healing including impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Bee venom (BV) has been used as an anti‐inflammatory agent for the treatment of several diseases. Nevertheless, the effect of BV on the healing of diabetic wounds has not been studied. Therefore, in this study, we investigated the impact of BV on diabetic wound closure in a type I diabetic mouse model. Three experimental groups were used: group 1, non‐diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice treated with BV. We found that the diabetic mice exhibited delayed wound closure characterized by a significant decrease in collagen production and prolonged elevation of inflammatory cytokines levels in wounded tissue compared to control non‐diabetic mice. Additionally, wounded tissue in diabetic mice revealed aberrantly up‐regulated expression of ATF‐3 and iNOS followed by a marked elevation in free radical levels. Impaired diabetic wound healing was also characterized by a significant elevation in caspase‐3, ‐8 and ‐9 activity and a marked reduction in the expression of TGF‐β and VEGF, which led to decreased neovascularization and angiogenesis of the injured tissue by impairing EPC mobilization. Interestingly, BV treatment significantly enhanced wound closure in diabetic mice by increasing collagen production and restoring the levels of inflammatory cytokines, free radical, TGF‐β and VEGF. Most importantly, BV‐treated diabetic mice exhibited mobilized long‐lived EPCs by inhibiting caspase activity in the wounded tissue. Our findings reveal the molecular mechanisms underlying improved diabetic wound healing and closure following BV treatment. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-30T06:46:42.007725-05:
      DOI: 10.1002/jcp.25328
       
  • Zebrafish as a Model for the Study of Chaperonopathies
    • Authors: Gianfranco Bellipanni; Francesco Cappello, Federica Scalia, Everly Conway de Macario, Alberto JL Macario, Antonio Giordano
      Abstract: There is considerable information on the clinical manifestations and mode of inheritance for many genetic chaperonopathies but little is known on the molecular mechanisms underlying the cell and tissue abnormalities that characterize them. This scarcity of knowledge is mostly due to the lack of appropriate animal models that mimic closely the human molecular, cellular, and histological characteristics. In this article we introduce zebrafish as a suitable model to study molecular and cellular mechanisms pertaining to human chaperonopathies. Genetic chaperonopathies manifest themselves from very early in life so it is necessary to examine the impact of mutant chaperone genes during development, starting with fertilization and proceeding throughout the entire ontogenetic process. Zebrafish is amenable to such developmental analysis as well as studies during adulthood. In addition, the zebrafish genome contains a wide range of genes encoding proteins similar to those that form the chaperoning system of humans. This, together with the availability of techniques for genetic manipulations and for examination of all stages of development, makes zebrafish the organism of choice for the analysis of the molecular features and pathogenic mechanisms pertaining to human chaperonopathies. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-27T06:46:27.446181-05:
      DOI: 10.1002/jcp.25319
       
  • Polyglucosan Molecules Induce Mitochondrial Impairment and Apoptosis in
           Germ Cells Without Affecting the Integrity and Functionality of Sertoli
           Cells
    • Abstract: Glycogen is the main storage form of glucose; however, the accumulation of glycogen‐like glucose polymers can lead to degeneration and cellular death. Previously, we reported that the accumulation of glycogen in testis of transgenic animals overexpressing a constitutively active form of glycogen synthase enhances the apoptosis of pre‐meiotic male germ cells and a complete disorganization of the seminiferous tubules. Here we sought to further identify the effects of glycogen storage in cells from the seminiferous tubules and the mechanism behind the pro‐apoptotic activity induced by its accumulation. Using an in vitro culture of Sertoli cells (line 42GPA9) and spermatocyte‐like cells (line GC‐1) expressing a superactive form of glycogen synthase or the Protein Targeting to Glycogen (PTG), we found that glycogen synthesized in both cell lines is poorly branched. In addition, the immunodetection of key molecules of apoptotic events suggests that cellular death induced by polyglucosan molecules affects GC‐1 cells, but not 42GPA9 cells by mitochondrial impairment and activation of an intrinsic apoptotic pathway. Furthermore, we analyzed the effects of glycogen deposition during the establishment of an in vitro blood‐testis barrier. The results using a non‐permeable fluorescent molecule showed that, in conditions of over‐synthesis of glycogen, 42GPA9 cells do not lose their capacity to generate an impermeable barrier and the levels of connexin43, occludin, and ZO1 proteins were not affected. These results suggest that the accumulation of polyglucosan molecules has a selective effect—triggered by the intrinsic activation of the apoptotic pathway—in germ cells without directly affecting Sertoli cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-21T06:54:39.640951-05:
      DOI: 10.1002/jcp.25315
       
  • IDO‐Expressing Fibroblasts Protect Islet beta Cells from
           Immunological Attack and Reverse Hyperglycemia in Non‐Obese Diabetic
           Mice
    • Abstract: Indoleamine 2,3‐dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: 1. Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non‐obese diabetic (NOD) mice; 2. Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 106 (10M) or 15 × 106 (15M) IDO‐expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8+ T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL‐ 1β and cleaved caspase‐3 levels were assessed in islets co‐cultured with IDO‐expressing fibroblasts. BGLs in 83% mice treated with 15M IDO‐expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO‐expressing cells were reversed to normoglycemia. 15M IDO‐expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8+ T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co‐cultured with IDO‐ expressing fibroblasts had reduced IL‐1β levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO‐expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8+ T cells and Th17 cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-07T08:24:47.026909-05:
      DOI: 10.1002/jcp.25301
       
