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Journal Cover Journal of Cellular Physiology
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   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  [1603 journals]   [SJR: 1.608]   [H-I: 118]
  • Genome‐wide transcriptional and functional analysis of endoglin
           isoforms in the human promonocytic cell line U937
    • Authors: Francisco J Blanco; Luisa Ojeda‐Fernandez, Mikel Aristorena, Eunate Gallardo‐Vara, Alberto Benguria, Ana Dopazo, Carmen Langa, Luisa M Botella, Carmelo Bernabeu
      Abstract: Endoglin is an auxiliary cell surface receptor for TGF‐β family members. Two different alternatively spliced isoforms, long (L)‐endoglin and short (S)‐endoglin, have been reported. S‐endoglin and L‐endoglin proteins vary from each other in their cytoplasmic tails that contain 14 and 47 amino acids, respectively. A critical role for endoglin in vascular development has primarily been studied in endothelial cells. In addition, endoglin expression is upregulated during monocyte‐to‐macrophage differentiation, however, little is known about its role in this myeloid context. To investigate the function of endoglin in monocytes, stable transfectants expressing the two endoglin isoforms in the promonocytic human cell line U937 were generated. The differential gene expression fingerprinting of these endoglin transfectants using DNA microarrays and further bioinformatics analysis showed a clear alteration in essential biological functions, mainly those related to “Cellular Movement”, including cell adhesion and transmigration. Interestingly, these cellular functions are highly dependent on adhesion molecules, including integrins α1 (CD49a, ITGA1 gene), αL (CD11a, ITGAL gene), αM (CD11b, ITGAM gene) and β2 (CD18, ITGB2 gene) and the chemokine receptor CCR2 (CD192, CCR2 gene), which are downregulated in endoglin transfectants. Moreover, activin A (INHBA gene), a TGF‐β superfamily member involved in macrophage polarization, was distinctly affected in each endoglin transfectant, and may contribute to the regulated expression of integrins. These data were confirmed by quantitative PCR, flow cytometry and functional tests. Taken together, these results provide new insight into endoglin function in monocytes. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:48:01.205103-05:
      DOI: 10.1002/jcp.24827
       
  • Novel Hedgehog Agonists Promote Osteoblast Differentiation in Mesenchymal
           Stem Cells
    • Authors: Takashi Nakamura; Masahiro Naruse, Yuta Chiba, Toshihisa Komori, Keiichi Sasaki, Masahiro Iwamoto, Satoshi Fukumoto
      Abstract: Hedgehog (Hh) family members are involved in multiple cellular processes including proliferation, migration, differentiation, and cell fate determination. Recently, the novel Hh agonists Hh‐Ag 1.3 and 1.7 were identified in a high‐throughput screening of small molecule compounds that activate the expression of Gli1, a target of Hh signaling. This study demonstrates that Hh‐Ag 1.3 and 1.7 strongly activate the expression of endogenous Gli1 and promote osteoblast differentiation in the mesenchymal stem cell line C3H10T1/2. Both compounds stimulated alkaline phosphatase activity in a dose‐dependent manner, and induced osteoblast marker gene expression in C3H10T1/2 cells, which indicated that they had acquired an osteoblast identity. Of the markers, the expression of osterix/Sp7, a downstream target of runt‐related transcription factor (Runx)2, was induced by Hh‐Ag 1.7, which also rescued the osteoblast differentiation defect ofRD‐127, a mesenchymal cell line from Runx2‐deficient mice. Hh‐Ags also activated canonical Wnt signaling and synergized with low doses of BMP‐2 to enhance osteoblastic potential. Thus, Hh‐Ag 1.7 could be useful for bone healing in individuals with abnormalities in osteogenesis, such as osteoporosis patients and the elderly, and can contribute to the development of novel therapeutics for the treatment of bone fractures and defects. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:47:21.000731-05:
      DOI: 10.1002/jcp.24823
       
  • MicroRNA‐302a Stimulates Osteoblastic Differentiation by Repressing
           COUP‐TFII Expression
    • Authors: In‐Hong Kang; Byung‐Chul Jeong, Sung‐Woong Hur, Hyuck Choi, Seung‐Ho Choi, Je‐Hwang Ryu, Yun‐Chan Hwang, Jeong‐Tae Koh
      Abstract: Chicken ovalbumin upstream promoter transcription factor II (COUP‐TFII) is a potent transcription factor that represses osteoblast differentiation and bone formation. Previously we observed that stimuli for osteoblast differentiation, such as bone morphogenetic protein 2 (BMP2), inhibits COUP‐TFII expression. This study was undertaken to identify BMP2‐regulated and COUP‐TFII‐targeting microRNAs (miRNAs), and to explore their regulatory roles in osteoblast differentiation. Based on in silico analysis, 12 miRNAs were selected and their expression in BMP2‐treated MC3T3‐E1 cells was examined. BMP2 induced miR‐302a expression in dose‐ and time‐dependent manners with the decrease in COUP‐TFII expression. Runx2, a BMP2‐downstream transcription factor, specifically regulated miR‐302a expression and its promoter activity. A computer‐based prediction algorithm led to the identification of two miR‐302a binding sites on the 3'‐untranslational region of COUP‐TFII mRNA (S1: 620‐626 bp, S2: 1016‐1022 bp), and a luciferase assay showed that miR‐302a directly targeted S1 and S2. Transfection of miR‐302a precursor significantly enhanced expression of osteogenic marker genes with decreasing COUP‐TFII mRNA and protein level, alkaline phosphatase activity and matrix mineralization. On the other hand, inhibition of miR‐302a significantly attenuated BMP2‐induced osteoblast specific gene expression, alkaline phosphatase activity, and matrix mineralization with increasing COUP‐TFII mRNA and protein level. These results indicate that miR‐302a is induced by osteogenic stimuli and promotes osteoblast differentiation by targeting COUP‐TFII. MiR‐302a could be a positive regulator for osteoblast differentiation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:45:31.80351-05:0
      DOI: 10.1002/jcp.24822
       
  • miR‐203 is a Direct Transcriptional Target of E2F1 and Causes G1
           Arrest in Esophageal Cancer Cells
    • Authors: Kun Zhang; Limeng Dai, Bo Zhang, Xueqing Xu, Jiazhong Shi, Liyuan Fu, Xuedan Chen, Juan Li, Yun Bai
      Abstract: miR‐203 act as tumor repressor by inhibiting cell proliferation and is repressed in a variety of human tumors, although the molecular mechanisms responsible have not been elucidated. Here, we reveal that miR‐203 is regulated by E2F1, an important transcription factor that can induce cell proliferation by controlling cell cycle progression. We found that miR‐203 expression was induced by cisplatin, which also induced E2F1 protein accumulation in esophageal squamous cell carcinoma (ESCC) cell lines. miR‐203 expression was elevated upon activation of ectopic E2F1, whereas this induction was abolished when the E2F1 gene was silenced. Moreover, with luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays, we demonstrated that E2F1 transactivates miR‐203 by directly binding to the miR‐203 gene promoter. In addition, we found that miR‐203 inhibited cell proliferation by inducing G1/S cell cycle arrest, but not apoptosis, in ESCC cell lines. Finally, we observed that miR‐203 negatively inhibited the expression of CDK6, subsequently decreasing E2F1 expression possibly through Rb phosphorylation. Taken together, our data show that cancer‐related miR‐203 is a novel transcriptional target of E2F1 and that it regulates cell cycle arrest by participating in a feedback loop with E2F1. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:43:18.257391-05:
      DOI: 10.1002/jcp.24821
       
  • FoxK2 is required for cellular proliferation and survival
    • Authors: Lars P. van der Heide; Patrick J. E. C. Wijchers, Lars von Oerthel, J. Peter H. Burbach, Marco F. M. Hoekman, Marten P. Smidt
      Abstract: FoxK2 is a forkhead transcription factor expressed ubiquitously in the developing murine central nervous system. Here we investigated the role of FoxK2 in vitro and focused on proliferation and cellular survival. Knockdown of FoxK2 results in a decrease in BrdU incorporation and H3 phosphorylation, suggesting attenuation of proliferation. In the absence of growth factors FoxK2 knockdown results in a dramatic increase in caspase 3 activity and propidium iodide positive cells, indicative of cell death. Additionally, knockdown of FoxK2 results in an increase in the mRNA of Gadd45α, Gadd45γ, as well as an increase in the phosphorylation of the mTOR dependent kinase p70S6K. Rapamycin treatment completely blocked the increase in p70S6K and synergistically potentiated the decrease in H3 phosphorylation upon FoxK2 knockdown. To gain more insight into the pro‐apoptotic effects upon FoxK2 knockdown we screened for changes in Bcl2 genes. Upon FoxK2 knockdown both Puma and Noxa were significantly upregulated. Both genes were not inhibited by rapamycin treatment, instead rapamycin increased Noxa mRNA. FoxK2 requirement in cellular survival is further emphasized by the fact that resistance to TGFβ‐induced cell death was greatly diminished after FoxK2 knockdown. Overall our data suggest FoxK2 is required for proliferation and survival, that mTOR is part of a feedback loop partly compensating for FoxK2 loss, possibly by upregulating Gadd45s, whereas cell death upon FoxK2 loss is induced in a Bcl2 dependent manner via Puma and Noxa. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:42:38.06738-05:0
      DOI: 10.1002/jcp.24828
       
  • Involvement of intracellular signalling in the IL‐1β inhibitory
           effect on fructose intestinal absorption
    • Authors: M Jesús Rodríguez‐Yoldi; Sonia Gascón, Cristina Barranquero, Alberto García‐Barrios, Jesús Osada
      Abstract: A variety of bacteria and their excreted/secreted products having direct effects on epithelial ion transport and permeability and the release of cytokines during bacterial infection may impact directly on epithelial function. Interleukin‐1β (IL‐1β) is a pleiotropic cytokine that affects the intestinal absorption of nutrients. The aim of this work was to study the intracellular signalling pathways involved in the inhibitory effect of IL‐1β on D‐fructose intestinal transport in rabbit jejunum and Caco‐2 cells. The results show that the cytokine inhibitory effect was completely reversed in presence of proteasome or PKC selective inhibitors in IL‐1β treated rabbits. In addition, the activation of PI3K abolished the IL‐1β effect. Likewise, these results were confirmed in Caco‐2 cells. In addition, p‐PI3K expression was increased by IL‐1β‐treatment whereas the expression of p‐PKCα was not significantly affected. In summary, the results suggest that IL‐1β could regulate the activation of pPKCα 73, pPI3K 55 and NF‐kB proteins. These events could exert an inhibitory effect on fructose intestinal absorption by a modification of GLUT5 insertion to brush‐border membrane and/or the functional transporter activity. This effect is independent of hormonal milieu and nervous stimuli. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:42:20.411333-05:
      DOI: 10.1002/jcp.24820
       
  • The dysfunction of the trabecular meshwork during glaucoma course
    • Authors: Sergio Claudio Saccà; Alessandra Pulliero, Alberto Izzotti
      Abstract: Primary open angle glaucoma is a multi‐tissue disease that targets, in an ascending order, the trabecular meshwork, the optic nerve head, the lateral geniculate nuclei and the visual cortex. Oxidative stress and vascular damage play major roles in triggering apoptotic cell loss in these tissues. Molecular alterations occurring in the ocular anterior chamber during the early course of glaucoma trigger this cell loss. These molecular events are mainly of endogenous origin and related to the long‐term accumulation of oxidative damages arising from mitochondrial failure and endothelial dysfunction. This situation results in decreased antioxidant defences in aqueous humour and apoptosis activation in trabecular meshwork cells as triggered by severe mitochondrial damage altering tissue function and integrity. The presence of neural proteins in glaucomatous aqueous humour indicate that a molecular interconnection exists between the anterior and the posterior chamber tissues. Trabecular meshwork and lamina cribrosa share a common neuro‐ectodermal embryological, which contribute to explain the interconnection between anterior and the posterior chamber during glaucoma pathogenesis. During glaucoma, proteins deriving from the damage occurring in endothelial trabecular meshwork cells are released into aqueous humour. Accordingly, aqueous humour composition is characterised in glaucomatous patients by the presence of proteins deriving from apoptosis activation, mitochondrial damage, loss of intercellular connections, antioxidant decrease. Many questions remain unanswered, but molecular events illuminate TM damage and indicate that trabecular cells protection plays a role in the treatment and prevention of glaucoma. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:41:42.077463-05:
      DOI: 10.1002/jcp.24826
       
  • Flt3 ligand induces monocyte proliferation and enhances the function of
           monocyte‐derived dendritic cells in vitro
    • Authors: Sung‐Whan Kim; Seong‐Mi Choi, Yee Shin Choo, Il‐Kwon Kim, Byeung‐Wook Song, Han‐Soo Kim
      Abstract: Flt3 ligand (FL), a potent hematopoietic cytokine, plays an important role in development and activation of dendritic cells (DCs) and natural killer cells (NK). Although some post‐receptor signaling events of FL have been characterized, the role of FL on Flt3 expressing human peripheral blood monocyte is unclear. In the current study, we examined the role of FL on cell survival and growth of peripheral blood monocytes and function of monocyte‐derived DCs. FL promoted monocyte proliferation in a dose‐dependent manner and prevented spontaneous apoptosis. FL induced ERK phosphorylation and a specific ERK inhibitor completely abrogated FL‐mediated cellular growth, while p38 MAPK, JNK and AKT were relatively unaffected. Addition of FL to GM‐CSF and IL‐4 during DCs generation from monocytes increased the yield of DCs through induction of cell proliferation. DCs generated in the presence of FL expressed more costimulatory molecules on their surfaces and stimulated allogeneic T cell proliferation in MLR to a higher magnitude. Furthermore, FL partially antagonized IL‐10‐mediated inhibition on DCs function. Further characterization of FL actions may provide new and important information for immunotherapeutic approaches utilizing DCs. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:41:13.443492-05:
      DOI: 10.1002/jcp.24824
       
  • Justicidin A‐Induced Autophagy Flux Enhances Apoptosis of Human
           Colorectal Cancer Cells via Class III PI3K and Atg5 Pathway
    • Authors: Shen‐Jeu Won; Cheng‐Hsin Yen, Hsiao‐Sheng Liu, Shan‐Ying Wu, Sheng‐Hui Lan, Ya‐Fen Jiang‐Shieh, Chun‐Nan Lin, Chun‐Li Su
      Abstract: Our previous reports showed that justicidin A (JA), a novel and pure arylnaphthalide lignan isolated from Justicia procumbens, induces apoptosis of human colorectal cancer cells and hepatocellular carcinoma cells, leading to the suppression of both tumor cell growth in NOD‐SCID mice. Here, we reveal that JA induces autophagy in human colorectal cancer HT‐29 cells by conversion of autophagic marker LC3‐I to LC3‐II. Furthermore, LC3 puncta and autophagic vesicle formation, and SQSTM1/p62 suppression were observed. Administration of autophagy inhibitor (bafilomycin A1 and chloroquine) and transfection of a tandem fluorescent‐tagged LC3 (mRFP‐GFP) reporter plasmid (ptfLC3) demonstrated that JA induces autophagy flux in HT‐29 cells. Expression of LC3, SQSTM1, Beclin 1, and nuclear DNA double‐strand breaks (representing apoptosis) were also detected in the tumor tissue of HT‐29 cells transplanted into NOD‐SCID mice orally administrated with JA. In addition, the expression of autophagy signaling pathway‐related molecules p‐PDK1, p‐mTOR, p‐p70S6k/p‐RPS6KB2 was decreased, whereas that of class III PI3K, Beclin 1, Atg5‐Atg12, and mitochondrial BNIP3 was increased in response to JA. Pre‐treatment of the cells with class III PI3K inhibitor 3‐methyladenine or Atg5 shRNA attenuated JA‐induced LC3‐II expression and LC3 puncta formation, indicating the involvement of class III PI3K and Atg5. A novel mechanism was demonstrated in the anticancer compound JA; pre‐treatment with 3‐methyladenine or Atg5 shRNA blocked JA‐induced suppression in cell growth and colony formation, respectively, via inhibition of apoptosis. In contrast, administration of apoptosis inhibitor Z‐VAD did not affect JA‐induced autophagy. Our data suggest the chemotherapeutic potential of JA for treatment of human colorectal cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-12T05:28:03.183994-05:
      DOI: 10.1002/jcp.24825
       
  • p27 and Leukemia: Cell cycle and beyond
    • Authors: Anita Roy; Subrata Banerjee
      Abstract: Cell division is the foundation to development and the regulation of cell cycle progression is therefore of paramount importance to the living organisms. Primary control of cell cycle is achieved by an array of cyclins and cyclin dependent kinases (CDKs). The functions of these cyclin‐CDK complexes are again regulated by a host of cyclin dependent kinase inhibitors (CDKI). Till date CDKIs are broadly classified into two groups – INK4 family (p15, p16, p18 and p19) and the cip/kip family (p21, p27 and p57). Collectively these CDKIs regulate the progression from G1 to S phase of cell cycle. This review summarizes the functions of p27 while highlighting its emerging roles in leukemia. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-10T00:28:21.380767-05:
      DOI: 10.1002/jcp.24819
       
  • Hesperetin induces apoptosis in breast carcinoma by triggering
           accumulation of ROS and activation of ASK1/JNK pathway
    • Authors: Shreyasi Palit; Susanta Kar, Gunjan Sharma, Pijush K. Das
      Abstract: Hesperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. In the present study hesperetin exhibited a significant cytotoxic effect in human breast carcinoma MCF‐7 cells in a concentration‐ and time‐dependent manner without affecting normal (HMEC) as well as immortalized normal mammary epithelial cells (MCF‐10A). The cytotoxic effect of hesperetin was due to the induction of apoptosis as evident from the phosphatidyl‐serine externalization, DNA fragmentation, caspase‐7 activation and PARP cleavage. Apoptosis was associated with caspase‐9 activation, mitochondrial membrane potential loss, release of cytochrome c and increase in Bax:Bcl‐2 ratio. Pre‐treatment with caspase‐9 specific inhibitor (Z‐LEHD‐fmk) markedly attenuated apoptosis suggesting an involvement of intrinsic mitochondrial apoptotic cascade. Further, DCFDA flow‐cytometric analysis revealed triggering of ROS in a time‐dependent manner. Pre‐treatment with ROS scavenger N‐acetylcysteine (NAC) and glutathione markedly abrogated hesperetin‐mediated apoptosis whereas carbonyl cyanide m‐chlorophenylhydrazone (CCCP) pretreatment along with DHR123‐based flow‐cytometry indicated the generation of cytosolic ROS. Profiling of MAPKs revealed activation of JNK upon hesperetin treatment which was abrogated upon NAC pretreatment. Additionally, inhibition of JNK by SP600125 significantly reversed hesperetinmediated apoptosis. The activation of JNK was associated with the activation of ASK1. Silencing of ASK1 resulted in significant attenuation of JNK activation as well as reversed the hesperetin‐mediated apoptosis suggesting that hesperetin‐mediated apoptosis of MCF‐7 cells involves accumulation of ROS and activation of ASK1/JNK pathway. In addition, hesperetin also induced apoptosis in triple negative breast cancer MDA‐MB‐231 cells via intrinsic pathway via activation of caspase ‐9 and ‐3 and increase in Bax:Bcl‐2 ratio. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-10T00:16:32.838662-05:
      DOI: 10.1002/jcp.24818
       
