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Journal Cover Journal of Cellular Physiology
  [SJR: 1.842]   [H-I: 139]   [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  [1612 journals]
  • Mechanical Stress Regulates Bone Metabolism through microRNAs
    • Authors: Yu Yuan; Lingli Zhang, Xiaoyang Tong, Miao Zhang, Yilong Zhao, Jianming Guo, Le Lei, Xi Chen, Jennifer Tickner, Jiake Xu, Jun Zou
      Abstract: There are many lines of evidence indicating that mechanical stress regulates bone metabolism and promotes bone growth. BMP, Wnt, ERK1/2 and OPG/RANKL are the main molecules thought to regulate the effects of mechanical loading on bone formation. Recently, microRNAs were found to be involved in bone cell proliferation and differentiation, regulating the balance of bone formation and bone resorption. Emerging evidence indicates that microRNAs also participate in mechanical stress-mediated bone metabolism, and is associated with disuse induced osteoporosis or osteopenia. Mechanical stress is able to induce expression of microRNAs that modulate the expression of osteogenic and bone resorption factors, leading to the positive impact of mechanical stress on bone. This review discusses the emerging evidence implicating an important role for microRNAs in the mechanical stress response in bone cells, as well as challenges translating microRNA research into potential treatment. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-28T12:00:24.433488-05:
      DOI: 10.1002/jcp.25688
  • Versatile Roles of Intracellularly Located TRPV1 Channel
    • Authors: Rui Zhao; Suk Ying Tsang
      Abstract: The ubiquitous expression in many organs throughout the body and the ability to respond to a wide variety of physical and chemical stimuli have brought transient receptor potential (TRP) channels to the vanguards of our sensory systems. TRP vanilloid-1 (TRPV1) is the founding member of the TRPV subfamily. TRPV1 can be activated by noxious heat, protons and vanilloids. Previous studies have shown that TRPV1 is located on the plasma membrane, serving to non-selectively permeate calcium ion from the extracellular region to the cytoplasm. Interestingly, increasing evidence suggests that TRPV1 is also located intracellularly in various cell types such as neurons, myocytes, and numerous cancer cells. By immunocytochemistry and/or fractionation followed by western blotting, TRPV1 was found to express on the endoplasmic reticulum/sarcoplasmic reticulum and the mitochondria. By using various pharmacological and molecular tools, intracellular TRPV1 was also found to functionally express to control calcium level both inside the organelles and in the cytoplasm. Recent studies have shown that intracellularly located TRPV1 serves versatile functions in various physiological and pathological conditions (e.g. exercise endurance, hypertrophy). In this review, we not only have summarized the well-characterized roles of TRPV1, but also have highlighted the increasing importance of intracellular TRPV1-mediated pathways. Lastly, we have pointed out future research direction for answering several important questions that have remained unanswered. Vigorous investigation of the emerging roles of intracellular TRPV1 can allow a better understanding of how TRPV1 controls the cellular calcium homeostasis and its role in various physiological and pathophysiological conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-27T23:26:12.731006-05:
      DOI: 10.1002/jcp.25704
  • NOR1 Suppresses Cancer Stem-Like Cells Properties of Tumor Cells via the
           Inhibition of the AKT-GSK-3β-Wnt/β-Catenin-ALDH1A1 Signal Circuit
    • Authors: Wei Wang; Mei Yi, Shengnan Chen, Junjun Li, Haijing Zhang, Wei Xiong, Guiyuan Li, Xiaoling Li, Bo Xiang
      Abstract: Cancer stem cells (CSCs) play a key role in tumor radiotherapy and chemotherapy resistance, relapse, and metastasis, and are primarily maintained in a resting state in vivo. The failure of conventional therapies to target CSCs is the main cause of treatment failure. The discovery of CSCs in nasopharyngeal carcinoma (NPC) tumors is becoming more prevalent; however, the understanding of the mechanisms underlying the maintenance of tumor stemness is still limited. We previously cloned NOR1, a tumor suppressor gene downregulated in NPC cell lines and tissues. In this study, we demonstrate that Wnt/β-catenin and ALDH1A1 form a signal circuit and that NOR1 antagonizes the tumor stem cell-like phenotype in NPC cell lines: the ectopic overexpression of NOR1 reduced β-catenin and ALDH1A1 expression; β-catenin/TCF4 targeted the regulation of ALDH1A1 transcription in NPC cells; silencing ALDH1A1 reduced AKT (total and phosphorylated) and GSK-3β (phosphorylated) expression; and eventually feedback decreased β-catenin expression levels. We also found that NOR1 expression decreased cancer stem-like cell properties of NPC cells, reduced their ability to form tumor spheroids in vitro, reduced tumorigenicity in nude mice in vivo, and increased sensitivity to chemotherapy agents. Taken together, our findings illustrated a new function of NOR1 that suppresses cancer stem-like cell properties in tumor cells by inhibiting the AKT-GSK-3β-Wnt/β-catenin-ALDH1A1 signal circuit. The study suggests that NOR1 deletion expression in NPC cells may be a potential molecular target for cancer stem cell therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-27T23:26:02.833545-05:
      DOI: 10.1002/jcp.25706
  • Celastrol Attenuates Cadmium-Induced Neuronal Apoptosis via Inhibiting
           Ca2+-CaMKII-Dependent Akt/mTOR Pathway
    • Authors: Ruijie Zhang; Yu Zhu, Xiaoqing Dong, Beibei Liu, Nana Zhang, Xiaoxue Wang, Lei Liu, Chong Xu, Shile Huang, Long Chen
      Abstract: Cadmium (Cd), an environmental and industrial pollutant, affects the nervous system and consequential neurodegenerative disorders. Recently we have shown that celastrol prevents Cd-induced neuronal cell death partially by suppressing Akt/mTOR pathway. However, the underlying mechanism remains to be elucidated. Here we show that celastrol attenuated Cd-elevated intracellular free calcium ([Ca2+]i) level and apoptosis in neuronal cells. Celastrol prevented Cd-induced neuronal apoptosis by inhibiting Akt-mediated mTOR pathway, as inhibition of Akt with Akt inhibitor X or ectopic expression of dominant negative Akt reinforced celastrol's prevention of Cd-induced phosphorylation of S6K1/4E-BP1 and cell apoptosis. Furthermore, chelating intracellular Ca2+ with BAPTA/AM or preventing [Ca2+]i elevation using EGTA potentiated celastrol's repression of Cd-induced [Ca2+]i elevation and consequential activation of Akt/mTOR pathway and cell apoptosis. Moreover, celastrol blocked Cd-elicited phosphorylation of CaMKII, and pretreatment with BAPTA/AM or EGTA enhanced celastrol's suppression of Cd-increased phosphorylation of CaMKII in neuronal cells, implying that celastrol hinders [Ca2+]i-mediated CaMKII phosphorylation. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of Akt/mTOR pathway and cell apoptosis, and this was strengthened by celastrol. Taken together, these data demonstrate that celastrol attenuates Cd-induced neuronal apoptosis via inhibiting Ca2+-CaMKII-dependent Akt/mTOR pathway. Our findings underscore that celastrol may act as a neuroprotective agent for the prevention of Cd-induced neurodegenerative disorders. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-27T23:19:55.560239-05:
      DOI: 10.1002/jcp.25703
  • Condensin Regulation of Genome Architecture
    • Authors: Vibhuti Rana; Giovanni Bosco
      Abstract: Progression through the cell cycle for both prokaryotes and eukaryotes is required for cell division. At almost every level of cellular organization structure and function of subcellular compartments and organelles must accommodate the progression of cell division. As cells progress through the cell division cycle, perhaps the most dramatic observable cellular change is the shape of chromosomes. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-26T01:35:40.940435-05:
      DOI: 10.1002/jcp.25702
  • Modulation of Chloride Currents in Human Lung Epithelial Cells Exposed to
           Exogenous Oxidative Stress
    • Authors: Rita Canella; Marta Martini, Roberta Borriello, Carlotta Cavicchio, Ximena M Muresan, Mascia Benedusi, Franco Cervellati, Giuseppe Valacchi
      Abstract: Air pollution continues to be a major public health concern affecting 9 out of 10 individuals living in urban areas worldwide. Respiratory tract is the organ most exposed to gas pollution and ozone has been shown to be one of the most noxious pollutants to which living organisms are exposed. In the present work, we have investigated the effects of 0.1 ppm of ozone on chloride currents in human lung epithelial cells (A549 line) and whether this effect could be modulated by vitamin E pre-treatment.Whole-cell patch clamp technique was applied to not excitable cells in order to obtain information about chloride currents behavior, important for epithelial lung cells homeostasis. Significant alteration of the I-V curve after ozone treatment was observed, with the appearance of a large outward rectifier component decreasing over time and returning to the basal state levels after 24 hours. Statistical analysis indicated a modification of the amount of ions passing the membrane in the unit of time as a possible cause of this difference. RT-qPCR analysis showed an increase in ClC-2 and ORCC mRNA after ozone exposure.In addition, pre-treatment with vitamin E was able to suppress the outward rectifier component induced by ozone, bringing back the current values to the control level and preventing ozone induced chloride channels up regulation.Our data suggest that ozone exposure is able to modify chloride current density and the use of vitamin E can prevent the above mentioned damage. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-25T08:05:22.13479-05:0
      DOI: 10.1002/jcp.25705
  • Supernatants of Adipocytes from Obese vs Normal Weight Women and Breast
           Cancer Cells: In Vitro Impact on Angiogenesis
    • Authors: L. Bougaret; L. Delort, H. Billard, C. Lequeux, N. Goncalves-Mendes, A. Mojallal, O. Damour, M.-P. Vasson, F. Caldefie-Chezet
      Abstract: Breast cancer is correlated with a higher risk of metastasis in obese postmenopausal women. Adipokines, whose plasma concentrations are modulated in obese subjects, surrounds mammary cells, suggesting that adipocyte secretions affect mammary tumorogenesis. We hypothsesis that matures adipocytes secretions from obese women conditioned or not by breast neoplasic cells, increase changes on the stages of angiogenesis.Supernatants of human matures adipocytes differentiated from stem cells of either adipose tissue of normal weight (MA20) or obese (MA30) women or obtained from co-cultures between MA20 or MA30 and breast cancer cell line MCF-7 were collected. The impact of these supernatants was investigated on proliferation, migration and tubes formation by endothelial cells (HUVEC).MA20 and MA30 showed a preservation of their “metabolic memory” (increase of Leptin, ObR, VEGF, CYP19A1 and decrease of Adiponectin expressions in MA30 compared to MA20).Supernatant from obese-adipocytes increased HUVEC proliferation, migration and sprouting like with supernatant obtained from co-cultures of MA/MCF-7 regardless the women's BMI. Additional analyses such as the use of neutralizing antibodies, analysis of supernatants (Milliplex®) and variations in gene expression (qRT-PCR), strongly suggest an implication of IL-6 or a synergistic action among adipokines, probably associated with that of VEGF or IL-6.As a conclusion, supernatant from co-cultures of MA30 and MCF-7 cells increase proliferation, migration and sprouting of HUVEC cells. These results provide insights into the interaction between adipocytes and epithelial cancer cells, particularly in case of obesity. The identification of synergistic action of adipokines would therefore be a great interest in developing preventive strategies. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-25T08:02:08.465987-05:
      DOI: 10.1002/jcp.25701
  • Interactive Effect of Corticosterone and Lactate on Regulation of
           Testosterone Production in Rat Leydig Cells
    • Authors: Chih-Chieh Chen; Chien-Wei Chen, Po-Han Lin, Jou-Chun Chou, Ting-Chun Weng, Cai-Yun Jian, Sindy Hu, Wei-Ho Lai, Fu-Kong Lieu, Shyi-Wu Wang, Paulus S. Wang
      Abstract: The increasing intensity of exercise enhanced corticosterone and lactate production in both humans and rodents. Our previous studies also demonstrated that lactate could stimulate testosterone production in vivo and in vitro. However, the production of testosterone in response to combined corticosterone and lactate on Leydig cells, and underlying molecular mechanisms are remained unclear. This study investigated the changes in testosterone levels of Leydig cells upon exposure to lactate, corticosterone or combination of both, and revealed the detailed mechanisms. Leydig cells were isolated from rat testes, and treated with different concentrations of lactate (2.5∼20 mM), cortiosterone (10−9∼10−4 M) and lactate plus corticosterone. The production of testosteroen were assayed by radioimmunoassay, and the key molecular proteins, including luteinizing hormone receptor (LHR), protein kinase A (PKA), steroidogenic acute regulatory protein (StAR) and cholesterol P450 side-chain cleavage enzyme (P450scc) involved in testosterone production were performed by western blot. Results showed that testosterone levels were significantly increased with lactate, while decresed with corticosterone and lactate plus corticosterone treatment. Protein expressions of LHR and P450scc were upregulated with lactate treatment. However, PKA and P450scc were downregulated by lactate plus corticosterone treatment. This downregulation was followed by decreased testoterone levels in Leydig cells. Furthermore, acetylated cAMP, which activates testosterone production was increased with lactate, but not altered by conrtiosterone. Our findings conclude that corticosterone may interfere with lactate, and restrict lactate-stimulated testosterone production in Leydig cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-25T08:02:06.068062-05:
      DOI: 10.1002/jcp.25700
  • Epigenetics in Reactive and Reparative Cardiac Fibrogenesis: The Promise
           of Epigenetic Therapy
    • Authors: Asish K Ghosh; Rahul Rai, Panagiotis Flevaris, Douglas E Vaughan
      Abstract: Epigenetic changes play a pivotal role in the development of a wide spectrum of human diseases including cardiovascular diseases, cancer, diabetes, and intellectual disabilities. Cardiac fibrogenesis is a common pathophysiological process seen during chronic and stress-induced accelerated cardiac aging. While adequate production of extracellular matrix (ECM) proteins is necessary for post-injury wound healing, excessive synthesis and accumulation of extracellular matrix protein in the stressed or injured hearts causes decreased or loss of lusitropy that leads to cardiac failure. This self-perpetuating deposition of collagen and other matrix proteins eventually alter cellular homeostasis; impair tissue elasticity and leads to multi-organ failure, as seen during pathogenesis of cardiovascular diseases, chronic kidney diseases, cirrhosis, idiopathic pulmonary fibrosis and scleroderma. In the last twenty five years, multiple studies have investigated the molecular basis of organ fibrosis and highlighted its multi- factorial genetic, epigenetic and environmental regulation. In this mini-review, we focus on five major epigenetic regulators and discuss their central role in cardiac fibrogenesis. Additionally, we compare and contrast the epigenetic regulation of hypertension-induced reactive fibrogenesis and myocardial infarction-induced reparative or replacement cardiac fibrogenesis. As microRNAs—one of the major epigenetic regulators—circulate in plasma, we also advocate their potential diagnostic role in cardiac fibrosis. Lastly, we discuss the evolution of novel epigenetic-regulating drugs and predict their clinical role in the suppression of pathological cardiac remodeling, cardiac aging and heart failure. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-24T08:25:31.386746-05:
      DOI: 10.1002/jcp.25699
  • TRPV4 Regulates Tight Junctions and Affects Differentiation in a Cell
           Culture Model of the Corneal Epithelium
    • Authors: Jacqueline Martínez-Rendón; Erika Sánchez-Guzmán, Angélica Rueda, James González, Rosario Gulias-Cañizo, Guillermo Aquino-Jarquín, Federico Castro-Muñozledo, Refugio García-Villegas
      Abstract: TRPV4 (Transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-21T07:15:23.334122-05:
      DOI: 10.1002/jcp.25698
  • Roles of Notch Signaling in Adipocyte Progenitor Cells and Mature
    • Authors: Tizhong Shan; Jiaqi Liu, Weiche Wu, Ziye Xu, Yizhen Wang
      Abstract: Adipose tissues, composed with mature adipocytes and preadipocytic stromal/stem cells, play crucial roles in whole body energy metabolism and regenerative medicine. Mature adipocytes are derived and differentiated from mesenchymal stem cells (MSCs) or preadipocytes. This differentiation process, also called adipogenesis, is regulated by several signaling pathways and transcription factors. Notch1 signaling is a highly conserved pathway that is indispensable for stem cell hemostasis and tissue development. In adipocyte progenitor cells, Notch1 signaling regulates the adipogenesis process including proliferation and differentiation of the adipocyte progenitor cells in vitro. Notably, the roles of Notch1 signaling in beige adipocytes formation, adipose development and function, and the whole body energy metabolism have been recently reported. Here, we mainly review and discuss the roles of Notch1 signaling in adipogenesis in vitro as well as in beige adipocytes formation, adipocytes dedifferentiation and function in vivo. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-21T07:10:20.732207-05:
      DOI: 10.1002/jcp.25697
  • Signaling in the Auditory System: Implications in Hair Cell Regeneration
           and Hearing Function
    • Authors: Rahul Mittal; Luca H. Debs, Desiree Nguyen, Amit P. Patel, M'hamed Grati, Jeenu Mittal, Denise Yan, Adrien A. Eshraghi, Xue Zhong Liu
      Abstract: Ear is a sensitive organ involved in hearing and balance function. The complex signaling network in the auditory system plays a crucial role in maintaining normal physiological function of the ear. The inner ear is comprised of a variety of host signaling pathways working in synergy to deliver clear sensory messages. Any disruption, as minor as it can be, has the potential to affect this finely tuned system with temporary or permanent sequelae including vestibular deficits and hearing loss. Mutations linked to auditory symptoms, whether inherited or acquired, are being actively researched for ways to reverse, silence, or suppress them. In this article, we discuss recent advancements in understanding the pathways involved in auditory system signaling, from hair cell development through transmission to cortical centers. Our review discusses Notch and Wnt signaling, cell to cell communication through connexin and pannexin channels, and the detrimental effects of reactive oxygen species on the auditory system. There has been an increased interest in the auditory community to explore the signaling system in the ear for hair cell regeneration. Understanding signaling pathways in the auditory system will pave the way for the novel avenues to regenerate sensory hair cells and restore hearing function. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-21T07:05:29.400192-05:
      DOI: 10.1002/jcp.25695
  • Roles of Multifunctional COP9 Signalosome Complex in Cell Fate and
           Implications for Drug Discovery
    • Authors: Ping Li; Longxiang Xie, Yinzhong Gu, Jiang Li, Jianping Xie
      Abstract: The eight subunits containing COP9 signalosome (CSN) complex, is highly conserved among eukaryotes. CSN, identified as a negative regulator of photomorphogenesis, has also been demonstrated to be important in proteolysis, cellular signal transduction and cell cycle regulation in various eukaryotic organisms. This review mainly summarizes the roles of CSN in cell cycle regulation, signal transduction and apoptosis, and its potential as diagnostic biomarkers, drug targets for cancer and infectious diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-21T07:05:21.153902-05:
      DOI: 10.1002/jcp.25696
  • Notch Signaling Pathway Regulates Angiogenesis via Endothelial Cell in 3D
           Co-Culture Model
    • Authors: Dan Zhao; Changyue Xue, Shiyu Lin, Sirong Shi, Qianshun Li, Mengting Liu, Xiaoxiao Cai, Yunfeng Lin
      Abstract: ObjectivesThis study aimed to investigate the role of Notch signaling pathway for angiogenesis in a three-dimensional (3D) collagen gel model with co-culture of adipose-derived stromal cells (ASCs) and endothelial cells (ECs).MethodsA 3D collagen gel model was established in vitro by implanting both ASCs from green fluorescent protein-labeled mouse and ECs from red fluorescent protein-labeled mouse, and the phenomena of angiogenesis with Notch signaling inducer Jagged1, inhibitor DAPT and PBS respectively were observed by confocal laser scanning microscopy. Semi-quantitative PCR and immunofluorescent staining were conducted to detect expressions of angiogenesis-related genes and proteins.ResultsAngiogenesis in the co-culture gels was promoted by Jagged1 treatment while attenuated by DAPT treatment, compared to control group. In co-culture system of ASCs and ECs, the gene expressions of VEGFA, VEGFB, Notch1, Notch2, Hes1, Hey1, VEGFR1and the protein expression of VEGFA, VEGFB, Notch1, Hes1, Hey1 were increased by Jagged1 treatment and decreased by DAPT treatment in ECs. And the result of VEGFR3 was the opposite. However, the same results did not appear completely in ASCs.ConclusionsThese results revealed the VEGFA/B-Notch1/2-Hes1/Hey1- VEGFR1/3 signal axis played an important role in angiogenesis when ASCs and ECs were co-cultured in a 3D collagen gel model. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-20T15:45:44.326649-05:
      DOI: 10.1002/jcp.25681
  • Tracing Males from Different Continents by Genotyping JC Polyomavirus in
           DNA from Semen Samples
    • Authors: John Charles Rotondo; Tommaso Candian, Rita Selvatici, Elisa Mazzoni, Gloria Bonaccorsi, Pantaleo Greco, Mauro Tognon, Fernanda Martini
      Abstract: The human JC polyomavirus (JCPyV) is an ubiquitous viral agent infecting approximately 60% of humans. Recently, JCPyV sequences have been detected in semen samples. The aim of this investigation was to test whether semen JCPyV genotyping can be employed to trace the origin continent of males. Semen DNA samples (n = 170) from males of different Continents, were investigated by PCR for the polymorphic JCPyV viral capsid protein 1 (VP1) sequences, followed by DNA sequencing. JCPyV sequences were detected with an overall prevalence of 27.6% (47/170). DNA sequencing revealed that European males carried JCPyV types 1A (71.4%), 4 (11.4%), 2B (2.9%), 2D1 (2.9%) and 3A (2.9%), Asians JCPyV type 2D1 (66.7%) and Africans JCPyV types 3A (33.3%) and 1A (33.3%). In 10.6% of males, two different JCPyV genotypes were detected suggesting that the second JCPyV genotype was acquired in the destination country. This study indicates that the majority of semen samples, found to be JCPyV-positive, were infected with the JCPyV genotype found in the geographic area of male origin. Therefore, semen JCPyV genotyping could be employed to trace the origin continent of males. Our findings could be applied to forensic investigations, in case of, for instance, sexual crimes. Indeed, JCPyV genotyping should enable investigators to make additional detailed profiling of the offender. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-20T15:45:36.45259-05:0
      DOI: 10.1002/jcp.25686
  • Wnt3A Induces GSK-3β Phosphorylation and β-Catenin Accumulation
           Through RhoA/ROCK
    • Authors: Jae-Gyu Kim; Myoung-Ju Kim, Won-Ji Choi, Mi-Young Moon, Hee-Jun Kim, Jae-Yong Lee, Jaebong Kim, Sung-Chan Kim, Seung Goo Kang, Goo-Young Seo, Pyeung-Hyeun Kim, Jae-Bong Park
      Abstract: In canonical pathway, Wnt3A has been known to stabilize β-catenin through the dissociation between β-catenin and glycogen synthase kinase-3β (GSK-3β) that suppresses the phosphorylation and degradation of β-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3β and accumulation of β-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3β and accumulation of β-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3β phosphorylation and β-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3β in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3β phosphorylation and β-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.Wnt3A activates RhoA and ROCK, which induces GSK-3beta phosphorylation and beta-catenin accumulation. RhoA and ROCK are involved in the regulation of cell proliferation and migration upon Wnt3A.