  • Table of Contents, Editor's Choice, Highlights
    • Pages: 1629 - 1634
      PubDate: 2016-04-18T08:25:07.054326-05:
      DOI: 10.1002/jcp.25376
       
  • Interactions Between Fatty Acid Transport Proteins, Genes That Encode for
           Them, and Exercise: A Systematic Review
    • Authors: Avindra F. Jayewardene; Yorgi Mavros, Anneliese Reeves, Dale P. Hancock, Tom Gwinn, Kieron B. Rooney
      Pages: 1671 - 1687
      Abstract: Long‐chain fatty acid (LCFA) movement into skeletal muscle involves a highly mediated process in which lipid rafts are utilized in the cellular membrane, involving numerous putative plasma membrane‐associated LCFA transport proteins. The process of LCFA uptake and oxidation is of particular metabolic significance both at rest and during light to moderate exercise. A comprehensive systematic search of electronic databases was conducted to investigate whether exercise alters protein and/or gene expression of putative LCFA transport proteins. There were 31 studies meeting all eligibility criteria, of these 13 utilized an acute exercise protocol and 18 examined chronic exercise adaptations. Seventeen involved a study design incorporating an exercise stimulus, while the remaining 14 incorporated a combined exercise and diet stimulus. Divergent data relating to acute exercise, as well as prolonged exercise training (≥3 weeks), on protein content (PC) response was identified for proteins CD36, FABPpm and CAV1. Messenger ribonucleic acid (mRNA) data did not always correspond to functional PC, supporting previous suggestions of a disconnect due to potentially limiting factors post gene expression. The large array of study designs, cohorts, and primary dependent variables within the studies included in the present review elucidate the complexity of the interaction between exercise and LCFA transport proteins. Summary of the results in the present review validate the need for further targeted investigation within this topic, and provide an important information base for such research. J. Cell. Physiol. 231: 1671–1687, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2016-02-02T05:18:02.208666-05:
      DOI: 10.1002/jcp.25281
       
  • Genetic Polymorphism of CHRM2 in COPD: Clinical Significance and
           Therapeutic Implications
    • Authors: Emanuela Cherubini; Maria Cristina Esposito, Davide Scozzi, Fabrizio Terzo, Giorgia Amira Osman, Salvatore Mariotta, Rita Mancini, Pierdonato Bruno, Alberto Ricci
      Pages: 1745 - 1751
      Abstract: Chronic Obstructive Pulmonary Disease (COPD) is a common preventable and treatable disease, characterized by persistent airflow limitation not fully reversible. However, a number of patients with COPD respond to bronchodilator agents. Some studies have shown polymorphisms in the b2‐adrenergic (ADRb2) and muscarinic M2 and M3 receptors (CHRM) that may participate in the modulation of the receptor responses. This study was designed to investigate the existence and the role of adrenergic and muscarinic receptor polymorphisms and their functional impact in COPD. Eighty‐two patients with COPD and 17 healthy smokers were recruited and screened for ADRb2 (T164I and R175R), for CHRM2 (rs1824024) and for CHRM3 (−513C/A and −492C/T). Among the polymorphisms studied our results was not able to demonstrate statistically significant association between the polymorphisms studied and COPD risk. Contrarily, we identified, in our COPD population, a significant association with the CHRM2 (rs1824024) polymorphism and disease severity, with lower lung function test values, frequent exacerbations, and poor response to anti‐cholinergic drugs. These results suggest the potential role of receptor polymorphism assessment to discriminate newly COPD phenotypes. J. Cell. Physiol. 231: 1745–1751, 2016. © 2015 Wiley Periodicals, Inc. Chronic Obstructive Pulmonary Disease (COPD) is a common preventable and treatable disease, characterized by persistent airflow limitation not fully reversible. This study was designed to investigate the existence and the role of muscarinic receptor polymorphisms and their functional impact in COPD. Our data indicated a significant association with the CHRM2 (rs1824024) polymorphism and disease severity, with lower lung function test values, frequent exacerbations, and poor response to anti‐cholinergic drugs.
      PubDate: 2016-01-15T11:13:37.671109-05:
      DOI: 10.1002/jcp.25277
       