  • The GPER agonist G‐1 induces mitotic arrest and apoptosis in human
           vascular smooth muscle cells independent of GPER
    • Authors: Yu Gui; Zhan Shi, Zeng Yong Wang, Jing‐Jing Li, Can Xu, Rui Juan Tian, Xin Xing Song, Michael P. Walsh, Dong Li, Jie Gao, Xi‐Long Zheng
      Abstract: The G protein‐coupled estrogen receptor (GPER) has been implicated in the regulation of smooth muscle cell (SMC) proliferation. The GPER selective agonist G‐1 has been a useful tool for exploring the biological roles of GPER in a variety of experimental settings, including SMC proliferation. The present study, originally designed to investigate cellular and signaling mechanisms underlying the regulatory role of GPER in vascular SMC proliferation using G‐1, unexpectedly revealed off‐target effects of G‐1. G‐1 (1‐10 μM) inhibited bromodeoxyuridine (BrdU) incorporation of human SMCs and caused G2/M cell accumulation. G‐1 treatment also increased mitotic index concurrent with a decrease in phosphorylation of Cdk1 (Tyr 15) and an increase in phosphorylation of the mitotic checkpoint protein BuBR1. Furthermore, G‐1 caused microtubule disruption, mitotic spindle damage, and tubulin depolymerization. G‐1 induced cell apoptosis as indicated by the appearance of TUNEL‐positive and annexin V‐positive cells with enhanced cleavage of caspases 3 and 9. However, neither the GPER antagonist G‐15 nor the MAPK kinase inhibitor PD98059 prevented these G‐1 effects. Down‐regulation of GPER or p44/42 MAPK with siRNA transfection also did not affect the G‐1‐induced apoptosis. We conclude that G‐1 inhibits proliferation of SMCs through mechanisms involving mitotic arrest and apoptosis, independent of GPER and the MAPK pathway. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-10T00:12:53.242319-05:
      DOI: 10.1002/jcp.24817
       
  • IL‐1β Promotes Malignant Transformation and Tumor
           Aggressiveness in Oral Cancer
    • Authors: Chia‐Huei Lee; Jeffrey Shu‐Ming Chang, Shih‐Han Syu, Thian‐Sze Wong, Jimmy Yu‐Wai Chan, Ya‐Chu Tang, Zhi‐Ping Yang, Wen‐Chan Yang, Chiung‐Tong Chen, Shao‐Chun Lu, Pei‐Hua Tang, Tzu‐Ching Yang, Pei‐Yi Chu, Jenn‐Ren Hsiao, Ko‐Jiunn Liu
      Abstract: Chronic inflammation, coupled with alcohol, betel quid, and cigarette consumption, is associated with oral squamous cell carcinoma (OSCC). Interleukin‐1 beta (IL‐1β) is a critical mediator of chronic inflammation and implicated in many cancers. In this study, we showed that increased pro‐IL‐1β expression was associated with the severity of oral malignant transformation in a mouse OSCC model induced by 4‐Nitroquinolin‐1‐oxide (4‐NQO) and arecoline, two carcinogens related to tobacco and betel quid, respectively. Using microarray and quantitative PCR assay, we showed that pro‐IL‐1β was upregulated in human OSCC tumors associated with tobacco and betel quid consumption. In a human OSCC cell line TW2.6, we demonstrated nicotine‐derived nitrosamine ketone (NNK) and arecoline stimulated IL‐1β secretion in an inflammasome‐dependent manner. IL‐1β treatment significantly increased the proliferation and dysregulated the Akt signaling pathways of dysplastic oral keratinocytes (DOKs). Using cytokine antibodies and inflammation cytometric bead arrays, we found that DOK and OSCC cells secreted high levels of IL‐6, IL‐8, and growth‐regulated oncogene‐α following IL‐1β stimulation. The conditioned medium of IL‐1β‐treated OSCC cells exerted significant proangiogenic effects. Crucially, IL‐1β increased the invasiveness of OSCC cells through the epithelial‐mesenchymal transition (EMT), characterized by downregulation of E‐cadherin, upregulation of Snail, Slug, and Vimentin, and alterations in morphology. These findings provide novel insights into the mechanism underlying OSCC tumorigenesis. Our study suggested that IL‐1β□ can be induced by tobacco and betel quid‐related carcinogens, and participate in the early and late stages of oral carcinogenesis by increasing the proliferation of dysplasia oral cells, stimulating oncogenic cytokines, and promoting aggressiveness of OSCC. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-10T00:11:17.930538-05:
      DOI: 10.1002/jcp.24816
       
  • Novel Insights into TRPM7 Function in Fibrotic Diseases: A Potential
           Therapeutic Target
    • Authors: Tao Xu; Bao‐ming Wu, Xiao‐ming Meng, Cheng Huang, Ming‐ming Ni, Jun Li
      Abstract: Transient receptor potential (TRP) channels are cellular sensors for a wide spectrum of physical and chemical stimuli. Activation of TRP channels changes the membrane potential, translocates important signaling ions crossing the cell membrane, alters enzymatic activity, and initiates endocytosis/exocytosis. Fibrosis is the leading cause of organ dysfunction in diseases, which is characterized by an imbalance in the turnover of extracellular matrix components. Accumulating evidence has demonstrated that TRPM7, a member of TRP channels superfamily, participates in the development and pathogenesis of fibrotic diseases, such as hepatic, pulmonary and cardiac fibrosis. In this review, we discuss the comprehensive role of TRPM7 in modulating profibrotic response and its potential as therapeutic target for fibrotic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T19:18:08.739893-05:
      DOI: 10.1002/jcp.24801
       
  • Epithelial‐to‐Mesenchymal Transition Induced by
           TGF‐β1 Is Mediated by AP1‐Dependent EpCAM Expression in
           MCF‐7 Cells
    • Authors: Jiujiao Gao; Qiu Yan, Jiao Wang, Shuai Liu, Xuesong Yang
      Abstract: The epithelial‐to‐mesenchymal transition (EMT), a process involving the breakdown of cell‐cell junctions and loss of epithelial polarity, is closely related to cancer metastasis and invasion. The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane protein expressed in the majority of normal epithelial tissues and overexpressed in the majority of human epithelial cancers including breast cancer. EpCAM plays an important role in cancer progression. We showed that EpCAM participated in TGF‐β1‐induced EMT. TGF‐β1 treatment of MCF‐7 breast cancer cells was shown to induce EpCAM expression, which promoted the EMT and cell migration. EpCAM overexpression further enhanced TGF‐β1‐induced EMT, and EpCAM knockdown inhibited TGF‐β1‐induced EMT. We further demonstrated that TGF‐β1 treatment induced the phosphorylation of JNK that was in turn responsible for the increased expression of Jun and Fos. This result suggests an important role of the JNK to AP‐1 signaling to EpCAM downstream of TGF‐β1 for the induction of EMT in the breast cancer cells. Collectively, our study highlights a novel function for EpCAM in TGF‐β1‐induced EMT process and suggests that targeting of EpCAM may be an attractive strategy to treat breast cancer. This study implicates the potential value of EpCAM as a molecular marker for breast cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T19:17:54.701705-05:
      DOI: 10.1002/jcp.24802
       
  • WW Domain of BAG3 is Required for the Induction of Autophagy in Glioma
           Cells
    • Authors: Nana Merabova; Ilker Kudret Sariyer, A Sami Saribas, Tijana Knezevic, Jennifer Gordon, M. Caterina Turco, Alessandra Rosati, Michael Weaver, Jacques Landry, Kamel Khalili
      Abstract: Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co‐chaperone family of proteins that associates with Hsp70 through a conserved BAG domain positioned near the C‐terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin‐1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin‐1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:57:46.553115-05:
      DOI: 10.1002/jcp.24811
       
  • A Group of Novel HIF‐1α Inhibitors, Glyceollins, Blocks
           HIF‐1α Synthesis and Decreases Its Stability via Inhibition of
           the PI3K/AKT/mTOR Pathway and Hsp90 Binding
    • Authors: Sun Hee Lee; Jun‐Goo Jee, Kwanghyun Ryu, You Mie Lee
      Abstract: Glyceollins, a group of phytoalexins isolated from soybean, are known to exhibit anticancer, antiestrogenic, and antiangiogenic activities. However, whether glyceollins regulate tumor growth through regulation of hypoxia‐inducible factor (HIF)‐1α has not been investigated. We determined whether and how glyceollins regulate the synthesis and stability of HIF‐1α. Quantitative real‐time PCR revealed that glyceollins inhibited the expression of HIF‐1‐induced genes such as vascular endothelial growth factor (VEGF) in cancer cells. Enzyme‐linked immunosorbent assay and reporter luciferase assay showed that glyceollins decreased VEGF secretion and its promoter activity, respectively. Treatment of various cancer cells with 0.5‐100 µM glyceollins under hypoxic conditions reduced the expression of HIF‐1α. Glyceollins blocked translation of HIF‐1α by inhibiting the PI3K/AKT/mTOR pathway under hypoxic conditions. Glyceollins decreased the stability of HIF‐1α after treatment with cycloheximide, a protein synthesis inhibitor, and increased the ubiquitination of HIF‐1α after treatment with MG132, a proteasome inhibitor. Glyceollins blocked the interaction of Hsp90 with HIF‐1α, as shown by immunoprecipitation assay. Chemical binding of Hsp90 with glyceollins, as confirmed by computational docking analysis, was stronger than that with geldanamycin at the HSP90 ATP‐binding pocket. We found that glyceollins decreased microvessel density, as well as expression of phosphorylated AKT/mTOR and the Hsp90 client protein CDK4, in solid tumor tissues. Glyceollins potently inhibited HIF‐1α synthesis and decreased its stability by blocking the PI3K/AKT/mTOR pathway and HSP90 binding activity, respectively. These results may provide new perspectives into potential therapeutic application of glyceollins for the prevention and treatment of hypervascularized diseases and into the mechanism of their anticancer activity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:51:50.62946-05:0
      DOI: 10.1002/jcp.24813
       
  • Gene Expression Changes in Progression of Cervical Neoplasia Revealed by
           Microarray Analysis of Cervical Neoplastic Keratinocytes
    • Authors: John Charles Rotondo; Silvia Bosi, Cristian Bassi, Manuela Ferracin, Giovanni Lanza, Roberta Gafà, Eros Magri, Rita Selvatici, Stefania Torresani, Roberto Marci, Paola Garutti, Massimo Negrini, Mauro Tognon, Fernanda Martini
      Abstract: To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large‐scale gene expression profile was carried out on HPV16‐CIN2, HPV16‐CIN3 and normal cervical keratinocytes derived from two HPV16‐CIN2, two HPV‐CIN3 lesions and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16‐CIN2 keratinocytes and in HPV16‐CIN2 keratinocytes compared with HPV16‐CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription‐polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:48:46.34043-05:0
      DOI: 10.1002/jcp.24808
       
  • The Coiled‐Coil Domain Containing 80 (ccdc80) Gene Regulates
           gadd45β2 Expression in the Developing Somites of Zebrafish as a New
           Player of the Hedgehog Pathway
    • Authors: Isabella Della Noce; Silvia Carra, Chiara Brusegan, Rosina Critelli, Andrea Frassine, Carlo De Lorenzo, Antonio Giordano, Gianfranco Bellipanni, Erica Villa, Franco Cotelli, Anna Pistocchi, Filippo Schepis
      Abstract: The Coiled‐Coil Domain Containing 80 (CCDC80) gene has been identified as strongly induced in rat thyroid PC CL3 cells immortalized by the adenoviral E1A gene. In human, CCDC80 is a potential oncosoppressor due to its down‐regulation in several tumor cell lines and tissues and it is expressed in almost all tissues. CCDC80 has homologous in mouse, chicken and zebrafish. We cloned the zebrafish ccdc80 and analyzed its expression and function during embryonic development. The in silico translated zebrafish protein shares high similarity with its mammalian homologous, with nuclear localization signals and a signal peptide. Gene expression analysis demonstrates that zebrafish ccdc80 is maternally and zygotically expressed throughout the development. In particular, ccdc80 is strongly expressed in the notochord and it is under the regulation of the Hedgehog pathway. In this work we investigated the functional effects of ccdc80‐loss‐of‐function during embryonic development and verified its interaction with gadd45β2 in somitogenesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:47:10.174514-05:
      DOI: 10.1002/jcp.24810
       
  • Vitamin D Attenuates Cytokine‐Induced Remodeling in Human Fetal
           Airway Smooth Muscle Cells
    • Authors: Rodney D. Britt; Arij Faksh, Elizabeth R. Vogel, Michael A. Thompson, Vivian Chu, Hitesh C. Pandya, Yassine Amrani, Richard J. Martin, Christina M. Pabelick, Y.S. Prakash
      Abstract: Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25‐dihydroxyvitamin D3; 1,25(OH)2D3), modulates responses to inflammation; however this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro‐inflammatory cytokines. Cells were pre‐treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP‐9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ‐induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:41:49.922249-05:
      DOI: 10.1002/jcp.24814
       
  • Isoform‐Specific SCFFbw7 Ubiquitination Mediates Differential
           Regulation of PGC‐1α
    • Authors: Julie S. Trausch‐Azar; Mona Abed, Amir Orian, Alan L. Schwartz
      Abstract: The E3 ubiquitin ligase and tumor suppressor SCFFbw7 exists as three isoforms that govern the degradation of a host of critical cell regulators, including c‐Myc, cyclin E, and PGC‐1α. Peroxisome proliferator activated receptor‐gamma coactivator 1α (PGC‐1α) is a transcriptional coactivator with broad effects on cellular energy metabolism. Cellular PGC‐1α levels are tightly controlled in a dynamic state by the balance of synthesis and rapid degradation via the ubiquitin‐proteasome system. Yet, isoform‐specific functions of SCFFbw7 are yet to be determined. Here, we show that the E3 ubiquitin ligase, SCFFbw7, regulates cellular PGC‐1α levels via two independent, isoform specific, mechanisms. The cytoplasmic isoform (SCFFbw7β) reduces cellular PGC‐1α levels via accelerated ubiquitin‐proteasome degradation. In contrast, the nuclear isoform (SCFFbw7α) increases cellular PGC‐1α levels and protein stability via inhibition of ubiquitin‐proteasomal degradation. When nuclear Fbw7α proteins are redirected to the cytoplasm, cellular PGC‐1α protein levels are reduced through accelerated ubiquitin‐proteasomal degradation. We find that SCFFbw7β catalyzes high molecular weight PGC‐1α‐ubiquitin conjugation, whereas SCFFbw7α produces low molecular weight PGC‐1α‐ubiquitin conjugates that are not effective degradation signals. Thus, selective ubiquitination by specific Fbw7 isoforms represents a novel mechanism that tightly regulates cellular PGC‐1α levels. Fbw7 isoforms mediate degradation of a host of regulatory proteins. The E3 ubiquitin ligase, Fbw7, mediates PGC‐1α levels via selective isoform‐specific ubiquitination. Fbw7β reduces cellular PGC‐1α via ubiquitin‐mediated degradation, whereas Fbw7α increases cellular PGC‐1α via ubiquitin‐mediated stabilization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:40:19.811917-05:
      DOI: 10.1002/jcp.24812
       
  • pRb2/p130 is Delocalized in Cytoplasm in Diffuse Gastric Cancer: A
           Preliminary Study
    • Authors: Letizia Cito; Paola Indovina, Iris Maria Forte, Domenico Di Marzo, Pasquale Somma, Daniela Barone, Antonella Penon, Danila Penon, Elisa Ceccherini, Pietro Micheli, Luca Saragoni, Marina Di Domenico, Antonia Feola, Franco Roviello, Eliseo Mattioli, Giovan Giacomo Giordano, Antonio Giordano
      Abstract: pRb2/p130 phosphorylation occurs at G0/G1 transition, when cells exit from quiescence. This phenomenon leads to E2F4 and E2F5 transcription factors releasing, allowing them to activate their target genes. Hence, pRb2/p130 represents one of the most important cell cycle controller because of transcription factors binding/releasing capability. It was previously shown that the loss of expression, or function, of pRb2/p130 is involved in different neoplasms development, due to related loss of control of E2F4 and E2F5 transcription activity. Here we analyzed gastric cancer tissue samples of diffuse histotype, comparing them with the normal counterpart. Yet, we did not find any change of expression levels. Rather, we found a cytoplasmic localization of pRb2/p130 in cancer tissue samples whereas, in normal counterparts, we found the expected nuclear localization. This occurrence lead to hypothesize an almost completely uncontrolled transcriptional activity of E2F4 and E2F5 in this kind of neoplasm, actually contributing to maintenance of transformed status. Although in limited number of cases, our data support the necessity of further investigations to verify the possibility of using pRb2/p130 as diagnostic marker of diffuse gastric cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:40:00.89997-05:0
      DOI: 10.1002/jcp.24805
       
  • GATA‐1 Deficiency Rescues Trabecular but not Cortical Bone in OPG
           Deficient Mice
    • Authors: Tomas E. Meijome; R. Adam Hooker, Ying‐Hua Cheng, Whitney Walker, Mark C. Horowitz, Robyn K. Fuchs, Melissa A. Kacena
      Abstract: GATA‐1low/low mice have an increase in megakaryocytes (MKs) and trabecular bone. The latter is thought to result from MKs directly stimulating osteoblastic bone formation while simultaneously inhibiting osteoclastogenesis. Osteoprotegerin (OPG) is known to inhibit osteoclastogenesis and OPG‐/‐ mice have reduced trabecular and cortical bone due to increased osteoclastogenesis. Interestingly, GATA‐1low/low mice have increased OPG levels. Here we sought to determine whether GATA‐1 knockdown in OPG‐/‐ mice could rescue the observed osteoporotic bone phenotype. GATA‐1low/low mice were bred with OPG‐/‐ mice and bone phenotype assessed. GATA‐1low/low X OPG‐/‐ mice have increased cortical bone porosity, similar to OPG‐/‐ mice. Both OPG‐/‐ and GATA‐1low/low X OPG‐/‐ mice, were found to have increased osteoclasts localized to cortical bone, possibly producing the observed elevated porosity. Biomechanical assessment indicates that OPG‐/‐ and GATA‐1low/low X OPG‐/‐ femurs are weaker and less stiff than C57BL/6 or GATA‐1low/low femurs. Notably, GATA‐1low/low X OPG‐/‐ mice had trabecular bone parameters that were not different from C57BL/6 values, suggesting that GATA‐1 deficiency can partially rescue the trabecular bone loss observed with OPG deficiency. The fact that GATA‐1 deficiency appears to be able to partially rescue the trabecular, but not the cortical bone phenotype suggests that MKs can locally enhance trabecular bone volume, but that MK secreted factors cannot access cortical bone sufficiently to inhibit osteoclastogenesis or that OPG itself is required to inhibit osteoclastogenesis in cortical bone. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:37:45.37493-05:0
      DOI: 10.1002/jcp.24803
       