      PubDate: 2016-11-20T15:45:29.212161-05:
      DOI: 10.1002/jcp.25572
  • Postnatal Hyperplasic Effects of Actriib Blockade in a Severely Dystrophic
    • Authors: C. Nielsen; R. M. Potter, C. Borowy, K. Jacinto, R. Kumar, C.G. Carlson
      Abstract: The efficacy of two ActRIIB ligand trapping agents (RAP-031, RAP-435) in treating muscular dystrophy was examined by determining their morphological effects on the severely dystrophic triangularis sterni (TS) muscle of the mdx mouse, a model for Duchenne muscular dystrophy. These agents trap all endogenous ligands to the ActRIIB receptor and thereby block myostatin signaling in a highly selective manner. Short (1 month) and long term (3 months) in vivo treatment of 1 month old mdx mice increased myonuclei and fiber cross section (FCS) density but did not alter individual fiber size. Vehicle-treated mdx mice exhibited age-dependent increases in myonuclei and FCS density, and age-dependent reductions in centronucleation that were each enhanced by treatment with RAP-435. Distributions of FCS area (FCSA) in the mdx TS were 90% identical to those from untreated age-matched nondystrophic mice and were unaltered by the substantial fiber hyperplasia observed with age and RAP-435 treatment. These results were inconsistent with injury-induced fiber regeneration which produces altered FCSA distributions characterized by a distinct class of smaller regenerated fibers. Nondystrophic mice exhibited a constant postnatal density of fiber cross sections and myonuclei, and RAP-435 treatment of nondystrophic mice increased TS mean FCSA but had no effects on myonuclei or FCS density. These results demonstrating a continual postnatal proliferation and fusion of satellite cells and a response to myostatin blockade characteristic of developing prenatal muscle suggest that the lack of dystrophin directly results in unrestrained postnatal satellite cell activation that is not necessarily dependent upon prior fiber degeneration. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-18T06:26:14.987783-05:
      DOI: 10.1002/jcp.25694
  • Homocysteine as a Pathological Biomarker for Bone Disease
    • Authors: Jyotirmaya Behera; Jyoti Bala, Mohammed Nuru, Suresh C. Tyagi, Neetu Tyagi
      Abstract: In the last few decades, perturbation in methyl-group and homocysteine (Hcy) balance have emerged as independent risk factors in a number of pathological conditions including neurodegenerative disease, cardiovascular dysfunction, cancer development, autoimmune disease and kidney disease. Recent studies report Hcy to be a newly recognized risk factor for osteoporosis. Elevated Hcy levels are known to modulate osteoclastgenesis by causing detrimental effects on bone via oxidative stress induced metalloproteinase-mediated extracellular matrix degradation and decrease in bone blood flow. Evidence from previous studies also suggests that the decreased chondrocytes mediated bone mineralization in chick limb-bud mesenchymal cells and during the gestational period of ossification in rat model. However, Hcy imbalance and its role in bone loss, regression in vascular invasion, and osteoporosis, are not clearly understood. More investigations are required to explore the complex interplay between Hcy imbalance and onset of bone disease progression. This article reviews the current body of knowledge on regulation of Hcy mediated oxidative stress and its role in bone remodeling, vascular blood flow and progression of bone disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-18T06:20:21.501959-05:
      DOI: 10.1002/jcp.25693
  • Cover Image, Volume 232, Number 3, March 2017
    • Authors: Antonio Celentano; Michele Davide Mignogna, Michael McCullough, Nicola Cirillo
      Abstract: Cover: The cover image, by Celentano et al., is based on the Review Article Pathophysiology of the Desmo-Adhesome,
      DOI : 10.1002/jcp.25515
      PubDate: 2016-11-17T09:27:53.584132-05:
  • Meeting Report: 4th European Seminars in Virology on Oncogenic and
           Oncolytic Viruses, in Bertinoro (Bologna), Italy
    • Authors: Alberto Reale; Lorenzo Messa, Adriana Vitiello, Arianna Loregian, Giorgio Palù
      Abstract: The 4th European Seminars in Virology (EuSeV), which was focused on oncogenic and oncolytic viruses, was held in Bertinoro (Bologna), Italy, from June 10 to 12, 2016. This article summarizes the plenary lectures and aims to illustrate the main topics discussed at 4th EuSeV, which brought together knowledge and expertise in the field of oncogenic and oncolytic viruses from all over the world. The meeting was divided in two parts, “Mechanisms of Viral Oncogenesis” and “Viral Oncolysis and Immunotherapy”, which were both focused on dissecting the complex and multi-factorial interplay between cancer and human viruses and on exploring new anti-cancer strategies. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-16T06:17:08.206426-05:
      DOI: 10.1002/jcp.25692
  • CD93 as a Potential Target in Neovascular Age-Related Macular Degeneration
    • Authors: Gian Marco Tosi; Elena Caldi, Barbara Parolini, Paolo Toti, Giovanni Neri, Federica Nardi, Claudio Traversi, Gabriele Cevenini, Davide Marigliani, Elisabetta Nuti, Tommaso Bacci, Federico Galvagni, Maurizio Orlandini
      Abstract: In patients with age-related macular degeneration (AMD), choroidal neovascularization is the major cause of severe visual loss. In these patients, the persistence of neovascular growth despite vascular endothelial growth factor-A blockage needs the discovery of new endothelial cell targets. The glycoprotein CD93, highly expressed in activated endothelial cells, has been recently involved in the regulation of the angiogenic process both as transmembrane and soluble protein.Choroidal neovascular membranes from patients affected by AMD were examined by immunofluorescence using anti-CD93 and anti-von Willebrand factor antibodies. Blood vessels within intraocular and extraocular neoplasias were used as controls for CD93 expression. All choroidal neovascular membranes displayed strong CD93 staining in the von Willebrand factor-positive endothelial cells, consistently with the analyses showing a high colocalization coefficient in the blood vessels. Intraocular and extraocular tumor vessels showed similar results, whereas the normal choroid displayed blood vessels with only faint CD93 staining. Additionally, the concentration of soluble CD93 was determined in the aqueous humor of patients affected by naïve neovascular AMD by enzyme-linked immunosorbent assays. Age-matched cataract patients served as controls. Soluble CD93 was significantly increased in the aqueous humor of naïve neovascular AMD patients and tended to decrease after treatment with an antiangiogenic drug. In conclusion, both transmembrane and soluble CD93 are overexpressed in patients with neovascular AMD, indicating that CD93 may represent a potential new antiangiogenic target in the treatment of choroidal neovascularization. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-15T03:31:09.290756-05:
      DOI: 10.1002/jcp.25689
  • Oligodendrocyte Progenitor Cells Directly Utilize Lactate for Promoting
           Cell Cycling and Differentiation
    • Authors: Yoshinori Ichihara; Toru Doi, Youngjae Ryu, Motoshi Nagao, Yasuhiro Sawada, Toru Ogata
      Abstract: Oligodendrocyte progenitor cells (OPCs) undergo marked morphological changes to become mature oligodendrocytes, but the metabolic resources for this process have not been fully elucidated. Although lactate, a metabolic derivative of glycogen, has been reported to be consumed in oligodendrocytes as a metabolite, and to ameliorate hypomyelination induced by low glucose conditions, it is not clear about the direct contribution of lactate to cell cycling and differentiation of OPCs, and the source of lactate for remyelination. Therefore, we evaluated the effect of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), an inhibitor of the glycogen catabolic enzyme glycogen phosphorylase, in a mouse cuprizone model. Cuprizone induced demyelination in the corpus callosum and remyelination occurred after cuprizone treatment ceased. This remyelination was inhibited by the administration of DAB. To further examine whether lactate affects proliferation or differentiation of OPCs, we cultured mouse primary OPC-rich cells and analyzed the effect of lactate. Lactate rescued the slowed cell cycling induced by 0.4 mM glucose, as assessed by the BrdU-positive cell ratio. Lactate also promoted OPC differentiation detected by monitoring the mature oligodendrocyte marker myelin basic protein, in the presence of both 36.6 mM and 0.4 mM glucose. Furthermore, these lactate-mediated effects were suppressed by the reported monocarboxylate transporter inhibitor, α-cyano-4-hydroxy-cinnamate. These results suggest that lactate directly promotes the cell cycling rate and differentiation of OPCs, and that glycogen, one of the sources of lactate, contributes to remyelination in vivo. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-15T03:31:01.611104-05:
      DOI: 10.1002/jcp.25690
  • A Real-World Multicentre Retrospective Study of Paclitaxel-Bevacizumab and
           Maintenance Therapy as First-Line for HER2-Negative Metastatic Breast
    • Authors: T. Gamucci; L. Mentuccia, C. Natoli, I. Sperduti, A. Cassano, A. Michelotti, L. Di Lauro, D. Sergi, A. Fabi, M.G. Sarobba, P. Marchetti, M. Barba, E. Magnolfi, M. Maugeri-Saccà, E. Rossi, V. Sini, A. Grassadonia, D. Pellegrini, A. Astone, C. Nisticò, F. Angelini, A. Vaccaro, A. Pellegrino, C. De Angelis, M. Palleschi, L. Moscetti, I. Bertolini, S. Buglioni, A. Giordano, L. Pizzuti, P. Vici
      Abstract: Bevacizumab in combination with taxanes in HER2-negative metastatic breast cancer (MBC) patients has shown improved progression-free survival (PFS), despite the lack of clear overall survival (OS) benefit. We performed a retrospective analysis to evaluate the impact of paclitaxel-bevacizumab and of maintenance therapy with bevacizumab (BM) and endocrine therapy (ET) in the real-world practice.We identified 314 HER2-negative MBC patients treated in 12 cancer centres.Overall, the median PFS and OS were 14 and 40 months, respectively. Among the 254 patients potentially eligible for BM, 183 received BM after paclitaxel discontinuation until progression/toxicity. PFS and OS were improved in patients who had received BM in comparison with those potentially eligible but who did not receive BM (p 
      PubDate: 2016-11-11T07:15:43.103661-05:
      DOI: 10.1002/jcp.25685
  • Lgr4 Expression in Osteoblastic Cells Is Suppressed by Hydrogen Peroxide
    • Authors: Chantida Pawaputanon Na Mahasarakham; Yayoi Izu, Katsuhiko Nishimori, Yuichi Izumi, Masaki Noda, Yoichi Ezura
      Abstract: LGR4 is expressed in bone and has been shown to be involved in bone metabolism. Oxidative stress is one of the key issues in pathophysiology of osteoporosis. However, the link between Lgr4 and oxidative stress has not been known. Therefore, effects of hydrogen peroxide on Lgr4 expression in osteoblasts were examined. Hydrogen peroxide treatment suppressed the levels of Lgr4 mRNA expression in an osteoblastic cell line, MC3T3E1. The suppressive effects were not obvious at 0.1mM while 1mM hydrogen peroxide suppressed Lgr4 expression by more than 50%. Hydrogen peroxide treatment suppressed Lgr4 expression within 12 hours and this suppression lasted at least up to 48 hours. Hydrogen peroxide suppression of Lgr4 expression was still observed in the presence of a transcription inhibitor but was no longer observed in the presence of a protein synthesis inhibitor. Although Lgr4 expression in osteoblasts is enhanced by BMP2 treatment as reported before, hydrogen peroxide treatment suppressed Lgr4 even in the presence of BMP2. Finally, hydrogen peroxide suppressed Lgr4 expression in primary cultures of osteoblasts similarly to MC3T3-E1 cells. These date indicate that hydrogen peroxide suppresses Lgr4 expression in osteoblastic cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-11T07:15:37.243966-05:
      DOI: 10.1002/jcp.25684
  • Role of Wnt5a in the Pathogenesis of Inflammatory Diseases
    • Authors: Mehran Pashirzad; Mojtaba Shafiee, Farzad Rahmani, Reihane Behnam-Rassouli, Fatemeh Hoseinkhani, Mikhail Ryzhikov, Maryam Moradi Binabaj, Mohammad Reza Parizadeh, Amir Avan, Seyed Mahdi Hassanian
      Abstract: Wnt5a initiates pro-inflammatory responses through activation of non-canonical Wnt signaling pathway. Pro-inflammatory functions of Wnt5a trigger pro-inflammatory signaling cascades and increase secretion of pro-inflammatory cytokines and chemokines. Wnt5a as a potent signaling molecule is strongly implicated in a number of diseases including cancer, diabetes, metabolic disorders, and of special interest in this review, inflammatory diseases. This review summarizes the role of Wnt5a in the pathogenesis of inflammatory diseases including atherosclerosis, rheumatoid arthritis, psoriasis vulgaris and sepsis, promoting greater understanding and clinical management of these diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-11T07:10:28.107554-05:
      DOI: 10.1002/jcp.25687
  • The Multifaceted Roles of Primary Cilia in the Regulation of Stem Cell
           Properties and Functions
    • Authors: Rui Lyu; Jun Zhou
      Abstract: Stem cells are a unique class of cells that are capable of self-renewal and differentiation into multiple lineages. An increasing number of studies have suggested that both embryonic and adult stem cells possess primary cilia, antenna-like structures protruding from cell surfaces that are critical for sensing and transducing environmental cues. The primary cilium appears to regulate stem cells in multiple aspects, such as lineage specification and stemness maintenance. Understanding the role of primary cilia in the control of stem cell behavior could lead to the identification of new targets for regenerative therapies. Here, we discuss recent studies investigating the diverse roles of primary cilia in the regulation of stem cell properties and functions. We also propose potential new avenues for exploration in this promising field. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-11T07:05:29.536643-05:
      DOI: 10.1002/jcp.25683
  • S100B + A1 CELISA: A Novel Potency Assay and Screening Tool for
           Redifferentiation Stimuli of Human Articular Chondrocytes
    • Authors: Jose Diaz-Romero; Sibylle Kürsener, Sandro Kohl, Dobrila Nesic
      Abstract: During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell-based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: 1) demonstrate a correlation between S100B + A1-positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; 2) develop an S100B + A1 cell-based ELISA, and 3) prove that S100B + A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high-osmolarity/BMP4 treatment in monolayer) using qRT-PCR, immunocytochemistry or immunohistochemistry. A cell-based ELISA (CELISA) was developed as a 96-well microplate multiplex assay to measure S100B + A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow-derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B + A1-positive HAC in monolayer and their neochondrogenesis in pellets validates S100B + A1 as a marker of chondrogenic potency. The S100B + A1-CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high-throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets - induced with high-osmolarity and BMP4 treatment in monolayer - suggests that cell instruction prior to implantation may improve cartilage repair. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-11T07:05:24.089704-05:
      DOI: 10.1002/jcp.25682
  • Sensitivity of TP53-Mutated Cancer Cells to the Phytoestrogen Genistein Is
           Associated with Direct Inhibition of Plk1 Activity
    • Authors: Sol-Bi Shin; Sang-Uk Woo, Young Won Chin, Young-Joo Jang, Hyungshin Yim
      Abstract: Polo-like kinase 1 (Plk1), a conserved Ser/Thr mitotic kinase, has been identified as a promising target for anticancer drug development because its overexpression is correlated with malignancy. Here, we found that genistein, an isoflavone, inhibits Plk1 kinase activity directly. Previously the mitotic disturbance phenomenon induced by treatment with genistein was not fully explained by its inhibitory effect on EGFR. In kinase profiling assays, it showed selectivity relative to a panel of kinases, including EGFR. Treatment with genistein induced cell death in a concentration-dependent manner in cancer cells from diverse tissue origins, but not in non-transformed cells such as hTERT-RPE or MCF10A cells. We also observed that genistein tended to be more selective against cancer cells with mutations in the TP53 gene. TP53-depeleted LNCaP and NCI-H460 cells using shRNA targeting human TP53 were more sensitive to cell death by treatment of genistein. Furthermore, genistein induced mitotic arrest by inhibiting Plk1 activity and, consequently, led to mitotic catastrophe and apoptosis. These data suggest that genistein may be a promising anticancer drug candidate due to its inhibitory activity against Plk1 as well as EGFR and effectiveness toward cancer cells, especially those with p53-mutation. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-09T15:46:18.521404-05:
      DOI: 10.1002/jcp.25680
  • Testosterone Rescues the De-Differentiation of Smooth Muscle Cells through
           Serum Response Factor/Myocardin
    • Authors: Carolina Leimgruber; Amado A. Quintar, Nahuel Peinetti, María V. Scalerandi, Juan P. Nicola, Joseph M. Miano, Cristina A. Maldonado
      Abstract: Prostatic smooth muscle cells (pSMCs) differentiation is a key factor for prostatic homeostasis, with androgens exerting multiple effects on these cells. Here, we demonstrated that the myodifferentiator complex Srf/Myocd is up-regulated by testosterone in a dose-dependent manner in primary cultures of rat pSMCs, which was associated to the increase in Acta2, Cnn1, and Lmod1 expressions. Blocking Srf or Myocd by siRNAs inhibited the myodifferentiator effect of testosterone. While LPS led to a dedifferentiated phenotype in pSMCs, characterized by down-regulation of Srf/Myocd and smooth muscle cell (SMC)-restricted genes, endotoxin treatment on Myocd-overexpressing cells did not result in phenotypic alterations.Testosterone at a physiological dose was able to restore the muscular phenotype by normalizing Srf/Myocd expression in inflammation-induced dedifferentiated pSMCs. Moreover, the androgen reestablished the proliferation rate and IL-6 secretion increased by LPS.These results provide novel evidence regarding the myodifferentiating role of testosterone on SMCs by modulating Srf/Myocd. Thus, androgens preserve prostatic SMC phenotype, which is essential to maintain the normal structure and function of the prostate. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-09T15:46:16.314765-05:
      DOI: 10.1002/jcp.25679
  • Exploring the Role of PGC-1α in Defining Nuclear Organisation in
           Skeletal Muscle Fibres
    • Authors: Jacob A. Ross; Adam Pearson, Yotam Levy, Bettina Cardel, Christoph Handschin, Julien Ochala
      Abstract: Muscle fibres are multinucleated cells, with each nucleus controlling the protein synthesis in a finite volume of cytoplasm termed the myonuclear domain (MND). What determines MND size remains unclear. In the present study, we aimed to test the hypothesis that the level of expression of the transcriptional coactivator PGC-1α and subsequent activation of the mitochondrial biogenesis are major contributors. Hence, we used two transgenic mouse models with varying expression of PGC-1α in skeletal muscles. We isolated myofibres from the fast twitch extensor digitorum longus (EDL) and slow twitch diaphragm muscles. We then membrane-permeabilised them and analysed the 3D spatial arrangements of myonuclei. In EDL muscles, when PGC-1α is over-expressed, MND volume decreases; whereas, when PGC-1α is lacking no change occurs. In the diaphragm no clear difference was noted. This indicates that PGC-1α and the related mitochondrial biogenesis programme are determinants of MND size. PGC-1α may facilitate the addition of new myonuclei in order to reach MND volumes that can support an increased mitochondrial density. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-07T06:47:31.67368-05:0
      DOI: 10.1002/jcp.25678
  • The Emerging Role of TRAF7 in Tumor Development
    • Authors: Tiziana Zotti; Ivan Scudiero, Pasquale Vito, Romania Stilo
      Abstract: The seven members of the tumor necrosis factor receptor (TNF-R)-associated factor (TRAF) family of intracellular proteins were originally discovered and characterized as signaling adaptor molecules coupled to the cytoplasmic regions of receptors of the TNF-R superfamily. Functionally, TRAFs act both as a scaffold and/or enzymatic proteins to regulate activation of mitogen-activated protein kinases (MAPKs) and transcription factors of nuclear factor-κB family (NF-κB). Given the wide variety of stimuli intracellularly conveyed by TRAF proteins, they are physiologically involved in multiple biological processes, including embryonic development, tissue homeostasis and regulation of innate and adaptive immune responses. In the last few years, it has become increasingly evident the involvement of TRAF7, the last member of the TRAF family to be discovered, in the genesis and progression of several human cancers, placing TRAF7 in the spotlight as a novel tumor suppressor protein. In this paper, we review and discuss the literature recently produced on this subject. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-03T05:59:27.377338-05:
      DOI: 10.1002/jcp.25676
  • E-Cigarette Vapor Induces an Apoptotic Response in Human Gingival
           Epithelial Cells Through the Caspase-3 Pathway
    • Authors: Mahmoud Rouabhia; Hyun Jin Park, Abdelhabib Semlali, Andrew Zakrzewski, Witold Chmielewski, Jamila Chakir
      Abstract: Electronic cigarettes represent an increasingly significant proportion of today's consumable tobacco products. E-cigarettes contain several chemicals which may promote oral diseases. The aim of this study was to investigate the effect of e-cigarette vapor on human gingival epithelial cells. Results show that e-cigarette vapor altered the morphology of cells from small cuboidal form to large undefined shapes. Both single and multiple exposures to e-cigarette vapor led to a bulky morphology with large faint nuclei and an enlarged cytoplasm. E-cigarette vapor also increased L-lactate dehydrogenase (LDH) activity in the targeted cells. This activity was greater with repeated exposures. Furthermore, e-cigarette vapor increased apoptotic/necrotic epithelial cell percentages compared to that observed in the control. Epithelial cell apoptosis was confirmed by TUNEL assay showing that exposure to e-cigarette vapor increased apoptotic cell numbers, particularly after two and three exposures. This negative effect involved the caspase-3 pathway, the activity of which was greater with repeated exposure and which decreased following the use of caspase-3 inhibitor. The adverse effects of e-cigarette vapor on gingival epithelial cells may lead to dysregulated gingival cell function and result in oral disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-03T05:59:00.705487-05:
      DOI: 10.1002/jcp.25677
  • Differential Expression of Estrogen Receptor Variants in Response to
           Inflammation Signals in Human Airway Smooth Muscle
    • Authors: Bharathi Aravamudan; Katelyn J Goorhouse, Ghanashyam Unnikrishnan, Michael A. Thompson, Christina M Pabelick, John R Hawse, YS Prakash, Venkatachalem Sathish
      Abstract: The prevalence of asthma is higher in pre-pubescent and aging males, and in post-pubertal females, strongly indicating that sex steroids (especially estrogen) may be an important modulator in lung disease. We recently demonstrated that airway smooth muscle (ASM) expresses both alpha and beta forms of the estrogen receptor (ERα and ERβ) in males and females, and that these receptors regulate intracellular [Ca2 +] and ASM contractility. While both ERα and ERβ have multiple splice variants, it is unclear if and how the expression of these variants is modulated under conditions such as chronic inflammation/ asthma. In order to test the hypothesis that the differential expression of ERα and ERβ variants contributes to the pathogenesis of asthma, we profiled the expression of various ERα and ERβ genes in asthmatic and inflamed (TNFα- or IL-13-treated) ASM. Gene expression was assessed at both the mRNA and protein levels in asthmatic ASM cells or non-asthmatic cells treated with TNFα (20ng/ml) or IL-13 (50ng/ml). We observed marked variation in the expression of ER isoforms in response to inflammatory stimuli, and in non-asthmatic vs. asthmatic ASM. Changes in protein levels of ERα and ERβ corresponded with the observed differential mRNA patterns. Pharmacological studies implicate cytosolic (p42/44 MAPK and PI3K) and nuclear (NFκB, STAT6 and AP-1) signaling pathways as putative mechanisms that mediate and/or regulate effects of inflammation on ER expression. We conclude that variations in ASM ER expression profiles occur with inflammation and that ER variants could contribute to estrogen signaling in airway diseases such as asthma. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-03T05:52:32.047268-05:
      DOI: 10.1002/jcp.25674
  • Cre Recombinase Strains Used For the Study of Adipose Tissue and Adipocyte
    • Authors: Jiaqi Liu; Ziye Xu, Weiche Wu, Yizhen Wang, Tizhong Shan
      Abstract: Adipose tissues play important roles in whole body energy homeostasis and lifer span. Understanding the mechanisms of controlling adipose tissues development is significant for providing useful information to treat the worldwide epidemic of obesity and its associated metabolic diseases. Several different Cre transgenes have been generated and used for determining the origin of adipose tissues and the function of individual gene in regulating adipose growth and development. Here, we mainly review and discuss the efficiency and specific of those Cre recombinase mouse strains used for the study of adipose tissue and adipocyte progenitors. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-03T05:52:29.646889-05:
      DOI: 10.1002/jcp.25675
  • Promoting Cancer Control in Africa With “Ubuntu”: A Report of the
           African Organization For Research and Training in Africa (AORTIC) 10th
           Conference, 2015 in Marrakech, Morocco
    • Authors: Kenneth O. Simbiri; Christopher K. Williams, Marcella Macaluso, Antonio Giordano
      Abstract: The objectives of the African Organization for Research and Training in Cancer (AORTIC), includes bringing products of decades of advances in cancer research to African populations through local and international collaboration. The consistent and huge growth in participation in the conferences and the diversity of the nations is a witness to the success of the organization thus far. The theme for the Tenth AORTIC International Conference on Cancer in Africa in Morocco in 2015 was “Road map to Cancer Control in Africa” and topics of discussion of paramount importance for low- and middle-income African countries included childhood cancers such as BL, cancers of the cervix, breast, and prostate; cancers associated with HIV-infection such as cervical, vulvar, and anal; as well as cancer care challenges associated with palliative care. The role of environmental factors that underlie some epigenetic changes in some of the cancers was emphasized. Oral and poster presentations from various parts of the continent indicate the growth of basic and translational science of cancer in the region, with studies revealing regional diversity in the frequencies of the triple-negative breast cancer, cervical cancer, prostate cancer, HCC, and Burkitt's lymphoma. There was a sign that Africa is trying to keep pace with the paradigm shift and focusing on translational medicine. This was shown by suggestions for application of genome-wide association studies, new generation sequencing, as well as the evaluation of single nucleotide polymorphisms that may be responsible for variable susceptibility in some of the prevalent cancers in people of African descent. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-01T02:11:16.008329-05:
      DOI: 10.1002/jcp.25671
  • Direct Delivery of Recombinant Pin1 Protein Rescued Osteoblast
           Differentiation of Pin1 Deficient Cells
    • Authors: Woo-Jin Kim; Rabia Islam, Bong-Soo Kim, Young-Dan Cho, Won-Joon Yoon, Jeong-Hwa Baek, Kyung-Mi Woo, Hyun-Mo Ryoo
      Abstract: Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine–proline or phosphothreonine–proline motifs of several proteins. We reported that Pin1 plays a critical role in the fate determination of Smad1/5, Runx2 and β-catenin that are indispensable nuclear proteins for osteoblast differentiation. Though several chemical inhibitors has been discovered for Pin1, no activator has been reported as of yet. In this study, we directly introduced recombinant Pin1 protein successfully into the cytoplasm via fibroin nanoparticle encapsulated in cationic lipid. This nanoparticle-lipid complex delivered its cargo with a high efficiency and a low cytotoxicity. Direct delivery of Pin1 leads to increased Runx2 and Smad signaling and resulted in recovery of the osteogenic marker genes expression and the deposition of mineral in Pin1 deficient cells. These result indicated that a direct Pin1 protein delivery method could be a potential therapeutics for the osteopenic diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-01T02:11:14.589265-05:
      DOI: 10.1002/jcp.25673
  • Lipocalin-2 (NGAL) Attenuates Autophagy to Exacerbate Cardiac Apoptosis
           Induced by Myocardial Ischemia
    • Authors: Hye Kyoung Sung; Yee Kwan Chan, Meng Han, James Won Suk Jahng, Erfei Song, Danielson Eric, Thorsten Berger, Tak W. Mak, Gary Sweeney
      Abstract: Lipocalin-2 (Lcn2; also termed neutrophil gelatinase-associated lipocalin (NGAL)) levels correlate positively with heart failure (HF) yet mechanisms via which Lcn2 contributes to the pathogenesis of HF remian unclear. In this study we used coronary artery ligation surgery to induce ischemia in wild type (wt) mice and this induced a significant increase in myocardial Lcn2. We then compared wt and Lcn2 knockout (KO) mice and observed that wt mice showed greater ischemia-induced caspase-3 activation and DNA damage measured by TUNEL than Lcn2KO mice. Analysis of autophagy by LC3 and p62 Western blotting, LC3 immunohistochemistry and transmission electron microscopy (TEM) indicated that Lcn2 KO mice had a greater ischemia-induced increase in autophagy. Lcn2KO were protected against ischemia-induced cardiac functional abnormalities measured by echocardiography. Upon treating a cardiomyocyte cell line (h9c2) with Lcn2 and examining AMPK and ULK1 phosphorylation, LC3 and p62 by Western blot as well as tandem fluorescent RFP/GFP-LC3 puncta by immunofluorescence, MagicRed assay for lysosomal cathepsin activity and TEM we demonstrated that Lcn2 suppressed autophagic flux. Lcn2 also exacerbated hypoxia-induced cytochromc c release from mitochondria and caspase-3 activation. We generated an autophagy-deficient H9c2 cell model by overexpressing dominant-negative Atg5 and found significantly increased apoptosis after Lcn2 treatment. In summary, our data indicate that Lcn2 can suppress the beneficial cardiac autophagic response to ischemia and that this contributes to enhanced ischemia-induced cell death and cardiac dysfunction. This article is protected by copyright. All rights reserved
      PubDate: 2016-11-01T02:11:05.376715-05:
      DOI: 10.1002/jcp.25672
  • Calcium Signaling During Meiotic Cell Cycle Regulation and Apoptosis in
           Mammalian Oocytes
    • Authors: Meenakshi Tiwari; Shilpa Prasad, Tulsidas G. Shrivastav, Shail K. Chaube
      Abstract: Calcium (Ca++) is one of the major signal molecules that regulate various aspects of cell functions including cell cycle progression, arrest and apoptosis in wide variety of cells. This review summarizes current knowledge on the differential roles of Ca++ in meiotic cell cycle resumption, arrest and apoptosis in mammalian oocytes. Release of Ca++ from internal stores and/or Ca++ influx from extracellular medium causes moderate increase of intracellular Ca++ ([Ca++]i) level and reactive oxygen species (ROS). Increase of Ca++ as well as ROS levels under physiological range trigger maturation promoting factor (MPF) destabilization thereby meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in oocytes. A sustained increase of [Ca++]i level beyond physiological range induces generation of ROS sufficient enough to cause oxidative stress (OS) in aging oocytes. The increased [Ca++]i triggers Fas ligand-mediated oocyte apoptosis. Further, OS triggers mitochondria-mediated oocyte apoptosis in several mammalian species. Thus, Ca++ exerts differential roles on oocyte physiology depending upon its intracellular concentration. A moderate increase of [Ca++]i as well as ROS mediate spontaneous resumption of meiosis from diplotene as well as M-II arrest, while their high levels cause meiotic cell cycle arrest and apoptosis by operating both mitochondria- as well as Fas ligand-mediated apoptotic pathways. Indeed, Ca++ regulates cellular physiology by modulating meiotic cell cycle and apoptosis in mammalian oocytes. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:53:27.761713-05:
      DOI: 10.1002/jcp.25670
  • SirT3 and p53 Deacetylation in Aging and Cancer
    • Authors: Jijun Chen; Aiqin Wang, Qingqi Chen
      Abstract: The mammalian Sirtuins are a family of highly conserved nicotinamide adenine dinucleotide (NAD +)-dependent histone deacetylase. The mammalian Sirtuins family has identified seven different types of sirtuin. Sirtuins 3(SirT3) is localized to mitochondria, and is a multiple functional protein deacylase through deacetylating a variety of substrates. Cellular senescence represents a permanent withdraw from cell cycle in response to diverse stress; it is controlled by the p53 and retinoblastoma protein (RB) tumor suppressors, and constitutes a potent anticancer mechanisms. p53 can be deacetylated by a protein complex containing histone deacetylases (HDAC1). There is possibility that SirT3 may also deacetylate p53. We introduce the recent research on the SirT3-p53 interplay directly and indirectly, and discuss the significance of p53 deacetylation by SirT3 in the aging and cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:52:10.443483-05:
      DOI: 10.1002/jcp.25669
  • Proteomics Applications in Dental Derived Stem Cells
    • Authors: Jie Li; Weidong Tian, Jinlin Song
      Abstract: At present, the existence of a variety of dental derived stem cells has been documented. These cells displayed promising clinical application potential not only for teeth and its surrounding tissue regeneration, but also for other tissues, such as nerve and bone regeneration. Proteomics is an unbiased, global informatics tool that provides information on all protein expression levels as well as post-translational modification in cells or tissues and is applicable to dental derived stem cells research. Over the last decade, considerable progress has been made to study the global proteome, secrotome, and membrane proteome of dental derived stem cells. Here we present an overview of the proteomics studies in the context of stem cell research. Particular attention is given to dental derived stem cell types as well as current challenges and opportunities. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:46:32.235162-05:
      DOI: 10.1002/jcp.25667
  • The Emerging Role of MORC Family Proteins in Cancer Development and Bone
    • Authors: Guoju Hong; Heng Qiu, Chao Wang, Gaurav Jadhav, Haibin Wang, Jennifer Tickner, Wei He, Jiake Xu
      Abstract: Microrchidia (MORC or MORC family CW-type zinc finger protein), a highly conserved nuclear protein superfamily, is an interesting new player in signaling-dependent chromatin remodeling and epigenetic regulation. MORC family proteins consist of MORC1, MORC2, MORC3 and MORC4 which display common structural determinants such as CW-type zinc finger and coiled-coil domains. They also exhibit unique structural motifs and tissue-specific expression profiles. Morc1 was first discovered as a key regulator for male meiosis and spermatogenesis. Accumulating biochemical and functional analyses unveil MORC proteins as key regulators for cancer development. More recently, using an ENU mutagenesis mouse model, MORC3 was found to play a role in regulating bone and calcium hemostasis. Here we discuss recent research progress on the emerging role of MORC proteins in cancer development and bone metabolism. Unravelling the cellular and molecular mechanisms by which MORC proteins carry out their functions in a tissue specific manner are important subjects for future investigation. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:46:21.015841-05:
      DOI: 10.1002/jcp.25665
  • Molecular Imaging of Huntington's Disease
    • Authors: Andrea Ciarmiello; Giampiero Giovacchini, Elisabetta Giovannini, Patrizia Lazzeri, Elisa Borsò, Antonio Mannironi, Luigi Mansi
      Abstract: The onset and the clinical progression of Huntington Disease (HD) is influenced by several events prompted by a genetic mutation that affects several organs tissues including different regions of the brain. In the last decades years, Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) helped to deepen the knowledge of neurodegenerative mechanisms that guide to clinical symptoms. Brain imaging with PET represents a tool to investigate the physiopathology occurring in the brain and it has been used to predict the age of onset of the disease and to evaluate the therapeutic efficacy of new drugs. This article reviews the contribution of PET and MRI in the research field on Huntington's disease, focusing in particular on some most relevant achievements that have helped recognize the molecular changes, the clinical symptoms and evolution of the disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:36:47.651368-05:
      DOI: 10.1002/jcp.25666
  • Contributions of 3D Cell Cultures For Cancer Research
    • Authors: Maddaly Ravi; Aarthi Ramesh, Aishwarya Pattabhi
      Abstract: Cancer cell lines have contributed immensely in understanding the complex physiology of cancers. They are excellent material for studies as they offer homogenous samples without individual variations and can be utilized with ease and flexibility. Also, the number of assays and end-points one can study is almost limitless; with the advantage of improvising, modifying or altering several variables and methods. Literally, a new dimension to cancer research has been achieved by the advent of 3Dimensional cell culture techniques. This approach increased many folds the ways in which cancer cell lines can be utilized for understanding complex cancer biology. 3D cell culture techniques are now the preferred way of using cancer cell lines to bridge the gap between the “absolute in vitro” and “true in vivo”. The aspects of cancer biology that 3D cell culture systems have contributed include morphology, microenvironment, gene and protein expression, invasion/migration/metastasis, angiogenesis, tumour metabolism and drug discovery, testing chemotherapeutic agents, adaptive responses and cancer stem cells. We present here, a comprehensive review on the applications of 3D cell culture systems for these aspects of cancers. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:36:43.990556-05:
      DOI: 10.1002/jcp.25664
  • Polydeoxyribonucleotides (PDRNs) From Skin to Musculoskeletal Tissue
           Regeneration Via Adenosine A2A Receptor Involvement: A Mini-Review
    • Authors: Francesca Veronesi; Dante Dallari, Giacomo Sabbioni, Chiara Carubbi, Lucia Martini, Milena Fini
      Abstract: Polydeoxyribonucleotides (PDRNs) are low molecular weight DNA molecules of natural origin that stimulate cell migration and growth, extracellular matrix (ECM) protein production and reduce inflammation. Most preclinical and clinical studies on tissue regeneration with PDRNs focused on skin, and only few are about musculoskeletal tissues.Starting from an overview on skin regeneration studies, through the analysis of in vitro, in vivo and clinical studies (1990–2016), the present review aimed at defining the effects of PDRN and their mechanisms of action in the regeneration of musculoskeletal tissues. This would also help future researches in this area.A total of 29 studies were found by PubMed and searches: 20 were on skin (6 in vitro, 6 in vivo, 1 vitro/vivo, 7 clinical studies), while the other 9 regarded bone (1 in vitro, 2 in vivo, 1 clinical studies), cartilage (1 in vitro, 1 vitro/vivo, 2 clinical studies) or tendon (1 clinical study) tissues regeneration.PDRNs improved cell growth, tissue repair, ECM proteins, physical activity and reduced pain and inflammation, through the activation of adenosine A2A receptor.PDRNs are currently used for bone, cartilage and tendon diseases, with a great variability regarding the PDRN dosage to be used in clinical practice, while the dosage for skin regeneration is well established. PDRNs are usually administered from a minimum of 3 to a maximum of 5 times and they act trough the activation of A2A receptor.Further studies are advisable to confirm the effectiveness of PDRNs and to standardize the PDRN dose. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-28T06:36:41.454992-05:
      DOI: 10.1002/jcp.25663
  • Pathophysiology of the Desmo-Adhesome
    • Authors: Antonio Celentano; Michele Davide Mignogna, Michael McCullough, Nicola Cirillo
      Abstract: Advances in our understanding of desmosomal diseases have provided a clear demonstration of the key role played by desmosomes in tissue and organ physiology, highlighting the importance of their dynamic and finely regulated structure. In this context, non-desmosomal regulatory molecules have acquired increasing relevance in the study of this organelle resulting in extending the desmosomal interactome, named the “desmo-adhesome.” Spatiotemporal changes in the expression and regulation of the desmo-adhesome underlie a number of genetic, infectious, autoimmune, and malignant conditions. The aim of the present article was to examine the structural and functional relationship of the desmosome, by providing a comprehensive, yet focused overview of the constituents targeted in human disease. The inclusion of the novel regulatory network in the desmo-adhesome pathophysiology opens new avenues to a deeper understanding of desmosomal diseases, potentially unveiling pathogenic mechanisms waiting to be explored. J. Cell. Physiol. 9999: 1–10, 2016. © 2016 Wiley Periodicals, Inc.The desmosome has been the subject of intense research over the last 20 years. While several reviews have recently been published on this topic, none has investigated the desmosomal interactome as a whole. This review is unique in that it not only takes into account the diseases that are linked to primary desmosomal components but, also, includes the potential role of accessory molecules in epithelial disease and cancer.
      PubDate: 2016-10-27T12:20:27.631002-05:
      DOI: 10.1002/jcp.25515
  • Novel Instruments for the Implementation of Electrochemotherapy Protocols:
           From Bench Side to Veterinary Clinic
    • Authors: Enrico P. Spugnini; Stefano Fais, Tommaso Azzarito, Alfonso Baldi
      Abstract: Electrochemotherapy (ECT) is a medical strategy that allows an increased efficacy of chemotherapy agents after the application of permeabilizing electric pulses having appropriate characteristics (form, voltage, frequency). In the past 10 years, the clinical efficacy of this therapeutic approach in several spontaneous models of tumors in animals has been shown. Moreover, some of the molecular and cellular mechanisms responsible for this phenomenon have been elucidated. Our group has been deeply involved in the development of new ECT protocols for companion animals, implementing the use of the technique as first line treatment, and evaluating different chemotherapy agents in laboratory animals as well as pets. This article summarizes the most important advances in veterinary ECT, including the development of novel equipment, therapeutic protocols, and their translation to humans. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 Wiley Periodicals, Inc.Electrochemotherapy (ECT) is a medical strategy that allows an increased efficacy of chemotherapy agents after the application of permeabilizing electric pulses having appropriate characteristics (form, voltage, frequency). Our group has been deeply involved in the development of new ECT protocols for companion animals, implementing the use of the technique as first line treatment, and evaluating different chemotherapy agents in laboratory animals as well as pets. This article summarizes the most important advances in veterinary ECT, including the development of novel equipment, therapeutic protocols, and their translation to humans.
      PubDate: 2016-10-27T12:20:24.360283-05:
      DOI: 10.1002/jcp.25505
  • Extracellular Matrix and Colorectal Cancer: How Surrounding
           Microenvironment Affects Cancer Cell Behavior?
    • Authors: Sara Crotti; Martina Piccoli, Flavio Rizzolio, Antonio Giordano, Donato Nitti, Marco Agostini
      Abstract: Colorectal cancer (CRC) whit more than a million of new cases per year is one of the most common registered cancers worldwide with few treatment options especially for advanced and metastatic patients.The tumor microenvironment is composed by extracellular matrix (ECM), cells and interstitial fluids. Among all these constituents, in the last years an increased interest around the ECM and its potential role in cancer tumorigenesis is arisen. During cancer progression the ECM structure and composition became disorganized, allowing cellular transformation and metastasis.Up to now, the focus has mainly been on the characterization of CRC microenvironment analyzing separately structural ECM components or cell secretome modifications. A more extensive view that interconnects these aspects should be addressed. In this review, biochemical (secretome) and biomechanical (structure and architecture) changes of tumor microenvironment will be discussed, giving suggestions on how these changes can affect cancer cell behavior. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-24T05:15:47.729256-05:
      DOI: 10.1002/jcp.25658
  • Functional Roles of Eph A-Ephrin A1 System in Endometrial Luminal
           Epithelial Cells During Early Pregnancy
    • Authors: Whasun Lim; Hyocheol Bae, Fuller W. Bazer, Gwonhwa Song
      Abstract: Eph and ephrin regulate diverse biological events such as proliferation, adhesion, migration, and angiogenesis through cell-to-cell interactions. However, little is known of their functional role and mechanisms of action in uterine endometrial cells. In the present study, we demonstrated the effects of the Eph and ephrin on interactions between blastocysts and endometrial luminal epithelial (pLE) cells in the pig that is regarded as an excellent biomedical animal model for research on the peri-implantation period of pregnancy. Results of this study indicated that among eight members of the Eph A family, expression of Eph A1, A2, A4 and A7 was strongly detected in endometrial epithelial cells during early pregnancy. Of these, for identification of signal transduction pathways induced by ephrin A1, a major ligand for Eph A, cell proliferation assays, and immunofluorescence and cell cycle regulation were analyzed following treatment of pLE cells with ephrin A1. Ephrin A1 stimulated proliferation of pLE cells as evidenced by abundant PCNA expression and an increase in the G2/M phase. Western blot analysis showed that ephrin A1 activated PI3K and MAPK signaling proteins in a time-dependent manner. Moreover, phosphorylation of AKT, ERK1/2, P38 and JNK proteins were suppressed by their inhibitors wortmannin, U0126, SB203580 and SP600125, respectively. Also, phosphor-AKT was reduced by ERK1/2 and P38 inhibitors. Ephrin A1-induced proliferation and migration of pLE cells was also blocked by those inhibitors. Collectively, these results suggest that ephrin A1 enhances interactions between porcine blastocysts and endometrial luminal epithelial cells by activating PI3K and MAPK signal transduction pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-24T05:15:41.367406-05:
      DOI: 10.1002/jcp.25659
  • Let-7I-Induced Atg4B Suppression is Essential for Autophagy of Placental
           Trophoblast in Preeclampsia
    • Authors: Yinyan Xu; Xinyan Huang, Juan Xie, Yanni Chen, Jing Fu, Li Wang
      Abstract: Autophagy, identified as type II programmed cell death, has already been known to be involved in the pathophysiology of preeclampsia (PE), which is a gestational disease with high morbidity. The present study aims to investigate the functional role of let-7i, a miRNA, in trophoblastic autophagy. Placental tissue used in this study was collected from patients with severe preeclampsia (SPE) or normal pregnant women. A decreased level of let-7i was found in placenta of SPE. In addition, autophagic vacuoles were observed in SPE and the expression of microtubule associated protein 1 light chain 3 (LC3) II/I was elevated. In vitro, let-7i mimics suppressed the autophagic activities in human HTR-8/SVneo trophoblast cell line (HTR-8) and human placental choriocarcinoma cell line JEG-3, whereas let-7i inhibitor enhanced the activities. As a potential target of let-7i, autophagy-related 4B cysteine peptidase(Atg4B) had an increased expression level in SPE. As expected, the increased expression of Atg4B was negatively regulated by let-7i using dual luciferase reporter assay. Furthermore, these trophoblast-like cells transfected with the let-7i mimic or inhibitors resulted in a significant change of Atg4B in both mRNA and protein level. More importantly, Atg4B overexpression could partly reverse let-7i mimic-reduced LC3II/I levels; whereas Atg4B silencing partly attenuated let-7i inhibitor-induced the level of LC3II/I expression. Taken together, these findings suggest that let-7i is able to regulate autophagic activity via regulating Atg4B expression, which might contribute to the pathogenesis of PE. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-22T14:50:32.447261-05:
      DOI: 10.1002/jcp.25661
  • Dermal-Epidermal Cross-Talk: Differential Interactions With Microvascular
           Endothelial Cells
    • Authors: Eleonora Bassino; Edoardo Vallariello, Franco Gasparri, Luca Munaron
      Abstract: The biological importance of circulatory blood supply and angiogenesis for hair growth is now well recognized, but the their regulatory mechanisms require more mechanistic investigation. In vitro cocultures and tricultures can be successfully employed to greatly improve our knowledge on paracrine crosstalk between cell types that populate the dermal-epidermal interface and cutaneous vasculature. Here we report that human dermal fibroblasts (NHDF) promote viability and proliferation of microvascular endothelial cells (HMVEC), while HMVEC are not mitogenic for NHDF. In triculture setup, conditioned media obtained by cocultures (HMVEC/NHDF or HMVEC/follicle fibroblasts) differently modulate growth and proliferation of keratinocytes and alter the expression of metabolic and pro-inflammatory markers. In conclusion, tricultures were successfully employed to characterize in vitro dermal-epithelial and endothelial interactions and could integrate ex vivo and in vivo approaches by the use of high-throughput and standardized protocols in controlled conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-20T18:36:10.658839-05:
      DOI: 10.1002/jcp.25657
  • ADP-Induced Ca2+ Signalling and Proliferation of Rat Ventricular
           Myofibroblasts Depend on Phospholipase C-Linked TRP Channels Activation
           Within Lipid Rafts
    • Authors: Mariana Certal; Adriana Vinhas, Aurora Barros-Barbosa, Fátima Ferreirinha, Maria Adelina Costa, Paulo Correia-de-Sá
      Abstract: Nucleotides released during heart injury affect myocardium electrophysiology and remodelling through P2 purinoceptors activation in cardiac myofibroblasts. ATP and UTP endorse [Ca2+]i accumulation and growth of DDR-2/a-SMA-expressing myofibroblasts from adult rat ventricles via P2Y4 and P2Y2 receptors activation, respectively. Ventricular myofibroblasts also express ADP-sensitive P2Yi, P2Yi2 and P2Yi3 receptors as demonstrated by immunofluorescence confocal microscopy and western blot analysis, but little information exists on ADP effects in these cells. ADP (0.003-3mM) and its stable analogue, ADPBS (100uM), caused fast [Ca2+]i transients originated from thapsigargin-sensitive internal stores, which partially declined to a plateau sustained by capacitative Ca2+ entry through transient receptor potential (TRP) channels inhibited by 2-APB (50uM) and flufenamic acid (100uM). Hydrophobic interactions between Gq/11-coupled P2Y purinoceptors and TRP channels were suggested by prevention of the ADP-induced [Ca2+]i plateau following PIP2 depletion with LiCl (10mM) and cholesterol removal from lipid rafts with methyl-B-cyclodextrin (2mM). ADP [Ca2+]i transients were insensitive to P2Y1, P2Y12 and P2Y13 receptor antagonists, MRS2179 (10uM), AR-C66096 (0.1uM) and MRS2211 (10uM), respectively, but were attenuated by suramin and reactive blue-2 (100uM) which also blocked P2Y4 receptors activation by UTP. Cardiac myofibroblasts growth and type I collagen production were favoured upon activation of MRS2179-sensitive P2Y1 receptors with ADP or ADPBS (30uM). In conclusion, ADP exerts a dual role on ventricular myofibroblasts: [Ca2+]i transients are mediated by fast-desensitizing P2Y4 receptors, whereas the pro-fibrotic effect of ADP involves the P2Y1 receptor activation. Data also show that ADP-induced capacitative Ca2+ influx depends on phospholipase C-linked TRP channels opening in lipid raft microdomains. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-18T11:55:22.363946-05:
      DOI: 10.1002/jcp.25656
  • Neuroprotection and Blood-Brain Barrier Restoration by Salubrinal After a
           Cortical Stab Injury
    • Authors: M. Asunción Barreda-Manso; Natalia Yanguas-Casás, Manuel Nieto-Sampedro, Lorenzo Romero-Ramírez
      Abstract: Following a central nervous system (CNS) injury, restoration of the blood-brain barrier (BBB) integrity is essential for recovering homeostasis. When this process is delayed or impeded, blood substances and cells enter the CNS parenchyma, initiating an additional inflammatory process that extends the initial injury and causes so-called secondary neuronal loss. Astrocytes and profibrotic mesenchymal cells react to the injury and migrate to the lesion site, creating a new glia limitans that restores the BBB. This process is beneficial for the resolution of the inflammation, neuronal survival and the initiation of the healing process. Salubrinal is a small molecule with neuroprotective properties in different animal models of stroke and trauma to the CNS. Here, we show that salubrinal increased neuronal survival in the neighbourhood of a cerebral cortex stab injury. Moreover, salubrinal reduced cortical blood leakage into the parenchyma of injured animals compared with injured controls. Adjacent to the site of injury, salubrinal induced immunoreactivity for platelet-derived growth factor subunit B (PDGF-B), a specific mitogenic factor for mesenchymal cells. This effect might be responsible for the increased immunoreactivity for fibronectin and the decreased activation of microglia and macrophages in injured mice treated with salubrinal, compared with injured controls. The immunoreactivity for PDGF-B colocalized with neuronal nuclei (NeuN), suggesting that cortical neurons in the proximity of the injury were the main source of PDGF-B. Our results suggest that after an injury, neurons play an important role in both, the healing process and the restoration of the BBB integrity. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-18T06:40:27.39664-05:0
      DOI: 10.1002/jcp.25655