  • The Lineage Specification of Mesenchymal Stem Cells Is Directed by the
           Rate of Fluid Shear Stress
    • Authors: Juan Lu; Yijuan Fan, Xiaoyuan Gong, Xin Zhou, Caixia Yi, Yinxing Zhang, Jun Pan
      Pages: 1752 - 1760
      Abstract: The effective regulation of fluid shear stress (FSS) on the lineage specification of mesenchymal stem cells (MSCs) remains to be addressed. We hypothesized that when MSCs are recruited to musculoskeletal system following stimulation, their differentiation into osteogenic or chondrogenic cells is directed by the rate of FSS (ΔSS) through modulation of the mechanosensitive, cation‐selective channels (MSCCs), intracellular calcium levels, and F‐actin. To this end, MSCs were exposed to laminar FSS linearly increased from 0 to 10 dyn/cm2 in 0, 2, or 20 min and maintained at 10 dyn/cm2 for a total of 20 min (termed as ΔSS 0‐0′, 0‐2′, and 0‐20′, respectively, representing more physiological (0‐0′) and non‐physiological (0‐2′ and 0‐20′) ΔSS treatments). Our results showed 0‐0′ facilitated MSC differentiation towards chondrogenic and not osteogenic phenotype, by promoting moderate intracellular calcium concentration ([Ca2+]i) increase from the calcium channels with the exception of MSCCs or intracellular calcium stores, and F‐actin organization. In contrast, 0‐2′ promoted MSCs towards osteogenic and not chondrogenic phenotype, by inducing significant [Ca2+]i increase mainly from the MSCCs, and F‐actin assembly. However, 0‐20′ elicited the modest osteogenic and chondrogenic phenotypes, as it induced the lowest [Ca2+]i increase mainly from MSCCs, and F‐actin assembly. Our results suggest that compared to the more physiological ΔSS, the non‐physiological ΔSS favors [Ca2+]i influx from MSCCs. An appropriate non‐physiological ΔSS (0‐2′) even elicits a large [Ca2+]i influx from the MSCCs that reverses the lineage specification of MSCs, providing validation for the high mechanosensitivity of MSCs and guidance for training osteoporosis and osteoarthritis patients. J. Cell. Physiol. 231: 1752–1760, 2016. © 2015 Wiley Periodicals, Inc. MSC differentiation into osteogenic or chondrogenic cells is directed by the rate of FSS (ΔSS) through modulation of the mechanosensitive, cation‐selective channels (MSCCs), intracellular calcium levels, and F‐actin. Our results suggest that compared to the more physiological ΔSS, the non‐physiological ΔSS favors [Ca2+]i influx from MSCCs. An appropriate non‐physiological ΔSS (0‐2′) even elicits a large [Ca2+]i influx from the MSCCs that reverses the lineage specification of MSCs.
      PubDate: 2016-01-15T11:14:31.808751-05:
      DOI: 10.1002/jcp.25278
       
  • Osteoactivin Promotes Migration of Oral Squamous Cell Carcinomas
    • Authors: Oneida A. Arosarena; Raul A. dela Cadena, Michael F. Denny, Evan Bryant, Eric W. Barr, Ryan Thorpe, Fayez F. Safadi
      First page: 1761
      Abstract: Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. Objectives To determine how integrin interactions modulate OA‐induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. Materials and Methods We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OA's interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co‐immunoprecipitation. We investigated OA‐mediated activation of mitogen‐activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA‐mediated cell migration were determined. We assessed effects of OA knock‐down on cell migration and proliferation. Results OA is overexpressed in OSCC cell lines, and serves as a migration‐promoting adhesion molecule. OA co‐localized with integrin subunits, and co‐immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the β1 subunit, inhibited cell adhesion (p = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin‐neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock‐down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. Conclusion OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-08T05:28:11.505983-05:
      DOI: 10.1002/jcp.25279
       
  • New Treatment of Medullary and Papillary Human Thyroid Cancer: Biological
           Effects of Hyaluronic Acid Hydrogel Loaded With Quercetin Alone or in
           Combination to an Inhibitor of Aurora Kinase
    • Authors: Vincenzo Quagliariello; Emilia Armenia, Caterina Aurilio, Francesco Rosso, Ottavia Clemente, Gabriele de Sena, Manlio Barbarisi, Alfonso Barbarisi
      Pages: 1784 - 1795
      Abstract: The aim of this paper is based on the use of a hyaluronic acid hydrogel of Quercetin tested alone and in combination to an inhibitor of Aurora Kinase type A and B (SNS‐314) on human medullary and papillary thyroid cancer cells. Biological investigations were focused on the cellular uptake of the hydrogel, cell viability, antioxidant, and cytokines secretion studies. Quercetin delivered from hydrogel show a time and CD44 dependent interaction with both cell lines with significant anti‐inflammatory effects. Combination of Quercetin and SNS‐314 leads to a synergistic cytotoxic effect on medullary TT and papillary BCPAP cell lines with a significant reduction of the IC50 value. These results, highlights the importance of synergistic effect of the hyaluronic acid hydrogel of Quercetin with SNS‐314 in the regulation of human thyroid cancer cell proliferation and emphasize the anti‐tumor activity of these molecules. J. Cell. Physiol. 231: 1784–1795, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2016-02-02T05:15:48.523024-05:
      DOI: 10.1002/jcp.25283
       
  • Electrically Activated Primary Human Fibroblasts Improve In Vitro and In
           Vivo Skin Regeneration
    • Authors: Mahmoud Rouabhia; Hyun Jin Park, Ze Zhang
      Pages: 1814 - 1821
      Abstract: Electrical stimulation (ES) changes cellular behaviors and thus constitutes a potential strategy to promote wound healing. However, well‐controlled in vitro findings have yet to be translated to in vivo trials. This study was to demonstrate the feasibility and advantages of transplanting electrically activated cells (E‐Cells) to help wound healing. Primary human skin fibroblasts were activated through well defined ES and cultured with keratinocytes to generate engineered human skin (EHS), which were transplanted to nu/nu mice. The electrically activated EHS grafts were analyzed at 20 and 30 days post‐grafting, showing faster wound closure, thick epidermis, vasculature, and functional basement membrane containing laminin and type IV collagen that were totally produced by the implanted human cells. Because a variety of cells can be electrically activated, E‐Cells may become a new cell source and the transplantation of E‐Cells may represent a new strategy in wound healing and tissue engineering. J. Cell. Physiol. 231: 1814–1821, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2016-01-15T11:13:27.590873-05:
      DOI: 10.1002/jcp.25289
       