  • Review: Cell Dynamics in Malignant Pleural Effusions
    • Authors: Enrico Giarnieri; Gianfranco Bellipanni, Marcella Macaluso, Rita Mancini, Adam Carl Holstein, Carla Milanese, Maria Rosaria Giovagnoli, Antonio Giordano, Giuseppe Russo
      Abstract: Malignant pleural effusions (MPEs) are a common manifestation found in patients with lung cancer. After cytological and histological confirmation of malignancy, talc pleurodesis still remains the treatment of choice in patients with MPEs resistant to chemotherapy. Despite this, primary challenges include reduced quality of life and life expectancy in general. Therefore, a better understanding of the cell biology of MPEs, along with improvements in treatment is greatly needed. It has recently been demonstrated that malignant pleural effusions (MPEs) may represent an excellent source for identification of molecular mechanisms within the tumor and its environment. The present review summarizes the current understanding of MPEs cells and tumor microenvironment, and particularly focuses on dissecting the cross‐talk between MPEs and epithelial to mesenchymal transition (EMT), inflammation and cancer stem cells. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:31:30.748438-05:
      DOI: 10.1002/jcp.24806
       
  • Role of Acinus in Regulating Retinoic Acid‐responsive Gene
           Pre‐mRNA Splicing
    • Authors: Fang Wang; Kenneth J. Soprano, Dianne Robert Soprano
      Abstract: Acinus‐S' is a co‐repressor for retinoic acid receptor (RAR)‐dependent gene transcription and has been suggested to be involved in RNA processing. In this study the role of Acinus isoforms in regulating pre‐mRNA splicing was explored using in vivo splicing assays. Both Acinus‐L and Acinus‐S', with the activity of Acinus‐L higher than that of Acinus‐S', increase the splicing of a retinoic acid (RA)‐responsive minigene containing a weak 5' splice site but not a RA‐responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing of the weak 5' splice site by Acinus in a dose‐ and time‐dependent manner, suggesting a RA‐dependent activity in addition to a RA‐independent activity of Acinus. The RA‐independent effect of Acinus occurs to varying degrees using minigene constructs containing several different promoters while the RA‐dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by a RA response element (RARE)‐containing promoter. This suggests that the ligand‐dependent splicing activity of Acinus is related to the RA‐activated RAR bound to the RARE. The RRM domain is necessary for the RA‐dependent splicing activity of Acinus and the RA‐independent splicing activity of Acinus is repressed by RNPS1. Importantly, measurement of the splicing of endogenous human RARβ and Bcl‐x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA‐dependent manner for RARβ and a RA‐independent manner for Bcl‐x. Taken together, these studies demonstrate that Acinus functions in both RAR‐dependent splicing and RAR‐dependent transcription. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T17:31:18.818058-05:
      DOI: 10.1002/jcp.24804
       
  • Palmitate‐stimulated monocytes induce adhesion molecule expression
           in endothelial cells via IL‐1 signaling pathway
    • Authors: Yosuke Shikama; Nanako Aki, Akiko Hata, Miho Nishimura, Seiichi Oyadomari, Makoto Funaki
      Abstract: Increased intake of saturated fatty acids (SFAs), such as palmitate (Pal), is linked to a higher risk of type 2 diabetes and cardiovascular disease. Although recent studies have investigated the direct effects of SFAs on inflammatory responses in vascular endothelial cells, it remains unknown whether SFAs also induce these responses mediated by circulating cells. In this study, especially focused on adhesion molecules and monocytes, we investigated the indirect effects of Pal on expression and release of ICAM‐1 and E‐selectin in vascular endothelial cells. Phorbol 12‐myristate 13‐acetate (PMA)‐treated THP‐1 (pTHP‐1) cells and human monocytes were stimulated with various free fatty acids (FFAs). SFAs, but not unsaturated fatty acids (UFAs), increased interleukin (IL)‐1β secretion and decreased IL‐1 receptor antagonist (IL‐1Ra) secretion, resulting in an increase in the IL‐1β/IL‐1Ra secretion ratio. UFAs dose‐dependently inhibited the increase in IL‐1β secretion and decrease in IL‐1Ra secretion induced by Pal. Moreover, in human aortic and vein endothelial cells, expression and release of ICAM‐1 and E‐selectin were induced by treatment with conditioned medium collected from Pal‐stimulated pTHP‐1 cells and human monocytes, but not by Pal itself. The up‐regulated expression and release of adhesion molecules by the conditioned medium were mostly abolished by recombinant human IL‐1Ra supplementation. These results suggest that the Pal‐induced increase in the ratio of IL‐1β/IL‐1Ra secretion in monocytes up‐regulates endothelial adhesion molecules, which could enhance leukocyte adhesion to endothelium. This study provides further evidence that IL‐1β neutralization through receptor antagonism may be useful for preventing the onset and development of cardiovascular disease. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T05:08:38.88911-05:0
      DOI: 10.1002/jcp.24797
       
  • microRNA‐151‐3p Regulates Slow Muscle Gene Expression by
           Targeting ATP2a2 in Skeletal Muscle Cells
    • Authors: Huan Wei; Zhongwen Li, Xiaobin Wang, Jie Wang, Weijun Pang, Gongshe Yang, Qingwu W. Shen
      Abstract: MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate the stability or translation of cognate mRNAs at the posttranscriptional level. Accumulating evidence indicates that miRNAs play important roles in many aspects of muscle function, including muscle growth and development, regeneration, contractility, and muscle fiber type plasticity. In the current study, we examined the function of miR‐151‐3p in myoblast proliferation and differentiation. Results show that overexpression of miR‐151‐3p not only upregulates myoblast proliferation, but also decreases slow muscle gene expression (such as MHC‐β/slow and slow muscle troponin I) in both C2C12 myotubes and in primary cultures. Alternatively, inhibition of miR‐151‐3p by antisense RNA was found to upregulate MHC‐β/slow expression, indicating that miR‐151‐3p plays a role in muscle fiber type determination. Further investigation into the underlying mechanisms revealed for the first time that miR‐151‐3p directly targets ATP2a2, a gene encoding for a slow skeletal and cardiac muscle specific Ca2+ ATPase, SERCA2 thus downregulating slow muscle gene expression. Mechanisms by which the alteration in SERCA2 expression induces changes in other slow muscle gene expression levels needs to be defined in future research. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T05:07:57.752466-05:
      DOI: 10.1002/jcp.24793
       
  • miR‐34c regulates the permeability of blood‐tumor barrier via
           MAZ‐mediated expression changes of ZO‐1, occludin and
           claudin‐5
    • Authors: Lini Zhao; Ping Wang, Yunhui Liu, Jun Ma, Yixue Xue
      Abstract: The purposes of this study were to investigate the potential roles of miR‐34c in regulating blood‐tumor barrier (BTB) functions and its possible molecular mechanisms. The over‐expression of miR‐34c significantly impaired the integrity and increased the permeability of BTB, which were detected in an in vitro BTB model by transendothelial electric resistance and horseradish peroxidase flux assays respectively. Meanwhile, real‐time quantitative PCR (qRT‐PCR), Western blot and immunofluorescence assays successively demonstrated down‐regulation of ZO‐1, occludin and claudin‐5. And miR‐34c silencing uncovered the opposite results. Dual‐luciferase reporter assays results revealed myc‐associated zinc‐finger protein (MAZ) is a target gene of miR‐34c. Besides, mRNA and protein expressions of MAZ were reversely regulated by miR‐34c. The down‐expression of MAZ significantly impaired the integrity and increased the permeability of BTB as well as down‐regulated the expressions of ZO‐1, occludin and claudin‐5. And chromatin immunoprecipitation verified that MAZ interacted with “GGGCGGG”, “CCCTCCC” and “GGGAGGG” DNA sequence of ZO‐1, occludin and claudin‐5 promoter respectively. The over‐expression or silencing of either miR‐34c or MAZ was performed simultaneously to further explore their functional relations, and results elucidated that miR‐34c and MAZ displayed reverse regulatory effects on the integrity and permeability of BTB as well as the expressions of ZO‐1, occludin and claudin‐5. In conclusion, our present study indicated that miR‐34c regulated the permeability of BTB via MAZ‐mediated expression changes of ZO‐1, occludin and claudin‐5. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:51:55.133271-05:
      DOI: 10.1002/jcp.24799
       
  • Sphingosine‐1‐phosphate mediates COX‐2 expression and
           PGE2/IL‐6 secretion via c‐Src‐dependent AP‐1
           activation
    • Authors: Chih‐Kai Hsu; I‐Ta Lee, Chih‐Chung Lin, Li‐Der Hsiao, Chuen‐Mao Yang
      Abstract: Sphingosine‐1‐phosphate (S1P) has been shown to regulate cyclooxygenase‐2 (COX‐2)/prostaglandin E2 (PGE2) expression and IL‐6 secretion in various respiratory diseases. However, the mechanisms underlying S1P‐induced COX‐2 expression and PGE2 production in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here we demonstrated that S1P markedly induced COX‐2 expression. S1P also induced PGE2 and IL‐6 secretion which were reduced by the inhibitors of COX‐2 (NS‐398 and celecoxib). Pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c‐Src (PP1), PYK2 (PF431396), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP‐1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c‐Src, PYK2, p38, p42, JNK2, c‐Jun, or c‐Fos reduced S1P‐induced COX‐2 expression and PGE2/IL‐6 secretion. Moreover, S1P induced c‐Src, PYK2, p42/p44 MAPK, JNK1/2, p38 MAPK, and c‐Jun phosphorylation. We observed that S1P‐induced p42/p44 MAPK and JNK1/2, but not p38 MAPK activation was mediated via a c‐Src/PYK2‐dependent pathway. S1P also enhanced c‐Fos, but not c‐Jun mRNA and protein expression and the AP‐1 promoter activity. S1P‐induced c‐Fos mRNA and protein expression, c‐Jun phosphorylation, and AP‐1 promoter activity was reduced by W123, CAY10444, PP1, PF431396, U0126, SP600125, or SB202190. These results demonstrated that S1P‐induced COX‐2 expression and PGE2/IL‐6 generation was mediated through S1PR1/3/c‐Src/PYK2/p42/p44 MAPK‐ or JNK1/2‐ and S1PR1/3/c‐Src/p38 MAPK‐dependent AP‐1 activation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:50:38.124258-05:
      DOI: 10.1002/jcp.24795
       
  • CRM197 in combination with shRNA interference of VCAM‐1 displays
           enhanced inhibitory effects on human glioblastoma cells
    • Authors: Yi Hu; Xing Lin, Ping Wang, Yi‐Xue Xue, Zhen Li, Li‐Bo Liu, Bo Yu, Tian‐Da Feng, Yun‐Hui Liu
      Abstract: CRM197 is a naturally nontoxic diphtheria toxin mutant that binds and inhibits heparin‐binding epidermal growth factor‐like growth factor. CRM197 serves as carrier protein for vaccine and other therapeutic agents. CRM197 also inhibits the growth, migration, invasion and induces apoptosis in various tumors. Vascular cell adhesion molecule‐1 (VCAM‐1) is an important cell surface adhesion molecule associated with malignancy of gliomas. In this work, we aimed to investigate the role and mechanism of CRM197 combined with shRNA interference of VCAM‐1 (shRNA‐VCAM‐1) on the migration, invasion and apoptosis of glioblastoma cells. U87 and U251 human glioblastoma cells were treated with CRM197 (10 μg/ml) and shRNA interfering technology was employed to silence VCAM‐1 expression. Cell viability, migration, invasiveness and apoptosis were assessed with CCK8, Transwell and Annexin V‐PE/7‐AAD staining. Activation of cleaved caspase‐3, 8 and 9, activity of matrix metalloproteinase‐2/9 (MMP‐2/9) and expression of phosphorylated Akt (p‐Akt) were also checked. Results showed that CRM197 and shRNA‐VCAM‐1 not only significantly inhibited the cell proliferation, migration, invasion, but also promoted the apoptosis of U87 and U251 cells. Combined treatment of both displayed enhanced inhibitory effects on the malignant biological behavior of glioma cells. The activation of cleaved caspase‐3, 8, 9 was promoted, activity of MMP‐2 and MMP‐9 and expression of p‐Akt were inhibited significantly by the treatment of CRM197 and shRNA‐VCAM‐1 alone or in combination, indicating that the combination of CRM197 with shRNA‐VCAM‐1 additively inhibited the malignant behavior of human glioblastoma cells via activating caspase‐3, 8, 9 as well as inhibiting MMP‐2, MMP‐9 and Akt pathway. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:47:53.443367-05:
      DOI: 10.1002/jcp.24798
       
  • MSCs modified with ACE2 restore endothelial function following LPS
           challenge by inhibiting the activation of RAS
    • Authors: Hong‐li He; Ling Liu, Qi‐hong Chen, Shi‐xia Cai, Ji‐bin Han, Shu‐ling Hu, Pan Chun, Yi Yang, Feng‐mei Guo, Ying‐zi Huang, Hai‐bo Qiu
      Abstract: Angiotensin (Ang) II plays an important role in the process of endothelial dysfunction in acute lung injury(ALI) and is degraded by angiotensin‐converting enzyme2 (ACE2). However, treatmentsthat target ACE2 to injured endothelium and promote endothelial repair of ALI are lacking. Mesenchymal stem cells (MSCs) are capable of homing to the injured site and delivering a protective gene. Our study aimed to evaluate the effects of genetically modified MSCs, which overexpress the ACE2 protein in a sustained manner via a lentiviral vector, on Ang II production in endothelium and in vitro repair of LPS‐induced endothelial injury.We found that the efficiency of lentiviral vector transduction of MSCs was as high as 97.8% and was well maintained over 30 passages. MSCs modified with ACE2 showed a sustained high expression of ACE2 mRNA and protein. The modified MSCs secreted soluble ACE2 protein into the culture medium, which reduced the concentration of Ang II and increased the production of Ang 1–7. MSCs modified with ACE2 were more effective at restoring endothelial function than were unmodified MSCs, as shown by the enhanced survival of endothelial cells; the downregulated production of inflammatory mediators, including ICAM‐1, VCAM‐1, TNF‐α, and IL‐6; reduced paracellular permeability; and increased expression of VE‐cadherin. These data demonstrate that MSCs modified to overexpress the ACE2 gene can produce biologically active ACE2 protein over a sustained period of time and have an enhanced ability to promote endothelial repair after LPS challenge.These results encourage further testing of the beneficial effects of ACE2‐modified MSCs in an ALI animal model. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:40:42.77059-05:0
      DOI: 10.1002/jcp.24794
       
  • Interleukin‐17 induces CC chemokine receptor 6 expression and cell
           migration in colorectal cancer cells
    • Authors: Chih‐Chien Chin; Cheng‐Nan Chen, Hsing‐Chun Kuo, Chung‐Sheng Shi, Meng Chiao Hsieh, Yi‐Hung Kuo, Shui‐Yi Tung, Kam‐Fai Lee, Wen‐Shih Huang
      Abstract: The CC chemokine receptor 6 (CCR6) and its ligand CCL20 are involved in human colorectal cancer (CRC) carcinogenesis and can promote the progression of CRC. In addition, interleukin‐17 (IL‐17), produced by a T cell subset named “Th17,” has been identified as an important player in inflammatory responses, and has emerged as a mediator in inflammation‐associated cancer. However, the relevance of IL‐17 in the development and progression of CRC still remains to be explored. This study aimed to investigate the effect of IL‐17 on the cell migration of CRC cells. Human CRC HCT‐116 cells were used to study the effect of IL‐17 on CCR6 expression and cell migration in CRC cells. IL‐17 treatment induced migration of HCT‐116 cells across the Boyden chamber membrane and increased the expression level of the CCR6. Inhibition of CCR6 by small interfering RNA (siRNA) and neutralizing antibody inhibited IL‐17‐induced cell migration. By using specific inhibitors and short hairpin RNA (shRNA), we demonstrated that the activation of ERK and p38 pathways are critical for IL‐17‐induced CCR6 expression and cell migration. Promoter activity and transcription factor ELISA assays showed that IL‐17 increased NF‐κB‐DNA binding activity in HCT‐116 cells. Inhibition of NF‐κB activation by specific inhibitors and siRNA blocked the IL‐17‐induced CCR6 expression. Our findings support the hypothesis that CCR6 up‐regulation stimulated by IL‐17 may play an active role in CRC cell migration. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:40:26.168474-05:
      DOI: 10.1002/jcp.24796
       
  • Acetylcholine Inhibits Tumor Necrosis Factor α Activated Endoplasmic
           Reticulum Apoptotic Pathway via EGFR‐PI3K Signaling in
           Cardiomyocytes
    • Authors: Yi Miao; Xue‐Yuan Bi, Mei Zhao, Hong‐Ke Jiang, Jin‐Jun Liu, Dong‐Ling Li, Xiao‐Jiang Yu, Yong‐Hua Yang, Ning Huang, Wei‐Jin Zang
      Abstract: Previous findings have shown that acetycholine (ACh) decreased hypoxia‐induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α‐induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase‐3 activation and terminal deoxynucleotidyl transferase mediated dUTP‐biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α‐induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol‐3‐kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh‐elicited anti‐apoptotic property. Together, these data indicate that EGFR‐PI3K/Akt signaling is involved in M2 AChR‐mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:35:37.86881-05:0
      DOI: 10.1002/jcp.24800
       