  • Src Family Kinase Links Insulin Signaling to Short Term Regulation of
           Na,K-ATPase in Nonpigmented Ciliary Epithelium
    • Authors: Mohammad Shahidullah; Amritlal Mandala, Nicholas A. Delamere
      Abstract: Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2 fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2 and p38 activation, all of which were prevented by a PI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates PI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-17T06:44:20.795008-05:
      DOI: 10.1002/jcp.25654
  • p62 Promotes Amino Acid Sensitivity of mTOR Pathway and Hepatic
           Differentiation in Adult Liver Stem/Progenitor Cells
    • Authors: Masakazu Sugiyama; Tomoharu Yoshizumi, Yoshihiro Yoshida, Yuki Bekki, Yoshihiro Matsumoto, Shohei Yoshiya, Takeo Toshima, Toru Ikegami, Shinji Itoh, Norifumi Harimoto, Shinji Okano, Yuji Soejima, Ken Shirabe, Yoshihiko Maehara
      Abstract: Autophagy is a homeostatic process regulating turnover of impaired proteins and organelles, and p62 (sequestosome-1, SQSTM1) functions as the autophagic receptor in this process. p62 also functions as a hub for intracellular signaling such as that in the mammalian target of rapamycin (mTOR) pathway. Liver stem/progenitor cells have the potential to differentiate to form hepatocytes or cholangiocytes. In this study, we examined effects of autophagy, p62 and associated signaling on hepatic differentiation. Adult stem/progenitor cells were isolated from the liver of mice with chemically-induced liver injury. Effects of autophagy, p62 and related signaling pathways on hepatic differentiation were investigated by silencing the genes for autophagy protein 5 (ATG5) and/or SQSTM1/p62 using small interfering RNAs. Hepatic differentiation was assessed based on increased albumin and hepatocyte nuclear factor 4α, as hepatocyte markers, and decreased cytokeratin 19 and SOX9, as stem/progenitor cell markers. These markers were measured using quantitative RT-PCR, immunofluorescence and western blotting. ATG5 silencing decreased active LC3 and increased p62, indicating inhibition of autophagy. Inhibition of autophagy promoted hepatic differentiation in the stem/progenitor cells. Conversely, SQSTM1/p62 silencing impaired hepatic differentiation. A suggested mechanism for p62-dependent hepatic differentiation in our study was activation of the mTOR pathway by amino acids. Amino acid activation of mTOR signaling was enhanced by ATG5 silencing and suppressed by SQSTM1/p62 silencing. Our findings indicated that promoting amino acid sensitivity of the mTOR pathway is dependent on p62 accumulated by inhibition of autophagy and that this process plays an important role in the hepatic differentiation of stem/progenitor cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-17T06:44:12.967902-05:
      DOI: 10.1002/jcp.25653
  • The Natural cAMP Elevating Compound Forskolin in Cancer Therapy: Is it
    • Authors: Luigi Sapio; Monica Gallo, Michela Illiano, Emilio Chiosi, Daniele Naviglio, Annamaria Spina, Silvio Naviglio
      Abstract: Cancer is a major public health problem and the second leading cause of mortality around the world. Although continuous advances in the science of oncology and cancer research are now leading to improved outcomes for many cancer patients, novel cancer treatment options are strongly demanded. Naturally occurring compounds from a variety of vegetables, fruits and medicinal plants have been shown to exhibit various anticancer properties in a number of in vitro and in vivo studies and represent an attractive research area for the development of new therapeutic strategies to fight cancer.Forskolin is a diterpene produced by the roots of the Indian plant Coleus forskohlii. The natural compound forskolin has been used for centuries in traditional medicine and its safety has also been documented in conventional modern medicine. Forskolin directly activates the adenylate cyclase enzyme, that generates cAMP from ATP, thus raising intracellular cAMP levels.Notably, cAMP signaling, through the PKA-dependent and/or independent pathways, is very relevant to cancer and its targeting has shown a number of antitumor effects, including the induction of mesenchymal-to-epithelial transition, inhibition of cell growth and migration and enhancement of sensitivity to conventional antitumor drugs in cancer cells.Here, we describe some features of cAMP signaling that are relevant to cancer biology and address the state of the art concerning the natural cAMP elevating compound forskolin and its perspectives as an effective anticancer agent. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-14T03:21:46.56839-05:0
      DOI: 10.1002/jcp.25650
  • Increased Chondrogenic Potential of Mesenchymal Cells from Adipose Tissue
           Versus Bone Marrow Derived Cells in Osteoarthritic In Vitro Models
    • Authors: Stefania Pagani; Veronica Borsari, Francesca Veronesi, Andrea Ferrari, Simona Cepollaro, Paola Torricelli, Giuseppe Filardo, Milena Fini
      Abstract: ObjectivesPrimarily, to compare the behavior of human mesenchymal stem cells (MSCs) derived from bone marrow (hBMSCs) and adipose tissue (hADSCs) in an osteoarthritic (OA) microenvironment; secondly, to investigate the reaction of these cell types in two alternative in vitro culture systems, obtained by using TNFα and/or IL1β as inflammation mediators, or by using synovial fluid harvested by OA patients (OSF) to simulate the complex inflamed knee microenvironment.Methods3D micromass cultures of hBMSCs or hADSCs were grown in chondrogenic medium (CTR), in the presence of TNFα and/or IL1β, or synovial fluid from OA patients. After 1 month of culture, the chondrogenic differentiation of micromasses was evaluated by gene expression, matrix composition and organization.ResultsBoth hMSCs types formed mature micromasses in CTR, but a better response of hADSCs to the inflammatory environment was documented by micromass area and Bern score evaluations. The addition of OSF elicited a milder reaction than with TNFα and/or IL1β by both cell types, probably due to the presence of both catabolic and protective factors. In particular, SOX9 and ACAN gene expression and GAG synthesis were more abundant in hADSCs than hBMSCs when cultured in OSF. The expression of MMP1 was increased for both hMSCs in inflammatory conditions, but in particular by hBMSCs.ConclusionhADSCs showed an increased chondrogenic potential in inflammatory culture systems, suggesting a better response of hADSCs in the OA environment, thus underlining the importance of appropriate in vitro models to study MSCs and potential advantages of using these cells for future clinical applications. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-14T03:21:41.163181-05:
      DOI: 10.1002/jcp.25651
  • Maintenance of Bone Homeostasis by DLL1-Mediated Notch Signaling
    • Authors: Yukari Muguruma; Katsuto Hozumi, Hiroyuki Warita, Takashi Yahata, Tomoko Uno, Mamoru Ito, Kiyoshi Ando
      Abstract: Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta-like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast-specific manner, we investigated the ligand-specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1-induced Notch signaling was responsible for the expansion of the bone-forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1-Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast-specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast-osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1-mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand-specific activation of Notch signaling in the maintenance of bone homeostasis. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:55:59.336038-05:
      DOI: 10.1002/jcp.25647
  • Role of microRNAs on the Regulation of Mitochondrial Biogenesis and
           Insulin Signaling in Skeletal Muscles
    • Authors: Tanes I. Lima; Hygor N. Araujo, Eveline S. Menezes, Carlos H. Sponton, Michel B. Araújo, Lucas H. M. Bomfim, André L. Queiroz, Madla A. Passos, Thais Amaral e Sousa, Sandro M. Hirabara, Amanda R. Martins, Helena C.L.B. Sampaio, Alice Rodrigues, Rui Curi, Everardo M. Carneiro, Antônio C. Boschero, Leonardo R. Silveira
      Abstract: Mitochondria play a critical role in several cellular processes and cellular homeostasis. Mitochondrion dysfunction has been correlated with numerous metabolic diseases such as obesity and type 2 diabetes. MicroRNAs are non-coding RNAs that have emerged as key regulators of cell metabolism. The microRNAs act as central regulators of metabolic gene networks by leading to the degradation of their target messenger RNA or repression of protein translation. In addition, vesicular and non-vesicular circulating miRNAs exhibit a potential role as mediators of the cross-talk between the skeletal muscle and other tissues/organs. In this review, we will focus on the emerging knowledge of miRNAs controlling mitochondrial function and insulin signaling in skeletal muscle cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:47:09.296418-05:
      DOI: 10.1002/jcp.25645
  • PPARα Antagonist AA452 Triggers Metabolic Reprogramming and Increases
           Sensitivity to Radiation Therapy in Human Glioblastoma Primary Cells
    • Authors: E Benedetti; M d'Angelo, A Ammazzalorso, G Gravina, C Laezza, A Antonosante, G Panella, B Cinque, L Cristiano, AC Dhez, C Astarita, R Galzio, MG Cifone, R Ippoliti, R Amoroso, E Di Cesare, A Giordano, A Cimini
      Abstract: Glioblastoma is the most-common cancer in the brain and with an increasing incidence. Despite major advances in the field, there is no curative therapy for GB to date. Many solid tumors, including GB, experienced metabolic reprogramming in order to sustain uncontrolled proliferation, hypoxic conditions and angiogenesis. PPARs, member of the steroid hormone receptor superfamily, are particularly involved in the control of energetic metabolism, particularly lipid metabolism, which has been reported deregulated in gliomas. PPARα was previously indicated by us as a potential therapeutic target for this neoplasm, due to the malignancy grade dependency of its expression, being particularly abundant in GB. In this work we used a new PPARα antagonist on patient-derived GB primary cells with particular focus on the effects on lipid metabolism and response to radiotherapy. The results obtained demonstrated that blocking PPARα results in cell death induction, increase of radiosensitivity and decrease of migration. Therefore, AA452 arises as a potent drug adjuvanting the gold standard therapies for GB, opening the possibility for pre-clinical and clinical trials for this class of compounds. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:47:07.048024-05:
      DOI: 10.1002/jcp.25648
  • Primary Cilium-Regulated EG-VEGF Signaling Facilitates Trophoblast
    • Authors: Chia-Yih Wang; Hui-Ling Tsai, Jhih-Siang Syu, Ting-Yu Chen, Mei-Tsz Su
      Abstract: Trophoblast invasion is an important event in embryo implantation and placental development. During these processes, endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is the key regulator mediating the crosstalk at the feto-maternal interface. The primary cilium is a cellular antenna receiving environmental signals and is crucial for proper development. However, little is known regarding the role of the primary cilium in early human pregnancy. Here, we demonstrate that EG-VEGF regulates trophoblast cell invasion via primary cilia. We found that EG-VEGF activated ERK1/2 signaling and subsequent upregulation of MMP2 and MMP9, thereby facilitating cell invasion in human trophoblast HTR-8/SVneo cells. Inhibition of ERK1/2 alleviated the expression of MMPs and trophoblast cell invasion after EG-VEGF treatment. In addition, primary cilia were observed in all the trophoblast cell lines tested and, more importantly, in human first-trimester placental tissue. The receptor of EG-VEGF, PROKR1, was detected in primary cilia. Depletion of IFT88, the intraflagellar transporter required for ciliogenesis, inhibited primary cilium growth, thereby ameliorating ERK1/2 activation, MMP upregulation, and trophoblast cell invasion promoted by EG-VEGF. These findings demonstrate a novel function of primary cilia in controlling EG-VEGF-regulated trophoblast invasion and reveal the underlying molecular mechanism. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:46:59.819024-05:
      DOI: 10.1002/jcp.25649
  • Arabinogalactan Proteins from Baobab and Acacia Seeds Influence Innate
           Immunity of Human Keratinocytes in vitro
    • Authors: Abderrakib Zahid; Julie Despres, Magalie Benard, Eric Nguema-Ona, Jerome Leprince, David Vaudry, Christophe Rihouey, Maité Vicré-Gibouin, Azeddine Driouich, Marie-Laure Follet-Gueye
      Abstract: Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However little is currently known regarding their potential activity towards skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-13T07:41:38.516217-05:
      DOI: 10.1002/jcp.25646
  • Osteogenic Differentiation of Periosteal Cells during Fracture Healing
    • Authors: Tao Wang; Xinping Zhang, Daniel D. Bikle
      Abstract: 5-10% of fractures fail to heal normally leading to additional surgery, morbidity, and altered quality of life. Fracture healing involves the coordinated action of stem cells primarily coming from the periosteum which differentiate into the chondrocytes and osteoblasts, forming first the soft (cartilage) callus followed by the hard (bone) callus. These stem cells are accompanied by a vascular invasion that appears critical for the differentiation process and which may enable the entry of osteoclasts necessary for the remodeling of the callus into mature bone. However, more research is needed to clarify the signaling events that activate the osteochondroprogenitor cells of periosteum and stimulate their differentiation into chondrocytes and osteoblasts. Ultimately a thorough understanding of the mechanisms for differential regulation of these osteochondroprogenitors will aid in the treatment of bone healing and the prevention of delayed union and nonunion of fractures. In this review, evidence supporting the concept that the periosteal cells are the major cell sources of skeletal progenitors for the fracture callus will be discussed. The osteogenic differentiation of periosteal cells manipulated by Wnt/β-catenin, TGF/BMP, Ihh/PTHrP, and IGF-1/PI3K–Akt signaling in fracture repair will be examined. The effect of physical (hypoxia and hyperoxia) and chemical factors (reactive oxygen species) as well as the potential coordinated regulatory mechanisms in the periosteal progenitor cells promoting osteogenic differentiation will also be discussed. Understanding the regulation of periosteal osteochondroprogenitors during fracture healing could provide insight into possible therapeutic targets and thereby help to enhance future fracture healing and bone tissue engineering approaches. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:40:55.646886-05:
      DOI: 10.1002/jcp.25641
  • New Roles of Lkb1 in Regulating Adipose Tissue Development and
    • Authors: Ziye Xu; Jiaqi Liu, Tizhong Shan
      Abstract: Adipose tissues regulate energy metabolism and reproduction. There are three types of adipocytes (brown, white and beige adipocytes) in mammals. White adipocytes store energy and are closely associated with obesity and other metabolic diseases. The beige and brown adipocytes have numerous mitochondria and high levels of UCP1 that dissipates lipid to generate heat and defend against obesity. The global epidemic of obesity and its associated metabolic diseases urge an imperative need for understating the regulation of adipogenesis. Liver kinase B1 (Lkb1), also called STK11, is a master kinase of the AMPK subfamily and plays crucial roles in regulating glucose and energy homeostasis in various metabolic tissues. In this review we focus on the regulatory roles of Lkb1 in regulating preadipocyte differentiation, adipose tissue development and thermogenesis. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:54.490641-05:
      DOI: 10.1002/jcp.25643
  • Low Shear Stress Attenuates COX-2 Expression Induced by Resistin in Human
           Osteoarthritic Chondrocytes
    • Authors: Yu-Ping Su; Cheng-Nan Chen, Hsin-I Chang, Kuo-Chin Huang, Chin-Chang Cheng, Fang-Yao Chiu, Ko-Chao Lee, Chun-Min Lo, Shun-Fu Chang
      Abstract: Low shear stress has been proposed to play a reparative role in modulating cartilage homeostasis. Recently, epidemiological studies have found a positive correlation between the resistin level in serum and synovial fluid and osteoarthritis (OA) severity in patients. However, the effect of moderate shear stress on the catabolic stimulation of resistin in OA chondrocytes remains unclear. Hence, this study was to investigate whether low shear stress could regulate resistin-induced catabolic cyclooxygenase (COX)-2 expression in human OA chondrocytes and the underlying mechanism. Human OA chondrocytes and SW1353 chondrosarcoma cells were used in this study. Two modes of low shear stress (2 dyn/cm2), pre-shear and post-shear, were applied to the chondrocytes. A specific activator and siRNAs were used to investigate the mechanism of low shear stress-regulated COX-2 expression of resistin induction. We found that human OA chondrocytes exposed to different modes of low shear stress elicit an opposite effect on resistin-induced COX-2 expression: pre-shear for a short duration attenuates the resistin effect by inhibiting the transcription factor nuclear factor (NF)-κB-p65 subunit and the cAMP response element binding protein; however, post-shear over a longer duration enhances the resistin effect by activating only the NF-κB-p65 subunit. Moreover, our results demonstrated that the regulation of both shear modes in resistin-stimulated COX-2 expression occurs through increasing AMP-activated protein kinase activation and then sirtuin 1 expression. This study elucidates the detailed mechanism of low shear stress regulating the resistin-induced catabolic COX-2 expression and indicates a possible reparative role of moderate shear force in resistin-stimulated OA development. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:50.402155-05:
      DOI: 10.1002/jcp.25644
  • Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is
           Involved in Cell Cycle Progression
    • Authors: Alessandro Poli; Roberta Fiume, Gianluca Baldanzi, Daniela Capello, Stefano Ratti, Marco Gesi, Lucia Manzoli, Andrea Graziani, Pann-Ghill Suh, Lucio Cocco, Matilde Y. Follo
      Abstract: Phosphatidylinositol (PI) signalling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signalling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:43.293952-05:
      DOI: 10.1002/jcp.25642
  • Peripheral Blood Mononuclear Cells Infiltration Downregulates Decidual
           FAAH Activity in an LPS-Induced Embryo Resorption Model
    • Authors: Manuel Luis Wolfson; Julieta Aisemberg, Fernando Correa, Ana María Franchi
      Abstract: Background and PurposeMaternal infections with Gram-negative bacteria are associated with miscarriage and are one of the most common complications during pregnancy. Previous studies from our group have shown that lipopolysaccharide (LPS)-activated infiltrating peripheral blood mononuclear cells (PBMC) into decidual tissue play an important role on the establishment of a local inflammatory process that result in embryo cytotoxicity and early embryo resorption. Moreover, we have also shown that an increased endocannabinoid tone mediates LPS-induced deleterious effects during early pregnancy loss. Here we sought to investigate whether the infiltrating PBMC modulates the decidual endocannabinoid tone and the molecular mechanisms involved.Experimental approachPBMC isolated from 7-days pregnant mice subjected to different treatments were co-cultured in a transwell system with decidual tissue from control 7-days pregnant mice. Decidual fatty acid amide hydrolase (FAAH) activity was measure by radioconvertion, total decidual protein nitration by western blot (WB) and decidual FAAH nitration by immuneprecipitation followed by WB.Key ResultsWe found that co-culture of PBMC obtained from LPS-treated mice increased the level of nitration of decidual FAAH, which resulted in a negative modulation of decidual FAAH activity. Interestingly, co-treatment with progesterone or aminoguanidine prevented this effect.Conclusions & ImplicationsWe found that LPS-treated PBMC release high amounts of nitric oxide (NO) which causes tyrosine nitration of decidual FAAH, diminishing its enzymatic activity. Inactivation of FAAH, the main degrading enzyme of anandamide and similar endocannabinoids, could lead to an increased decidual endocannabinoid tone with embryotoxic effects. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:36:34.357677-05:
      DOI: 10.1002/jcp.25640
  • The Role of Cardiac Myosin Binding Protein C3 in Hypertrophic
           Cardiomyopathy- Progress and Novel Therapeutic Opportunities
    • Authors: Iman A. Mohamed; Navaneethakrishnan T. Krishnamoorthy, Gheyath K. Nasrallah, Sahar Da'as
      Abstract: Hypertrophic cardiomyopathy (HCM) is a common autosomal dominant genetic cardiovascular disorder marked by genetic and phenotypic heterogeneity. Mutations in the gene encodes the cardiac myosin-binding protein C, cMYBPC3 is amongst the various sarcomeric genes that are associated with HCM. These mutations produce mutated mRNAs and truncated cMyBP-C proteins. In this review, we will discuss the implications and molecular mechanisms involved in MYBPC3 different mutations. Further, we will highlight the novel targets that can be developed into potential therapeutics for the treatment of HMC. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-12T06:31:00.403813-05:
      DOI: 10.1002/jcp.25639
  • Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) Regulates
           Osteoclast Differentiation via the Ca2+ /NFATc1 Axis
    • Authors: Ya-Hui Chiu; Edward Schwarz, Dongge Li, Yuexin Xu, Tzong-Ren Sheu, Jinbo Li, Karen L. de Mesy Bentley, Changyong Feng, Baoli Wang, Jhih-Cheng Wang, Liz Albertorio-Saez, Ronald Wood, Minsoo Kim, Wensheng Wang, Christopher T. Ritchlin
      Abstract: DC-STAMP is a multi-pass transmembrane protein essential for cell-cell fusion of osteoclast precursors during osteoclast (OC) development. DC-STAMP-/- mice have mild osteopetrosis and form mononuclear cells with limited resorption capacity. The identification of an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) on the cytoplasmic tail of DC-STAMP suggested a potential signaling function but the absence of known DC-STAMP ligand has hindered examination of downstream signaling pathways. To address this problem, we engineered a light-activatable DC-STAMP chimeric molecule in which light exposure mimics ligand engagement that can be traced by downstream Ca2+ signaling. Deletion of the cytoplasmic ITIM resulted in a significant elevation in the amplitude and duration of intracellular Ca2+ flux. We also found that decreased NFATc1 expression in DC-STAMP-/- cells was restored by DC-STAMP over-expression. Multiple biological phenotypes including cell-cell fusion, bone erosion, cell mobility, DC-STAMP cell surface distribution, and NFATc1 nuclear translocation were altered by deletion of the ITIM and adjacent amino acids. In contrast, mutations on each of tyrosine residues in the ITIM showed no effect on DC-STAMP function. Collectively, our results suggest that ITIM on DC-STAMP is a functional motif that regulates osteoclast differentiation through the NFATc1 / Ca2+ axis. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-10T06:31:04.676478-05:
      DOI: 10.1002/jcp.25638
  • Dact1, a Wnt-Pathway Inhibitor, Mediates Human Mesangial Cell
           TGF-β1-Induced Apoptosis
    • Authors: Daniele Pereira Jardim; Paula Cristina Eiras Poço, Alexandre Holthausen Campos
      Abstract: Chronic kidney disease (CKD) is a worldwide public health problem that affects millions of men and women of all ages and racial groups. Loss of mesangial cells (MC) represents an early common feature in the pathogenesis of CKD. Transforming growth factor-β1 (TGF-β1) is a key inducer of kidney damage and triggers several pathological changes in renal cells, notably MC apoptosis. However, the mechanism of MC apoptosis induced by TGF-β1 remains elusive. Here we demonstrate for the first time a novel regulatory pathway in which the disheveled-binding antagonist of beta-catenin 1 (Dact1) gene is upregulated by TGF-β1, inducing MC apoptosis. We also show that the inhibitory effect of Dact1 and TGF-β1 on the transcriptional activation of the pro-survival Wnt pathway is the mechanism of death induction. In addition, Dact1 mRNA/protein levels are increased in kidney remnants from 5/6 nephrectomized rats and strongly correlate with TGF-β1 expression. Together, our results point to Dact1 as a novel element controlling MC survival that is causally related to CKD progression. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:49:27.48411-05:0
      DOI: 10.1002/jcp.25636
  • Inhibitory Effects of Quercetin on Progression of Human Choriocarcinoma
    • Authors: Whasun Lim; Changwon Yang, Sunwoo Park, Fuller W. Bazer, Gwonhwa Song
      Abstract: As a major dietary flavonol, quercetin mitigates proliferation and progression of cancer due to its anti-angiogenic, anti-inflammatory, anti-oxidant and apoptotic biological effects on cells. Although its apoptotic effects have been reported for various cancers, little is known of the functional role of quercetin in gestational choriocarcinoma. Results of the present study indicated that quercetin reduced proliferation and induced cell death in two choriocarcinoma cell lines, JAR and JEG3 cells, with an increase in the sub-G1 phase of the cell cycle. In addition, quercetin induced mitochondrial dysfunction significantly reduced mitochondrial membrane potential (MMP) and increased production of reactive oxygen species (ROS) in both JAR and JEG3 cells. Further, quercetin inhibited phosphorylation of AKT, P70S6K and S6 proteins whereas it increased phosphorylation of ERK1/2, P38, JNK and P90RSK proteins in JAR and JEG3 cells. The decrease in viability of choriocarcinoma cells treated with quercetin was confirmed by using combinations of quercetin and pharmacological inhibitors of the PI3K and MAPK signaling pathways. Classical chemotherapeutic agents, cisplatin (a platinum-based drug) and paclitaxel (a taxene-based drug), inhibited proliferation of JAR and JEG3 cells and when combined with quercetin, the antiproliferative effects of cisplatin and paclitaxel were enhanced for both choriocarcinoma cell lines. Collectively, these results suggest that quercetin prevents development of choriocarcinoma and may be a valuable therapeutic agent for treatment of choriocarcinoma through its regulation of PI3K and MAPK signal transduction pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:49:16.88812-05:0
      DOI: 10.1002/jcp.25637
  • Osteoclast Fusion: Time-Lapse Reveals Involvement of CD47 and Syncytin-1
           at Different Stages of Nuclearity
    • Authors: Anaïs Marie Julie Møller; Jean-Marie Delaissé, Kent Søe
      Abstract: Investigations addressing the molecular keys of osteoclast fusion are primarily based on end-point analyses. No matter if investigations are performed in vivo or in vitro the impact of a given factor is predominantly analyzed by counting the number of multinucleated cells, the number of nuclei per multinucleated cell or TRAcP activity. But end-point analyses do not show how the fusion came about. This would not be a problem if fusion of osteoclasts is a random process and occurs by the same molecular mechanism from beginning to end. However, we and others have in the recent period published data suggesting that fusion partners may specifically select each other and that heterogeneity between the partners seems to play a role. Therefore we set out to directly test the hypothesis that fusion factors have a heterogenic involvement at different stages of nuclearity. Therefore, we have analyzed individual fusion events using time-lapse and antagonists of CD47 and syncytin-1. All time-lapse recordings have been studied by two independent observers. A total of 1808 fusion events were analyzed. The present study shows that CD47 and syncytin-1 have different roles in osteoclast fusion depending on the nuclearity of fusion partners. While CD47 promotes cell fusions involving mono-nucleated pre-osteoclasts, syncytin-1 promotes fusion of two multi-nucleated osteoclasts, but also reduces the number of fusions between mono-nucleated pre-osteoclasts. Furthermore, CD47 seems to mediate fusion mostly through broad contact surfaces between the partners' cell membrane while syncytin-1 mediate fusion through phagocytic-cup like structure. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:36:16.164329-05:
      DOI: 10.1002/jcp.25633
  • Essential Role of CFTR in PKA-Dependent Phosphorylation, Alkalinization
           and Hyperpolarization During Human Sperm Capacitation
    • Authors: Lis C. Puga Molina; Nicolás A. Pinto, Paulina Torres Rodríguez, Ana Romarowski, Alberto Vicens Sanchez, Pablo E. Visconti, Alberto Darszon, Claudia L. Treviño, Mariano G. Buffone
      Abstract: Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3- is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm and membrane hyperpolarization. However, how HCO3- is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3--entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:36:13.303942-05:
      DOI: 10.1002/jcp.25634
  • Short- and Long-Term Hindlimb Immobilization and Reloading: Profile of
           Epigenetic Events in Gastrocnemius
    • Authors: Alba Chacon-Cabrera; Joaquim Gea, Esther Barreiro
      Abstract: Skeletal muscle dysfunction and atrophy are characteristic features accompanying chronic conditions. Epigenetic events regulate muscle mass and function maintenance. We hypothesized that the pattern of epigenetic events (muscle-enriched microRNAs and histone acetylation) and acetylation of transcription factors known to signal muscle wasting may differ between early- and late-time points in skeletal muscles of mice exposed to hindlimb immobilization (I) and recovery following I. Body and muscle weights, grip strength, muscle-enriched microRNAs, histone deacetylases (HDACs), acetylation of proteins, histones, and transcription factors (TF), myogenic TF factors, and muscle phenotype were assessed in gastrocnemius of mice exposed to periods (1, 2, 3, 7, 15 and 30 days, I groups) of hindlimb immobilization, and in those exposed to reloading for different periods of time (1, 3, 7, 15, and 30 days, R groups) following 7-day immobilization. Compared to non-immobilized controls, muscle weight, limb strength, microRNAs, especially miR-486, SIRT1 levels, and slow- and fast-twitch cross-sectional areas were decreased in mice of I groups, whereas Pax7, and acetylated FoxO1 and FoxO3 levels were increased. Muscle reloading following splint removal improved muscle mass loss, strength, and fiber atrophy, by increasing microRNAs, particularly miR-486, and SIRT1 content, while decreasing acetylated FoxO1 and FoxO3 levels. In this mouse model of disuse muscle atrophy, muscle-enriched microRNAs, especially miR-486, through Pax7 regulation delayed muscle cell differentiation following unloading of gastrocnemius muscle. Acetylation of FoxO1 and 3 seemed to drive muscle mass loss and atrophy, while deacetylation of these factors through SIRT1 would enable the muscle fibers to regenerate. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:36:06.967146-05:
      DOI: 10.1002/jcp.25635
  • Challenging of AS160/TBC1D4 Alters Intracellular Lipid Milieu in L6
           Myotubes Incubated with Palmitate
    • Authors: Agnieszka Mikłosz; Bartłomiej Łukaszuk, Małgorzata Żendzian-Piotrowska, Justyna Brańska-Januszewska, Halina Ostrowska, Adrian Chabowski
      Abstract: The Akt substrate of 160 kDa (AS160) is a key regulator of GLUT4 translocation from intracellular depots to the plasma membrane in myocytes. Likely, AS160 also controls LCFAs transport, which requires relocation of fatty acid transporters. The aim of the present study was to determine the impact of AS160 knockdown on lipid milieu in L6 myotubes incubated with palmitate (PA). Therefore, we compared two different settings, namely: 1) AS160 knockdown prior to palmitate incubation (pre-PA-silencing, AS160-/PA); 2) palmitate incubation with subsequent AS160 knockdown (post-PA-silencing, PA/AS160-). The efficiency of AS160 silencing was checked at mRNA and protein levels. The expression and localization of FA transporters were determined using Western Blot and immunofluorescence analyses. Intracellular lipid content (FFA, DAG, TAG and PL) and FA composition were estimated by GLC, whereas basal palmitate uptake was analyzed by means of scintigraphy. Both groups with silenced AS160 were characterized by a greater expression of FA transporters (FAT/CD36, FATP-1, 4) which had contributed to an increased FA cellular influx. Accordingly, we observed that post-PA-silencing of AS160 resulted in a marked decrement in DAG, TAG and PL contents, but increased FFA content (PA/AS160- vs. PA). The opposite effect was observed in the group with pre-PA-silencing of AS160 in which AS160 knockdown did not affect the lipid pools (AS160-/PA vs. PA). Our results indicate that post-PA-silencing of AS160 has a capacity to decrease the lipotoxic effect(s) of PA by decreasing the content of lipids (DAG and PL) that promote insulin resistance in myotubes. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-07T06:35:57.765285-05:
      DOI: 10.1002/jcp.25632
  • Bone Shaft Revascularization After Marrow Ablation is Dramatically
           Accelerated in BSP-/- Mice, Along With Faster Haematopoietic
    • Authors: Wafa Bouleftour; Renata Neves Granito, Arnaud Vanden-Bossche, Odile Sabido, Bernard Roche, Mireille Thomas, Marie Thérèse Linossier, Jane E Aubin, Marie-Hélène Lafage-Proust, Laurence Vico, Luc Malaval
      Abstract: The bone organ integrates the activity of bone tissue, bone marrow and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the Small Integrin Binding Ligand N-Linked Glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP-/- mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP + /+ and BSP-/- mice. At d3, the ablated area in BSP-/- femurs showed higher vessel density (x4) and vascular volume (x7) than BSP + / +. Vessel numbers in the shaft of ablated BSP + /+ mice reached BSP-/- values only by d8, but with a vascular volume which was twice the value in BSP-/-, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin– (x3) as well as LSK (Lin- IL-7Rα- Sca-1hi c-Kithi, x2) and hematopoietic stem cells (HSC: Flt3- LSK, x2) were counted in BSP-/- marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin- was lower in BSP-/- and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP-/- femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants.In conclusion, bone marrow ablation in BSP-/- mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-05T05:11:10.605707-05:
      DOI: 10.1002/jcp.25630
  • Indispensable Role of Ion Channels and Transporters in the Auditory System
    • Authors: Rahul Mittal; Mayank Aranke, Luca H. Debs, Desiree Nguyen, Amit P. Patel, M'hamed Grati, Jeenu Mittal, Denise Yan, Prem Chapagain, Adrien A. Eshraghi, Xue Zhong Liu
      Abstract: Ear is a complex system where appropriate ionic composition is essential for maintaining the tissue homeostasis and hearing function. Ion transporters and channels present in the auditory system plays a crucial role in maintaining proper ionic composition in the ear. The extracellular fluid, called endolymph, found in the cochlea of the mammalian inner ear is particularly unique due to its electrochemical properties. At an endocochlear potential of about +80 mV, signaling initiated by acoustic stimuli at the level of the hair cells is dependent on the unusually high potassium (K+) concentration of endolymph. There are ion channels and transporters that exists in the ear to ensure that K+ is continually being cycled into the stria media endolymph. This review is focused on the discussion of the molecular and genetic basis of previously and newly recognized ion channels and transporters that support sensory hair cell excitation based on recent knock-in and knock-out studies of these channels. This article also addresses the molecular and genetic defects and the pathophysiology behind Meniere's disease as well as how the dysregulation of these ion transporters can result in severe defects in hearing or even deafness. Understanding the role of ion channels and transporters in the auditory system will facilitate in designing effective treatment modalities against ear disorders including Meniere's disease and hearing loss. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-05T05:11:04.350582-05:
      DOI: 10.1002/jcp.25631
  • Increased Amyloid Precursor Protein and Tau Expression Manifests as Key
           Secondary Cell Death in Chronic Traumatic Brain Injury
    • Authors: Sandra A. Acosta; Naoki Tajiri, Paul R. Sanberg, Yuji Kaneko, Cesar V. Borlongan
      Abstract: In testing the hypothesis of Alzheimer's disease (AD)-like pathology in late stage traumatic brain injury (TBI), we evaluated AD pathological markers in late stage TBI model. Sprague-Dawley male rats were subjected to moderate controlled cortical impact (CCI) injury, and 6 months later euthanized and brain tissues harvested. Results from H&E staining revealed significant 33% and 10% reduction in the ipsilateral and contralateral hippocampal CA3 interneurons, increased MHCII-activated inflammatory cells in many gray matter (8-20 fold increase) and white matter (6-30 fold increased) regions of both the ipsilateral and contralateral hemispheres, decreased cell cycle regulating protein marker by 1.6- and 1-fold in the SVZ and a 2.3 and 1.5 fold reductions in the ipsilateral and contralateral dentate gyrus, diminution of immature neuronal marker by 2- and 1- fold in both the ipsilateral and contralateral SVZ and dentate gyrus, and amplified amyloid precursor protein (APP) distribution volumes in white matter including corpus callosum, fornix, and internal capsule (4-38 fold increase), as well as in the cortical gray matter, such as the striatum hilus, SVZ and dentate gyrus (6-40 fold increase) in TBI animals compared to controls (p's 
      PubDate: 2016-10-04T06:03:23.777999-05:
      DOI: 10.1002/jcp.25629
  • MicroRNA-494 Activation Suppresses Bone Marrow Stromal Cell-Mediated Drug
           Resistance in Acute Myeloid Leukemia Cells
    • Authors: Chen Tian; Guoguang Zheng, Hongqing Zhuang, Xubin Li, Dongzhi Hu, Lei Zhu, Wang Tengteng, M. James You, Yizhuo Zhang
      Abstract: Acute myeloid leukemia (AML) is not sensitive to chemotherapy partially because of the protection of AML cells by mesenchymal stromal cells (MSCs). Our previous studies found that MSCs protected AML cells from apoptosis through the c-Myc-dependent pathway. However, the mechanism by which MSCs regulate c-Myc in AML cells is still unknown. To elucidate the mechanism, we performed microRNA array analysis of AML cell lines and validated by TaqMan realtime PCR. The results showed that the expression of microRNA-494 (miR-494) in AML cells after coculture with MSCs was down-regulated. Reporter gene analysis confirmed miR-494 as one of the regulators of c-Myc. In the coculture system, activation of miR-494 in AML cells suppressed proliferation and induced apoptosis of AML cells in vitro. After addition of mitoxantrone to the coculture system, the proliferation of AML cells with miR-494 activation was suppressed more than that of control cells. After subcutaneous injection of AML cell lines in combination with MSC, tumor growth was suppressed in mice injected with miR-494-overexpressing AML cells. The rate of tumor formation was even lower after mitoxantrone treatment in the miR-494 overexpressing group. Moreover, miR-494 activation resulted in a decrease of leukemic cell counts in peripheral blood and bone marrow and prolonged survival in mice injected with miR-494-overexpressing AML cellls and MSCs compared to the control mice. Our results indicate that miR-494 suppresses drug resistance in AML cells by down-regulating c-Myc through interaction with MSCs and that miR-494 therefore is a potential therapeutic target. This article is protected by copyright. All rights reserved
      PubDate: 2016-10-03T06:35:39.113816-05:
      DOI: 10.1002/jcp.25628
  • Epigenetic Signatures at the RUNX2-P1 and Sp7 Gene Promoters Control
           Osteogenic Lineage Commitment of Umbilical Cord-Derived Mesenchymal Stem
    • Authors: Hugo Sepulveda; Rodrigo Aguilar, Catalina P. Prieto, Francisco Bustos, Sócrates Aedo, José Lattus, Brigitte van Zundert, Veronica Palma, Martin Montecino
      Abstract: Wharton's Jelly mesenchymal stem cells (WJ-MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ-MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies here we analyze the ability of WJ-MSCs to engage osteoblast lineage. In undifferentiated WJ-MSCs, RUNX2/P57 P1 and SP7 promoters are found deprived of significant levels of the histone post-translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ-MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together these results point to critical regulatory steps during epigenetic control of WJ-MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-30T16:25:24.511202-05:
      DOI: 10.1002/jcp.25627
  • Differences in Activation of HIV-1 Replication by Superinfection With
           HIV-1 and HIV-2 in U1 Cells
    • Authors: Xue Wang; Bing Sun, Christelle Mbondji, Santanu Biswas, Jiangqin Zhao, Indira Hewlett
      Abstract: Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir that serve as a viral source for the infection of CD4 T cells. The relationship between HIV-1 latent infection and superinfection in macrophages has not been well studied. Using susceptible U1 cells chronically infected with HIV-1, we studied the effects of HIV superinfection on latency and differences in superinfection with HIV-1 and HIV-2 in macrophages. We found that HIV-1 (MN) superinfection displayed increased HIV-1 replication in a time-dependent manner; while cells infected with HIV-2 (Rod) initially showed increased HIV-1 replication, followed by a decrease in HIV-1 RNA production. HIV-1 superinfection upregulated/activated NF-ĸB, NFAT, AP-1, SP-1, and MAPK Erk through expression/activation of molecules, CD4, CD3, TCRβ, Zap-70, PLCγ1, and PKCΘ in T cell receptor-related signaling pathways; while HIV-2 superinfection initially increased expression/activation of these molecules followed by decreased protein expression/activation. HIV superinfection initially downregulated HDAC1 and upregulated acetyl-histone H3 and histone H3 (K4), while HIV-2 superinfection demonstrated an increase in HDAC1 and a decrease in acetyl-histone H3 and histone H3 (K4) relative to HIV-1 superinfection. U1 cells superinfected with HIV-1 or HIV-2 showed differential expression of proteins, IL-2, PARP-1, YB-1, and LysRS. These findings indicate that superinfection with HIV-1 or HIV-2 has different effects on reactivation of HIV-1 replication. HIV-1 superinfection with high load of viral replication may result in high levels of cytotoxicity relative to HIV-2 superinfection. Cells infected with HIV-2 showed lower level of HIV-1 replication, suggesting that co-infection with HIV-2 may result in slower progression toward AIDS. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-09-30T09:20:42.86365-05:0
      DOI: 10.1002/jcp.25614
  • Hypoglycemia Enhances Epithelial-Mesenchymal Transition and Invasiveness,
           and Restrains the Warburg Phenotype, in Hypoxic HeLa Cell Cultures and
    • Authors: Álvaro Marín-Hernández; Juan Carlos Gallardo-Pérez, Ileana Hernández-Reséndiz, Isis Del Mazo-Monsalvo, Diana Xochiquetzal Robledo-Cadena, Rafael Moreno-Sánchez, Sara Rodríguez-Enríquez
      Abstract: The accelerated growth of solid tumors leads to episodes of both hypoxia and hypoglycemia (HH) affecting their intermediary metabolism, signal transduction, and transcriptional activity. A previous study showed that normoxia (20% O2) plus 24 h hypoglycemia (2.5 mM glucose) increased glycolytic flux whereas oxidative phosphorylation (OxPhos) was unchanged versus normoglycemia in HeLa cells. However, the simultaneous effect of HH on energy metabolism has not been yet examined. Therefore, the effect of hypoxia (0.1–1% O2) plus hypoglycemia on the energy metabolism of HeLa cells was analyzed by evaluating protein content and activity, along with fluxes of both glycolysis and OxPhos. Under hypoxia, in which cell growth ceased and OxPhos enzyme activities, ΔΨm and flux were depressed, hypoglycemia did not stimulate glycolytic flux despite increasing H-RAS, p-AMPK, GLUT1, GLUT3, and HKI levels, and further decreasing mitochondrial enzyme content. The impaired mitochondrial function in HH cells correlated with mitophagy activation. The depressed OxPhos and unchanged glycolysis pattern was also observed in quiescent cells from mature multicellular tumor spheroids, suggesting that these inner cell layers are similarly subjected to HH. The principal ATP supplier was glycolysis for HH 2D monolayer and 3D quiescent spheroid cells. Accordingly, the glycolytic inhibitors iodoacetate and gossypol were more effective than mitochondrial inhibitors in decreasing HH-cancer cell viability. Under HH, stem cell-, angiogenic-, and EMT-biomarkers, as well as glycoprotein-P content and invasiveness, were also enhanced. These observations indicate that HH cancer cells develop an attenuated Warburg and pronounced EMT- and invasive-phenotype. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.Hypoglycemia enhances (i) epithelial-mesenchymal transition, (ii) invasiveness, and (iii) restrains the Warburg phenotype in hypoxic HeLa cells.
      PubDate: 2016-09-30T09:16:31.539215-05:
      DOI: 10.1002/jcp.25617
  • PACAP and VIP Inhibit HIF-1α-Mediated VEGF Expression in a Model of
           Diabetic Macular Edema
    • Authors: Grazia Maugeri; Agata Grazia D'Amico, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata
      Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) exert a protective role against retinal injuries, including diabetic macular edema (DME). The macular damage is induced by hyperglycemia, which damages vessels supplying blood to the retina and induces hypoxia. The microenvironmental changes stimulate the expression of hypoxia-inducible factors (HIFs), which promote the choroidal endothelial cell transmigration across the retinal pigmented epithelium (RPE) into neurosensory retina, where they proliferate into new vessels under stimulation of the vascular endothelial growth factor (VEGF). In the present study, we have investigated whether PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors. In accord to our hypothesis, we have shown that both peptides are able to significantly reduce HIF-1α and increase HIF-3α expression in ARPE-19 cells exposed to hyperglycemic/hypoxic insult. This effect is also related to a reduction of VEGF and its receptors expression. Moreover, both peptides also reduce the activation of p38 mitogen-activated protein kinase (MAPK), a pro-apoptotic signaling pathway, which is activated by VEGFR-1 and 2 receptors. In conclusion, our study has further elucidated the protective role performed by PACAP and VIP, against the harmful combined effect of hyperglycemia/hypoxia characterizing the DME microenvironment. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF, and its receptors; PACAP and VIP act against the harmful combined effect of hyperglycemia/hypoxia.
      PubDate: 2016-09-30T09:16:20.837141-05:
      DOI: 10.1002/jcp.25616
  • MED28 Regulates Epithelial–Mesenchymal Transition Through NFκB in Human
           Breast Cancer Cells
    • Authors: Chun-Yin Huang; Nien-Tsu Hsieh, Chun-I Li, Yu-Ting Weng, Hsiao-Sheng Liu, Ming-Fen Lee
      Abstract: MED28, a mammalian Mediator subunit, was found highly expressed in several types of malignancy, including breast cancer. Recently, we have identified a role of MED28 in regulating both cell growth and migration in human breast cancer cells. In epithelium-derived solid tumor, migration and invasion are preceded by the progression of epithelial–mesenchymal transition (EMT) which calls for downregulation of epithelial markers as well as upregulation of mesenchymal markers, among other features. The objective of this study was to investigate a putative role of MED28 in the progression of EMT in human breast cancer cells. In fibroblast-like MDA-MB-231 cells, suppression of MED28 attenuated the mesenchymal morphology, concomitantly with a reduction of several mesenchymal biomarkers and Snail, a transcriptional repressor of E-cadherin. The suppression effect was also accompanied by downregulation of p-NFκB/p65. However, overexpression of MED28 exhibited in an opposite manner. In epithelial MCF7 cells, administration of Adriamycin®, an experimental EMT induction system, led to a mesenchyme-like appearance correlated with increased expression of MED28, p-p65, and Snail, and a reciprocal change of epithelial and mesenchymal markers. Furthermore, suppression of MED28 attenuated the experimental EMT effect and restored the original expression status of E-cadherin and MMP9 in MCF7 cells. Our data indicate that MED28 modulates the development of EMT through NFκB in human breast cancer cells, further reinforcing the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.MED28 regulates the expression of epithelial markers and mesenchymal markers through NFκB, thereby modulating the development of epithelial–mesenchymal transition. These findings further reinforce the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration.
      PubDate: 2016-09-30T09:09:02.969848-05:
      DOI: 10.1002/jcp.25610
  • Hyperexcitability of Mesencephalic Trigeminal Neurons and Reorganization
           of Ion Channel Expression in a Rett Syndrome Model
    • Authors: Max F. Oginsky; Ningren Cui, Weiwei Zhong, Christopher M. Johnson, Chun Jiang
      Abstract: People with Rett syndrome (RTT) have defects in motor function also seen in Mecp2-null mice. Motor function depends on not only central motor commands but also sensory feedback that is vulnerable to changes in excitability of propriosensory neurons. Here we report evidence for hyperexcitability of mesencephalic trigeminal (Me5) neurons in Mecp2-null mice and a novel cellular mechanism for lowering its impact. In in vitro brain slices, the Me5 neurons in both Mecp2−/Y male and symptomatic Mecp2+/− female mice were overly excitable showing increased firing activity in comparison to their wild-type (WT) male and asymptomatic counterparts. In Mecp2−/Y males, Me5 neurons showed a reduced firing threshold. Consistently, the steady-state activation of voltage-gated Na+ currents (INa) displayed a hyperpolarizing shift in the Mecp2-null neurons with no change in the INa density. This seems to be due to NaV1.1, SCN1B and SCN4B overexpression and NaV1.2 and SCN3B under-expression. In contrast to the hyperexcitability, the sag potential and postinhibitory rebound (PIR) were reduced in Mecp2-null mice. In voltage-clamp, the IH density was deficient by ∼33%, and the steady-state half-activation had a depolarizing shift of ∼10 mV in the Mecp2-null mice. Quantitative PCR analysis indicated that HCN2 was decreased, HCN1 was upregulated with no change in HCN4 in Mecp2−/Y mice compared to WT. Lastly, blocking IH reduced the firing rate much more in WT than in Mecp2-null neurons. These data suggest that the Mecp2 defect causes an increase in Me5 neuronal excitability likely attributable to alterations in INa, meanwhile IH is reduced likely altering neuronal excitability as well. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.Mesencephalic trigeminal V neurons are hyperexcitable in Mecp2-null mice. Our data suggest that the Mecp2 defect that causes an increase in Me5 neuronal excitability is likely attributable to alterations in INa, meanwhile IH is reduced likely altering neuronal excitability as well. Further, the changes in these currents seem to be due to alterations in channel subunit composition.
      PubDate: 2016-09-30T09:09:00.806058-05:
      DOI: 10.1002/jcp.25589
  • Inflammatory Events and Oxidant Production in the Diaphragm,
           Gastrocnemius, and Blood of Rats Exposed to Chronic Intermittent Hypoxia:
           Therapeutic Strategies
    • Authors: Marisol Domínguez-Álvarez; Joaquim Gea, Esther Barreiro
      Abstract: We hypothesized that inflammatory events and reactive oxygen species (ROS) production may be differentially expressed in respiratory and limb muscles, and blood of a chronic intermittent hypoxia (CIH) experimental model and that antioxidants and TNF-alpha blockade may influence those events. In blood, diaphragm, and gastrocnemius of rats non-invasively exposed to CIH (10% hypoxia, 2 h/day, 14 consecutive days) with/without concomitant treatment with either anti-TNF-alpha antibody (infliximab) or N-acetyl cysteine (NAC), inflammatory cytokines, superoxide anion production, muscle structural abnormalities, and fiber-type composition were assessed. Compared to non-exposed controls, in CIH-exposed rats, body weight gain was reduced, TNF-alpha, IL-1beta, IL-6, and interferon-gamma levels were increased in diaphragm, TNF-alpha, and IL-1 beta plasma levels were greater, systemic and muscle superoxide anion production was higher, diaphragm and gastrocnemius inflammatory cells and internal nuclei were higher, and muscle fiber-type and morphometry remained unmodified. CIH rats treated with infliximab further increased TNF-alpha, IL-1beta, IL-6, and interferon-gamma diaphragm levels, whereas NAC induced a reduction only in TNF-alpha and IL-1beta levels in diaphragm and plasma. Infliximab and NAC elicited a significant decline in superoxide anion production in diaphragm, gastrocnemius, and plasma, while inducing a further increase in inflammatory cells and internal nuclei in both muscles. Proinflammatory cytokines are differentially expressed in respiratory and limb muscles and plasma of CIH-exposed rats, while superoxide anion production increased in both muscle types and blood. Infliximab and NAC exerted different effects. These findings may help understand the biology underlying CIH in skeletal muscles and blood of patients with chronic respiratory diseases. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Proinflammatory cytokines are differentially expressed in respiratory and limb muscles and plasma of CIH-exposed rats, while superoxide anion production increased in both muscle types and blood. Infliximab and NAC exerted different effects. These findings may help understand the biology underlying CIH in skeletal muscles and blood of patients with chronic respiratory diseases.
      PubDate: 2016-09-30T09:08:52.834967-05:
      DOI: 10.1002/jcp.25600
  • Role of Protein Kinase G and Reactive Oxygen Species in the Regulation of
           Podocyte Function in Health and Disease
    • Authors: Agnieszka Piwkowska
      Abstract: Podocytes and their foot processes form an important cellular layer of the glomerular barrier involved in the regulation of glomerular permeability. Disturbing the function of podocytes plays a central role in the development of proteinuria in diabetic nephropathy. Retraction of the podocyte foot processes that form slit diaphragms is a common feature of proteinuria; although, the correlation between these events in not well understood. Notably, it is unclear whether podocyte foot processes are able to regulate slit diaphragm permeability and glomerular ultrafiltration. The occurrence of reactive oxygen species generation, insulin resistance, and hyperglycemia characterizes early stages of type 2 diabetes. Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. It is activated in both cGMP-dependent and cGMP-independent manners. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI-dependent signaling pathways in podocytes. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI-dependent signaling pathways in podocytes.