  • Focal Adhesion Assembly Induces Phenotypic Changes and Dedifferentiation
           in Chondrocytes
    • Authors: Hyunjun Shin; Mi Nam Lee, Jin Seung Choung, Sanghee Kim, Byung Hyune Choi, Minsoo Noh, Jennifer H. Shin
      Pages: 1822 - 1831
      Abstract: The expansion of autologous chondrocytes in vitro is used to generate sufficient populations for cell‐based therapies. However, during monolayer culture, chondrocytes lose inherent characteristics and shift to fibroblast‐like cells as passage number increase. Here, we investigated passage‐dependent changes in cellular physiology, including cellular morphology, motility, and gene and protein expression, as well as the role of focal adhesion and cytoskeletal regulation in the dedifferentiation process. We found that the gene and protein expression levels of both the focal adhesion complex and small Rho GTPases are upregulated with increasing passage number and are closely linked to chondrocyte dedifferentiation. The inhibition of focal adhesion kinase (FAK) but not small Rho GTPases induced the loss of fibroblastic traits and the recovery of collagen type II, aggrecan, and SOX9 expression levels in dedifferentiated chondrocytes. Based on these findings, we propose a strategy to suppress chondrogenic dedifferentiation by inhibiting the identified FAK or Src pathways while maintaining the expansion capability of chondrocytes in a 2D environment. These results highlight a potential therapeutic target for the treatment of skeletal diseases and the generation of cartilage in tissue‐engineering approaches. J. Cell. Physiol. 231: 1822–1831, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2016-02-04T04:23:19.064156-05:
      DOI: 10.1002/jcp.25290
       
  • Let‐7d miRNA Shows Both Antioncogenic and Oncogenic Functions in
           Osteosarcoma‐Derived 3AB‐OS Cancer Stem Cells
    • Pages: 1832 - 1841
      Abstract: Osteosarcoma (OS), an aggressive highly invasive and metastatic bone‐malignancy, shows therapy resistance and recurrence, two features that likely depend on cancer stem cells (CSCs), which hold both self‐renewing and malignant potential. So, effective anticancer therapies against OS should specifically target and destroy CSCs. We previously found that the let‐7d microRNA was downregulated in the 3AB‐OS‐CSCs, derived from the human OS‐MG63 cells. Here, we aimed to assess whether let‐7d modulation affected tumorigenic and stemness properties of these OS‐CSCs. We found that let‐7d‐overexpression reduced cell proliferation by decreasing CCND2 and E2F2 cell‐cycle‐activators and increasing p21 and p27 CDK‐inhibitors. Let‐7d also decreased sarcosphere‐and‐colony forming ability, two features associated with self‐renewing, and it reduced the expression of stemness genes, including Oct3/4, Sox2, Nanog, Lin28B, and HMGA2. Moreover, let‐7d induced mesenchymal‐to‐epithelial‐transition, as shown by both N‐Cadherin‐E‐cadherin‐switch and decrease in vimentin. Surprisingly, such switch was accompanied by enhanced migratory/invasive capacities, with a strong increase in MMP9, CXCR4 and VersicanV1. Let‐7d‐ overexpression also reduced cell sensitivity to apoptosis induced by both serum‐starvation and various chemotherapy drugs, concomitant with decrease in caspase‐3 and increase in BCL2 expression. Our data suggest that let‐7d in 3AB‐OS‐CSCs could induce plastic‐transitions from CSCs‐to‐non‐CSCs and vice‐versa. To our knowledge this is the first study to comprehensively examine the expression and functions of let‐7d in OS‐CSCs. By showing that let‐7d has both tumor suppressor and oncogenic functions in this context, our findings suggest that, before prospecting new therapeutic strategies based on let‐7d modulation, it is urgent to better define its multiple functions. J. Cell. Physiol. 231: 1832–1841, 2016. © 2015 Wiley Periodicals, Inc. Osteosarcoma (OS) is an aggressive highly invasive and metastatic bone‐malignancy showing therapy resistance and recurrence that likely depend on cancer stem cells (CSCs). So, effective anticancer therapy against OS should destroy CSCs. We found that in the human 3AB‐OS‐CSCs, where the let‐7d microRNA was strongly downregulated, let‐7d‐overexpression decreased cell proliferation, sarcosphere‐and‐colony forming ability and the expression of stemness genes, by also inducing mesenchymal‐to‐epithelial‐transition. Surprisingly these changes were accompanied by enhanced migratory/invasive capacities suggesting that in 3AB‐OS‐CSCs let‐7d could induce plastic‐transitions from CSCs‐to‐non‐CSCs and vice‐versa. By showing that let‐7d has both tumor suppressor and oncogenic functions in this context, our findings suggest that, before prospecting new therapeutic strategies based on let‐7d modulation, it is urgent to better define its multiple functions.
      PubDate: 2016-01-28T04:42:07.921937-05:
      DOI: 10.1002/jcp.25291
       