  • Roles of SATB2 in Site‐Specific Stemness, Autophagy and Senescence
           of Bone Marrow Mesenchymal Stem Cells
    • Authors: WEIJIE DONG; PING ZHANG, YU FU, JIE GE, JIE CHENG, HUA YUAN, HONGBING JIANG
      Abstract: Craniofacial bone marrow mesenchymal stem cells (BMSCs) display some site‐specific properties that differ from those of BMSCs derived from the trunk and appendicular skeleton, but the characteristics of craniofacial BMSCs and the mechanisms that underlie their properties are not completely understood. Previous studies indicated that special AT‐rich binding protein 2 (SATB2) may be a potential regulator of craniofacial skeletal patterning and site‐specific osteogenic capacity. Here, we investigated the stemness, autophagy, and anti‐aging capacity of mandible‐derived BMSCs (M‐BMSCs) and tibia‐derived BMSCs (T‐BMSCs) and explored the role of SATB2 in regulating these properties. M‐BMSCs not only possessed stronger expression of SATB2 and stemness markers (pluripotency genes, such as Nanog, OCT‐4, Sox2, and Nestin) but also exhibited stronger autophagy and anti‐aging capacities under normal or hypoxia/serum deprivation conditions compared to T‐BMSCs. Exogenous expression of SATB2 in T‐BMSCs significantly enhanced the expression of pluripotency genes as well as autophagy and anti‐aging capacity. Moreover, SATB2 markedly enhanced osteogenic differentiation of BMSCs in vitro, and promoted bone defect regeneration and the survival of BMSCs that were transplanted into mandibles with critical size defects. Mechanistically, SATB2 upregulates pluripotency genes and autophagy‐related genes, which in turn activate the mechanistic target of rapamycin signaling pathway. Collectively, our results provide novel evidence that site‐specific BMSCs have distinct biological properties and suggest that SATB2 plays a potential role in regulating the stemness, autophagy, and anti‐aging properties of craniofacial BMSCs. The application of SATB2 to manipulate stem cells for the reconstruction of bone defects might represent a new approach. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-09T04:34:09.725711-05:
      DOI: 10.1002/jcp.24792
       
  • Gene therapies for cancer: strategies, challenges and successes
    • Authors: Swadesh K Das; Mitchell E. Menezes, Shilpa Bhatia, Xiang‐Yang Wang, Luni Emdad, Devanand Sarkar, Paul B. Fisher
      Abstract: Gene therapy, which involves replacement of a defective gene with a functional, healthy copy of that gene, is a potentially beneficial cancer treatment approach particularly over chemotherapy, which often lacks selectivity and can cause non‐specific toxicity. Despite significant progress pre‐clinically with respect to both enhanced targeting and expression in a tumor‐selective manner several hurdles still prevent success in the clinic, including non‐specific expression, low‐efficiency delivery and biosafety. Various innovative genetic approaches are under development to reconstruct vectors/transgenes to make them safer and more effective. Utilizing cutting‐edge delivery technologies, gene expression can now be targeted in a tissue‐ and organ‐specific manner. With these advances, gene therapy is poised to become amenable for routine cancer therapy with potential to elevate this methodology as a first line therapy for neoplastic diseases. This review discusses recent advances in gene therapy and their impact on a pre‐clinical and clinical level. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-06T03:27:34.725787-05:
      DOI: 10.1002/jcp.24791
       
  • The Cooperation of CREB and NFAT is Required for PTHrP‐Induced RANKL
           Expression in Mouse Osteoblastic Cells
    • Authors: Hyun‐Jung Park; Kyunghwa Baek, Jeong‐Hwa Baek, Hyung‐Ryong Kim
      Pages: n/a - n/a
      Abstract: Parathyroid hormone‐related protein (PTHrP) is known to induce the expression of receptor activator of NF‐κB ligand (RANKL) in stromal cells/osteoblasts. However, the signaling pathways involved remain controversial. In the present study, we investigated the role of cAMP/protein kinase A (PKA) and calcineurin/NFAT pathways in PTHrP‐induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. PTHrP‐mediated induction of RANKL expression was significantly inhibited by H89 and FK506, an inhibitor of PKA and calcineurin, respectively. PTHrP upregulated CREB phosphorylation and the transcriptional activity of NFAT. Knockdown of CREB or NFATc1 blocked PTHrP‐induced RANKL expression. PTHrP increased the activity of the RANKL promoter reporter that contains approximately 2 kb mouse RANKL promoter DNA sequences. Insertions of mutations in CRE‐like element or in NFAT‐binding element abrogated PTHrP‐induced RANKL promoter activity. Chromatin immunoprecipitation assays showed that PTHrP increased the binding of CREB and NFATc1/NFATc3 to their cognate binding elements in the RANKL promoter. Inhibition of cAMP/PKA and its downstream ERK activity suppressed PTHrP‐induced expression and transcriptional activity of NFATc1. CREB knockdown prevented PTHrP induction of NFATc1 expression. Furthermore, NFATc1 and CREB were co‐immunoprecipitated. Mutations in CRE‐like element completely blocked NFATc1‐induced transactivation of the RANKL promoter reporter; however, mutations in NFAT‐binding element partially suppressed CREB‐induced RANKL promoter activity. Overexpression of CREB increased NFATc1 binding to the RANKL promoter and vice versa. These results suggest that PTHrP‐induced RANKL expression depends on the activation of both cAMP/PKA and calcineurin/NFAT pathways, and subsequently, CREB and NFAT cooperate to transactivate the mouse RANKL gene. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-04T05:04:05.489794-05:
      DOI: 10.1002/jcp.24790
       
  • Influence of Nuclear Blebs and Micronuclei Status on the Growth Kinetics
           of Human Mesenchymal Stem Cells
    • Authors: Shikha Sharma; Ramesh Bhonde
      Pages: n/a - n/a
      Abstract: Therapeutic potential of mesenchymal stem cells (MSCs) demands assurance of the quality, safety, and genetic stability. Nuclear blebs (NBs) and Micronuclei (MNs) are considered as biomarkers for cancer and an increase in their numbers is associated with malignancy and other pathologic disorders. However, the status of NBs and MNs in MSCs is not known. Hence we examined the frequency of NBs and MNs in MSCs from umbilical cord (UC‐MSCs) and placenta (PD‐MSCs) and found a difference in the number of NBs and MNs depending on the source of the MSCs. The number of NBs and MNs was always found to be less in UC‐MSCs as compared to PD‐MSCs obtained from the same patient. Furthermore, we observed that the number of NBs was inversely proportional to the proliferation rate of the cells. The cryopreservation of these MSCs over 6 months also led to increase in the number of NBs and MNs thus slowing down their rate of proliferation on revival. MSCs from both the sources exhibiting high NBs and MNs showed longer S phase and G2‐M arrest and increase in senescent cells without altering their CD‐marker profile and differentiation potential. This feature was consistent with the upregulation of cell cycle checkpoint genes (p53, p21, Gadd45, ATM, ATR, chek2, p27, p16, and p10). In conclusion, our data demonstrates for the first time the importance of checking the occurrence of NBs and MNs in MSCs before using them for cellular therapy as a quality control measure to check their genetic stability. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-04T05:03:32.484741-05:
      DOI: 10.1002/jcp.24789
       
  • Pin1 Plays a Critical Role as a Molecular Switch in Canonical BMP
           Signaling
    • Authors: Won‐Joon Yoon; Rabia Islam, Young‐Dan Cho, Kyung‐Min Ryu, Hye‐Rim Shin, Kyung‐Mi Woo, Jeong‐Hwa Baek, Hyun‐Mo Ryoo
      Pages: n/a - n/a
      Abstract: Pin1 is a peptidyl prolyl cis‐trans isomerase that specifically binds to the phosphoserine–proline or phosphothreonine–proline motifs of numerous proteins. Previously, we reported that Pin1 deficiency resulted in defects in osteoblast differentiation during early bone development. In this study, we found that adult Pin1‐deficient mice developed osteoporotic phenotypes compared to age‐matched controls. Since BMP2 stored in the bone matrix plays a critical role in adult bone maintenance, we suspected that BMP R‐Smads (Smad1 and Smad5) could be critical targets for Pin1 action. Pin1 specifically binds to the phosphorylated linker region of Smad1, which leads to structural modification and stabilization of the Smad1 protein. In this process, Pin1‐mediated conformational modification of Smad1 directly suppresses the Smurf1 interaction with Smad1, thereby promoting sustained activation of the Smad1 molecule. Our data demonstrate that post‐phosphorylational prolyl isomerization of Smad1 is a converging signal to stabilize the Smad1 molecule against the ubiquitination process mediated by Smurf1. Therefore, Pin1 is a critical molecular switch in the determination of Smad1 fate, opposing the death signal transmitted to the Smad1 linker region by phosphorylation cascades after its nuclear localization and transcriptional activation. Thus, Pin1 could be developed as a major therapeutic target in many skeletal diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-04T04:55:34.928623-05:
      DOI: 10.1002/jcp.24787
       
  • Methotrexate‐Induced Bone Marrow Adiposity is Mitigated by Folinic
           Acid Supplementation Through the Regulation of Wnt/β‐Catenin
           Signalling
    • Authors: Kristen R. Georgiou; Rethi Raghu Nadhanan, Chia‐Ming Fan, Cory J. Xian
      Pages: n/a - n/a
      Abstract: Antimetabolite Methotrexate (MTX) is commonly used in childhood oncology. As a dihydrofolate reductase inhibitor it exerts its action through the reduction of cellular folate, thus its intensive use is associated with damage to soft tissues, bone marrow and bone. In the clinic, MTX is administered with folinic acid (FA) supplementation to alleviate some of this soft tissue damage. However, whether and how FA alleviates damage to the bone and bone marrow requires further investigation. As the Wnt/β‐catenin signalling pathway is critical for commitment and differentiation of mesenchymal stem cells down the osteogenic or adipogenic lineage, its deregulation has been found associated with increased marrow adiposity following MTX treatment. In order to elucidate whether FA supplementation prevents MTX‐induced bone marrow adiposity by regulating Wnt/β‐catenin signalling, young rats were given saline or 0.75mg/kg MTX once daily for 5 days, receiving saline or 0.75mg/kg FA 6h after MTX. FA rescue alleviated the MTX‐induced bone marrow adiposity, as well as well as inducing up‐regulation of Wnt10b mRNA and β‐catenin protein expression in the bone. Furthermore, FA blocked up‐regulation of the secreted Wnt antagonist sFRP‐1 mRNA expression. Moreover, secreted sFRP‐1 protein in the bone marrow and its expression by osteoblasts and adipocytes was found increased following MTX treatment. This potentially indicates that sFRP‐1 is a major regulator of defective Wnt/β‐catenin signalling following MTX treatment. This study provides evidence that folate depletion caused by MTX chemotherapy results in increased bone marrow adiposity, and that FA rescue alleviates these defects by up‐regulating Wnt/β‐catenin signalling in the bone. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-09-04T04:44:20.12122-05:0
      DOI: 10.1002/jcp.24788
       
  • Palmitate induces ER stress and autophagy in H9c2 cells: implications for
           apoptosis and adiponectin resistance
    • Authors: Min Park; Anna Sabetski, Yee Kwan Chan, Subat Turdi, Gary Sweeney
      Abstract: The association between obesity and heart failure is well documented and recent studies have indicated that understanding the physiological role of autophagy will be of great significance. Cardiomyocyte apoptosis is one component of cardiac remodeling which leads to heart failure and in this study we used palmitate‐treated H9c2 cells as an in vitro model of lipotoxicity to investigate the role of autophagy in cell death. Temporal analysis revealed that palmitate (100μM) treatment induced a gradual increase of intracellular lipid accumulation as well as apoptotic cell death. Palmitate induced autophagic flux, determined via increased LC3‐II formation and p62 degradation as well as by detecting reduced colocalization of GFP with RFP in cells overexpressing tandem fluorescent GFP/RFP‐LC3. The increased level of autophagy indicated by these measures were confirmed using transmission electron microscopy (TEM). Upon inhibiting autophagy using bafilomycin we observed an increased level of cell death assessed by Annexin V/PI staining, detection of active caspase‐3 and MTT cell viability assay. Interestingly, using TEM and p‐PERK or p‐eIF2α detection we observed increased endoplasmic reticulum (ER) stress in response to palmitate. Autophagy was induced as an adaptive response against ER stress since it was sensitive to ER stress inhibition. Palmitate‐induced ER stress also induced adiponectin resistance, assessed via AMPK phosphorylation, via reducing APPL1 expression. This effect was independent of palmitate‐induced autophagy. In summary, our data indicate that palmitate induces autophagy subsequent to ER stress and that this confers a prosurvival effect against lipotoxicity‐induced cell death. Palmitate‐induced ER stress also led to adiponecin resistance. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-28T04:18:15.283126-05:
      DOI: 10.1002/jcp.24781
       
  • Thymoquinone restores radiation induced TGF‐β expression and
           abrogate s EMT in chemoradiotherapy of breast cancer cells
    • Authors: Shashi Rajput; B. N. Prashanth Kumar, Payel Banik, Sheetal Parida, Mahitosh Mandal
      Abstract: Radiotherapy remains a prime approach to adjuvant therapies in patients with early and advanced breast cancer. In spite of therapeutic success, metastatic progression in patients undergoing therapy, limits its application. However, effective therapeutic strategies to understand the cellular and molecular machinery in inhibiting radiation induced metastatic progression, which is poorly understood so far, need to be strengthened. Ionizing radiation was known to prompt cancer cell's metastatic ability by eliciting Transforming Growth Factor‐beta (TGF‐ beta), a key regulator in epithelial‐mesenchymal transdifferentiation and radio‐resistance. In this viewpoint, we employed thymoquinone as a radiosensitizer to investigate its migration and invas ion reversal abilities in irradiated breast cancer cell lines by assessing their respective attributes. The role of metastasis regulatory molecules like TGF‐β, E‐cadherin and integrin aV and its downstream molecules were determined using RT‐PCR, western blotting, immunofluorescence and extracellular TGF‐β levels affirmed through ELISA assays. These studies affirmed the TGF‐β restoring ability of thymoquinone in radiation driven migration and invasion. Also, results demonstrated that the epithelial markers E‐cadherin and cytokeratin 19 were down‐regulated whereas mesenchymal markers like integrin aV, MMP9 and MMP2 were upregulated by irradiation treatment, however thymoquinone pre‐sensitization has reverted the expression of these proteins backs to control proteins expression. Here, paclitaxel was chosen as an apoptosis inducer in TGF‐β restored cells and confirmed its cytotoxic effects in radiation alone and thymoquinone sensitized irradiated cells. We conclude that this therapeutic modality is effective in preventing radiation induced epithelial‐mesenchymal transdifferentiation and concomitant induction of apoptosis in breast cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-28T04:16:32.026714-05:
      DOI: 10.1002/jcp.24780
       
  • Editor's Choice
    • PubDate: 2014-08-27T15:58:45.197513-05:
      DOI: 10.1002/jcp.24782
       
  • Highlights: Volume 229, Number 12
    • PubDate: 2014-08-27T15:58:41.966133-05:
      DOI: 10.1002/jcp.24786
       
  • Table of Contents: Volume 229, Number 12
    • PubDate: 2014-08-27T15:58:34.356236-05:
      DOI: 10.1002/jcp.24784
       
  • Journal of Cellular Physiology: Volume 229, Number 12, December 2014
    • Abstract: Cover: A comparison of 2D (left) and 3D (right) heatmaps for summarizing the results of an artificial intelligence analysis of genome‐wide genetic data. The 3D heatmap was implemented using the Unity 3D video game engine and is ideal for visualizing big data because it allows several additional layers of information to be represented and explored. See article by Greene et al. on pages 1896–1900.
      PubDate: 2014-08-27T15:58:33.809834-05:
      DOI: 10.1002/jcp.24785
       
  • PLCâ1a and PLCâ1b selective regulation and cyclin D3 modulation
           reduced by Kinamycin F during K562 cell differentiation
    • Authors: Alberto Bavelloni; Gary I. Dmitrienko, Valerie J. Goodfellow, Ahmad Ghavami, Manuela Piazzi, William Blalock, Francesca Chiarini, Lucio Cocco, Irene Faenza
      Abstract: Here we report that both PLCâ1a and PLCâ1b are relevant regulators of erythropoiesis in that kinamycin F, a potent inducer of γ‐globin production in K562 cells, caused a selectively reduction of both PLCâ1 isozymes even though the results point out that the effect of the drug is mainly directed toward the expression of the PLCâ1a isoform. We have identified a different role for the two isozymes as regulators of K562 differentiation process induced by kinamycin F. The overexpression of PLCâ1b induced an increase in γ‐globin expression even in the absence of kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCâ1 signaling pathway. Namely the amplification of the expression of the PLCâ1a, but not of PLCâ1b, is able to maintain high levels of expression of cyclin D3 even after treatment with kinamycin F. This could be due to their different distribution in the cell compartments since the amount of PLCâ1b is mainly present in the nucleus in respect to PLCâ1a. Our data indicate that the amplification of PLCâ1a expression, following treatment with kinamycin F, confers a real advantage to K562 cells viability and protects cells themselves from apoptosis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T06:10:17.697556-05:
      DOI: 10.1002/jcp.24776
       
  • Initial Characterization of Osteoblast Differentiation and Loss of RUNX2
           Stability in the Newly Established SK11 Human Embryonic Stem
           Cell‐Derived Cell Line
    • Authors: Jiali Yu; Helty Adisetiyo, Gillian H. Little, C.Thomas Vangsness, Jianjie Jiang, Hal Sternberg, Michael D. West, Baruch Frenkel
      Abstract: We describe a novel model for investigation of genetically normal human osteoblasts in culture. SK11 is a clonal progenitor cell line derived from human embryonic stem cells. Initially selected based on the expression of chondrogenic markers when differentiated in micromass culture, SK11 cultures display typical mRNA expression patterns of bone phenotypic genes under osteogenic conditions. These include Osterix, α1(I) collagen, Alkaline phosphatase, Osteonectin, Osteopontin and Osteocalcin. Similar to well‐characterized murine osteoblast cultures, the osteoblast master regulator RUNX2 was present during the first few days after plating, but the protein disappeared during the first week of culture. Loss of RUNX2 expression is considered an important regulatory feature for osteoblast maturation. Indeed, following ∼2 weeks of differentiation, SK11 cultures exhibited robust calcium deposition, evidenced by alizarin red staining. We also introduced a lentiviral vector encoding doxycycline (dox)‐inducible FLAG‐tagged RUNX2 into SK11 cells. Dox‐mediated enhancement of RUNX2 expression resulted in accelerated mineralization, which was further increased by co‐treatment with BMP‐2. Like the endogenous RUNX2, expression of the virally coded FLAG‐RUNX2 was lost during the first week of culture despite persistent dox treatment. By following RUNX2 decay after dox withdrawal from day‐5 versus day‐3 cultures, we demonstrated a developmentally regulated decrease in RUNX2 stability. Availability of culture models for molecular investigation of genetically normal human osteoblasts is important because differences between murine and human osteoblasts, demonstrated here for the regulation of Matrix Gla Protein, may have significant biomedical implications. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T06:05:41.538286-05:
      DOI: 10.1002/jcp.24773
       