      PubDate: 2016-09-30T08:58:57.845176-05:
      DOI: 10.1002/jcp.25613
  • Risk Differences Between Prediabetes And Diabetes According To Breast
           Cancer Molecular Subtypes
    • Authors: A. Crispo; L.S.A. Augustin, M. Grimaldi, F. Nocerino, A. Giudice, E. Cavalcanti, M. Di Bonito, G. Botti, M. De Laurentiis, M. Rinaldo, E. Esposito, G. Riccardi, A. Amore, M. Libra, G. Ciliberto, D.J.A. Jenkins, M. Montella
      Abstract: Hyperglycemia and hyperinsulinemia may play a role in breast carcinogenesis and prediabetes and diabetes have been associated with increased breast cancer (BC) risk. However, whether BC molecular subtypes may modify these associations is less clear. We therefore investigated these associations in all cases and by BC molecular subtypes among women living in Southern Italy. Cases were 557 patients with non-metastatic incident BC and controls were 592 outpatients enrolled during the same period as cases and in the same hospital for skin-related non-malignant conditions. Adjusted multivariate logistic regression models were built to assess the risks of developing BC in the presence of prediabetes or diabetes. The analyses were repeated by strata of BC molecular subtypes: Luminal A, Luminal B, HER2+, and Triple Negative (TN). Prediabetes and diabetes were significantly associated with higher BC incidence after controlling for known risk factors (OR = 1.94, 95% CI 1.32–2.87 and OR = 2.46, 95% CI 1.38–4.37, respectively). Similar results were seen in Luminal A and B while in the TN subtype only prediabetes was associated with BC (OR = 2.43, 95% CI 1.11–5.32). Among HER2+ patients, only diabetes was significantly associated with BC risk (OR = 3.04, 95% CI 1.24–7.47). Furthermore, when postmenopausal HER2+ was split into hormone receptor positive versus negative, the association with diabetes remained significant only in the former (OR = 5.13, 95% CI 1.53–17.22). These results suggest that prediabetes and diabetes are strongly associated with BC incidence and that these metabolic conditions may be more relevant in the presence of breast cancer molecular subtypes with positive hormone receptors. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-09-30T08:12:46.992363-05:
      DOI: 10.1002/jcp.25579
  • Mitochondria-Targeted Antioxidants and Uncouplers of Oxidative
           Phosphorylation in Treatment of the Systemic Inflammatory Response
           Syndrome (SIRS)
    • Authors: Vlada V. Zakharova; Olga Yu. Pletjushkina, Roman A. Zinovkin, Ekaterina N. Popova, Boris V. Chernyak
      Abstract: Systemic inflammatory response syndrome (SIRS) development is accompanied by mitochondrial dysfunction and excessive ROS production. Mitochondrial dysfunctions also occur in many SIRS-related diseases and may be critical for their pathogenesis; therefore a use of mitochondria-targeted drugs is a promising trend in SIRS research and therapy. Here we review recent studies concerning the application of the mitochondria-targeted antioxidants and uncouplers of oxidative phosphorylation in animal models of SIRS and related diseases. We propose that a new class of uncouplers of oxidative phosphorylation, lipophilic cations could be a base for a new generation of drugs for SIRS treatment. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-29T14:05:12.571562-05:
      DOI: 10.1002/jcp.25626
  • Basic Signaling in Cardiac Fibroblasts
    • Authors: Stéphanie Chacar; Nassim Farès, Patrick Bois, Jean-François Faivre
      Abstract: Cardiac fibroblasts are commonly known as supporting cells of the cardiac network and exert many essential functions that are fundamental for normal cardiac growth as well as for cardiac remodeling process during pathological conditions. This review focuses on the roles of cardiac fibroblasts in the formation and regulation of the extracellular matrix components, and in maintaining structural, biochemical and mechanical properties of the heart. Additionally, though considered as non-excitable cells, we review the functional expression in cardiac fibroblasts of a wide variety of transmembrane ion channels which activity may contribute to key regulation of cardiac physiological processes. All together, cardiac fibroblasts which actively participate to fundamental regulation of cardiac physiology and physiopathology processes may represent pertinent targets for pharmacological approaches of cardiac diseases and lead to new tracks of therapeutic strategies. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-29T14:05:11.464802-05:
      DOI: 10.1002/jcp.25624
  • Exhaustive Training Leads to Hepatic Fat Accumulation
    • Authors: Alisson L. da Rocha; Ana P. Pinto, Giovana R. Teixeira, Bruno C. Pereira, Luciana C. Oliveira, Adriana C. Silva, Gustavo P. Morais, Dennys E. Cintra, José R. Pauli, Adelino S. R. da Silva
      Abstract: Recently, we demonstrated that an overtraining (OT) protocol for mice based on downhill running sessions increased the hepatic phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (S6K1; Thr389), a downstream target of the mammalian target of rapamycin complex 1 (mTORC1). In liver, the overactivation of the Akt/mTORC1 pathway induces lipogenesis via regulation of the action of sterol regulatory element binding protein-1 (SREBP-1) at multiple steps. Herein, we verified the effects of three running OT models with same external load (i.e., the product between intensity and volume of training), but performed in downhill, uphill and without inclination, on the proteins related to the mTORC1 signaling pathway, the protein content of the SREBP-1, ACC and FAS, and the morphological characteristics of C57BL/6 mouse livers. In summary, the downhill running-induced OT model up-regulated the levels of major proteins of the mTORC1 signaling pathway, the protein levels of SREBP-1 (p125 precursor) and induced signs of cell swelling accompanied by acute inflammation. The other two OT protocols performed uphill and without inclination did not modulate the most analyzed molecular proteins, but induced hepatic morphological alterations, suggesting an acute pathological adaptation. The three OT models induced hepatic fat accumulation. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-29T14:05:10.503024-05:
      DOI: 10.1002/jcp.25625
  • New Insights on the Regulation of Ca2+-Activated Chloride Channel TMEM16A
    • Authors: Ke Ma; Hui Wang, Jiankun Yu, Minjie Wei, Qinghuan Xiao
      Abstract: TMEM16A, also known as anoctamin 1, is a recently identified Ca2+-activated chloride channel and the first member of a 10-member TMEM16 family. TMEM16A dysfunction is implicated in many diseases such as cancer, hypertension, and cystic fibrosis. TMEM16A channels are well known to be dually regulated by voltage and Ca2+. In addition, recent studies have revealed that TMEM16A channels are regulated by many molecules such as calmodulin, protons, cholesterol, and phosphoinositides, and a diverse range of stimuli such as thermal and mechanical stimuli. A better understanding of the regulatory mechanisms of TMEM16A is important to understand its physiological and pathological role. Recently, the crystal structure of a TMEM16 family member from the fungus Nectria haematococcaten (nhTMEM16) is discovered, and provides valuable information for studying the structure and function of TMEM16A. In this review, we discuss the structure and function of TMEM16A channels based on the crystal structure of nhTMEM16A and focus on the regulatory mechanisms of TMEM16A channels. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:13.785451-05:
      DOI: 10.1002/jcp.25621
  • Understanding the Pathogenesis of Neurotrophic Keratitis: The Role of
           Corneal Nerves
    • Authors: Leonardo Mastropasqua; Giacomina Massaro-Giordano, Mario Nubile, Marta Sacchetti
      Abstract: Neurotrophic keratitis (NK) is a rare degenerative disease of the cornea caused by trigeminal nerve damage, which leads to loss of corneal sensitivity, corneal epithelium breakdown, and poor healing. Though extremely uncommon, NK is increasingly recognized for its characteristics as a distinct and well-defined clinical entity rather than a rare complication of various diseases that can disrupt trigeminal innervation. Indeed, the defining feature of NK is loss of corneal sensitivity, and its clinical findings do not correlate with the wide range of systemic or ocular conditions that underlie trigeminal nerve damage. Despite increasing awareness of NK as a distinct condition, its management continues to be challenged by the lack of treatments that target nerve regeneration. This review focuses on the role of corneal nerves in maintaining ocular surface homeostasis, the consequences (such as alterations in neuromediators and corneal cell morphology/function) of impaired innervation, and advances in NK diagnosis and management. Novel therapeutic strategies should aim to improve corneal innervation in order support corneal renewal and healing. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:09.942628-05:
      DOI: 10.1002/jcp.25623
  • Identification of a Novel Human E-Cadherin Splice Variant and Assessment
           of its Effects upon EMT-Related Events
    • Authors: María Laura Matos; Lara Lapyckyj, Arina Rosso, María José Besso, María Victoria Mencucci, Clara Isabel Marín Briggiler, Silvina Giustina, Laura Inés Furlong, Mónica Hebe Vazquez-Levin
      Abstract: Epithelial Cadherin (E-cadherin) is involved in calcium-dependent cell-cell adhesion and signal transduction. The E-cadherin decrease/loss is a hallmark of Epithelial to Mesenchymal Transition (EMT), a key event in tumor progression. The underlying molecular mechanisms that trigger E-cadherin loss and consequent EMT have not been completely elucidated. This study reports the identification of a novel human E-cadherin variant mRNA produced by alternative splicing. A bioinformatics evaluation of the novel mRNA sequence and biochemical verifications suggest its regulation by Nonsense-Mediated mRNA Decay (NMD). The novel E-cadherin variant was detected in 29/42 (69%) of human tumor cell lines, expressed at variable levels (E-cadherin variant expression relative to the wild type mRNA = 0.05%-11.6%). Stable transfection of the novel E-cadherin variant in MCF-7 cells (MCF7Ecadvar) resulted in downregulation of wild type E-cadherin expression (transcript/protein) and EMT-related changes, among them cell acquisition of fibroblastic-like phenotype, increased expression of Twist, Snail, Zeb1 and Slug transcriptional repressors and decreased expression of ESRP1 and ESRP2 RNA binding proteins. Moreover, loss of cytokeratins and gain of vimentin, N-cadherin and Dysadherin/FXYD5 proteins was observed. Dramatic changes in cell behavior were found in MCF7Ecadvar, as judged by the decreased cell-cell adhesion (Hanging-drop assay), increased cell motility (Wound Healing) and increased cell migration (Transwell) and invasion (Transwell w/Matrigel). Some changes were found in MCF-7 cells incubated with culture medium supplemented with conditioned medium from HEK-293 cells transfected with the E-cadherin variant mRNA. Further characterization of the novel E-cadherin variant will help understanding molecular basis of tumor progression and improve cancer diagnosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:07.213371-05:
      DOI: 10.1002/jcp.25622
  • p120-Catenin Is Required for Dietary Calcium Suppression of Oral
           Carcinogenesis in Mice
    • Authors: Zhongjian Xie; Yuan Yuan, Yi Jiang, Chandrama Shrestha, Ying Chen, Liyan Liao, Shangli Ji, Xiaoge Deng, Eryuan Liao, Daniel D. Bikle
      Abstract: Previous studies have shown that dietary calcium suppresses oral carcinogenesis, but the mechanism is unclear. p120-catenin (p120) is a cytoplasmic protein closely associated with E-cadherin to form the E-cadherin-β-catenin complex and may function as a tumor suppressor in the oral epithelium. To determine whether p120 is involved in the mechanism by which dietary calcium suppresses oral carcinogenesis, The normal, low or high calcium diet was fed control mice (designated as floxed p120 mice) or mice in which p120 was specifically deleted in the oral squamous epithelium during the adult stage (designated as p120cKO mice). All mice were exposed to a low dose of oral cancer carcinogen 4-nitroquinoline 1-oxide and rates of oral squamous cell carcinoma (OSCC) and proliferation and differentiation in the cancerous and non-cancerous oral epithelium of these mice were examined. The results showed that the low calcium diet increased rates of OSCC and proliferation of the non-cancerous oral epithelium and decreased differentiation of the non-cancerous oral epithelium, but had no effect on cancerous oral epithelium. In contrast, the high calcium diet had opposite effects. However, the effect of the dietary calcium on the rates of OSCC, proliferation and differentiation of the non-cancerous epithelium were not seen in p120cKO mice. Based on these results, we conclude that p120 is required for dietary calcium suppression of oral carcinogenesis and oral epithelial proliferation and dietary calcium induction of oral epithelial differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-28T15:52:02.467485-05:
      DOI: 10.1002/jcp.25620
  • Diverse Functions and Signal Transduction of the Exocyst Complex in Tumor
    • Authors: Toshiaki Tanaka; Kaoru Goto, Mitsuyoshi Iino
      Abstract: The exocyst complex is a large conserved hetero-oligomeric complex that consists of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 subunits. It has been implicated in the targeting of vesicles for regulated exocytosis in various cell types, and is also important for targeted exocytosis of post-Golgi transport vesicles to the plasma membrane. The exocyst complex is essential for membrane growth, secretion and function during exocytosis and endocytosis. Moreover, the individual components of the complex are thought to act on specific biological processes, such as cytokinesis, ciliogenesis, apoptosis, autophagy and epithelial-mesenchymal transition (EMT). As a result, recent studies suggest that the exocyst complex may be involved in several diseases such as kidney disease, neuropathogenesis, diabetes, and cancer. In this review, we focus on the diverse functions and cellular signaling pathways of the exocyst complex in various tumors. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-26T12:15:22.951594-05:
      DOI: 10.1002/jcp.25619
  • Nephroprotective Effects of Metformin in Diabetic Nephropathy
    • Authors: Sreenithya Ravindran; Vinitha Kuruvilla, Kerry Wilbur, Shankar Munusamy
      Abstract: Metformin, a well-known anti-diabetic agent, is very effective in lowering blood glucose in patients with type 2 diabetes with minimal side-effects. Metformin is also being recommended in the treatment of obesity and polycystic ovary syndrome. Metformin elicits its therapeutic effects mainly via activation of AMP-activated kinase (AMPK) pathway. Renal cells under hyperglycemic or proteinuric conditions exhibit inactivation of cell defense mechanisms such as AMPK and autophagy, and activation of pathologic pathways such as mammalian target of rapamycin (mTOR), endoplasmic reticulum (ER) stress, epithelial-to-mesenchymal transition (EMT), oxidative stress, and hypoxia. As these pathologic pathways are intertwined with AMPK signaling, the potential benefits of metformin therapy in patients with type 2 diabetes would extend beyond its anti-hyperglycemic effects. However, since metformin is eliminated unchanged through the kidneys and some studies have shown the incidence of lactic acidosis with its use during severe renal dysfunction, the use of metformin was contraindicated in patients with renal disease until recently. With more studies indicating the relatively low incidence of lactic acidosis and revealing the additional benefits with metformin therapy, the US FDA has now approved metformin to be administered in patients with established renal disease based on their renal function. The purpose of this review is to highlight the various mechanisms by which metformin protects renal cells that have lost its functionality in a diabetic or non-diabetic setting and to enlighten the advantages and therapeutic potential of metformin as a nephroprotectant for patients with diabetic nephropathy and other non-diabetic forms of chronic kidney disease. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.Metformin, a popular anti-diabetic drug and an AMPK activator, modulates various signaling pathways altered in diabetic nephropathy and exhibits nephroprotective properties in various animal models of diabetes. Additional studies in humans are needed to ascertain its therapeutic potential as a nephroprotectant to treat nephropathy in patients with diabetes.
      PubDate: 2016-09-26T11:05:22.881493-05:
      DOI: 10.1002/jcp.25598
  • MnTBAP Inhibits Bleomycin-Induced Pulmonary Fibrosis by Regulating VEGF
           and Wnt Signaling
    • Authors: Rajkumar Venkatadri; Anand Krishnan V. Iyer, Vani Ramesh, Clayton Wright, Carlos A. Castro, Juan S. Yakisich, Neelam Azad
      Abstract: Cellular oxidative stress is implicated not only in lung injury but also in contributing to the development of pulmonary fibrosis. We demonstrate that a cell-permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger, manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly inhibited bleomycin-induced fibrogenic effects both in vitro and in vivo. Further investigation into the underlying mechanisms revealed that MnTBAP targets canonical Wnt and non-canonical Wnt/Ca2+ signaling pathways, both of which were upregulated by bleomycin treatment. The effect of MnTBAP on canonical Wnt signaling was significant in vivo but inconclusive in vitro and the non-canonical Wnt/Ca2+ signaling pathway was observed to be the predominant pathway regulated by MnTBAP in bleomycin-induced pulmonary fibrosis. Furthermore, we show that the inhibitory effects of MnTBAP involve regulation of VEGF which is upstream of the Wnt signaling pathway. Overall, the data show that the superoxide scavenger MnTBAP attenuates bleomycin-induced pulmonary fibrosis by targeting VEGF and Wnt signaling pathways. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-09-26T10:55:22.029958-05:
      DOI: 10.1002/jcp.25608
  • G Protein-Coupled Receptor Endosomal Signaling and Regulation of Neuronal
           Excitability and Stress Responses: Signaling Options and Lessons from the
           PAC1 Receptor
    • Authors: Victor May; Rodney L. Parsons
      Abstract: Our understanding of G protein coupled receptor (GPCR) mechanisms and functions have evolved considerably. Among the many conceptual realignments, GPCRs can exist in an ensemble of active microstates that have the potential to differentially engage specific downstream signaling events. Furthermore, among GPCR dynamics, GPCR internalization and vesicular trafficking are no longer solely mechanisms for desensitization, but now appreciated to form intricate endosomal signaling complexes that can potentially target second messengers to intracellular compartments with high temporal and spatial resolution. The PACAPergic system is important in the maintenance of physiological homeostasis in the central and peripheral nervous systems and activation of the PACAP-selective PAC1 receptor can generate differential but coordinate plasma membrane and endosomal signals for cellular responses. The integration of these signals can modulate PACAP-induced changes in ionic conductances that gate neuronal excitability. PACAP/PAC1 receptor generation of endosomal ERK signals participate in chronic pain and anxiety-like responses which can be attenuated with endocytosis inhibitors. From the abilities of ligands to stabilize the different GPCR microstates for biased downstream signaling, the development of biased PAC1 receptor agonists and antagonists may provide opportunities to dissociate the homeostatic regulatory signals of PACAP from the maladaptive effects. In particular, the development of biased antagonists to PAC1 receptor-mediated endosomal signaling may offer therapeutic options for chronic pain and stress-related disorders. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-23T17:41:35.805915-05:
      DOI: 10.1002/jcp.25615
  • The Role of Calcium-Sensing Receptors in Endothelin-1-Dependent Effects on
           Adult Rat Ventricular Cardiomyocytes: Possible Contribution to Adaptive
           Myocardial Hypertrophy
    • Authors: Elena Dyukova; Rolf Schreckenberg, Christoph Arens, Guzel Sitdikova, Klaus-Dieter Schlüter
      Abstract: Nitric oxide (NO)-deficiency as it occurs during endothelial dysfunction activates the endothelin-1 system and increases the expression of receptor activity modifying protein (RAMP)-1 that acts as a chaperon for calcium-sensing receptors (CaR) that have recently been identified to improve cardiac function. Here we hypothesized that endothelin-1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP-1, endothelin receptors, and CaR were analyzed by RT-PCR in left ventricular tissues of L-NAME-treated rats. Effects of ET-1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP-1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1, ETB2, and ETA receptors. Non-specific NO synthase inhibition with L-Nitro arginine methyl ester (L-NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of endothelin-1 on cardiomyocytes. Indeed, endothelin-1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Down-regulation of RAMP-1 or selective inhibition of ETA and ETB1 receptors led endothelin-1 reducing cell shortening and attenuates up-regulation of CaR. In conclusion, endothelin-1 causes an adaptive type of hypertrophy by up-regulation of CaR in cardiomyocytes via ETA receptors. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-23T17:41:28.634426-05:
      DOI: 10.1002/jcp.25612
  • Anti-Tumor Effects of Cardiac Glycosides on Human Lung Cancer Cells and
           Lung Tumorspheres
    • Authors: Vivek Kaushik; Juan Sebastian Yakisich, Neelam Azad, Yogesh Kulkarni, Rajkumar Venkatadri, Clayton Wright, Yon Rojanasakul, Anand Krishnan V. Iyer
      Abstract: Lung cancer is a leading cause of cancer-related death in the United States. While several drugs have been developed that target individual biomarkers, their success has been limited due to intrinsic or acquired resistance for the specific targets of such drugs. A more effective approach is to target multiple pathways that dictate cancer progression. Cardiac glycosides demonstrate such multimodal effects on cancer cell survival, and our aim was to evaluate the effect of two naturally occurring monosaccaridic cardiac glycosides – Convallatoxin and Peruvoside on lung cancer cells. While both drugs had significant anti-proliferative effects on H460 and Calu-3 lung cancer cells, Convallatoxin demonstrated two-fold higher activity as compared to Peruvoside using both viability and colony forming assays, suggesting a role for the aglycone region in dictating drug potency. The tumor suppressor p53 was found to be important for action of both drugs – p53-underexpressing cells were less sensitive as compared to p53-positive H460 cells. Further assessment of p53-underexpressing H460 cells showed that drugs were able to arrest cells in the G0/G1 phase of the cell cycle in a dose-dependent manner. Both drugs significantly inhibited migration and invasion of cancer cells and decreased the viability of floating tumorspheres. An assessment of intracellular pathways indicated that both drugs were able to modulate proteins that are involved in apoptosis, autophagy, cell cycle, proliferation and EMT. Our data suggests a promising role for cardiac glycosides in lung cancer treatment, and provides impetus for further investigation of the anti-cancer potential of this class of drugs. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-23T17:35:52.30901-05:0
      DOI: 10.1002/jcp.25611
  • Interfering of the Reelin/ApoER2/PSD95 Signaling Axis Reactivates
           Dendritogenesis of Mature Hippocampal Neurons
    • Authors: Estibaliz Ampuero; Nur Jury, Steffen Härtel, María-Paz Marzolo, Brigitte van Zundert
      Abstract: Reelin, an extracellular glycoprotein secreted in embryonic and adult brains, participates in neuronal migration and neuronal plasticity. Extensive evidence shows that reelin via activation of the ApoER2 and VLDLR receptors promotes dendrite and spine formation during early development. Further evidence suggests that reelin signaling is needed to maintain a stable architecture in mature neurons, but, direct evidence is lacking. During activity-dependent maturation of the neuronal circuitry, the synaptic protein PSD95 is inserted into the postsynaptic membrane to induce structural refinement and stability of spines and dendrites. Given that ApoER2 interacts with PSD95, we tested if reelin signaling interference in adult neurons reactivates the dendritic architecture. Unlike findings in developing cultures, the presently obtained in vitro and in vivo data show, for the first time, that reelin signaling interference robustly increase dendritogenesis and reduce spine density in mature hippocampal neurons. In particular, the expression of a mutant ApoER2 form (ApoER2-tailless), which is unable to interact with PSD95 and hence cannot transduce reelin signaling, resulted in robust dendritogenesis in mature hippococampal neurons in vitro. These results indicate that reelin/ApoER2/PSD95 signaling is important for neuronal structure maintenance in mature neurons. Mechanistically, obtained immunofluorescent data indicate that reelin signaling impairment reduced synaptic PSD95 levels, consequently leading to synaptic re-insertion of NR2B-NMDARs. Our findings underscore the importance of reelin in maintaining adult network stability and reveal a new mode for reactivating dendritogenesis in neurological disorders where dendritic arbor complexity is limited, such as in depression, Alzheimer's disease, and stroke. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-21T12:25:21.899058-05:
      DOI: 10.1002/jcp.25605
  • HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1
    • Authors: Eleonora Foglio; Giovanni Puddighinu, Antonia Germani, Matteo A. Russo, Federica Limana
      Abstract: Exogenous High Mobility Group Box-1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex. In particular, its effect on ischemic cardiomyocytes has been poorly investigated. Aim of the present study was to verify whether and how autophagy and apoptosis were involved in HMGB1-induced heart repair following myocardial infarction (MI). HMGB1 (200 ng) or denatured HMGB1 were injected in the peri-infarcted region of mouse hearts following acute MI. Three days after treatment, an upregulation of autophagy was detected in infarcted HMGB1 treated hearts compared to controls. Specifically, HMGB1 induced autophagy by significantly upregulating the protein expression of LC3, Beclin-1, and Atg7 in the border zone. To gain further insights into the molecular mechanism of HMGB1-mediated autophagy, WB analysis were performed in cardiomyocytes isolated from 3 days infarcted hearts in the presence and in the absence of HMGB1 treatment. Results showed that upregulation of autophagy by HMGB1 treatment was potentially related to activation of AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Accordingly, in these hearts, phospho-Akt signaling pathway was inhibited. The induction of autophagy was accompanied by reduced cardiomyocyte apoptotic rate and decreased expression levels of Bax/Bcl-2 and active caspase-3 in the border zone of 3 days infarcted mice following HMGB1 treatment. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP-activated protein kinase-dependent autophagy. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.Exogenous High Mobility Group Box-1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex; in particular, its effect on ischemic cardiomyocytes has been poorly investigated. HMGB1 (200 ng) or denatured HMGB1 were injected in the peri-infarcted region of mouse hearts following acute MI. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP-activated protein kinase-dependent autophagy.
      PubDate: 2016-09-21T10:55:22.031754-05:
      DOI: 10.1002/jcp.25576
  • VIP Family Members Prevent Outer Blood Retinal Barrier Damage in a Model
           of Diabetic Macular Edema
    • Authors: Grazia Maugeri; Agata Grazia D'Amico, Caterina Gagliano, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata
      Abstract: Diabetic macular edema (DME), characterized by an increase of thickness in the eye macular area, is due to breakdown of the blood-retinal barrier (BRB). Hypoxia plays a key role in the progression of this pathology by activating the hypoxia-inducible factors. In the last years, various studies have put their attention on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in retinal dysfunction. However, until now, no study has investigated their protective role against the harmful combined effect of both hyperglycemia and hypoxia on outer BRB. Therefore, in the present study, we have analyzed the role of these peptides on permeability, restoration of tight junctions expression and inhibition of hyperglycemia/hypoxia-induced apoptosis, in an experimental in vitro model of outer BRB. Our results have demonstrated that the peptides' treatment have restored the integrity of outer BRB induced by cell exposure to hyperglycemia/hypoxia. Their effect is mediated through the activation of phosphoinositide 3 kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling pathways. In conclusion, our study further clarifies the mechanism through which PACAP and VIP perform the beneficial effect on retinal damage induced by hyperglycemic/hypoxic insult, responsible of DME progression. J. Cell. Physiol. 9999: 1–7, 2016. © 2016 Wiley Periodicals, Inc.PACAP and VIP restore the integrity of outer BRB exposed to hyperglycemia/hypoxia. PACAP and VIP perform their beneficial effect on retinal damage through activation of PI3K/Akt and MAPK/ERK signaling cascades.
      PubDate: 2016-09-21T10:45:25.121149-05:
      DOI: 10.1002/jcp.25510
  • Emerging Roles of Transforming Growth Factor β Signaling in Diabetic
    • Authors: Sarah E. Wheeler; Nam Y. Lee
      Abstract: Diabetic retinopathy (DR) is a serious complication of diabetes mellitus affecting about one third of diabetic adults. Despite its prevalence, treatment options are limited and often implemented only in the later stages of the disease. To date, the pathogenesis of DR has been extensively characterized in the context of elevated glucose, insulin, and VEGF signaling, although a growing number of other growth factors and molecules, including transforming growth factor β (TGF-β) are being recognized as important contributors and/or therapeutic targets. Here, we review the complex roles of TGF-β signaling in DR pathogenesis and progression. J. Cell. Physiol. 9999: 1–4, 2016. © 2016 Wiley Periodicals, Inc.Transforming growth factor β (TGF-β) has a clear role in the pathogenesis of diabetic retinopathy, although the underlying mechanisms are poorly established. Here, we discuss our current but limited understanding of how signaling by TGF-β superfamily ligands contributes to the pathogenesis and progression of diabetic retinopathy.
      PubDate: 2016-09-21T10:40:24.736228-05:
      DOI: 10.1002/jcp.25506
  • Functional Effects of Cigarette Smoke-Induced Changes in Airway Smooth
           Muscle Mitochondrial Morphology
    • Authors: Bharathi Aravamudan; Michael Thompson, Gary C. Sieck, Robert Vassallo, Christina M. Pabelick, Y. S. Prakash
      Abstract: Long-term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). We previously showed that CS destabilizes the networked morphology of mitochondria in human ASM by regulating the expression of mitochondrial fission and fusion proteins via multiple signaling mechanisms. Emerging data link regulation of mitochondrial morphology to cellular structure and function. We hypothesized that CS-induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling. Here, ASM cells were exposed to 1% cigarette smoke extract (CSE) for 48 h to alter mitochondrial fission/fusion, or by inhibiting the fission protein Drp1 or the fusion protein Mfn2. Mitochondrial function was assessed via changes in bioenergetics or altered rates of proliferation and apoptosis. Our results indicate that both exposure to CS and inhibition of mitochondrial fission/fusion proteins affect mitochondrial function (i.e., energy metabolism, proliferation, and apoptosis) in ASM cells. In vivo, the airways in mice chronically exposed to CS are thickened and fibrotic, and the expression of proteins involved in mitochondrial function is dramatically altered in the ASM of these mice. We conclude that CS-induced changes in mitochondrial morphology (fission/fusion balance) correlate with mitochondrial function, and thus may control ASM proliferation, which plays a central role in airway health. J. Cell. Physiol. 9999: 1–16, 2016. © 2016 Wiley Periodicals, Inc.Long-term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). Previous studies show that CS destabilizes the networked morphology of mitochondria in human ASM. We hypothesized that CS-induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling.