  • Research Needs for Understanding the Biology of Overdiagnosis in Cancer
           Screening
    • Authors: Sudhir Srivastava; Brian J. Reid, Sharmistha Ghosh, Barnett S. Kramer
      Abstract: Many cancers offer an extended window of opportunity for early detection and therapeutic intervention that could lead to a reduction in cause‐specific mortality. The pursuit of early detection in screening settings has resulted in decreased incidence and mortality for some cancers (eg, colon and cervical cancers), and increased incidence with only modest or no effect on cause‐specific mortality in others (eg, breast and prostate). Whereas highly sensitive screening technologies are better at detecting a number of suspected “cancers” that are indolent and likely to remain clinically unimportant in the lifetime of a patient, defined as overdiagnosis, they often miss cancers that are aggressive and tend to present clinically between screenings, known as interval cancers. Unrecognized overdiagnosis leads to overtreatment with its attendant (often long‐lasting) side effects, anxiety, and substantial financial harm. Existing methods often cannot differentiate indolent lesions from aggressive ones or understand the dynamics of neoplastic progression. To correctly identify the population that would benefit the most from screening and identify the lesions that would benefit most from treatment, the evolving genomic and molecular profiles of individual cancers during the clinical course of progression or indolence must be investigated, while taking into account an individual's genetic susceptibility, clinical and environmental risk factors, and the tumor microenvironment. Practical challenges lie not only in the lack of access to tissue specimens that are appropriate for the study of natural history, but also in the absence of targeted research strategies. This commentary summarizes the recommendations from a diverse group of scientists with expertise in basic biology, translational research, clinical research, statistics, and epidemiology and public health professionals convened to discuss research directions. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-27T17:47:33.070998-05:
      DOI: 10.1002/jcp.25227
       
  • 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
       
  • SGLT2 Inhibitors: Glucotoxicity and Tumorigenesis Downstream the Renal
           Proximal Tubule?
    • Pages: 1635 - 1637
      Abstract: At present, diabetes mellitus is the main cause of end‐stage renal disease. Effective glycaemic management is the most powerful tool to delay the establishment of diabetic complications, such as diabetic kidney disease. Together with reducing blood glucose levels, new anti‐diabetic agents are expected not only to control the progression but also to restore known defects of the diabetic kidney. Sodium–glucose co‐transporter 2 (SGLT2) inhibitors are promising anti‐diabetic agents that reduce hyperglycaemia by impairing glucose reabsorption in proximal tubule of the kidney and increasing glucosuria. SGLT2 inhibitors have shown to reduce glucotoxicity in isolated proximal tubule cells and also to attenuate expression of markers of overall kidney damage in experimental animal models of diabetes, but the actual renoprotective effect for downstream nephron segments is still unknown and deserves further attention. Here, we briefly discuss possible undesired effects of enhanced glucosuria and albuminuria in nephron segments beyond the proximal tubule after SGLT2 inhibitor treatment, offering new lines of research to further understand the renoprotective action of these anti‐diabetic agents. Strategies blocking glucose reabsorption by renal proximal tubule epithelial cells (RPTEC) may be protective for RPTEC, but downstream nephron segments will still be exposed to high glucose and albumin levels through the luminal face. The actual effect of constant enhanced glucosuria over distal nephron segments remains to be established. J. Cell. Physiol. 231: 1635–1637, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-12-30T03:07:10.140697-05:
      DOI: 10.1002/jcp.25286
       
  • MicroRNAs and Endothelial (Dys) Function
    • Authors: Gaetano Santulli
      Pages: 1638 - 1644
      Abstract: Accumulating evidence indicates that microRNAs (miRs)—non‐coding RNAs that can regulate gene expression via translational repression and/or post‐transcriptional degradation—are becoming one of the most fascinating areas of physiology, given their fundamental roles in countless pathophysiological processes. The relative roles of different miRs in vascular biology as direct or indirect post‐transcriptional regulators of fundamental genes implied in vascular remodeling designate miRs as potential biomarkers and/or promising drug targets. The mechanistic importance of miRs in modulating endothelial cell (EC) function in physiology and in disease is addressed here. Drawbacks of currently available therapeutic options are also discussed, pointing at the challenges and clinical opportunities provided by miR‐based treatments. J. Cell. Physiol. 231: 1638–1644, 2016. © 2015 Wiley Periodicals, Inc. Given their fundamental roles in several pathophysiological processes, microRNAs (miRs) are becoming one of the most fascinating areas of physiology. The relative role of different miRs in vascular biology as direct or indirect post‐transcriptional regulators of fundamental genes implied in vascular remodeling designate miRs as potential biomarkers and/or promising drug targets.
      PubDate: 2015-12-30T03:23:57.436402-05:
      DOI: 10.1002/jcp.25276
       
  • Nuclear Phosphatidylinositol Signaling: Focus on Phosphatidylinositol
           Phosphate Kinases and Phospholipases C
    • Pages: 1645 - 1655
      Abstract: Phosphatidylinositol (PI) metabolism represents the core of a network of signaling pathways which modulate many cellular functions including cell proliferation, cell differentiation, apoptosis, and membrane trafficking. An array of kinases, phosphatases, and lipases acts on PI creating an important number of second messengers involved in different cellular processes. Although, commonly, PI signaling was described to take place at the plasma membrane, many evidences indicated the existence of a PI cycle residing in the nuclear compartment of eukaryotic cells. The discovery of this mechanism shed new light on many nuclear functions, such as gene transcription, DNA modifications, and RNA expression. As these two PI cycles take place independently of one another, understanding how nuclear lipid signaling functions and modulates nuclear output is fundamental in the study of many cellular processes. J. Cell. Physiol. 231: 1645–1655, 2016. © 2015 Wiley Periodicals, Inc. Phosphatidylinositol (PI) metabolism represents the core of a network of signaling pathways which modulate many cellular functions including cell proliferation, cell differentiation, apoptosis, and membrane trafficking. In the cell, there are two independent PI cycles that take place at the plasma membrane and in the nuclear compartment respectively. The comprehension of how nuclear lipid signaling works and modulates nuclear functions is fundamental in the study of many cellular processes.
      PubDate: 2015-12-28T04:42:48.594412-05:
      DOI: 10.1002/jcp.25273
       