  • Recovery and cultivation of keratinocytes from shipped mouse skin
    • Authors: Hsin‐ya Yang; Thi Dinh La, Janna Gurenko, Pieter Steenhuis, Wei Liu, R. Rivkah Isseroff
      Abstract: Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 hours at 0°C. The cultured keratinocytes from the control, non‐shipped skin and the 2‐day shipped skin were 43.6 +/‐ 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta‐1. However, under the same shipping conditions, the 3‐day shipped tissue failed to establish colonies in the culture. Therefore, this 2‐day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long‐distance collaborations. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T05:45:33.828001-05:
      DOI: 10.1002/jcp.24775
       
  • The Differential Palmitoylation States of N‐Ras and H‐Ras
           Determine Their Distinct Golgi Sub‐compartment Localizations
    • Authors: Stephen J. Lynch; Harriet Snitkin, Iwona Gumper, Mark R. Philips, David Sabatini, Angel Pellicer
      Abstract: Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C‐terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H‐Ras and NRas) localize on the Golgi apparatus whereas K‐Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub‐organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H‐Ras is distributed throughout the Golgi stacks, the singly palmitoylated N‐Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants we show that the different sub‐Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane but also their distribution within the Golgi stacks. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T05:43:35.450171-05:
      DOI: 10.1002/jcp.24779
       
  • Derivation of endodermal progenitors from pluripotent stem cells
    • Authors: Laertis Ikonomou; Darrell N. Kotton
      Abstract: Stem and progenitor cells play important roles in organogenesis during development and in tissue homeostasis and response to injury postnatally. As the regenerative capacity of many human tissues is limited, cell replacement therapies hold great promise for human disease management. Pluripotent stem cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are prime candidates for the derivation of unlimited quantities of clinically relevant cell types through development of directed differentiation protocols, i.e. the recapitulation of developmental milestones in in vitro cell culture. Tissue‐specific progenitors, including progenitors of endodermal origin, are important intermediates in such protocols since they give rise to all mature parenchymal cells. In this review, we focus on the in vivo biology of embryonic endodermal progenitors in terms of key transcription factors and signaling pathways. We critically review the emerging literature aiming to apply this basic knowledge to achieve the efficient and reproducible in vitro derivation of endodermal progenitors such as pancreas, liver and lung precursor cells. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T05:41:36.36009-05:0
      DOI: 10.1002/jcp.24771
       
  • Blocking the expression of both bone sialoprotein (BSP) and osteopontin
           (OPN) impairs the anabolic action of PTH in mouse calvaria bone
    • Authors: Wafa Bouleftour; Guénaëlle Bouet, Renata Neves Granito, Mireille Thomas, Marie Thérèse Linossier, Arnaud Vanden Bossche, Jane E Aubin, Marie‐Hélène Lafage‐Proust, Laurence Vico, Luc Malaval
      Abstract: Osteopontin (OPN) and bone sialoprotein (BSP) are coexpressed in osteoblasts and osteoclasts, and display overlapping properties. We used daily injection of parathyroid hormone 1‐84 (iPTH) over the calvaria of BSP knockout (‐/‐) mice to investigate further their functional specificity and redundancy. iPTH stimulated bone formation in both +/+ and ‐/‐ mice, increasing to the same degree periosteum, osteoid and total bone thickness. Expression of OPN, osterix, osteocalcin (OCN) and DMP1 was also increased by iPTH in both genotypes. In contrast to +/+, calvaria cell cultures from ‐/‐ mice revealed few osteoblast colonies, no mineralization and little expression of OCN, MEPE or DMP1. In contrast, OPN levels were 5x higher in ‐/‐ vs +/+ cultures. iPTH increased alkaline phosphatase (ALP) activity in cell cultures of both genotypes, with higher OCN and the induction of mineralization in ‐/‐ cultures. siRNA blocking of OPN expression did not alter the anabolic action of the hormone in BSP+/+ calvaria, while it blunted iPTH effects in ‐/‐ mice, reduced to a modest increase in periosteum thickness. In ‐/‐ (not +/+) cell cultures, siOPN blocked the stimulation by iPTH of ALP activity and OCN expression, as well as the induction of mineralization. Thus, full expression of either OPN or BSP is necessary for the anabolic effect of PTH at least in the ectopic calvaria injection model. This suggests that OPN may compensate for the lack of BSP in the response to this hormonal challenge, and provides evidence of functional overlap between these cognate proteins. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T05:39:39.199422-05:
      DOI: 10.1002/jcp.24772
       
  • Modulation of mast cell proliferative and inflammatory responses by
           Leukotriene D4 and Stem Cell Factor signaling interactions
    • Authors: Nosayba Al‐Azzam; Vinay Kondeti, Ernest Duah, Farai Gombedza, Charles K. Thodeti, Sailaja Paruchuri
      Abstract: Mast cells (MCs) are important effector cells in asthma and pulmonary inflammation, and their proliferation and maturation is maintained by stem cell factor (SCF) via its receptor, c‐Kit. Cysteinyl leukotrienes (cys‐LTs) are potent inflammatory mediators that signal through CysLT1R and CysLT2R located on the MC surface, and they enhance MC inflammatory responses. However, it is not known if SCF and cys‐LTs cross‐talk and influence MC hyperplasia and activation in inflammation. Here, we report the concerted effort of the growth factor SCF and the inflammatory mediator LTD4 in MC activation. Stimulation of MCs by LTD4 in the presence of SCF enhances c‐Kit‐mediated proliferative responses. Similarly, SCF synergistically enhances LTD4‐induced calcium, c‐fos expression and phosphorylation, as well as MIP1β generation in MCs. These findings suggest that integration of SCF and LTD4 signals may contribute to MC hyperplasia and hyper‐reactivity during airway hyper‐response and inflammation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T05:38:38.356789-05:
      DOI: 10.1002/jcp.24777
       
  • Signaling pathways involved in megakaryocyte‐mediated proliferation
           of osteoblast lineage cells
    • Authors: Ying‐Hua Cheng; Drew A. Streicher, David L. Waning, Brahmananda R. Chitteti, Rita Gerard‐O'Riley, Mark C. Horowitz, Joseph P. Bidwell, Fredrick M. Pavalko, Edward F. Srour, Lindsey D. Mayo, Melissa A. Kacena
      Abstract: Recent studies suggest that megakaryocytes (MKs) may play a significant role in skeletal homeostasis, as evident by the occurrence of osteosclerosis in multiple MK related diseases (Thiele, et al., 1999, Lennert, et al., 1975, Chagraoui, et al., 2006). We previously reported a novel interaction whereby MKs enhanced proliferation of osteoblast lineage/osteoprogenitor cells (OBs) by a mechanism requiring direct cell‐cell contact. However, the signal transduction pathways and the downstream effector molecules involved in this process have not been characterized. Here we show that MKs contact with OBs, via beta1 integrin, activate the p38/MAPKAPK2/p90RSK kinase cascade in the bone cells, which causes Mdm2 to neutralizes p53/Rb ‐mediated check point and allows progression through the G1/S. Interestingly, activation of MAPK (ERK1/2) and AKT, collateral pathways that regulate the cell cycle, remained unchanged with MK stimulation of OBs. The MK‐to‐OB signaling ultimately results in significant increases in the expression of c‐fos and cyclin A, necessary for sustaining the OB proliferation. Overall, our findings show that OBs respond to the presence of MKs, in part, via an integrin‐mediated signaling mechanism, activating a novel response axis that de‐represses cell cycle activity. Understanding the mechanisms by which MKs enhance OB proliferation will facilitate the development of novel anabolic therapies to treat bone loss associated with osteoporosis and other bone ‐related diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T05:36:25.937558-05:
      DOI: 10.1002/jcp.24774
       
  • Inhibition of metalloproteinase activity in FANCA is linked to altered
           oxygen metabolism
    • Authors: Silvia Ravera; Cristina Capanni, Danika Tognotti, Roberta Bottega, Marta Columbaro, Carlo Dufour, Enrico Cappelli, Paolo Degan
      Abstract: Bone marrow (BM) failure, increased risk of myelodysplastic syndrome, acute leukaemia and solid tumors, endocrinopathies and congenital abnormalities are the major clinical problems in Fanconi Anemia patients (FA). Chromosome instability and DNA repair defects are the cellular characteristics used for the clinical diagnosis. However, these biological defects are not sufficient to explain all the clinical phenotype of FA patie nts. The known defects are structural alteration in cell cytoskeleton , altered structural organization for intermediate filaments, nuclear lamina and mitochondria. These are associated with different expression and/or maturation of the structural proteins vimentin, mitofilin and lamin A/C. suggesting the involvement of metalloproteinases (MPs). Matrix metalloproteinases (MMP) are involved in normal physiological processes such as human skeletal tissue development, maturation and hematopoietic reconstitution after bone marrow suppression. Current observations upon the eventual role of MPs in FA cells are largely inconclusive. We evaluated the overall MPs activity in FA complementation group A (FANCA) cells by exposing them to the antioxidants N‐acetyl cysteine (NAC) and resveratrol (RV). This work supports the hypothesis that treatment of Fanconi patients with antioxidants may be important in FA therapy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T05:34:31.884249-05:
      DOI: 10.1002/jcp.24778
       
  • LPS promote the odontoblastic differentiation of human dental pulp stem
           cells via MAPK, but not NF‐KB signaling pathway
    • Authors: Wenxi He; Zhihua Wang, Zhirong Luo, Qing Yu, Yong Jiang, Yaqing Zhang, Zeyuan Zhou, Anthony J. Smith, Paul R. Cooper
      Abstract: Human dental pulp stem cells (hDPSCs) show significant potential for exploitation in novel regeneration strategies, although lack of understanding of their responses to bacterial challenge constrains their application. The present study aimed to investigate whether lipopolysaccharide (LPS), the major pathogenic factor of Gram‐negative bacteria, regulates the differentiation of hDPSCs and which intracellular signaling pathways may be involved. LPS treatment significantly promoted the differentiation of hDPSCs demonstrable by increased mineralized nodule formation and mRNA expression of several odontoblastic markers in a dose‐dependent manner. While inhibition of TLR4, p38, and ERK signaling markedly antagonized LPS‐mediated differentiation of hDPSCs. The inhibition of JNK and NF‐κB signaling had no detectable effect on LPS activation of hDPSCs. LPS stimulation resulted in phosphorylation of NF‐κB p65, IκB‐α, extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (MAPK) in DPSCs in a time‐dependent manner, which was markedly suppressed by their specific inhibitors, respectively. Data demonstrated that LPS promoted odontoblastic differentiation of hDPSCs via TLR4, ERK, and P38 MAPK signaling pathways, but not NF‐κB signaling. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-07T15:41:01.136015-05:
      DOI: 10.1002/jcp.24732
       
  • VEGF receptor 2 (VEGFR2) activation is essential for osteocyte survival
           induced by mechanotransduction
    • Authors: L F de Castro; M Maycas, B Bravo, P Esbrit, AR Gortazar
      Abstract: Mechanical loading plays a key role in bone formation and maintenance. While unloading induces osteocyte apoptosis and bone loss in vivo, mechanical stimuli prevents osteocyte death through a mechanism involving β‐catenin accumulation and ERK nuclear translocation. Vascular endothelial growth factor (VEGF) has a crucial role in bone formation, but its interaction with osteocytes is not completely understood. Of interest, VEGF receptor 2 (VEGFR2) has recently been shown to mediate the mechanical response of endothelial cells. The present study aimed to evaluate the putative role of the VEGF system in osteocyte mechanosensing. We show that either short (10 min) mechanical stimulus by pulsatile fluid flow (FF) (10 dyn/cm2, 8 Hz) or exogenous VEGF165 (6 ng/ml) similarly stimulated cell viability, ERK phosphorylation, and β‐catenin membrane translocation. A VEGFR2 antagonist (SU5416) or transfection with specific VEGFR2 siRNAs (siVEGFR2) decreased these events. FF for 10 min increased VEGFR2 phosphorylation at both Tyr‐1059 and Tyr‐1175; an effect that was mimicked by VEGF165 but was unaffected by a VEGF neutralizing antibody. Subsequently (at 6 h), this mechanical stimulus induced VEGF gene overexpression, which was prevented by siVEGFR2 transfection. Depletion of the structural protein caveolin‐1 by using siRNA technology impaired FF‐induced VEGFR2 phosphorylation. In conclusion, these in vitro findings point to caveolin‐1‐dependent VEGFR2 activation as an important mechanism whereby mechanical stimuli promote osteocyte viability. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-07T15:25:52.868833-05:
      DOI: 10.1002/jcp.24734
       
  • Detection of Phosphorylated Insulin Receptor in Colorectal Adenoma and
           Adenocarcinoma: Implications for Prognosis and Clinical Outcome
    • Authors: Claudia Abbruzzese; Maria Grazia Diodoro, Isabella Sperduti, Anna Maria Mileo, Giada Pattaro, Laura De Salvo, Maurizio Cosimelli, Nicola Perrotti, Marco G. Paggi
      Abstract: Colorectal carcinoma remains among the most frequent causes of cancer death. Besides the well‐known genetic predisposition, a key role in colorectal adenoma and adenocarcinoma etio‐pathogenesis, mainly in sporadic cases, is played by definite risk factors, such as obesity, type 2 diabetes, insulin resistance, hyper‐insulinemia, and insulin therapy. These epidemiological data motivated us to determine, by means of immunohistochemistry, the amount of activated (phosphorylated) insulin receptor in archival samples from 22 colorectal adenoma and 117 adenocarcinoma patients, with the objective to estimate the role of this factor in colorectal epithelium transformation and cancer progression. Statistical analysis of the results clearly showed that positive staining for phosphorylated insulin receptor was significantly more frequent in adenomas than adenocarcinomas (P 
      PubDate: 2014-08-07T15:25:41.185516-05:
      DOI: 10.1002/jcp.24733
       
  • Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane
           Reveals Roles of Acrosomal Proteins in ZP3 Binding
    • Authors: Kessiri Kongmanas; Hathairat Kruevaisayawan, Arpornrad Saewu, Clarissa Sugeng, Jason Fernandes, Puneet Souda, Jonathan B. Angel, Kym F. Faull, R. John Aitken, Julian Whitelegge, Daniel Hardy, Trish Berger, Mark A. Baker, Nongnuj Tanphaichitr
      Abstract: The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm‐ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non‐capacitated and capacitated sperm. The results revealed that ZP‐binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far‐western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non‐capacitated and capacitated sperm, but the complexes (~750‐1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome‐intact sperm during capacitation. Co‐immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with antizonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm‐ZP binding during capacitation. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-07-30T17:09:02.901656-05:
      DOI: 10.1002/jcp.24728
       
  • Non‐random Patterns in the Distribution of NOR‐bearing
           Chromosome Territories in Human Fibroblasts: A Network Model of
           Interactions
    • Authors: Artem Pliss; Andrew J. Fritz, Branislav Stojkovic, Hu Ding, Lopamudra Mukherjee, Sambit Bhattacharya, Jinhui Xu, Ronald Berezney
      Abstract: We present a 3‐D mapping in WI38 human diploid fibroblast cells of chromosome territories (CT) 13,14,15,21, and 22, which contain the nucleolar organizing regions (NOR) and participate in the formation of nucleoli. The nuclear radial positioning of NOR‐CT correlated with the size of chromosomes with smaller CT more interior. A high frequency of pairwise associations between NOR‐CT ranging from 52% (CT13‐21) to 82% (CT15‐21) was detected as well as a triplet arrangement of CT15‐21‐22 (72%). The associations of homologous CT were significantly lower (24‐36%). The arrangements of each pairwise CT varied from CT13‐14 and CT13‐22, which had a majority of cells with single associations, to CT13‐15 and CT13‐21 where a majority of cells had multiple interactions. In cells with multiple nucleoli, one of the nucleoli (termed “dominant”) always associated with a higher number of CT. Moreover, certain CT pairs more frequently contributed to the same nucleolus than to others. This nonrandom pattern suggests that a large number of the NOR‐chromsomes are poised in close proximity during the postmitotic nucleolar recovery and through their NORs may contribute to the formation of the same nucleolus. A global data mining program termed the chromatic median determined the most probable interchromosomal arrangement of the entire NOR‐CT population. This interactive network model was significantly above randomized simulation and was composed of 13 connections among the NOR‐CT. We conclude that the NOR‐CT form a global interactive network in the cell nucleus that may be a fundamental feature for the regulation of nucleolar and other genomic functions. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-07-30T17:00:50.88451-05:0
      DOI: 10.1002/jcp.24726
       
  • Mouse Matrix Metalloprotease‐1a (Mmp1a) Gives New Insight Into MMP
           Function
    • Authors: Caitlin J. Foley; Athan Kuliopulos
      First page: 1875
      Abstract: Matrix metalloprotease‐1 (MMP1) has been implicated in many human disease processes, however the lack of a well characterized murine homologue has significantly limited the study of MMP1 and the development of MMP‐targeted therapeutics. The discovery of murine Mmp1a in 2001, the functional mouse homologue of MMP1, offers a valuable tool for modeling MMP1‐mediated processes in mice. Variation in physiologic expression levels of Mmp1a in mice as compared to MMP1 in humans highlights the importance of understanding the similarities and differences between the homologues. Recent studies have demonstrated tumor growth‐, invasion‐, and angiogenesis‐promoting functions of Mmp1a in lung cancer models, consistent with the analogous functions observed for human MMP1. Biochemical investigations have shown that point mutations in the pro‐domain of mouse Mmp1a weaken docking between the pro‐ and catalytic domains, generating an unstable zymogen primed for activation. The difficulty to effectively maintain Mmp1a in the zymogen form may account for the tight control of Mmp1a expression and reduced expression in normal tissue as compared to inflammatory states or cancer. This discovery raises important questions about the activation mechanisms and regulation of the MMP family in general. J. Cell. Physiol. 229: 1875–1880, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:40.698707-05:
      DOI: 10.1002/jcp.24650
       
  • Spindle Microtubule Dysfunction and Cancer Predisposition
    • Authors: Jason Stumpff; Prachi N. Ghule, Akiko Shimamura, Janet L. Stein, Marc Greenblatt
      First page: 1881
      Abstract: Chromosome segregation and spindle microtubule dynamics are strictly coordinated during cell division in order to preserve genomic integrity. Alterations in the genome that affect microtubule stability and spindle assembly during mitosis may contribute to genomic instability and cancer predisposition, but directly testing this potential link poses a significant challenge. Germ‐line mutations in tumor suppressor genes that predispose patients to cancer and alter spindle microtubule dynamics offer unique opportunities to investigate the relationship between spindle dysfunction and carcinogenesis. Mutations in two such tumor suppressors, adenomatous polyposis coli (APC) and Shwachman–Bodian–Diamond syndrome (SBDS), affect multifunctional proteins that have been well characterized for their roles in Wnt signaling and interphase ribosome assembly, respectively. Less understood, however, is how their shared involvement in stabilizing the microtubules that comprise the mitotic spindle contributes to cancer predisposition. Here, we briefly discuss the potential for mutations in APC and SBDS as informative tools for studying the impact of mitotic spindle dysfunction on cellular transformation. J. Cell. Physiol. 229: 1881–1883, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:37.840525-05:
      DOI: 10.1002/jcp.24691
       