      PubDate: 2016-09-21T10:15:28.976808-05:
      DOI: 10.1002/jcp.25508
  • Effects of Conjugated Linoleic Acid Associated With Endurance Exercise on
           Muscle Fibres and Peroxisome Proliferator-Activated Receptor γ
           Coactivator 1 α Isoforms
    • Authors: Rosario Barone; Claudia Sangiorgi, Antonella Marino Gammazza, Daniela D'Amico, Monica Salerno, Francesco Cappello, Cristoforo Pomara, Giovanni Zummo, Felicia Farina, Valentina Di Felice, Filippo Macaluso
      Abstract: Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre-type-specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 μl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6-week experimental period, whereas the placebo groups were gavaged with 35 μl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota-Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2, and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre-type-specific hypertrophy induced by endurance exercise (of types I and IIb muscle fibres). J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres.
      PubDate: 2016-09-21T10:15:23.022164-05:
      DOI: 10.1002/jcp.25511
  • Endothelial STAT3 Activation Increases Vascular Leakage Through
           Downregulating Tight Junction Proteins: Implications for Diabetic
    • Authors: Jang-Hyuk Yun; Sung Wook Park, Kyung-Jin Kim, Jong-Sup Bae, Eun Hui Lee, Sun Ha Paek, Seung U. Kim, Sangkyu Ye, Jeong-Hun Kim, Chung-Hyun Cho
      Abstract: Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro-inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL-6-induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO-1 and occludin. In a co-culture model with microglia and endothelial cells under a high glucose condition, the microglia-derived IL-6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL-6-induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO-1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL-6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.Vascular inflammation is characteristic feature of diabetic retinopathy. We demonstrated that STAT3 in retinal endothelial cells plays a causative role in downregulating ZO-1 and occludin levels and increasing the endothelial permeability through VEGF production in retinal endothelial cells. These results suggest the potential importance of IL-6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy.
      PubDate: 2016-09-21T10:05:28.489171-05:
      DOI: 10.1002/jcp.25575
  • Heat Stress Modulates both Anabolic and Catabolic Signaling Pathways
           Preventing Dexamethasone-Induced Muscle Atrophy In Vitro
    • Authors: Wakako Tsuchida; Masahiro Iwata, Takayuki Akimoto, Shingo Matsuo, Yuji Asai, Shigeyuki Suzuki
      Abstract: It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid-induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid-induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel-like factor 15 (KLF15). Heat stress recovered the dexamethasone-induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress-induced protection against glucocorticoid-induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-20T14:20:23.775655-05:
      DOI: 10.1002/jcp.25609
  • Human Telomerase Reverse Transcriptase (hTERT) Positively Regulates 26S
           Proteasome Activity
    • Authors: Eunju Im; Jong Bok Yoon, Han-Woong Lee, Kwang Chul Chung
      Abstract: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, an RNA-dependent DNA polymerase that elongates telomeric DNA. hTERT displays several extra-telomeric functions that are independent of its telomere-regulatory function, including tumor progression and neuronal cell death regulation. In this study, we evaluated these additional hTERT non-telomeric functions. We determined that hTERT interacts with several 19S and 20S proteasome subunits. The 19S regulatory particle and 20S core particle are part of 26S proteasome complex, which plays a central role in ubiquitin-dependent proteolysis. In addition, hTERT positively regulated 26S proteasome activity independent of its enzymatic activity. Moreover, hTERT enhanced subunit interactions, which may underlie hTERT's ability of hTERT to stimulate the 26S proteasome. Furthermore, hTERT displayed cytoprotective effect against ER stress via the activation of 26S proteasome in acute myeloid leukemia cells. Our data suggest that hTERT acts as a novel chaperone to promote 26S proteasome assembly and maintenance. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-20T14:15:22.299963-05:
      DOI: 10.1002/jcp.25607
  • Pulsed Electromagnetic Field Exposure Reduces Hypoxia and Inflammation
           Damage in Neuron-Like and Microglial Cells
    • Authors: Fabrizio Vincenzi; Annalisa Ravani, Silvia Pasquini, Stefania Merighi, Stefania Gessi, Stefania Setti, Ruggero Cadossi, Pier Andrea Borea, Katia Varani
      Abstract: In the present study the effect of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) has been investigated by using different cell lines derived from neuron-like cells and microglial cells. In particular, the primary aim was to evaluate the effect of PEMF exposure in inflammation- and hypoxia-induced injury in two different neuronal cell models, the human neuroblastoma-derived SH-SY5Y cells and rat pheochromocytoma PC12 cells and in N9 microglial cells. In neuron-like cells, live/dead and apoptosis assays were performed in hypoxia conditions from 2 to 48 h. Interestingly, PEMF exposure counteracted hypoxia damage significantly reducing cell death and apoptosis. In the same cell lines, PEMFs inhibited the activation of the hypoxia-inducible factor 1α (HIF-1α), the master transcriptional regulator of cellular response to hypoxia. The effect of PEMF exposure on reactive oxygen species (ROS) production in both neuron-like and microglial cells was investigated considering their key role in ischemic injury. PEMFs significantly decreased hypoxia-induced ROS generation in PC12, SH-SY5Y and N9 cells after 24 or 48 h of incubation. Moreover, PEMFs were able to reduce some of the most well-known pro-inflammatory cytokines such as tumor necrosis factor -α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 release in N9 microglial cells stimulated with different concentrations of LPS for 24 or 48 h of incubation time. These results show a protective effect of PEMFs on hypoxia damage in neuron-like cells and an anti-inflammatory effect in microglial cells suggesting that PEMFs could represent a potential therapeutic approach in cerebral ischemic conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:30:51.474977-05:
      DOI: 10.1002/jcp.25606
  • Valproic Acid and Lithium Meditate Anti-Inflammatory Effects by
           Differentially Modulating Dendritic Cell Differentiation and Function
    • Authors: Sy-Jye Leu; Yi-Yuan Yang, Hsing-Cheng Liu, Chieh-Yu Cheng, Yu-Chen Wu, Ming-Chyi Huang, Yuen-Lun Lee, Chi-Ching Chen, Winston W. Shen, Ko-Jiunn Liu
      Abstract: Valproic acid (VPA), with inhibition activity mainly towards histone deacetylase (HDAC) and Glycogen Synthase Kinase (GSK)-3, and lithium, with inhibition activity mainly toward GSK-3, are both prescribed in clinical as mood-stabilizers and anticonvulsants for the control of bipolar disorder. This study aims to compare the immuno-modulation activities of VPA and lithium, especially on the differentiation and functions of dendritic cells (DC). Our data show that treatment with VPA or lithium effectively alleviated the severity of collagen-induced arthritis triggered by LPS in mice. Both agents reduced the serum level of IL-6 and IL-10 after LPS challenge in mice. VPA and lithium both induce significant down-regulation of group I CD1 expression and secretion of IL-6 during differentiation of human monocyte-derived immature DC, while they differ in the induction of CD83 and CD86 expression, secretion of IL-8, IL-10, and TNF-α. Upon stimulation of immature DC with LPS, VPA and lithium both reduced the secretion of IL-6 and TNF-α. However, only lithium significantly increased the production of IL-10, while VPA increased the production of IL-8 but substantially reduce the secretion of IL-10 and IL-23. Treatment with VPA resulted in a reduced capacity of LPS-stimulated DC to promote the differentiation of T helper 17 cells that are critical in the promotion of inflammatory responses. Taken together, our results suggest that VPA and lithium may differentially modulate inflammation through regulating the capacity of DC to mediate distinct T cell responses, and they may provide a complementary immunomodulatory effects for the treatment of inflammation-related diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:30:50.294937-05:
      DOI: 10.1002/jcp.25604
  • Human Brain Malignant Glioma (BMG-1) 3D Aggregate Morphology and Screening
           for Cytotoxicity and Anti-Proliferative Effects
    • Authors: Maddaly Ravi; Aarthi Ramesh, Aishwarya Pattabhi
      Abstract: Two interesting aspects of cell lines grown in 3D conditions are their distinct morphology and production of extracellular matrix (ECM). Also, it is known that 3D aggregates have different susceptibilities to damage-inducing agents compared to their 2D monolayer counterparts. We describe the effect of ECM on 3D aggregate morphology, the effect of cisplatin, bleomycin and UV on the 3D aggregates and 2D monolayers of the BMG-1 cell line. We also present a rapid method for analyzing cytotoxicity and anti-proliferative effects of 3D aggregates in 96-well plates. We utilized a single-step protocol using the dye resazurin.BMG-1 cells formed floating aggregates on 1% agarose hydrogels. The extent of ECM formed by them was dependent on number of cells seeded irrespective of the seeding density, which in turn directed the 3D aggregates compactness. The 3D aggregates were less susceptible to cisplatin and UV-induced cytotoxicity compared to 2D counterparts. The IC 50 value of cisplatin was elevated at 210 µg/ml for the aggregates compared to 170µg/ml for the monolayers. Exposure to UV for 10, 20 and 30 minutes gave inhibition values of 2.98%, 8%, 22.99% and 31.8% for the aggregates as compared to 3.06%, 7.5%, 39.4% and 46.7% for the monolayers. While bleomycin-induced effects were unapparent when analyzed by vital staining for the doses used, the rapid, single-step method in 96-well plates was able to provide a dose-response for cytotoxicity and anti-proliferative effects. Also, comparative analysis of results obtained from vital staining and the single-step method demonstrates the reliability of the assay described. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:26:20.762476-05:
      DOI: 10.1002/jcp.25603
  • Early Subchondral Bone Loss at Arthritis Onset Predicted Late Arthritis
           Severity in a Rat Arthritis Model
    • Authors: Guillaume Courbon; Damien Cleret, Marie-Thérèse Linossier, Laurence Vico, Hubert Marotte
      Abstract: Synovitis is usually observed before loss of articular function in rheumatoid arthritis (RA). In addition to the synovium and according to the “Inside-Outside” theory, bone compartment is also involved in RA pathogenesis. Then, we investigated time dependent articular bone loss and prediction of early bone loss to late arthritis severity on the rat adjuvant-induced arthritis (AIA) model. Lewis female rats were longitudinally monitored from arthritis induction (day 0), with early (day 10) and late (day 17) steps. Trabecular and cortical microarchitecture parameters of 4 ankle bones were assessed by microcomputed tomography. Gene expression was determined at sacrifice. Arthritis occurred at day 10 in AIA rats. At this time, bone erosions were detected on 4 ankle bones, with cortical porosity increase (+67%) and trabecular alterations including bone volume fraction (BV/TV: -13%) and trabecular thickness decrease. Navicular bone assessment was the most reproducible and sensitive. Furthermore, strong correlations were observed between bone alterations at day 10 and arthritis severity or bone loss at day 17, including predictability of day 10 BV/TV to day 17 articular index (R2 = 0.76). Finally, gene expression at day 17 confirmed massive osteoclast activation and interestingly provided insights on strong activation of bone formation inhibitor markers at the joint level. In rat AIA, bone loss was already observed at synovitis onset and was predicted late arthritis severity. Our results reinforced the key role of subchondral bone in arthritis pathogenesis, in favour to the “Inside-Outside” theory. Mechanisms of bone loss in rat AIA involved resorption activation and formation inhibition changes. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-16T17:05:52.041277-05:
      DOI: 10.1002/jcp.25601
  • Store-Operated Ca2+ Entry in Oocytes Modulate the Dynamics of
           IP3-Dependent Ca2+ Release From Oscillatory to Tonic
    • Authors: Raphaël Courjaret; Maya Dib, Khaled Machaca
      Abstract: Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal, and amplitude features. Here, we investigate the role of store-operated Ca2+ entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release leads to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low-amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here, we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3R) results in a pump-leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER-plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3Rs at distant sites, a process we refer to as “Ca2+ teleporting.” Therefore, sustained SOCE activation in the presence of an IP3-dependent “leak” pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.Calcium influx through SOCE inhibits calcium oscillations and favors a tonic mode of signaling. This is achieved through focal calcium entry at SOCE clusters, followed by calcium uptake into the ER through SERCA and re-release through open IP3 Receptors. A process referred to as “calcium teleporting.”
      PubDate: 2016-09-07T14:25:44.356793-05:
      DOI: 10.1002/jcp.25513
  • MiR-145 Regulates Lipogenesis in Goat Mammary Cells Via Targeting INSIG1
           and Epigenetic Regulation of Lipid-Related Genes
    • Authors: Hui Wang; Huaiping Shi, Jun Luo, Yongqing Yi, Dawei Yao, Xueying Zhang, Gongzhen Ma, Juan J. Loor
      Abstract: MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post-transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid-lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs, and 821 pre-miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR-145) had significant differences in expression over the lactation cycle. Further studies revealed that miR-145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR-145. Overexpression of miR-145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR-145 impaired fatty acid synthesis. Inhibition of miR-145 increased methylation levels of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), peroxisome proliferator-activated receptor gamma (PPARG), and sterol regulatory element binding transcription factor 1 (SREBF1). Luciferase reporter assays confirmed that insulin induced gene 1 (INSIG1) is a direct target of miR-145. These findings underscore the need for further studies to evaluate the potential for targeting miR-145 for improving beneficial milk components in ruminant milk. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.Mammary lipogenesis is partly regulated by epigenetic mechanisms through miR-145 and INSIG1.
      PubDate: 2016-09-07T14:25:37.979785-05:
      DOI: 10.1002/jcp.25499
  • Early Growth Response 3 (Egr3) Contributes a Maintenance of C2C12 Myoblast
    • Authors: Mitsutoshi Kurosaka; Yuji Ogura, Toshiya Funabashi, Tatsuo Akema
      Abstract: Satellite cell proliferation is a crucially important process for adult myogenesis. However, its regulatory mechanisms remain unknown. Early growth response 3 (Egr3) is a zinc-finger transcription factor that regulates different cellular functions. Reportedly, Egr3 interacts with multiple signaling molecules that are also known to regulate satellite cell proliferation. Therefore, it is possible that Egr3 is involved in satellite cell proliferation. Results of this study have demonstrated that Egr3 transcript levels are upregulated in regenerating mouse skeletal muscle after cardiotoxin injury. Using C2C12 myoblast culture (a model of activated satellite cells), results show that inhibition of Egr3 by shRNA impairs the myoblast proliferation rate. Results also show reduction of NF-кB transcriptional activity in Egr3-inhibited cells. Inhibition of Egr3 is associated with an increase in annexin V+ cell fraction and apoptotic protein activity including caspase-3 and caspase-7, and Poly-ADP ribose polymerase. By contrast, the reduction of cellular proliferation by inhibition of Egr3 was partially recovered by treatment of pan-caspase inhibitor Z-VAD-FMK. Collectively, these results suggest that Egr3 is involved in myoblast proliferation by interaction with survival signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-30T17:45:37.199268-05:
      DOI: 10.1002/jcp.25574
  • Role of thrombin in the pathogenesis of central nervous system
           inflammatory diseases
    • Authors: Safieh Ebrahimi; Najme Jaberi, Amir Avan, Mikhail Ryzhikov, Mohammad Reza Keramati, Mohammad Reza Parizadeh, Seyed Mahdi Hassanian
      Abstract: Thrombin initiates proinflammatory signaling responses through activation of protease-activated receptors (PARs) in in vitro and in vivo systems. Proinflammatory signaling function of thrombin increases secretion of proinflammatory cytokines and chemokines, triggers vascular permeability, promotes leukocyte migration, and induces adhesion molecule expression. Thrombin as a potent signaling molecule is strongly implicated in a number of proinflammatory disorders including severe sepsis, cancer, cardiovascular disease, and of special interest in this review neurodegenerative disorders. This review summarizes the role of thrombin in the pathogenesis of central nervous system (CNS) inflammatory diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), promoting greater understanding and clinical management of these diseases. J. Cell. Physiol. 9999: 1–4, 2016. © 2016 Wiley Periodicals, Inc.Proinflammatory signaling function of thrombin increases secretion of proinflammatory cytokines and chemokines, triggers vascular permeability, promotes leukocyte migration, and induces adhesion molecule expression. This review summarizes the role of thrombin in the pathogenesis of central nervous system (CNS) inflammatory diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), promoting greater understanding and clinical management of these diseases.
      PubDate: 2016-08-29T03:00:22.583247-05:
      DOI: 10.1002/jcp.25501
  • Quantitative Analysis of Endocytic Recycling of Membrane Proteins by
           Monoclonal Antibody-Based Recycling Assays
    • Authors: Gordana Blagojević Zagorac; Hana Mahmutefendić, Senka Maćešić, Ljerka Karleuša, Pero Lučin
      Abstract: In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 9999: 1–14, 2016. © 2016 Wiley Periodicals, Inc.In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules.
      PubDate: 2016-08-22T02:21:41.323098-05:
      DOI: 10.1002/jcp.25503
  • Neuronatin Protein in Health and Disease
    • Authors: Priyamvada M. Pitale; Wayne Howse, Marina Gorbatyuk
      Abstract: Neuronatin (NNAT) was first identified as a brain-specific gene crucial for brain development. Over the years, NNAT has been studied in different developing and post-developed tissues and organs. While NNAT manifests functional and structural similarities to the phospholamban gene, its physiological and pathological roles in healthy and diseased tissues have not been precisely identified. Ca2+ signaling, glucose transport, insulin secretion, and inflammation modulated at different pathological conditions have been proposed to be governed by NNAT. This review describes the current findings of cellular molecular pathways known to be modified concomitantly with an alteration in NNAT expression, and it highlights the need to conduct extensive investigation regarding the role of NNAT in health and disease. J. Cell. Physiol. 9999: 1–5, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-07-27T10:50:23.162398-05:
      DOI: 10.1002/jcp.25498
  • Table of Contents, Editor's Choice, Highlights
    • First page: 457
      PubDate: 2016-11-17T09:27:48.236766-05:
      DOI: 10.1002/jcp.25662
  • Non-Metabolic Role of PKM2 in Regulation of the HIV-1 LTR
    • Authors: Satarupa Sen; Satish L. Deshmane, Rafal Kaminski, Shohreh Amini, Prasun K. Datta
      First page: 517
      Abstract: Identification of cellular proteins, in addition to already known transcription factors such as NF-κB, Sp1, C-EBPβ, NFAT, ATF/CREB, and LEF-1, which interact with the HIV-1 LTR, is critical in understanding the mechanism of HIV-1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells, a macrophage model of latency. We observed that HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA-induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA-induced U1 cells in comparison to PMA-induced U937 cells. We focused on understanding the potential role of PKM2 in HIV-1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA-activated U1 and TZM-bl cells demonstrated the interaction of PKM2 with the HIV-1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV-1 LTR-luciferase reporter in U937, U-87 MG, and TZM-bl cells. Using various truncated constructs of the HIV-1 LTR, we mapped the region spanning −120 bp to −80 bp to be essential for PKM2-mediated transactivation. This region contains the NF-κB binding site and deletion of this site attenuated PKM2-mediated activation of HIV-1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF-κB. These observations demonstrate for the first time that PKM2 is a transcriptional co-activator of HIV-1 LTR. J. Cell. Physiol. 232: 517–525, 2017. © 2016 Wiley Periodicals, Inc.PKM2 is a transcriptional co-activator of HIV-1 LTR.
      PubDate: 2016-06-10T10:01:42.21665-05:0
      DOI: 10.1002/jcp.25445
  • T11TS Treatment Augments Apoptosis of Glioma Associated Brain Endothelial
           Cells, Hint Toward Anti-Angiogenic Action in Glioma
    • Authors: Debanjan Bhattacharya; Manoj Kumar Singh, Suhnrita Chaudhuri, Ankur Datta, Swapna Chaudhuri
      First page: 526
      Abstract: Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor-associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11-target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma-associated brain endothelial cells. In the present study, we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma-associated brain endothelial cells. Annexin-V/PI staining showed that T11TS treatment in glioma-induced rats increases apoptosis of glioma-associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC-1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma-associated brain endothelial cells. Flowcytometry, immunoblotting, and in situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment-upmodulated expression of p53, Bax, Fas, FasL, and FADD in glioma associated endothelial cells and downregulated Bcl-2 protein. T11TS therapy induced cytochrome-c release into cytosol, activated caspase −9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma-associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas-dependent extrinsic pathway. The pro-apoptotic action of T11TS on glioma-associated endothelial cells provides crucial insight into how T11TS exerts its anti-angiogenic function in glioma. J. Cell. Physiol. 232: 526–539, 2017. © 2016 Wiley Periodicals, Inc.T11TS induced apoptosis of glioma-associated brain endothelial cells and its effect on glioma angiogenesis.
      PubDate: 2016-06-21T10:15:40.99355-05:0
      DOI: 10.1002/jcp.25447
  • Ethanol Extract of Cissus quadrangularis Enhances Osteoblast
           Differentiation and Mineralization of Murine Pre-Osteoblastic MC3T3-E1
    • Authors: Raazia Tasadduq; Jonathan Gordon, Khalid A. Al-Ghanim, Jane B. Lian, Andre J. Van Wijnen, Janet L. Stein, Gary S. Stein, Abdul Rauf Shakoori
      First page: 540
      Abstract: Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti-osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre-osteoblast cell line, MC3T3-E1. Ethanolic extract of CQ (CQ-E) was found to affect growth kinetics of MC3T3-E1 cells in a dosage-dependent manner. High concentrations of CQ-E (more than 10 μg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non-toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ-E. CQ-E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose-dependent effect of CQ-E with lower concentrations exhibiting anabolic and osteogenic properties. J. Cell. Physiol. 232: 540–547, 2017. © 2016 Wiley Periodicals, Inc.Ethanolic extract of a herb, Cissus quadrangularis (CQ-E) has dose dependent effect on the differentiation of murine pre-osteoblast MC3T3-E1 cells. Lower concentrations exhibit anabolic and osteogenic properties. We have demonstrated that changes in cellular activity during osteogenesis is accompanied by corresponding changes in expression of osteoblast differentiation specific marker genes. CQ-E has the potential to be used for healing fractures and to prevent and treat osteoporosis.
      PubDate: 2016-06-15T11:55:47.466708-05:
      DOI: 10.1002/jcp.25449
  • In Vitro and In Vivo Differentiation of Progenitor Stem Cells Obtained
           After Mechanical Digestion of Human Dental Pulp
    • Authors: Manuela Monti; Antonio Graziano, Silvana Rizzo, Cesare Perotti, Claudia Del Fante, Riccardo d'Aquino, Carlo Alberto Redi, Ruggero Rodriguez y Baena
      First page: 548
      Abstract: Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity. The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical-grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate toward different cell types and to generate new bone in patients. We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio-maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. J. Cell. Physiol. 232: 548–555, 2017. © 2016 Wiley Periodicals, Inc.This paper presents a new approach to obtain a clinical-grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. Micro-grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate toward different cell types and to generate new bone in patients.
      PubDate: 2016-06-21T10:27:22.598555-05:
      DOI: 10.1002/jcp.25452
  • Leucine-Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast Requires
           Flotillin-1 Mediated Endocytosis
    • Authors: Luciane Martins; Adriana Franco Paes Leme, Kamila Rosamilia Kantovitz, Em nome de Luciane Martins, Enilson Antonio Sallum, Márcio Zaffalon Casati, Francisco Humberto Nociti
      First page: 556
      Abstract: Basic, pre-clinical, and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine-Rich Amelogenin Peptide—LRAP), which is supposed to act as a signaling molecule in epithelial–mesenchymal interactions. LRAP-binding protein complexes from immortalized murine cementoblasts (OCCM-30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin-1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co-precipitation and co-localization assays as a protein-binding partner for LRAP in OCCM-30 cells. In addition, we found that exogenously added GST-LRAP recombinant protein was internalized by OCCM-30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1-dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM-30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp), and runt-related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin-assisted endocytosis, which suggests an involvement of LRAP in lipid-raft-dependent signaling pathways which are mediated by flotillin-1. J. Cell. Physiol. 232: 556–565, 2017. © 2016 Wiley Periodicals, Inc.Flotillin-1, a lipid raft microdomain-associated protein involved in intracellular trafficking pathways, endocytosis, and signal transduction, was identified as a protein-binding partner for Leucine-Rich Amelogenin Peptide (LRAP) impacting on LRAP-mediated expression of key genes involved in cementoblastic differentiation.