  • Molecular Structure and Regulation of P2X Receptors With a Special
           Emphasis on the Role of P2X2 in the Auditory System
    • Authors: Rahul Mittal; Brandon Chan, M'hamed Grati, Jeenu Mittal, Kunal Patel, Luca H. Debs, Amit P. Patel, Denise Yan, Prem Chapagain, Xue Zhong Liu
      Pages: 1656 - 1670
      Abstract: The P2X purinergic receptors are cation‐selective channels gated by extracellular adenosine 5′‐triphosphate (ATP). These purinergic receptors are found in virtually all mammalian cell types and facilitate a number of important physiological processes. Within the past few years, the characterization of crystal structures of the zebrafish P2X4 receptor in its closed and open states has provided critical insights into the mechanisms of ligand binding and channel activation. Understanding of this gating mechanism has facilitated to design and interpret new modeling and structure–function experiments to better elucidate how different agonists and antagonists can affect the receptor with differing levels of potency. This review summarizes the current knowledge on the structure, activation, allosteric modulators, function, and location of the different P2X receptors. Moreover, an emphasis on the P2X2 receptors has been placed in respect to its role in the auditory system. In particular, the discovery of three missense mutations in P2X2 receptors could become important areas of study in the field of gene therapy to treat progressive and noise‐induced hearing loss. J. Cell. Physiol. 231: 1656–1670, 2016. © 2015 Wiley Periodicals, Inc. In this review article, we discussed the current knowledge on the structure, activation, allosteric modulators, function, and location of the different P2X receptors. Moreover, an emphasis on the P2X2 receptors has been placed in respect to its role in the auditory system.
      PubDate: 2015-12-30T03:25:54.492201-05:
      DOI: 10.1002/jcp.25274
       
  • Human Microbiome and Its Association With Health and Diseases
    • First page: 1688
      Abstract: Human microbiota are distinct communities of microorganisms that resides at different body niches. Exploration of the human microbiome has become a reality due to the availability of powerful metagenomics and metatranscriptomic analysis technologies. Recent advances in sequencing and bioinformatics over the past decade help provide a deep insight into the nature of the host‐microbial interactions and identification of potential deriver genes and pathways associated with human health, well‐being, and predisposition to different diseases. In the present review, we outline recent studies devoted to elucidate the possible link between the microbiota and various type of diseases. The present review also highlights the potential utilization of microbiota as a potential therapeutic option to treat a wide array of human diseases. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-11T04:12:58.062992-05:
      DOI: 10.1002/jcp.25284
       
  • Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and
           Thyroglobulin Internalization in a Thyroid Cell Line
    • Pages: 1695 - 1708
      Abstract: Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over‐expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over‐expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over‐expressing cells, Rab7 and LC3B‐II co‐localized and co‐localization in large circular structures occurred only in Rab7 over‐expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co‐localization of Rab7 with cortactin and f‐actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over‐expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP‐stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over‐expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695–1708, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-12-10T04:08:58.636712-05:
      DOI: 10.1002/jcp.25267
       
  • Molecular Mechanism of Transcriptional Regulation of Matrix
           Metalloproteinase‐9 in Diabetic Retinopathy
    • Authors: Manish Mishra; Jadwiga Flaga, Renu A. Kowluru
      Pages: 1709 - 1718
      Abstract: Increase in matrix metalloproteinase‐9 (MMP‐9) is implicated in retinal capillary cell apoptosis, a phenomenon which precedes the development of diabetic retinopathy. MMP‐9 promoter has multiple sites for binding the transcriptional factors, including two for activator protein 1 (AP‐1). The binding of AP‐1, a heterodimer of c‐Jun and c‐Fos, is regulated by posttranslational modifications, and in diabetes, deacetylating enzyme, Sirt1, is inhibited. Our aim, is to investigate the molecular mechanism of MMP‐9 transcriptional regulation in diabetes. Binding of AP‐1 (c‐Jun, c‐Fos) at the MMP‐9 promoter, and AP‐1 acetylation were analyzed in retinal endothelial cells incubated in normal or high glucose by chromatin‐immunoprecipitation and co‐immunoprecipitation respectively. Role of AP‐1 in MMP‐9 regulation was confirmed by c‐Jun or c‐Fos siRNAs, and that of its acetylation, by Sirt1 overexpression. In vitro results were validated in the retina from diabetic mice overexpressing Sirt1, and in the retinal microvessels from human donors with diabetic retinopathy. In experimental models, AP‐1 binding was increased at the proximal and distal sites of the MMP‐9 promoter, and similar phenomenon was confirmed in the retinal microvessels from human donors with diabetic retinopathy. Silencing of AP‐1, or overexpression of Sirt1 ameliorated glucose‐induced increase in MMP‐9 expression and cell apoptosis. Thus, in diabetes, due to Sirt1 inhibition, AP‐1 is hyperacetylated, which increases its binding at MMP‐9 promoter, and hence, activation of Sirt1 could inhibit the development of diabetic retinopathy by impeding MMP‐9‐mediated mitochondrial damage. J. Cell. Physiol. 231: 1709–1718, 2016. © 2015 Wiley Periodicals, Inc. Due to inhibition of retinal Sirt1 in diabetes, AP‐1 is hyperacetylated, which facilitates its binding at the MMP‐9 promoter and increases its expression. Increased MMP‐9 damages mitochondria and accelerates cell apoptosis, resulting in the development of diabetic retinopathy.
      PubDate: 2015-12-22T05:36:54.583902-05:
      DOI: 10.1002/jcp.25268
       