  • Disruption of Crosstalk between Mesenchymal Stromal and Tumor Cells in
           Bone Marrow as a Therapeutic Target to Prevent Metastatic Bone Disease
    • Authors: Jonathan A. R. Gordon; Jennifer W. Lisle, Benjamin A. Alman, Jane B. Lian
      First page: 1884
      Abstract: Skeletal metastasis is a serious complication of many primary cancers. A common feature of tumor cells that metastasize to the bone marrow microenvironment is that they initiate a cascade of events, recruiting and presumably/potentially altering the phenotype of bone marrow mesenchymal stromal cells (MSC) to produce an environment that allows for tumor growth and in some cases, drug‐resistant dormancy of latent cancer cells. Consequently the MSC population can contribute to metastatic disease through several distinct mechanisms by differentiating into cancer‐associated fibroblasts (CAFs). Understanding the expression and epigenetic changes that occur as normal MSCs become associated with metastatic tumors would reveal possible therapeutic targets for treating skeletal metastasis. J. Cell. Physiol. 229: 1884–1886, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:37.145476-05:
      DOI: 10.1002/jcp.24692
       
  • Isoforms of Receptors of Fibroblast Growth Factors
    • Authors: Siew‐Ging Gong
      First page: 1887
      Abstract: The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high‐throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases. J. Cell. Physiol. 229: 1887–1895, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:44.512378-05:
      DOI: 10.1002/jcp.24649
       
  • Big Data Bioinformatics
    • Authors: Casey S. Greene; Jie Tan, Matthew Ung, Jason H. Moore, Chao Cheng
      First page: 1896
      Abstract: Recent technological advances allow for high throughput profiling of biological systems in a cost‐efficient manner. The low cost of data generation is leading us to the “big data” era. The availability of big data provides unprecedented opportunities but also raises new challenges for data mining and analysis. In this review, we introduce key concepts in the analysis of big data, including both “machine learning” algorithms as well as “unsupervised” and “supervised” examples of each. We note packages for the R programming language that are available to perform machine learning analyses. In addition to programming based solutions, we review webservers that allow users with limited or no programming background to perform these analyses on large data compendia. J. Cell. Physiol. 229: 1896–1900, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:34.525682-05:
      DOI: 10.1002/jcp.24662
       
  • Negative Regulators of Brown Adipose Tissue (BAT)‐Mediated
           Thermogenesis
    • Authors: Bal Krishan Sharma; Mallikarjun Patil, Ande Satyanarayana
      First page: 1901
      Abstract: Brown adipose tissue (BAT) is specialized for energy expenditure, a process called adaptive thermogenesis. PET‐CT scans recently demonstrated the existence of metabolically active BAT in adult humans, which revitalized our interest in BAT. Increasing the amount and/or activity of BAT holds tremendous promise for the treatment of obesity and its associated diseases. PGC1α is the master regulator of UCP1‐mediated thermogenesis in BAT. A number of proteins have been identified to influence thermogenesis either positively or negatively through regulating the expression or transcriptional activity of PGC1α. Therefore, BAT activation can be achieved by either inducing the expression of positive regulators of PGC1α or by inhibiting the repressors of the PGC1α/UCP1 pathway. Here, we review the most important negative regulators of PGC1α/UCP1 signaling and their mechanism of action in BAT‐mediated thermogenesis. J. Cell. Physiol. 229: 1901–1907, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:39.366576-05:
      DOI: 10.1002/jcp.24664
       
  • Wnt5a Suppresses Colon Cancer by Inhibiting Cell Proliferation and
           Epithelial–Mesenchymal Transition
    • Authors: Runfen Cheng; Baocun Sun, Zhiyong Liu, Xiulan Zhao, Lisha Qi, Yixian Li, Qiang Gu
      First page: 1908
      Abstract: Colon cancer remains one of the lethal malignancies in the world. Aberrant activation of canonical Wnt/β‐catenin signaling pathway has been observed in colon cancer. In contrast, the non‐canonical Wnt signaling functions remain obscure. Wnt5a is a representative non‐canonical Wnt ligand which has gained extensive attention nowadays. Wnt5a has been shown to play an important role in EMT in prostate cancer and melanoma, but its role in colon cancer is still ambiguous. Here we have evaluated Wnt5a expression in a large cohort of 217 colon cancers by immunohistochemistry and analyzed its correlation with clinicopathologic characteristics. We found that expression of Wnt5a was diminished significantly in majority of primary colon cancers and negatively related with EMT biomarkers. To further enlighten the mechanism which Wnt5a regulates EMT in vitro, we established ectopic Wnt5a expression models. Protein analysis demonstrated that Wnt5a inhibited EMT and antagonized canonical Wnt signaling in colon cancer cells. Overexpression of Wnt5a impaired cell motility and invasion and inhibited cell proliferation by manipulating Bax. Moreover, Wnt5a suppressed the tumor growth in nude mice and impaired tumorigenicity in vivo. Wnt5a also induced intracellular calcium and activated non‐canonical Wnt/Ca2+ signaling in colon cancer. In summary, although Wnt5a was down‐regulated in majority of colon cancers, enhanced Wnt5a expression predict preferable outcome in colon cancer patients. Our findings indicate that Wnt5a might act as tumor suppressor by inhibiting cell proliferation and attenuating EMT in colon cancer cells. Wnt5a could be used as a novel prognostic marker and/or therapeutic target for colon cancer in the future. J. Cell. Physiol. 229: 1908–1917, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:38.867872-05:
      DOI: 10.1002/jcp.24566
       
  • Hypomethylating Agent 5‐Aza‐2′‐deoxycytidine (DAC)
           Ameliorates Multiple Sclerosis in Mouse Models
    • Authors: Katia Mangano; Paolo Fagone, Klaus Bendtzen, Pier Luigi Meroni, Cinzia Quattrocchi, Santa Mammana, Michelino Di Rosa, Lucia Malaguarnera, Marinella Coco, Gaetano Magro, Roberto Di Marco, Ferdinando Nicoletti
      First page: 1918
      Abstract: Increasing evidence supports the role of epigenetics in the development of autoimmune disorders and the possibility of using epigenetic modifying drugs in the context of MS has not yet been investigated. We have explored the effect of the hypomethylating agent 5‐aza‐2′‐deoxycytidine (DAC) in two murine models of experimental allergic encephalomyelitis (EAE). DAC treatment was associated with a significant amelioration of the clinical and histological hallmarks of EAE in both models. These effects were observed both in prophylactic and therapeutic regimens. The milder course of the disease was associated with a reduction in the number of spinal cord infiltrating lymphocytes and amelioration of the histopathological signs associated with EAE. In addition, increased transcript levels of anti‐inflammatory cytokines and decreased mRNA expression of pro‐inflammatory mediators were also observed. Finally, DAC treatment increased the percentage of circulating regulatory T cells by inducing Foxp3 expression via demethylation of a CpG island in Foxp3. J. Cell. Physiol. 229: 1918–1925, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:42.515526-05:
      DOI: 10.1002/jcp.24641
       
  • Primary Cilium Regulates CaV1.2 Expression Through Wnt Signaling
    • Authors: Brian S. Muntean; Xingjian Jin, Frederick E. Williams, Surya M. Nauli
      First page: 1926
      Abstract: Primary cilia are sensory organelles that provide a feedback mechanism to restrict Wnt signaling in the absence of endogenous Wnt activators. Abnormal Wnt signaling has been shown to result in polycystic kidney disease (PKD) although the exact mechanism has been debated. Previously, we reported that the calcium channel CaV1.2 functions in primary cilia. In this study, we show that CaV1.2 expression level is regulated by Wnt signaling. This occurs through modulation of mitochondrial mass and activity resulting in increased reactive oxygen species which generate oxidative DNA lesions. We found that the subsequent cellular DNA damage response triggers increased CaV1.2 expression. In the absence of primary cilia where Wnt signaling is upregulated, we found that CaV1.2 is overexpressed as a compensatory mechanism. We show for the first time that CaV1.2 knockdown in zebrafish results in classic primary cilia defects including renal cyst formation, hydrocephalus, and left‐right asymmetry defects. Our study shows that suppressed Wnt signaling prevents CaV1.2 expression ultimately resulting in PKD phenotypes. Thus, CaV1.2 expression is tightly regulated through Wnt signaling and plays an essential sensory role in primary cilia necessary for cellular homeostasis. J. Cell. Physiol. 229: 1926–1934, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:45.228701-05:
      DOI: 10.1002/jcp.24642
       
  • CCL27 Is Downregulated by Interferon Gamma via Epidermal Growth Factor
           Receptor in Normal Human Epidermal Keratinocytes
    • Authors: Masaru Karakawa; Mayumi Komine, Yasushi Hanakawa, Hidetoshi Tsuda, Koji Sayama, Kunihiko Tamaki, Mamitaro Ohtsuki
      First page: 1935
      Abstract: The cutaneous T cell‐attracting chemokine (CTACK)/CCL27 is indispensable in skin inflammation. CTACK/CCL27 is exclusively produced by epidermal keratinocytes to attract CCR10‐expressing T lymphocytes to the skin. We investigated the mechanism of CTACK/CCL27 production from normal human epidermal keratinocytes (NHEKs) by the proinflammatory cytokines TNFα and IFNγ. CTACK/CCL27 production was induced by TNFα via ERK, JNK, p38, and NFκB. The induction of CTACK/CCL27 by TNFα was suppressed by IFNγ via a pathway dependent on JAK, STAT1, and STAT3. Our results also demonstrated that IFNγ and TNFα induced the phosphorylation of EGFR and the following phosphorylation of ERK, which is partly responsible for the suppressive effect of IFNγ on TNFα‐induced production of CTACK/CCL27. Peri‐lesional skin of psoriasis demonstrates early inflammatory changes as we have previously reported. CTACK/CCL27 expression was diffuse in the peri‐lesional epidermis, while it was restricted to basal layer in lesional epidermis, suggesting that CTACK/CCL27 expression was induced in the early stage of psoriatic plaque formation, and IFNγ could participate in the suppression of CTACK/CCL27 expression in the lesional epidermis, reflecting the later stage of psoriatic plaque formation. Our study suggests that CTACK/CCL27 may have a pivotal role in the early stage of psoriasis plaque formation, but should be downregulated in the later stage to induce inflammation characteristic for chronic psoriasis plaques. J. Cell. Physiol. 229: 1935–1945, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:38.421322-05:
      DOI: 10.1002/jcp.24643
       
  • Inactivation of the LOX‐1 Pathway Promotes the Golgi Apparatus
           during Cell Differentiation of Mural Granulosa Cells
    • Authors: J.M. Weitzel; A. Vernunft, B. Krüger, C. Plinski, T. Viergutz
      First page: 1946
      Abstract: In female mammals, granulosa cells of the ovarian follicle differentiate into the corpus luteum after ovulation of the pregnable oocyte into the fallopian tube. During these differentiation processes several morphological alterations have to occur and the molecular basis is not fully understood. As an endpoint estradiol production from granulosa cells has to switch off in favor for progesterone production from the proceeding corpus luteum to sustain the developing embryo. Previously, we demonstrated that the multiligand receptor LOX‐1 plays a critical role in steroid hormone synthesis of granulosa cells via intracellular calcium release from endoplasmic (ER)‐dependent and ER‐independent calcium pools. In the present study, we show that inhibition of LOX‐1 leads to a rearrangement of ceramide from the basal membrane toward the Golgi apparatus. This activity is accomplished by a calcium‐dependent phosphorylation of aromatase, the key step in estradiol production. Phosphorylated aromatase increased estradiol production in a dose‐dependent manner. Our data indicate that the ceramide cascade is essential for proper granulosa cell function and ceramide redistribution serves as a first step in order to proceed with the prosperous differentiation into a corpus luteum. J. Cell. Physiol. 229: 1946–1951, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:47.471396-05:
      DOI: 10.1002/jcp.24644
       
  • Analysis of Global Changes in Gene Expression Induced by Human
           Polynucleotide Phosphorylase (hPNPaseold‐35)
    • Authors: Upneet K. Sokhi; Manny D. Bacolod, Luni Emdad, Swadesh K. Das, Catherine I. Dumur, Michael F. Miles, Devanand Sarkar, Paul B. Fisher
      First page: 1952
      Abstract: As a strategy to identify gene expression changes affected by human polynucleotide phosphorylase (hPNPaseold‐35), we performed gene expression analysis of HeLa cells in which hPNPaseold‐35 was overexpressed. The observed changes were then compared to those of HO‐1 melanoma cells in which hPNPaseold‐35 was stably knocked down. Through this analysis, 90 transcripts, which positively or negatively correlated with hPNPaseold‐35 expression, were identified. The majority of these genes were associated with cell communication, cell cycle, and chromosomal organization gene ontology categories. For a number of these genes, the positive or negative correlations with hPNPaseold‐35 expression were consistent with transcriptional data extracted from the TCGA (The Cancer Genome Atlas) expression datasets for colon adenocarcinoma (COAD), skin cutaneous melanoma (SKCM), ovarian serous cyst adenocarcinoma (OV), and prostate adenocarcinoma (PRAD). Further analysis comparing the gene expression changes between Ad.hPNPaseold‐35 infected HO‐1 melanoma cells and HeLa cells overexpressing hPNPaseold‐35 under the control of a doxycycline‐inducible promoter, revealed global changes in genes involved in cell cycle and mitosis. Overall, this study provides further evidence that hPNPaseold‐35 is associated with global changes in cell cycle‐associated genes and identifies potential gene targets for future investigation. J. Cell. Physiol. 229: 1952–1962, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:33.846445-05:
      DOI: 10.1002/jcp.24645
       
  • Beclin‐1 Is Required for RANKL‐Induced Osteoclast
           Differentiation
    • Authors: Yeon‐Ho Chung; Youngsaeng Jang, Bongkun Choi, Da‐Hyun Song, Eun‐Jin Lee, Sang‐Min Kim, Youngsup Song, Sang‐Wook Kang, Seung‐Yong Yoon, Eun‐Ju Chang
      First page: 1963
      Abstract: Beclin‐1 plays a critical role in autophagy; however, it also contributes to other biological processes in a non‐autophagic manner. Although studies have examined the non‐autophagic role of autophagy proteins in the secretory function of osteoclasts (OC), the role of Beclin‐1 is unclear. Here, we examined the role of Beclin‐1 in OC differentiation, and found that mouse bone marrow macrophages (BMMs) showed increased expression of Beclin‐1 upon RANKL stimulation in a p38‐ and NF‐kappa B‐dependent manner. During OC differentiation, Beclin‐1 localized to the mitochondria, where it was involved in the production of mitochondrial intracellular reactive oxygen species. Knockdown of Beclin‐1 in RANKL‐primed BMMs led to a significant reduction in RANKL‐dependent osteoclastogenesis, which was accompanied by reduced NFATc1 induction. Furthermore, knockdown of Beclin‐1 inhibited RANKL‐mediated activation of JNK and p38, both of which act downstream of reactive oxygen species, resulting in the suppression of NFATc1 induction. Finally, overexpression of constitutively active NFATc1 rescued the phenotype induced by Beclin‐1 knockdown, indicating that Beclin‐1 mediates RANKL‐induced osteoclastogenesis by regulating NFATc1 expression. These findings show that Beclin‐1 plays a non‐autophagic role in RANKL‐induced osteoclastogenesis by inducing the production of reactive oxygen species and NFATc1. J. Cell. Physiol. 229: 1963–1971, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:38.060717-05:
      DOI: 10.1002/jcp.24646
       
  • Advanced Glycation End Products Induce Lipogenesis: Regulation by Natural
           Xanthone through Inhibition of ERK and NF‐κB
    • Authors: Sidhartha K. Mahali; Neeharika Verma, Sunil K. Manna
      First page: 1972
      Abstract: Advanced glycation end products (AGE) accumulate in diabetic patients and aged persons due to high amounts of 3‐ or 4‐carbon derivatives of glucose. Understanding the mechanism of AGE‐mediated signaling leading to these consequences, like oxidative stress, inflammation, apoptosis, etc. and its regulation would be a viable strategy to control diabetic complication and age‐related diseases. We have detected the probable mechanism by which AGE increases lipogenesis, the cause of fatty liver in diabetic patients. AGE increased lipid accumulation in different cells as shown by Oil Red O staining. AGE‐mediated regulation of several transcription factors was determined by gel shift assay. Antioxidants like NAC, PDTC, and vitamin C, except mangiferin, were unable to protect AGE‐induced activation of SREBP and subsequent lipid accumulation. AGE increased the phosphorylation of ERK, and IKK and also DNA binding ability of SREBP, thereby its dependent gene transcription. AGE induces NF‐κB which might suppress PPARγ activity, in turn reducing lipid breakdown and mobilization. Mangiferin not only inhibits AGE‐mediated ROI generation that requires NF‐κB activation, but also inhibits ERK and IKK activity, thereby suppression of SREBP activity and lipogenesis. Mangiferin has shown a double‐edged sword effect to suppress AGE‐mediated ailments by reducing ROI‐mediated responses as antioxidant and inhibiting SREBP activation thereby lipogenesis, suggesting its potential efficacy against diabetes and obesity‐related diseases. J. Cell. Physiol. 229: 1972–1980, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:34.850008-05:
      DOI: 10.1002/jcp.24647
       
  • Mg2+‐Dependent Modulation of BKCa Channels by Genistein in Rat
           Arteriolar Smooth Muscle Cells
    • Authors: Xiaoran Wang; Tingting Zhao, Shanshan Zhou, Lina Sun, Liming Zhang, Guichun Yu
      First page: 1981
      Abstract: Genistein, a protein tyrosine kinase (PTK) inhibitor, regulates ion channel activities. However, the mechanism of action of genistein on large‐conductance calcium‐activated potassium (BKCa) channels is unclear. This study aimed to investigate whether the mechanism of Mg2+‐dependent modulation of BKCa channel activity in vascular smooth muscle cells involved inhibition of phosphorylation by genistein or direct interaction between genistein and BKCa channels. The whole‐cell and inside‐out patch‐clamp techniques were used to measure BKCa currents and the effects of genistein on BKCa channel activities in rat mesenteric arteriolar smooth muscle cells. We found that the effects of genistein on BKCa currents were Mg2+‐dependent. Genistein (50 μM) inhibited BKCa currents if the intracellular free magnesium concentration ([Mg2+]i) was 2 μM or 20 μM, but amplified BKCa currents if [Mg2+]i was 200 μM or 2000 μM. The inhibitory effect of genistein on BKCa currents was reversed by the protein tyrosine phosphatase inhibitor sodium orthovanadate (0.5 mM). Daidzein (50 μM), an inactive analogue of genistein, also amplified BKCa currents, and its amplification was insensitive to orthovanadate. Another PTK inhibitor, tyrphostin 23 (50 μM), reduced the open probability of BKCa channels. This inhibitory effect was weaker at 200 μM [Mg2+]i than at 2 μM [Mg2+]i, and was countered by orthovanadate. Our results suggest that genistein amplifies BKCa currents at a high [Mg2+]i, but inhibits BKCa currents at a low [Mg2+]i. The mechanism of this biphasic effects involves PTK‐independent amplification and [Mg2+]i‐PTK‐dependent inhibition. J. Cell. Physiol. 229: 1981–1989, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:43.523214-05:
      DOI: 10.1002/jcp.24648
       