      PubDate: 2016-06-20T11:40:31.584039-05:
      DOI: 10.1002/jcp.25453
  • Affinity Selection of FGF2-Binding Heparan Sulfates for Ex Vivo Expansion
           of Human Mesenchymal Stem Cells
    • Authors: Sampath Jeewantha Wijesinghe; Ling Ling, Sadasivam Murali, Yeong Hui Qing, Simon F.R. Hinkley, Susan M. Carnachan, Tracey J. Bell, Kunchithapadam Swaminathan, James H. Hui, Andre J. van Wijnen, Victor Nurcombe, Simon M. Cool
      First page: 566
      Abstract: The future of human mesenchymal stem cells (hMSCs) as a successful cell therapy relies on bioprocessing strategies to improve the scalability of these cells without compromising their therapeutic ability. The culture-expansion of hMSCs can be enhanced by supplementation with growth factors, particularly fibroblast growth factor 2 (FGF2). The biological activity of FGF2 is controlled through interactions with heparan sulfate (HS) that facilitates ligand-receptor complex formation. We previously reported on an FGF2-interacting HS variant (termed HS2) isolated from embryonic tissue by anionic exchange chromatography that increased the proliferation and potency of hMSCs. Here, we detail the isolation of an FGF2 affinity-purified HS variant (HS8) using a scalable platform technology previously employed to generate HS variants with increased affinity for BMP-2 or VEGF165. This process used a peptide sequence derived from the heparin-binding domain of FGF2 as a substrate to affinity-isolate HS8 from a commercially available source of porcine mucosal HS. Our data show that HS8 binds to FGF2 with higher affinity than to FGF1, FGF7, BMP2, PDGF-BB, or VEGF165. Also, HS8 protects FGF2 from thermal destabilization and increases FGF signaling and hMSC proliferation through FGF receptor 1. Long-term supplementation of cultures with HS8 increased both hMSC numbers and their colony-forming efficiency without adversely affecting the expression of hMSC-related cell surface antigens. This strategy further exemplifies the utility of affinity-purifying HS variants against particular ligands important to the stem cell microenvironment and advocates for their addition as adjuvants for the culture-expansion of hMSCs destined for cellular therapy. J. Cell. Physiol. 232: 566–575, 2017. © 2016 Wiley Periodicals, Inc.We here report a novel strategy for ex vivo expansion of human mesenchymal stem cells that replies on supplementation of growth factor-binding heparan sulfates, instead of the growth factor itself into the culture. The advantage of such approach is to avoid the reported adverse effect of FGF2 on MSCs upon long-term exogenous supplementation, and more importantly, the quality of hMSCs expanded is not compromised after long-term culture. The work is of particular importance given the wide therapeutic use of hMSCs and the key role of FGF2 in the technology of hMSC scaling-up.
      PubDate: 2016-06-28T11:30:29.02415-05:0
      DOI: 10.1002/jcp.25454
  • Post-Transcriptional Regulation of the Human Mu-Opioid Receptor (MOR) by
           Morphine-Induced RNA Binding Proteins hnRNP K and PCBP1
    • Authors: Kyu Young Song; Hack Sun Choi, Ping-Yee Law, Li-Na Wei, Horace H. Loh
      First page: 576
      Abstract: Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5′-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). J. Cell. Physiol. 232: 576–584, 2017. © 2016 Wiley Periodicals, Inc.During stimulation, the vast majority of MOR mRNA partitions to the polysomal fraction for synthesis of MOR proteins. These effects are more evident in NMB1 cells, which stably express MOR.
      PubDate: 2016-06-24T11:50:23.799907-05:
      DOI: 10.1002/jcp.25455
  • Inhibition of mTOR Signaling Pathway Delays Follicle Formation in Mice
    • Authors: Jing Zhang; Wenwen Liu, Xinhui Sun, Feifei Kong, Ye Zhu, Yue Lei, Youqiang Su, Yiping Su, Jing Li
      First page: 585
      Abstract: In mammalian ovaries, follicle assembly requires proper germ cell cyst breakdown and the invasion of somatic cells to encapsulate individual oocytes. Abnormalities in this process lead to a number of pathologies such as premature ovarian failure and infertility. As a conserved pathway regulating cell growth and metabolism in response to growth factors and nutrients, the roles of mTOR signaling in follicular development have been extensively studied in recent years. However, its functions during follicle formation remain unknown. In this study, the expression of p-rpS6 (phospho-ribosomal proteinS6), a downstream marker of mTORC1, showed dynamic changes in perinatal ovaries. When E18.5 ovaries, which mainly contained germ cell nests, were incubated with the mTOR inhibitors Rapamycin and Torin1 for 24 h, follicle assembly was delayed with differential somatic cell invasion into germ cell cyst among the groups. After transplanting treated or untreated ovaries into kidney capsules of recipient ovariectomized mice, follicular development was blocked in treated ovaries, as shown by fewer antral follicles and a higher proportion of primordial follicles. Further studies showed a significant decrease in somatic cell proliferation and the expression of marker genes related to follicular development (Kitl, Kit, Gdf9, Bmp15, Zp3, and Amhr2) in treated ovaries. Moreover, the addition of KITL, a growth factor that is mainly produced by pregranulosa cells during germ cell nest breakdown, rescued the extension of follicle formation induced by mTOR inhibitors. These results suggest that KITL functions downstream of mTOR in somatic cells to regulate their communication with oocytes during follicle formation. J. Cell. Physiol. 232: 585–595, 2017. © 2016 Wiley Periodicals, Inc.In mammalian ovaries, follicle assembly requires proper germ cell cyst breakdown and the invasion of somatic cells to encapsulate individual oocytes. In the study, follicle formation was delayed after inhibiting the mTOR signaling pathway by mTOR inhibitors. Moreover, KITL functions downstream of mTOR in somatic cells to regulate their communication with oocytes during follicle formation.
      PubDate: 2016-06-24T12:00:44.764926-05:
      DOI: 10.1002/jcp.25456
  • Cholesterol and Its Derivatives Reversibly Inhibit Proteinase K
    • Authors: Namrata Singh; Debasish Bhattacharyya
      First page: 596
      Abstract: Microorganisms express a variety of proteases that degrade many proteins of the host body and subvert host immune response. While elucidating the mechanism/s of an immune stimulatory drug that contains bile lipid, regulation of proteolytic activity was investigated. The drug and bile lipids both stabilize Proteinase K, an aggressive protease of fungal origin against auto-digestion. Among the components of bile lipids, only cholesterol and its derivatives stabilize the enzyme. Biophysical evidences such as scattering of light, intrinsic and extrinsic fluorescence emission spectra, circular dichroism spectra, atomic force microscopy, and transmission electron microscopy images indicated that cholesterol and its derivatives interact with Proteinase K. Inhibition kinetics using esterolysis of ATEE revealed non-competitive inhibition by cholesterol. Surface Plasmon Resonance and mass spectrometric analysis indicated 1:1 stoichiometry of binding and with dissociation constant in the μM range. Further, the presence of four cholesterol recognition amino acid consensus motifs (CRAC motifs I–IV) was identified in Proteinase K. Bioinformatics analysis revealed that the whole stretch of cholesterol interacts very well with the hydrophobic groove of motif II only among the four CRAC motifs. Variation of cholesterol content of HepG2 human liver carcinoma cells showed positive correlation with binding of fluorescence tagged Proteinase K. Under these conditions, binding of Proteinase K to the cells did not affect their morphology, viability and growth kinetics. Cell bound Proteinase K could be released by an excess of its substrate, thereby restoring reversibly its proteolytic activity. J. Cell. Physiol. 232: 596–609, 2017. © 2016 Wiley Periodicals, Inc.Cholesterol and its derivatives reversibly inactivates Proteinase K, an aggressive protease of fungal origin against its auto-digestion. In ex-vivo studies, confocal images indicated that membrane cholesterol reversibly binds with the enzyme.
      PubDate: 2016-07-09T09:15:31.18314-05:0
      DOI: 10.1002/jcp.25457
  • FGF23 Neutralizing Antibody Ameliorates Hypophosphatemia and Impaired FGF
           Receptor Signaling in Kidneys of HMWFGF2 Transgenic Mice
    • Authors: E. Du; L. Xiao, M.M. Hurley
      First page: 610
      Abstract: High molecular weight FGF2 transgenic mice (HMWTg) phenocopy the Hyp mouse, homolog of human X-linked hypophosphatemic rickets with phosphate wasting and abnormal fibroblast growth factor (FGF23), fibroblast growth factor receptor (FGFR), Klotho and mitogen activated protein kinases (MAPK) signaling in kidney. In this study, we assessed whether short-term (24 h) in vivo administration of FGF23 neutralizing antibody (FGF23Ab) could rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg male mice. Bone mineral density and bone mineral content in 1-month-old HMWTg mice were significantly reduced compared with Control/VectorTg mice. Serum FGF23 was significantly increased in HMWTg compared with VectorTg. Serum phosphate was significantly reduced in HMWTg and was rescued by FGF23Ab. Serum parathyroid hormone (PTH) was significantly increased in HMWTg but was not reduced by FGF23Ab. 1, 25(OH)2 D was inappropriately normal in serum of HMWTg and was significantly increased in both Vector and HMWTg by FGF23Ab. Analysis of HMWTg kidneys revealed significantly increased mRNA expression of the FGF23 co-receptor Klotho, transcription factor mRNAs for early growth response-1 transcription factor (Egr-1), and c-fos were all significantly decreased by FGF23Ab. A significant reduction in the phosphate transporter Npt2a mRNA was also observed in HMWTg kidneys, which was increased by FGF23Ab. FGF23Ab reduced p-FGFR1, p-FGFR3, KLOTHO, p-ERK1/2, C-FOS, and increased NPT2A protein in HMWTg kidneys. We conclude that FGF23 blockade rescued hypophosphatemia by regulating FGF23/FGFR downstream signaling in HMWTg kidneys. Furthermore, HMWFGF2 isoforms regulate PTH expression independent of FGF23/FGFR signaling. J. Cell. Physiol. 232: 610–616, 2017. © 2016 Wiley Periodicals, Inc.A schematic model for the role of HMWFGF2 in the regulation of Pi by FGF23 signaling. Overexpression of HMWFGF2 in osteoblast linage cells induced excess FGF23 in blood. Elevated FGF23 can bind to its co-receptors Fgfr1c/Fgfr3c and Klotho on kidney to activate ERK1/2 signaling pathway in distal tubule, this signaling was transferred to Npt2a in proximal tubules, resulting in Pi wasting. Blockade of FGF23 with FGF23Ab can rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg mice. Activated ERK1/2 can lead to hypophosphatemia by suppression of Cyp27b1 and induction of Cyp24.
      PubDate: 2016-06-30T10:15:27.700269-05:
      DOI: 10.1002/jcp.25458
  • DGKζ Downregulation Enhances Osteoclast Differentiation and Bone
           Resorption Activity Under Inflammatory Conditions
    • Authors: Kiyoshi Iwazaki; Toshiaki Tanaka, Yasukazu Hozumi, Masashi Okada, Rieko Tsuchiya, Ken Iseki, Matthew K. Topham, Kaneyuki Kawamae, Michiaki Takagi, Kaoru Goto
      First page: 617
      Abstract: Bone homeostasis is maintained by a balance between resorption of the bone matrix and its replacement by new bone. Osteoclasts play a crucially important role in bone metabolism. They are responsible for bone resorption under pathophysiological conditions. Differentiation of these cells, which are derived from bone marrow cells, depends on receptor activator of NF-κB ligand (RANKL). RANKL-induced osteoclastogenesis is regulated by the phosphoinositide (PI) signaling pathway, in which diacylglycerol (DG) serves as a second messenger in signal transduction. In this study, we examined the functional implications of DG kinase (DGK), an enzyme family responsible for DG metabolism, for osteoclast differentiation and activity. Of DGKs, DGKζ is most abundantly expressed in osteoclast precursors such as bone marrow-derived monocytes/macrophages. During osteoclast differentiation from precursor cells, DGKζ is downregulated at the protein level. In this regard, we found that DGKζ deletion enhances osteoclast differentiation and bone resorption activity under inflammatory conditions in an animal model of osteolysis. Furthermore, DGKζ deficiency upregulates RANKL expression in response to TNFα stimulation. Collectively, results suggest that DGKζ is silent under normal conditions, but it serves as a negative regulator in osteoclast function under inflammatory conditions. Downregulation of DGKζ might be one factor predisposing a person to osteolytic bone destruction in pathological conditions. J. Cell. Physiol. 232: 617–624, 2017. © 2016 Wiley Periodicals, Inc.The DGK family is involved in cellular signal transduction. Of DGKs, DGKζ serves as a negative regulator in osteoclast function. Downregulation of DGKζ enhances bone destruction in pathological conditions.
      PubDate: 2016-06-30T10:20:47.424435-05:
      DOI: 10.1002/jcp.25461
  • MicroRNA-30b Suppresses Epithelial-Mesenchymal Transition and Metastasis
           of Hepatoma Cells
    • Authors: Xiaolin Sun; Shuhua Zhao, Huanan Li, Hanwen Chang, Zhen Huang, Zhi Ding, Lei Dong, Jiangning Chen, Yuhui Zang, Junfeng Zhang
      First page: 625
      Abstract: Epithelial–mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis in HCC. Growing evidence indicates that upregulation of Snail, the major EMT inducer, significantly correlates with the metastasis and poor prognosis of HCC. Here, we investigate the underlying mechanism of miR-30b in suppressing metastasis of hepatoma cells by targeting Snail. In this study, we found that miR-30b was significantly downregulated and negatively associated with Snail production in HCC cell lines with higher metastatic potentials. Gain- and loss-of-function studies revealed that miR-30b could dramatically inhibit in vitro HCC cell migration and invasion. In vivo orthotopic liver xenograft model further demonstrated that stable over-expression of miR-30b significantly repressed the local invasion and lung metastasis of hepatoma cells. Meanwhile, the restoration of miR-30b expression suppressed the distant colonization of hepatoma cells. Both gain- and loss-of-function studies showed that miR-30b suppressed the EMT of hepatoma cells as indicated by the morphology changes and deregulation of epithelial and mesenchymal markers. Using RNAi, we further investigated the role of Snail in HCC cell EMT and demonstrated that knockdown of Snail significantly inhibited the EMT and cancer cell metastasis. Additionally, miR-30b exhibited inhibitory effects on HCC cell proliferation in vitro and in vivo. In conclusion, our findings highlight the significance of miR-30b downregulation in HCC tumor metastasis and invasiveness, and implicate a new potential therapeutic target for HCC metastasis. J. Cell. Physiol. 232: 625–634, 2017. © 2016 Wiley Periodicals, Inc.miR-30b was significantly downregulated and negatively associated with Snai1 production in HCC cell lines with higher metastatic potentials. miR-30b exerts its suppressive function on HCC metastasis by inhibiting the Snail-driven EMT.
      PubDate: 2016-06-30T10:25:27.648539-05:
      DOI: 10.1002/jcp.25466
  • SCD1 Alters Long-Chain Fatty Acid (LCFA) Composition and Its Expression Is
           Directly Regulated by SREBP-1 and PPARγ 1 in Dairy Goat Mammary Cells
    • Authors: Dawei Yao; Jun Luo, Qiuya He, Hengbo Shi, Jun Li, Hui Wang, Huifen Xu, Zhi Chen, Yongqing Yi, Juan J. Loor
      First page: 635
      Abstract: Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme for the synthesis of the monounsaturated fatty acids (MUFA) palmitoleic acid and oleic acid. In non-ruminant species, SCD1 expression is known to be tightly regulated by a variety of transcription factors. Although the role of SCD1 and the transcriptional regulatory mechanism by SREBP-1 and PPARs in other species is clear, changes in lipid metabolism related to SCD1 and via the regulation of SREBP-1 or PPARG1 in ruminant mammary tissue remain largely unknown. Here, we demonstrated that SCD1 expression in goat mammary tissue is higher during lactation than the dry period. Overexpression of SCD1 increased the intracellular MUFA content and lipid accumulation, whereas SCD1 silencing resulted in a significant decrease in oleic acid concentration and triacylglycerol (TAG) accumulation. The overexpression of SREBF1 in goat mammary epithelial cells (GMEC) enhanced SCD1 expression and its promoter activity, but that effect was abolished when SREBF1 was silenced. Furthermore, deletion of sterol regulatory element (SRE) and the nuclear factor (NF-Y)-binding sites within a −1713 to +65-base pair region of the SCD1 promoter completely abolished SREBP-1-induced SCD1 transcription. Otherwise, PPARG1 overexpression also stimulated the expression of SCD1 and its transcriptional activity directly via a PPAR response element (PPRE) in the SCD1 promoter. Together, these results indicate that SCD1 could markedly affect the fatty acid composition and rate of TAG synthesis through direct regulation via SREBP-1 and PPARG1, hence, underscoring an important role of the enzyme and this transcription regulator in controlling mammary gland lipid synthesis in the goat. J. Cell. Physiol. 232: 635–649, 2017. © 2016 Wiley Periodicals, Inc.The function of stearoyl-CoA desaturase 1 gene in goat mammary cells. The regulatory mechanism of SCD1 by SREBP1 and PPARG in dairy goat.
      PubDate: 2016-07-18T11:30:35.5889-05:00
      DOI: 10.1002/jcp.25469
  • Differential Expression of miR-4520a Associated with Pyrin Mutations
           Suggesting a Role of Autophagy in Familial Mediterranean Fever (FMF)
    • Abstract: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (p = 0.032), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (p = 0.00548). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results support a role of deregulated autophagy in the pathogenesis of FMF. This article is protected by copyright. All rights reserved
  • Association Between Aerobic Exercise and Rosiglitazone Avoided the NAFLD
           and Liver Inflammation Exacerbated in PPAR-α Knockout Mice
    • Abstract: Nonalcoholic fatty liver disease (NAFLD) is one of the main liver diseases today, and may progress to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Some studies have shown the beneficial effects of aerobic exercise on reversing NAFLD. To verify whether chronic aerobic exercise improves the insulin resistance, liver inflammation, and steatohepatitis caused by a high fat diet (HF) and whether PPARα is involved in these actions. C57BL6 wild type (WT) and PPAR-α knockout (KO) mice were fed with a standard diet (SD) or HF during 12 weeks; the HF mice were trained on a treadmill during the last 8 weeks. Serum glucose and insulin tolerances, serum levels of aspartate aminotransferase, hepatic content of triacylglycerol, cytokines, gene expression, and protein expression were evaluated in all animals. Chronic exposure to HF diet increased triacylglycerol accumulation in the liver, leading to NAFLD, increased aminotransferase in the serum, increased peripheral insulin resistance, and higher adiposity index. Exercise reduced all these parameters in both animal genotypes. The liver lipid accumulation was not associated with inflammation; trained KO mice, however, presented a huge inflammatory response that was probably caused by a decrease in PPAR-γ expression. We conclude that exercise improved the damage caused by a HF independently of PPARα, apparently by a peripheral fatty acid oxidation in the skeletal muscle. We also found that the absence of PPARα together with exercise leads to a decrease in PPAR-γ and a huge inflammatory response. J. Cell. Physiol. 9999: 1–12, 2016. © 2016 Wiley Periodicals, Inc.Exercise training improved lipids toxicity in the liver; PPAR-alpha KO developed liver inflammation after training protocol Roziglitazone reduced liver inflammation independent of PPAR-alpha.
  • Cardiotrophin-1 Regulates Adipokine Production in 3T3-L1 Adipocytes and
           Adipose Tissue from Obese Mice
    • Abstract: Cardiotrophin-1 (CT-1) belongs to the IL-6 family of cytokines. Previous studies of our group revealed that CT-1 is a key regulator of glucose and lipid metabolism. The aim of the present study was to analyze the in vitro and in vivo effects of CT-1 on the production of several adipokines involved in body weight regulation, nutrient metabolism and inflammation. For this purpose, 3T3-L1 adipocytes were incubated with recombinant protein CT-1 (rCT-1) (1- 40 ng/mL) for 1 and 18 h. Moreover, the acute effects of rCT-1 administration (0.2 mg/kg, i.v.) for 30 min and 3 h on adipokines levels were also evaluated in high-fat fed obese mice. In 3T3-L1 adipocytes, rCT-1 treatment downregulated the expression and secretion of leptin, resistin and visfatin. However, rCT-1 significantly stimulated apelin mRNA and secretion. rCT- 1 (18 h) also promoted the activation by phosphorylation of AKT, ERK 1/2 and STAT3. Interestingly, pre-treatment with the PI3K inhibitor LY294002 reversed the stimulatory effects of rCT-1 on apelin expression, suggesting that this pathway could be mediating the effects of rCT-1 on apelin production. In contrast, acute administration of rCT-1 (30 min and 3 h) to diet- induced obese mice downregulated leptin and resistin, without significantly modifying apelin or visfatin mRNA in adipose tissue. Furthermore, CT-1 null mice exhibited altered expression of adipokines in adipose tissue. The present study demonstrates that rCT-1 modulates the production of adipokines in vitro and in vivo, suggesting that the regulation of the secretory function of adipocytes could be involved in the metabolic actions of this cytokine. This article is protected by copyright. All rights reserved
  • Overexpression of Large-Conductance Calcium-Activated Potassium Channels
           in Human Glioblastoma Stem-Like Cells and Their Role in Cell Migration
    • Abstract: Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large-conductance, calcium-activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem-like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem-like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87-MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem-like cells (U87-NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co-expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87-NS cells migration was much more sensitive to BK channel block than U87-MG cells. Our data show that BK channels are highly expressed in GBM stem-like cells, and participate to their high migratory activity. This article is protected by copyright. All rights reserved
  • Tight Junction Protein Abundance and Apoptosis During Involution of Rat
           Mammary Glands
    • Abstract: To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement was induced by teat-sealing with an adhesive for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. In non-sealed control glands, histological analysis confirmed a lactating phenotype, indicating suckling by pups throughout the experiment. In contrast, alveoli of teat-sealed glands were distended within 6 h, with maximal luminal size observed by 12 h of non-suckling. By 18 h following teat-sealing, an involuting phenotype was observed, indicated by alveolar lumina engorged with milk vesicles and increased leukocytes. Relative to non-sealed glands, mammary apoptosis was increased in engorged glands 18 h following teat-sealing. The abundance of ZO-1 and occludin proteins was decreased in engorged glands by 12 and 18 h, respectively, following teat-sealing. In contrast, the claudin-1 22 kDa band was increased by 6 h and peaked at 12-18 h, whereas the 28 kDa band declined by 36 h, relative to controls. There were no temporal changes in ZO-1, occludin, and claudin-1 22 kDa proteins within control glands, although there were minor differences in claudin-1 28 kDa. These data indicate that intramammary milk accumulation due to cessation of milk removal is associated with mammary apoptosis. The apoptotic event is preceded by a rapid loss of abundance of ZO-1, occludin and an initial increase in claudin-1. The loss of cell-cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non-suckled glands. This article is protected by copyright. All rights reserved
  • Cyclic Tensile Strain Reduces TNF-α Induced Expression of MMP-13 by
           Condylar Temporomandibular Joint Cells
    • Abstract: ObjectiveTo investigate whether the disproportionate degradation of mandibular condyle cartilage in arthritic juvenile temporomandibular joint (TMJ) is related to distinctive responses of TMJ-derived cells to tumor necrosis factor-α (TNF-α), and whether mechanical loading affects this response.MethodsThe effect of TNF-α (0.1-10ng/ml) was tested on juvenile porcine TMJ cells isolated from the condyle, fossa, and disc, grown in 3D agarose gels. Expression of anabolic and catabolic factors was quantified by RT-qPCR and/or immunohistochemistry. Condylar cells were stimulated for 12h with TNF-α (10ng/ml), followed by 8h of 6% cyclic tensile strain, and gene expression of MMPs was quantified.ResultsTNF-α (10ng/ml) reduced the expression of the matrix proteins collagen type I and type II after 6h of incubation. Aggrecan gene expression was increased in the presence of 0.1ng/ml TNF-α. The fossa and disc cells responded to TNF-α with an increased expression of the aggrecanase ADAMTS4. TNF-α enhanced MMP-13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect.ConclusionCells isolated from the different cartilaginous structures reacted differently to TNF-α. Since the disc and fossa contain a very low level of proteoglycans in comparison to the condyle, the role played by ADAMTS4 in degradation of the fossa and disc might be limited. TNF-α induced MMP-13 expression by condylar cells might be involved in the degradation of the juvenile condyle. Since this expression was reduced by mechanical loading, functional loading with oral physiotherapy or orthodontic activators may help to reduce the catabolic effect of TNF-α. This article is protected by copyright. All rights reserved
  • Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal
           Epithelial Cells
    • Abstract: Intestinal epithelial cell culture is important for biological, functional and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered-cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio-membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL-25 and TSLP), inhibitory (IL-10 and TGF-β) or pro-inflammatory mediators (IL-1β and TNF) were induced. In conclusion, this novel protocol using cow's collagen-ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell-microorganism interaction studies and stem cell expansion. This article is protected by copyright. All rights reserved
  • Identifying Nuclear Matrix–Attached DNA Across the Genome
    • Abstract: Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method-specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR-Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF-10A mammary epithelial-like cells and MDA-MB-231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene-regulatory histone modifications (ChIP-Seq). In the normal-like cells, nuclear matrix–attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non-expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR-Seq approach, we provide the first genome-wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix–associated genome is highly cell-context dependent. This article is protected by copyright. All rights reserved
  • RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease
           Modeling and Therapy
    • Abstract: Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogrammed iPSCs lines may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic and translational research. This article is protected by copyright. All rights reserved
  • A Novel Skin Splint for Accurately Mapping Dermal Remodeling and
           Epithelialization During Wound Healing
    • Abstract: The mouse excisional dorsal full-thickness wound model with a silicon splint fixed on the skin has been widely used to mimic human wound healing. However, the method cannot accurately quantify dermal remodeling, since the initial point of epithelialization on the wound surface is unclear. To overcome this limitation, we have developed a novel mouse excisional wound model to assess the degree of epithelial extension and regeneration, using a plastic ring-shaped splint fixed beneath the surrounding epidermal tissue. At the end of the experiment, tissue samples were fixed in formalin, the splint was excised, and paraffin sections were prepared. Splint holes, corresponding to the prior location of the splint, were evident on the tissue cross-sections, and the epidermis above the holes was considered the initial excision site. The epidermal contraction and epithelial regeneration, as independent essential tissue alterations in wound healing, could be distinguishable and quantified. Compared with previous splint models, this method provides an accurate evaluation of epidermal processes in wound healing, and can be a platform to assess the effects of various wound healing factors. This article is protected by copyright. All rights reserved
  • A MicroRNA Cluster miR-23∼24∼27 Is Upregulated by Aldosterone in the
           Distal Kidney Nephron where It Alters Sodium Transport
    • Abstract: The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23∼24∼27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3'-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR-27 in aldosterone's action via a novel target. This article is protected by copyright. All rights reserved
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