  • Valproic Acid Enhances iPSC Induction From Human Bone Marrow‐Derived
           Cells Through the Suppression of Reprogramming‐Induced Senescence
    • Pages: 1719 - 1727
      Abstract: Reprogramming of human somatic cells into pluripotent cells (iPSCs) by defined transcription factors is an extremely inefficient process. Treatment with the histone deacetylase inhibitor valproic acid (VPA) during reprogramming can improve the induction of iPSCs. To examine the specific mechanism underlying the role of VPA in reprogramming, we transfected human bone marrow‐derived cells (HSC‐J2 and HSC‐L1) with lentiviruses carrying defined factors (OCT4, SOX2, KLF4, and c‐MYC, OSKM) in the presence of VPA. We found that, OSKM lentiviruses caused significant senescence in transfected cells. Administration of VPA, however, significantly suppressed this reprogramming‐induced stress. Notably, VPA treatment improved cell proliferation in the early stages of reprogramming, and this was related to the down‐regulation of the activated p16/p21 pathway. In addition, VPA also released the G2/M phase blockade in lentivirus‐transfected cells. This study demonstrates a new mechanistic role of the histone deacetylase inhibitor in enhancing the induction of pluripotency. J. Cell. Physiol. 231: 1719–1727, 2016. © 2015 Wiley Periodicals, Inc. Histone deacetylase inhibitor valproic acid enhances iPSC induction by suppressing OSKM reprogramming‐induced senescence stress. Valproic acid down‐regulates the p16/p21 pathway in reprogramming.
      PubDate: 2015-12-28T04:45:35.560076-05:
      DOI: 10.1002/jcp.25270
       
  • Fibronectin Fiber Extension Decreases Cell Spreading and Migration
    • Pages: 1728 - 1736
      Abstract: The extracellular matrix (ECM) is present in a range of molecular conformations and intermolecular arrangements. Fibronectin (Fn) molecules that constitute fibers within the ECM can exist in a variety of conformations that result from both mechanical stress and chemical factors such as allosteric binding partners. The long‐standing hypothesis that conformational changes regulate the binding of cells to Fn fibers has only been tested for mutated molecules of Fn and has yet to be fully evaluated with Fn fibers. Using time‐lapse microscopy we examined how mechanical extension of single fibers of Fn affects the adhesion and migration of endothelial cells. Using this single fiber adhesion technique, we show that high levels of mechanical strain applied to Fn fibers decreases the rates of both cell spreading and cell migration. These data indicate a fundamental cellular response to mechanical strain in the ECM that might have important implications for understanding how cells are recruited during tissue development and repair. J. Cell. Physiol. 231: 1728–1736, 2016. © 2015 Wiley Periodicals, Inc. Cell contractile forces applied to fibronectin (Fn) fibers in the extracellular matrix (ECM) stretch molecules within the fibers, which leads to changes in the conformation of Fn molecules. It has been proposed that these conformational changes may impact cell adhesion to Fn fibers. We used a single‐fiber technique to determine whether cell adhesion and migration are affected by the strain state of Fn and found that, indeed, cell spreading and cell migration are greatly reduced on stretched fibers. These findings indicate a fundamental cellular response to mechanical strain in the ECM.
      PubDate: 2015-12-28T04:45:04.543959-05:
      DOI: 10.1002/jcp.25271
       
  • Thymosin β4 Prevents Angiotensin II‐Induced Cardiomyocyte
           Growth by Regulating Wnt/WISP Signaling
    • First page: 1737
      Abstract: Thymosin beta‐4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. However, the role of Tβ4 in cardiomyocyte hypertrophy is currently unknown. The purpose of this study was to determine the cardio‐protective effect of Tβ4 in angiotensin II (Ang II)‐induced cardiomyocytes growth. Neonatal rat ventricular cardiomyocytes (NRVM) were pretreated with Tβ4 followed by Ang II stimulation. Cell size, hypertrophy marker gene expression and Wnt signaling components, β‐catenin and Wnt‐induced secreted protein‐1 (WISP‐1) were evaluated by quantitative real‐time PCR, Western blotting and fluorescent microscopy. Pre‐treatment of Tβ4 resulted in reduction of cell size, hypertrophy marker genes and Wnt‐associated gene expression and protein levels; induced by Ang II in cardiomyocytes. WISP‐1 was overexpressed in NRVM and, the effect of Tβ4 in Ang II‐induced cardiomyocytes growth was evaluated. WISP‐1 overexpression promoted cardiomyocytes growth and was reversed by pretreatment with Tβ4. This is the first report which demonstrates that Tβ4 targets Wnt/WISP‐1 to protect Ang II‐induced cardiomyocyte growth. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-02T08:11:29.042492-05:
      DOI: 10.1002/jcp.25275
       