  • Statin‐Induced Impairment of Monocyte Migration Is
           Gender‐Related
    • Authors: Anna Ruggieri; Lucrezia Gambardella, Angela Maselli, Rosa Vona, Simona Anticoli, Alessia Panusa, Walter Malorni, Paola Matarrese
      First page: 1990
      Abstract: Statins, widely used for treatment of hypercholesterolemia, have been demonstrated to exert pleiotropic beneficial effects independently of their cholesterol‐lowering action, such as anti‐inflammatory activity. A gender disparity has been observed in their cholesterol lowering activity as well as in response to these “off label” effects. Monocytes play a central role in atherosclerotic disease and, more in general, in inflammatory responses, through their chemotactic function and cytokine production. On these bases, in the present work, we examined the effect of statins on homeostasis and migration properties of freshly isolated monocytes from male and female healthy donors. Two prototypic natural and synthetic statins with different polarity, that is, type 1 and type 2 statins, have been considered: simvastatin and atorvastatin. Freshly isolated monocytes from peripheral blood of male and female healthy donors were treated with these drugs in the absence or presence of lipopolysaccharide (LPS) stimulation. Results obtained indicated that the polar statin efficiently inhibited chemotaxis of monocytes more than the apolar statin and that this effect was more significantly induced in cells from females than in cells from males. Dissecting the mechanisms involved, we found that these results could mainly be due to differential effects on: (i) the release of key cytokines, for example, MCP‐1 and TNF‐α; (ii) the maintenance of the redox homeostasis; (iii) a target activity on microfilament network integrity and function. All in all these results could suggest a reappraisal of “off‐label” effects of statins taking into account either their chemical structure, that is, molecular polarity, or the gender issue. J. Cell. Physiol. 229: 1990–1998, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:35.793521-05:
      DOI: 10.1002/jcp.24657
       
  • The Paracrine Feedback Loop Between Vitamin D3 (1,25(OH)2D3) and PTHrP in
           Prehypertrophic Chondrocytes
    • Authors: Frances C. Bach; Kirsten Rutten, Kristyanne Hendriks, Frank M. Riemers, Peter Cornelissen, Alain de Bruin, Ger J. Arkesteijn, Richard Wubbolts, William A. Horton, Louis C. Penning, Marianna A. Tryfonidou
      First page: 1999
      Abstract: The endocrine feedback loop between vitamin D3 (1,25(OH)2D3) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH‐related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)2D3 and PTH. This was investigated in ATDC5 cells treated with 10−8 M 1,25(OH)2D3 or PTHrP, Col2‐pd2EGFP transgenic mice, and primary Col2‐pd2EGFP growth plate chondrocytes isolated by FACS, using RT‐qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)2D3 receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)2D3 decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α‐ and 24‐hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)2D3 treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)2D3 and PTHrP in the growth plate. 1,25(OH)2D3 decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)2D3 by increasing VDR production. In light of the role of 1,25(OH)2D3 and PTHrP in modulating chondrocyte differentiation, 1,25(OH)2D3 in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 229: 1999–2014, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:43.935156-05:
      DOI: 10.1002/jcp.24658
       
  • Reactive Oxygen Species‐Dependent Nitric Oxide Production in
           Reciprocal Interactions of Glioma and Microglial Cells
    • Authors: Shing‐Chuan Shen; Ming‐Shun Wu, Hui‐Yi Lin, Liang‐Yo Yang, Yi‐Hsuan Chen, Yen‐Chou Chen
      First page: 2015
      Abstract: Conditioned mediums (CMs) from glioma cells U87, GBM‐8401, and C6 significantly induced iNOS protein and NO production by microglial cells BV‐2 but without altering the cell viability or cell‐cycle progression of BV2 microglia. Significant increases in intracellular peroxide by U87‐CM and C6‐CM were detected by a DCHF‐DA assay, and vitamin (Vit) C and N‐acetyl cysteine (NAC)‐reduced intracellular peroxide levels elicited by CMs lead to inhibition of iNOS/NO production The extracellular signal‐regulated kinase (ERK) inhibitor, U0126, and c‐Jun N‐terminal kinase (JNK) inhibitor, SP600125, suppressed U87‐CM‐ and C6‐CM‐induced iNOS/NO production by respectively blocking phosphorylated ERK (pERK) and JNK (pJNK) protein expressions stimulated by U87‐CM and C6‐CM. Increased migration of U87 and C6 glioma cells by a co‐culture with BV‐2 microglial cells or adding the nitric oxide (NO) donor, sodium nitroprusside (SNP) was observed, and that was blocked by adding an NO synthase (NOS) inhibitor, N‐nitro L‐arginine methyl ester (NAME). Contributions of ROS, pERK, and pJNK to the migration of glioma cells was further demonstrated in a transwell coculture system of U87 and C6 gliomas with BV‐2 microglial cells. Furthermore, expressions of tumor necrosis factor (TNF)‐α and monocyte chemoattractant protein (MCP)‐1 messenger (m)RNA in U87 and C6 cells were detected by an RT‐PCR, and TNF‐α and MCP‐1 induced iNOS protein expression in time‐ and concentration‐dependent manners. Neutralization of TNF‐α or MCP‐1 in U87‐CM and C6‐CM using a TNF‐α or MCP‐1 antibody inhibited iNOS protein expression, and increased intracellular peroxide by TNF‐α or MCP‐1 was identified in BV‐2 cells. The reciprocal activation of glioma cells and microglia via ROS‐dependent iNOS/NO elevation at least partially mediated by TNF‐α and MCP‐1 is elucidated. J. Cell. Physiol. 229: 2015–2026, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:45.596026-05:
      DOI: 10.1002/jcp.24659
       
  • Expression Variability and Function of the RET Gene in Adult Peripheral
           Blood Mononuclear Cells
    • Authors: Marta Rusmini; Paola Griseri, Ivana Matera, Elena Pontarini, Roberto Ravazzolo, Domenico Mavilio, Isabella Ceccherini
      First page: 2027
      Abstract: RET is a gene playing a key role during embryogenesis and in particular during the enteric nervous system development. High levels of RET gene expression are maintained in different human tissues also in adulthood, although their physiological role remains unclear. In particular, collected evidences of a RET contribution in the development and maintenance of the immune system prompted us to investigate its levels of surface expression on peripheral blood mononuclear cells (PBMCs) from adult healthy donors. Despite variability among samples, RET expression was conserved at similar levels in the different immune cell subsets, with higher correlations in similar lymphocyte populations (i.e. CD4+ and CD8+ T cells). Conversely, no correlation was found between the amount of RET receptor, the expression of its putative ligands and co‐receptors and the genotypes at the RET locus. Moreover, we investigated the RET‐associated inflammatory pathways in PBMCs from healthy donors both in resting conditions and upon glial cell derived neurotrophic factor (GDNF) and GPI‐linked co‐receptors alpha 1 (GFRα1) mediated RET activation. RET mRNA levels positively correlated with the transcript amount of interleukin‐8 (IL‐8), a cytokine produced by monocytes and macrophages, though we could not demonstrate its direct effect on RET expression by in vitro experiments on THP1 human monocytic cells. These results imply that RET expression might be influenced by either cis‐ and/or trans‐factors, which together would account for its high variability within the general population, and suggest a putative functional role of the RET gene in modulating immune cell responses during inflammation and carcinogenesis. J. Cell. Physiol. 229: 2027–2037, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:41.065133-05:
      DOI: 10.1002/jcp.24660
       
  • Sub‐Toxic Nicotine Concentrations Affect Extracellular Matrix and
           Growth Factor Signaling Gene Expressions in Human Osteoblasts
    • Authors: Lorella Marinucci; Maria Bodo, Stefania Balloni, Paola Locci, Tiziano Baroni
      First page: 2038
      Abstract: Exposure to nicotine and other compounds contained in cigarette smoking affects human health. This study examined the effects of exposure to a single or multiple sub‐toxic nicotine concentrations on human osteoblasts. Cell growth and expression of genes involved in bone differentiation, extracellular matrix (ECM) metabolism, and growth factor signaling pathways were investigated in nicotine‐treated cells compared to untreated cells. Depending on osteoblast concentration and maturation stages, nicotine differently regulated cell growth. Real‐time PCR showed regulated expressions of genes expressed by nicotine‐treated osteoblasts compared to untreated cells. Among ECM genes, type I collagen was down‐regulated and osteonectin was up‐regulated in nicotine‐treated osteoblasts; similarly, fibroblast growth factor‐1 (FGF1) and fibroblast growth factor‐2 (FGF2), two members of FGF signaling system, were discordantly modulated; genes involved in osteoblast maturation and differentiation such as alkaline phosphatase (ALP), runt‐related transcription factor‐2 (RUNX2), and bone sialoprotein (BSP) were over‐expressed after drug treatment. Our results show a positive association between nicotine exposure and osteoblast phenotype and illustrate for the first time a mechanism whereby acute or chronic exposure to sub‐toxic nicotine concentrations may affect bone formation through the impairment of growth factor signaling system and ECM metabolism. J. Cell. Physiol. 229: 2038–2048, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:42.076084-05:
      DOI: 10.1002/jcp.24661
       
  • RUNX3 Facilitates Growth of Ewing Sarcoma Cells
    • Authors: Krista L. Bledsoe; Meghan E. McGee‐Lawrence, Emily T. Camilleri, Xiaoke Wang, Scott M. Riester, Andre J. van Wijnen, Andre M. Oliveira, Jennifer J. Westendorf
      First page: 2049
      Abstract: Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage‐essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt‐domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal‐derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX‐responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI‐responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma. J. Cell. Physiol. 229: 2049–2056, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:37.412906-05:
      DOI: 10.1002/jcp.24663
       
  • Bisphenol‐A Induces Podocytopathy With Proteinuria in Mice
    • Authors: Nuria Olea‐Herrero; María Isabel Arenas, Carmen Muñóz‐Moreno, Rafael Moreno‐Gómez‐Toledano, Marta González‐Santander, Ignacio Arribas, Ricardo J. Bosch
      First page: 2057
      Abstract: Bisphenol‐A, a chemical used in the production of the plastic lining of food and beverage containers, can be found in significant levels in human fluids. Recently, bisphenol‐A has been associated with low‐grade albuminuria in adults as well as in children. Since glomerular epithelial cells (podocytes) are commonly affected in proteinuric conditions, herein we explored the effects of bisphenol‐A on podocytes in vitro and in vivo. On cultured podocytes we first observed that bisphenol‐A—at low or high concentrations—(10 nM and 100 nM, respectively) was able to induce hypertrophy, diminish viability, and promote apoptosis. We also found an increase in the protein expression of TGF‐β1 and its receptor, the cyclin‐dependent kinase inhibitor p27Kip1, as well as collagen‐IV, while observing a diminished expression of the slit diaphragm proteins nephrin and podocin. Furthermore, mice intraperitoneally injected with bisphenol‐A (50 mg/Kg for 5 weeks) displayed an increase in urinary albumin excretion and endogenous creatinine clearance. Renal histology showed mesangial expansion. At ultrastructural level, podocytes displayed an enlargement of both cytoplasm and foot processes as well as the presence of condensed chromatin, suggesting apoptosis. Furthermore, immunohistochemistry for WT‐1 (specific podocyte marker) and the TUNEL technique showed podocytopenia as well as the presence of apoptosis, respectively. In conclusion, our data demonstrate that Bisphenol‐A exposure promotes a podocytopathy with proteinuria, glomerular hyperfiltration and podocytopenia. Further studies are needed to clarify the potential role of bisphenol‐A in the pathogenesis as well as in the progression of renal diseases. J. Cell. Physiol. 229: 2057–2066, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:33.101106-05:
      DOI: 10.1002/jcp.24665
       
  • Short‐Term Hypoxia Enhances the Migratory Capability of Dendritic
           Cell Through HIF‐1α and PI3K/Akt Pathway
    • Authors: Irene Filippi; Emilia Morena, Carlo Aldinucci, Fabio Carraro, Silvano Sozzani, Antonella Naldini
      First page: 2067
      Abstract: Hypoxia represents an inadequate oxygen supply to tissues, which can modulate cell functions, primarily through the hypoxia‐inducible transcription factor HIF‐1α. Dendritic cells (DC) are professional antigen‐presenting cells and their migration maybe affected by hypoxia, since the local microenvironment in lymphoid organs, as well as in inflamed and tumor tissues, is characterized by low oxygen tensions. In this study we observed an enhanced migratory capability of human monocyte‐derived DC, using in vitro migration assays performed under hypoxic conditions. Such enhancement was independent on either the chemoattractants involved or the maturation level of DC. However, HIF‐1α appeared to be crucial for the migration only of immature DC and not for mature DC under hypoxia, as indicated by HIF‐1α siRNA approaches. Furthermore, we observed that while Akt phosphorylation was enhanced in both immature and mature DC exposed to hypoxia, other signaling pathways, such as p38 and p42/p44 MAPK, were differently affected during hypoxic treatment. More interestingly, aspecific and specific inhibition of PI3K/Akt indicated that such pathway was relevant for the migration of both immature and matured DC under hypoxia, even when DC were transfected with HIF‐1α siRNA. Our results indicate that, besides HIF‐1α, several other pathways, including PI3K/Akt, may be involved in the response to hypoxia of immature and, more specifically, of mature DC to sustain their trafficking and functions within hypoxic microenvironments. J. Cell. Physiol. 229: 2067–2076, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:40.320561-05:
      DOI: 10.1002/jcp.24666
       
  • High Insulin‐Induced Down‐Regulation of
           Erk‐1/IGF‐1R/FGFR‐1 Signaling Is Required for Oxidative
           Stress‐Mediated Apoptosis of Adipose‐Derived Stem Cells
    • Authors: Maria Giovanna Scioli; Valerio Cervelli, Gaetano Arcuri, Pietro Gentile, Elena Doldo, Alessandra Bielli, Elena Bonanno, Augusto Orlandi
      First page: 2077
      Abstract: Homeostasis of adipose tissue requires highly coordinated response between circulating factors and cell population. Human adult adipose‐derived stem cells (ASCs) display multiple differentiation properties and are sensitive to insulin stimulation. Insulin resistance and high level of circulating insulin characterize patients with type 2 diabetes and obesity. At physiological concentration, insulin promoted proliferation and survival of ASCs in vitro, whereas high insulin level induced their dose‐dependent proliferative arrest and apoptosis. Insulin‐induced apoptotic commitment depended on the down‐regulation of Erk‐1, insulin growth factor‐1 receptor (IGF‐1R), and fibroblast growth factor receptor‐1 (FGFR‐1)‐mediated signaling. Specific inhibition of Erk‐1/2, IGF‐1R, and FGFR activity promoted ASC apoptosis but did not increase insulin effects, whereas EGFR and ErbB2 inhibition potentiated insulin‐induced apoptosis. FGFRs and EGFR inhibition reduced ASC adipogenic differentiation, whereas Erk‐1/2 and IGF‐1R inhibition was ineffective. Insulin‐induced apoptosis associated to reactive oxygen species (ROS) accumulation and inhibition of NADPH oxidase 4 (Nox4) activity prevented ASC apoptosis. Moreover, specific inhibition of Erk‐1/2, IGF‐1R, and FGFR‐1 activity promoted ROS generation and this effect was not cumulative with that of insulin alone. Our data indicate that insulin concentration is a critical regulatory switch between proliferation and survival of ASCs. High insulin level‐induced apoptotic machinery involves Nox4‐generated oxidative stress and the down‐regulation of a complex receptor signaling, partially distinct from that influencing adipogenic differentiation of ASCs. J. Cell. Physiol. 229: 2077–2087, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:35.238782-05:
      DOI: 10.1002/jcp.24667
       
  • Bradykinin Regulates Osteoblast Differentiation by Akt/ERK/NFκB
           Signaling Axis
    • Authors: Swati Srivastava; Kirti Sharma, Narender Kumar, Partha Roy
      First page: 2088
      Abstract: Bradykinin (BK), a well known mediator of pain and inflammation, is also known to be involved in the process of bone resorption. The present study therefore evaluated the role of BK in osteoblast lineage commitment. Our data showed that BK inhibits the migration of bone marrow mesenchymal stem cells, but does not affect their viability. Moreover, BK also inhibits osteoblastic differentiation by significantly downregulating the levels of mRNAs for osteopontin, runX2, col24, osterix, osteocalcin genes and bone mineralization (P 
      PubDate: 2014-08-27T15:58:36.20662-05:0
      DOI: 10.1002/jcp.24668
       
  • A p53‐Dependent Tumor Suppressor Network Is Induced by Selective
           miR‐125a‐5p Inhibition in Multiple Myeloma Cells
    • Authors: Marzia Leotta; Lavinia Biamonte, Lavinia Raimondi, Domenica Ronchetti, Maria Teresa Di Martino, Cirino Botta, Emanuela Leone, Maria Rita Pitari, Antonino Neri, Antonio Giordano, Pierosandro Tagliaferri, Pierfrancesco Tassone, Nicola Amodio
      First page: 2106
      Abstract: The analysis of deregulated microRNAs (miRNAs) is emerging as a novel approach to disclose the regulation of tumor suppressor or tumor promoting pathways in tumor cells. Targeting aberrantly expressed miRNAs is therefore a promising strategy for cancer treatment. By miRNA profiling of primary plasma cells from multiple myeloma (MM) patients, we previously reported increased miR‐125a‐5p levels associated to specific molecular subgroups. On these premises, we aimed at investigating the biological effects triggered by miR‐125a‐5p modulation in MM cells. Expression of p53 pathway‐related genes was down‐regulated in MM cells transfected with miR‐125a‐5p mimics. Luciferase reporter assays confirmed specific p53 targeting at 3′UTR level by miR‐125a‐5p mimics. Interestingly, bone marrow stromal cells (BMSCs) affected the miR‐125a‐5p/p53 axis, since adhesion of MM cells to BMSCs strongly up‐regulated miR‐125a‐5p levels, while reduced p53 expression. Moreover, ectopic miR‐125a‐5p reduced, while miR‐125‐5p inhibitors promoted, the expression of tumor suppressor miR‐192 and miR‐194, transcriptionally regulated by p53. Lentiviral‐mediated stable inhibition of miR‐125a‐5p expression in wild‐type p53 MM cells dampened cell growth, increased apoptosis and reduced cell migration. Importantly, inhibition of in vitro MM cell proliferation and migration was also achieved by synthetic miR‐125a‐5p inhibitors and was potentiated by the co‐expression of miR‐192 or miR‐194. Taken together, our data indicate that miR‐125a‐5p antagonism results in the activation of p53 pathway in MM cells, underlying the crucial role of this miRNA in the biopathology of MM and providing the molecular rationale for the combinatory use of miR‐125a inhibitors and miR‐192 or miR‐194 mimics for MM treatment. J. Cell. Physiol. 229: 2106–2116, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:46.131002-05:
      DOI: 10.1002/jcp.24669
       
  • mTORC1 Is Involved in Hypoxia‐Induced Pulmonary Hypertension Through
           the Activation of Notch3
    • Authors: Wang Wang; Jie Liu, Aiping Ma, Ran Miao, Yuling Jin, Hongbing Zhang, Kaifeng Xu, Chen Wang, Jun Wang
      First page: 2117
      Abstract: Hypoxia‐induced pulmonary hypertension (HPH) is a clinical syndrome associated with high morbidity and mortality. However, the underlying mechanisms remain unclear. Both the mammalian target of rapamycin (mTOR) and the Notch3 signaling pathways have been reported to be involved in HPH; however, it is unknown whether there is a connection between these two signaling pathways in HPH. This study was designed to investigate the relationship between mTOR and Notch3 in HPH. After treatment with 10% O2 for 4 weeks, male C57BL/6 mice developed HPH with gradually increased right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary arteriolar remodeling accompanied by the activation of mTOR complex 1 (mTORC1) and Notch3 in the lung tissue and pulmonary arterioles. Pretreatment with the mTORC1 inhibitor rapamycin not only alleviated pulmonary arterial pressure and pulmonary arteriolar remodeling but also suppressed hypoxia‐induced mTORC1 and Notch3 activation. Prophylactic N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) administration, a Notch signaling inhibitor, protected against the effects of hypoxia. These in vivo data were confirmed by in vitro experiments on human pulmonary arterial smooth muscle cell (PASMC) exposed to 3% O2. Furthermore, overexpression of Notch3 intracellular domain partially abrogated the inhibitory effects of rapamycin on human PASMC proliferation. These data indicate that both mTORC1 and Notch3 signaling are involved in HPH and the downstream effects of mTORC1 activation in HPH are partially dependent on the activation of Notch3 signaling. J. Cell. Physiol. 229: 2117–2125, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:46.617185-05:
      DOI: 10.1002/jcp.24670
       
  • Expression of Adenine Nucleotide Translocase (ANT) Isoform Genes Is
           Controlled by PGC‐1α Through Different Transcription Factors
    • Authors: Aleix Gavaldà‐Navarro; Josep A. Villena, Anna Planavila, Octavi Viñas, Teresa Mampel
      First page: 2126
      Abstract: Adenine nucleotide translocase (ANT) isoforms are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in the cytosol. The aim of this study was to determine the role of the transcriptional coactivator PGC‐1α (peroxisome proliferator‐activated receptor‐γ [PPAR‐γ] coactivator 1α), a master regulator of mitochondrial oxidative metabolism, in the regulation of the expression of ANT isoform genes and to identify the transcription factors involved. We found that PGC‐1α overexpression induced the expression of all ANT human and mouse isoforms but to different degrees. The transcription factor ERRα was involved in PGC‐1α‐induced expression of all human ANT isoforms (hANT1‐3) in HeLa cells as well as in the regulation of mouse isoforms (mANT1‐2) in C2C12 myotubes and 3T3‐L1 adipocytes, even though ANT isoforms have important physiological differences and are regulated in a tissue‐specific manner. In addition to ERRα, PPARδ and mTOR pathways were involved in the induction of mANT1‐2 by PGC‐1α in C2C12 myotubes, while PPARγ was involved in PGC‐1α‐regulation of mANT1‐2 in 3T3‐L1 adipocytes. Furthermore, the regulation of mANT genes by PGC‐1α was also observed in vivo in knockout mouse models lacking PGC‐1α. In summary, our results show that the regulation of genes encoding ANT isoforms is controlled by PGC‐1α through different transcription factors depending on cell type. J. Cell. Physiol. 229: 2126–2136, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:41.564498-05:
      DOI: 10.1002/jcp.24671
       
  • Ras Oncoprotein Disrupts the TSH/CREB Signaling Upstream Adenylyl Cyclase
           in Human Thyroid Cell
    • Authors: Marcella Salzano; Eleonora Russo, Salvatore Salzano, Maurizio Bifulco, Mario Vitale
      First page: 2137
      Abstract: Activating mutations in RAS genes and p21 Ras overactivation are common occurrences in a variety of human tumors. p21 Ras oncoproteins deregulate a number of signaling pathways, dedifferentiating the thyroid cell, and negatively regulating the expression of thyroid specific genes. In rat thyroid cells, Ras oncoproteins inhibit the TSH pathway by reducing PKA activity and thus the expression of thyroid specific genes, while in mouse melanocytes, Ras oncoproteins reduce the αMSH‐stimulated cAMP signaling by increasing the expression of the phosphodiesterase‐4B. Given these cell‐dependent differences, we investigated if and how the TSH/CREB pathway is modulated by Ras oncoprotein in a human thyroid cell line. CREB phosphorylation was stimulated by TSH and forskolin in TAD‐2 cells. RasV12 expression negatively regulated the TSH‐stimulated CREB phosphorylation but was ineffective on forskolin‐stimulated CREB phosphorylation. Phosphodiesterase inhibition by IBMX enhanced TSH‐stimulated CREB phosphorylation, but did not restore TSH‐stimulated CREB phosphorylation inhibited by Ras oncoprotein. These data indicate that Ras oncoprotein disrupts the TSH/CREB pathway, upstream adenylyl cyclase, and highlight the existence of mechanisms of interaction between Ras and the cAMP pathway different in human and in rat thyroid cells. J. Cell. Physiol. 229: 2137–2141, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:36.779814-05:
      DOI: 10.1002/jcp.24672
       
  • Heparin Responses in Vascular Smooth Muscle Cells Involve
           cGMP‐Dependent Protein Kinase (PKG)
    • Authors: Albert C. Gilotti; Wutigri Nimlamool, Raymond Pugh, Joshua B. Slee, Trista C. Barthol, Elizabeth A. Miller, Linda J. Lowe‐Krentz
      First page: 2142
      Abstract: Published data provide strong evidence that heparin treatment of proliferating vascular smooth muscle cells results in decreased signaling through the ERK pathway and decreases in cell proliferation. In addition, these changes have been shown to be mimicked by antibodies that block heparin binding to the cell surface. Here, we provide evidence that the activity of protein kinase G is required for these heparin effects. Specifically, a chemical inhibitor of protein kinase G, Rp‐8‐pCPT‐cGMS, eliminates heparin and anti‐heparin receptor antibody effects on bromodeoxyuridine incorporation into growth factor‐stimulated cells. In addition, protein kinase G inhibitors decrease heparin effects on ERK activity, phosphorylation of the transcription factor Elk‐1, and heparin‐induced MKP‐1 synthesis. Although transient, the levels of cGMP increase in heparin treated cells. Finally, knock down of protein kinase G also significantly decreases heparin effects in growth factor‐activated vascular smooth muscle cells. Together, these data indicate that heparin effects on vascular smooth muscle cell proliferation depend, at least in part, on signaling through protein kinase G. J. Cell. Physiol. 229: 2142–2152, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:39.851478-05:
      DOI: 10.1002/jcp.24677
       
  • Hypoxia/Hypercapnia‐Induced Adaptation Maintains Functional Capacity
           of Cord Blood Stem and Progenitor Cells at 4°C
    • Authors: Marija Vlaski; Luc Negroni, Milica Kovacevic‐Filipovic, Christelle Guibert, Philippe Brunet de la Grange, Rodrigue Rossignol, Jean Chevaleyre, Pascale Duchez, Xavier Lafarge, Vincent Praloran, Jean‐Marie Schmitter, Zoran Ivanovic
      First page: 2153
      Abstract: We analyzed the effect of exposure to hypoxic/hypercapnic (HH) gas mixture (5% O2/9% CO2) on the maintenance of functional cord blood CD34+ hematopoietic stem and progenitor cells in severe hypothermia (4°C) employing the physiological and proteomic approaches. Ten‐day exposure to HH maintained the Day 0 (D‐0) level of hematopoietic stem cells as detected in vivo on the basis of hematopoietic repopulation of immunodeficient mice—short‐term scid repopulating cells (SRC). Conversely, in the atmospheric air (20% O2/0.05% CO2), usual condition used for cell storage at 4°C, stem cell activity was significantly decreased. Also, HH doubled the survival of CD34+ cells and committed progenitors (CFCs) with respect to the atmospheric air (60% vs. 30%, respectively). Improved cell maintenance in HH was associated with higher proportion of aldehyde dehydrogenase (ALDH) positive cells. Cell‐protective effects are associated with an improved maintenance of the plasma and mitochondrial membrane potential and with a conversion to the glycolytic energetic state. We also showed that HH decreased apoptosis, despite a sustained ROS production and a drop of ATP amount per viable cell. The proteomic study revealed that the global protein content was better preserved in HH. This analysis identified: (i) proteins sensitive or insensitive to hypothermia irrespective of the gas phase, and (ii) proteins related to the HH cell‐protective effect. Among them are some protein families known to be implicated in the prolonged survival of hibernating animals in hypothermia. These findings suggest a way to optimize short‐term cell conservation without freezing. J. Cell. Physiol. 229: 2153–2165, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:42.922792-05:
      DOI: 10.1002/jcp.24678
       
  • Pin1 Regulates Osteoclast Fusion Through Suppression of the Master
           Regulator of Cell Fusion DC‐STAMP
    • Authors: Rabia Islam; Han‐Sol Bae, Won‐Joon Yoon, Kyung‐Mi Woo, Jeong‐Hwa Baek, Hong‐Hee Kim, Takafumi Uchida, Hyun‐Mo Ryoo
      First page: 2166
      Abstract: Cell fusion is a fundamental biological event that is essential for the development of multinucleated cells such as osteoclasts. Fusion failure leads to the accumulation of dense bone such as in osteopetrosis, demonstrating the importance of fusion in osteoclast maturity and bone remodeling. In a recent study, we reported that Pin1 plays a role in the regulation of bone formation and Runx2 regulation. In this study, we explored the role of Pin1 in osteoclast formation and bone resorption. Pin1 null mice have low bone mass and increased TRAP staining in histological sections of long bones, compared to Pin1 wild‐type mice. In vitro osteoclast forming assays with bone marrow‐derived monocyte/macrophage revealed that Pin1‐deficient osteoclasts are larger than wild‐type osteoclasts and have higher nuclei numbers, indicating greater extent of fusion. Pin1 deficiency also highly enhanced foreign body giant cell formation both in vitro and in vivo. Among the known fusion proteins, only DC‐STAMP was significantly increased in Pin1−/− osteoclasts. Immunohistochemistry showed that DC‐STAMP expression was also significantly increased in tibial metaphysis of Pin1 KO mice. We found that Pin1 binds and isomerizes DC‐STAMP and affects its expression levels and localization at the plasma membrane. Taken together, our data indicate that Pin1 is a determinant of bone mass through the regulation of the osteoclast fusion protein DC‐STAMP. The identification of Pin1 as a factor involved in cell fusion contributes to the understanding of osteoclast‐associated diseases, including osteoporosis, and opens new avenues for therapeutic targets. J. Cell. Physiol. 229: 2166–2174, 2014. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-08-27T15:58:46.971466-05:
      DOI: 10.1002/jcp.24679
       
  • UCP2‐related mitochondrial pathway participates in oroxylin
           A‐induced apoptosis in human colon cancer cells
    • Abstract: Oroxylin A is a flavonoid extracted from the root of Scutellaria baicalensis Georgi. Our previous research demonstrated that oroxylin A have various anti‐tumor effects including apoptosis, cell cycle arrest, drug‐resistant reversion and others. This paper explores the mechanism how oroxylin A induce apoptosis by regulating uncoupling protein 2 (UCP2) in human colon cancer cells. We found that the inhibition of UCP2 by UCP2 siRNA significantly increased the sensitivity of cells to drugs, reactive oxygen species (ROS) generation and the opening of mitochondrial permeability transition pore (MPTP) of CaCo‐2 cells. We also found that UCP2 inhibition could lead to ROS‐mediated MPTP activation. Furthermore, we demonstrated that oroxylin A triggered MPTP‐dependent pro‐apoptotic protein release from mitochondria to matrix and then induced apoptotic cascade by inhibiting UCP2. Intriguingly, the inhibition of UCP2 by oroxylin A was able to block Bcl‐2 translocation to the mitochondria, keeping MPTP at open‐state. In conclusion, we have demonstrate that UCP2 play a key role in mitochondrial apoptotic pathway; UCP2's inhibition by oroxylin A triggers the MPTP opening, and promotes the apoptosis in CaCo‐2 cells. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
  • Alpha‐synuclein as a Pathological Link between Chronic Traumatic
           Brain Injury and Parkinson's disease
    • Abstract: The long‐term consequences of traumatic brain injury (TBI) are closely associated with the development of histopathological deficits. Notably, TBI may predispose long‐term survivors to age‐related neurodegenerative diseases, such as Parkinson's disease (PD), which is characterized by a gradual degeneration of the nigrostriatal dopaminergic neurons. However, preclinical studies on the pathophysiological changes in substantia nigra (SN) after chronic TBI are lacking. In the present in vivo study, we examined the pathological link between PD‐associated dopaminergic neuronal loss and chronic TBI. Sixty days post TBI, rats were euthanized and brain tissues harvested. Immunostaining was performed using tyrosine hydroxylase (TH), an enzyme required for the synthesis of dopamine in neurons, á‐synuclein, a presynaptic protein that plays a role in synaptic vesicle recycling, and major histocompatibility complex II (MHCII), a protein found in antigen presenting cells such as inflammatory microglia cells, all key players in PD pathology. Unbiased stereology analyses revealed significant decrease of TH‐positive expression in the surviving dopaminergic neurons of the SN pars compacta (SNpc) relative to sham control. In parallel, increased á‐synuclein accumulation was detected in the ipsilateral SN compared to the contralateral SN in TBI animals or sham control. In addition, exacerbation of MHCII+ cells was recognized in the SN and cerebral peduncle ipsilateral to injury relative to contralateral side and sham control. These results suggest á‐synuclein as a pathological link between chronic effects of TBI and PD symptoms as evidenced by significant overexpression and abnormal accumulation of á‐synuclein in inflammation‐infiltrated SN of rats exposed to chronic TBI. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
  • LIGHT is a crucial mediator of airway remodeling
    • Abstract: Chronic inflammatory airway diseases like asthma and chronic obstructive pulmonary disease are major health problems globally. Airway epithelial cells play important role in airway remodeling, which is a critical process in the pathogenesis of diseases. This study aimed to demonstrate that LIGHT, an inflammatory factor secreted by T cells after allergen exposure, is responsible for promoting airway remodeling. LIGHT increased primary human bronchial epithelial cells (HBECs) undergoing epithelial‐mesenchymal transition (EMT) and expressing MMP‐9. The induction of EMT was associated with increased NF‐κB activation and p300/NF‐κB association. The interaction of NF‐κB with p300 facilitated NF‐κB acetylation, which in turn, was bound to the promoter of ZEB1, resulting in E‐cadherin downregulation. LIGHT also stimulated HBECs to produce numerous cytokines/chemokines that could worsen airway inflammation. Furthermore, LIGHT enhanced HBECs to secrete activin A, which increased bronchial smooth muscle cell (BSMC) migration. In contrast, depletion of activin A decreased such migration. The findings suggest a new molecular determinant of LIGHT‐mediated pathogenic changes in HBECs and that the LIGHT‐related vicious cycle involving HBECs and BSMCs may be a potential target for the treatment of chronic inflammation airway diseases with airway remodeling. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
  • Lkb1 deletion promotes ectopic lipid accumulation in muscle progenitor
           cells and mature muscles
    • Abstract: Excessive intramyocellular triglycerides (muscle lipids) are associated with reduced contractile function, insulin resistance and Type 2 diabetes, but what governs lipid accumulation in muscle is unclear. Here we report a role of Lkb1 in regulating lipid metabolism in muscle stem cells and their descendent mature muscles. We used MyodCre and Lkb1flox/flox mice to specifically delete Lkb1 in myogenic cells including stem and differentiated cells, and examined the lipid accumulation and gene expression of myoblasts cultured from muscle stem cells (satellite cells). Genetic deletion of Lkb1 in myogenic progenitors led to elevated expression of lipogenic genes and ectopic lipid accumulation in proliferating myoblasts. Interestingly, the Lkb1‐deficient myoblasts differentiated into adipocyte‐like cells upon adipogenic induction. However, these adipocyte‐like cells maintained myogenic gene expression with reduced ability to form myotubes efficiently. Activation of AMPK by AICAR prevented ectopic lipid formation in the Lkb1 null myoblasts. Notably, Lkb1‐deficient muscles accumulated excessive lipids in vivo in response to high‐fat diet feeding. These results demonstrate that Lkb1 acts through AMPK to limit lipid deposition in muscle stem cells and their derivative mature muscles, and point to the possibility of controlling muscle lipid content using AMPK activating drugs. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
  • Could lncRNAs be the Missing Links in Control of Mesenchymal Stem Cell
           Differentiation'
    • Abstract: Long suspected, recently recognized, and increasingly studied, non protein‐coding RNAs (ncRNAs) are emerging as key drivers of biological control and pathology. Since their discovery in 1993, microRNAs (miRNAs) have been the subject of intense research focus and investigations have revealed striking findings, establishing that these molecules can exert a shocking level of biological control in numerous tissues. More recently, long ncRNAs (lncRNAs), the lesser‐studied siblings of miRNA, have been suggested to have a similar robust role in developmental and adult tissue regulation. Mesenchymal stem cells (MSCs) are an important source of multipotent cells for normal and therapeutic tissue repair. Much is known about the critical role of miRNAs in biogenesis and differentiation of MSCs however; recent studies have suggested lncRNAs may play an equally important role in the regulation of these cells. Here we highlight the role of lncRNAs in the regulation of mesenchymal stem cell lineages including adipocytes, chondrocytes, myoblasts, and osteoblasts. In addition, the potential for these noncoding RNAs to be used as biomarkers for disease or therapeutic targets is also discussed. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
       
 
 
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