  • MicroRNA Expression Signature Is Altered in the Cardiac Remodeling Induced
           by High Fat Diets
    • Pages: 1771 - 1783
      Abstract: Recent studies have revealed the involvement of microRNAs (miRNAs) in the control of cardiac hypertrophy and myocardial function. In addition, several reports have demonstrated that high fat (HF) diet induces cardiac hypertrophy and remodeling. In the current study, we investigated the effect of diets containing different percentages of fat on the cardiac miRNA expression signature. To address this question, male C57Bl/6 mice were fed with a low fat (LF) diet or two HF diets, containing 45 kcal% fat (HF45%) and 60 kcal% fat (HF60%) for 10 and 20 weeks. HF60% diet promoted an increase on body weight, fasting glycemia, insulin, leptin, total cholesterol, triglycerides, and induced glucose intolerance. HF feeding promoted cardiac remodeling, as evidenced by increased cardiomyocyte transverse diameter and interstitial fibrosis. RNA sequencing analysis demonstrated that HF feeding induced distinct miRNA expression patterns in the heart. HF45% diet for 10 and 20 weeks changed the abundance of 64 and 26 miRNAs in the heart, respectively. On the other hand, HF60% diet for 10 and 20 weeks altered the abundance of 27 and 88 miRNAs in the heart, respectively. Bioinformatics analysis indicated that insulin signaling pathway was overrepresented in response to HF diet. An inverse correlation was observed between cardiac levels of GLUT4 and miRNA‐29c. Similarly, we found an inverse correlation between expression of GSK3β and the expression of miRNA‐21a‐3p, miRNA‐29c‐3p, miRNA‐144‐3p, and miRNA‐195a‐3p. In addition, miRNA‐1 overexpression prevented cardiomyocyte hypertrophy. Taken together, our results revealed differentially expressed miRNA signatures in the heart in response to different HF diets. J. Cell. Physiol. 231: 1771–1783, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-12-30T03:24:20.920844-05:
      DOI: 10.1002/jcp.25280
       
  • All Trans‐Retinoic Acid Mediates MED28/HMG Box‐Containing
           Protein 1 (HBP1)/β‐Catenin Signaling in Human Colorectal Cancer
           Cells
    • Pages: 1796 - 1803
      Abstract: Vitamin A is required for normal body function, including vision, epithelial integrity, growth, and differentiation. All trans‐retinoic acid (ATRA), a family member of vitamin A, has been explored in treating acute promyelocytic leukemia and other types of cancer. Dysregulated Wnt/β‐catenin signaling and disrupted cadherin‐catenin complex often contribute to colorectal malignancy. MED28, a mammalian Mediator subunit, is found highly expressed in breast and colorectal cancers. Our laboratory has also reported that MED28 regulates cell growth, migration, and invasion in human breast cancer cells. In the current study we investigated the effect of ATRA on MED28 and Wnt/β‐catenin signaling in colorectal cancer. HCT116, HT29, SW480, and SW620, four human colorectal cancer cell lines representing different stages of carcinogenesis and harboring critical genetic changes, were employed. Our data indicated that regardless of genetic variations among these cells, suppression of MED28 reduced the expression of cyclin D1, c‐Myc, and nuclear β‐catenin, but increased the expression of E‐cadherin and HMG box‐containing protein 1 (HBP1) where HBP1 has been described as a negative regulator of the Wnt/β‐catenin signaling. The reporter activity of an HBP1 promoter increased upon MED28 knockdown, but decreased upon MED28 overexpression. ATRA reduced the expression of MED28 and mimicked the effect of MED28 suppression in down‐regulating Wnt/β‐catenin signaling. Taken together, ATRA can reverse the suppressive effect of MED28 on HBP1 and E‐cadherin and inactivate the Wnt/β‐catenin pathway in colorectal cancer, suggesting a protective effect of ATRA against colorectal cancer. J. Cell. Physiol. 231: 1796–1803, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-12-30T03:24:43.050634-05:
      DOI: 10.1002/jcp.25285
       
  • Cellular Metabolism and Dose Reveal Carnitine‐Dependent and
           ‐Independent Mechanisms of Butyrate Oxidation in Colorectal Cancer
           Cells
    • Authors: Anna Han; Natalie Bennett, Amber MacDonald, Megan Johnstone, Jay Whelan, Dallas R. Donohoe
      Pages: 1804 - 1813
      Abstract: Dietary fiber has been suggested to suppress colorectal cancer development, although the mechanisms contributing to this beneficial effect remain elusive. Butyrate, a fermentation product of fiber, has been shown to have anti‐proliferative and pro‐apoptotic effects on colorectal cancer cells. The metabolic fate of butyrate in the cell is important in determining whether, it acts as an HDAC inhibitor or is consumed as a short‐chain fatty acid. Non‐cancerous colonocytes utilize butyrate as the primary energy source whereas cancerous colonocytes increase glucose utilization through the Warburg effect. In this study, we show that butyrate oxidation is decreased in cancerous colonocytes compared to non‐cancerous colonocytes. We demonstrate that colorectal cancer cells utilize both a carnitine‐dependent and carnitine‐independent mechanism that contributes to butyrate oxidation. The carnitine‐dependent mechanism is contingent on butyrate concentration. Knockdown of CPT1A in colorectal cancer cells abolishes butyrate oxidation. In terms of selectivity, the carnitine‐dependent mechanism only regulated butyrate oxidation, as acetate and propionate oxidation were carnitine‐independent. Carnitine decreased the action of butyrate as an HDAC inhibitor and suppressed induction of H3 acetylation by butyrate in colorectal cancer cells. Thus, diminished oxidation of butyrate is associated with decreased HDAC inhibition and histone acetylation. In relation to the mechanism, we find that dichloroacetate, which decreases phosphorylation of pyruvate dehydrogenase, increased butyrate oxidation and that this effect was carnitine‐dependent. In conclusion, these data suggest that colorectal cancer cells decrease butyrate oxidation through inhibition of pyruvate dehydrogenase, which is carnitine‐dependent, and provide insight into why butyrate shows selective effects toward colorectal cancer cells. J. Cell. Physiol. 231: 1804–1813, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-12-28T04:41:26.695139-05:
      DOI: 10.1002/jcp.25287
       
 
 
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