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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  [1604 journals]   [SJR: 1.608]   [H-I: 118]
  • Insights Into RNA Transcriptome Profiling of Cardiac Tissue in Obesity and
           Hypertension Conditions
    • Authors: Alzenira Costa; Octavio Luiz Franco
      Pages: n/a - n/a
      Abstract: Several epidemiological studies suggest that obesity and hypertension are associated with cardiac transcriptome modifications that could be further associated with inflammatory processes and cardiac hypertrophy. In this field, transcriptome studies have demonstrated their importance to elucidate physiological mechanisms, pathways or genes involved in many biological processes. Over the past decade, RNA microarray and RNA‐seq analysis has become an essential component to examine metabolic pathways in terms of mRNA expression in cardiology. In this review, cardiac muscle gene expression in response to effects of obesity and hypertension will be focused, providing a broad view on cardiac transcriptome and physiological and biochemical mechanisms involved in gene expression changes produced by these events, emphasizing the use of new technologies for gene expression analyses. This article is protected by copyright. All rights reserved
      PubDate: 2014-11-12T18:27:22.005069-05:
      DOI: 10.1002/jcp.24807
  • The synergistic effect of SAHA and Parthenolide in MDA‐MB231 breast
           cancer cells
    • Authors: Daniela Carlisi; Marianna Lauricella, Antonella D'Anneo, Giuseppina Buttitta, Sonia Emanuele, Riccardo di Fiore, Roberta Martinez, Christian Rolfo, Renza Vento, Giovanni Tesoriere
      Abstract: The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA‐MB231 cells, a triple‐negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA‐MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down‐regulation of DNMT1 expression induced by PN. Inhibition of the DNA‐binding activity of NF‐kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-11-05T05:37:12.554035-05:
      DOI: 10.1002/jcp.24863
  • A tissue specific magnetic resonance contrast agent, Gd‐AMH, for
           diagnosis of stromal endometriosis lesions: A phase I study
    • Authors: Pietro G. Signorile; Alfonso Baldi
      Abstract: The anti‐mullerian hormone (AMH) is a homodimeric glycoprotein member of the transforming growth factor ƒÀ (TGF‐ƒÀ) superfamily, and is secreted by Sertoli cells in the embryonic testes and is responsible of the regression of the mullerian duct. The physiological functions of this protein remain largely unknown, and its expression in human tissues has yet to be completely determined. [objective]. Firstly, we analyzed AMH expression in human tissues by immunohistochemistry. It was found that AMH was distributed in many organs, although with different tissue and cell localization and various expression levels; we also demonstrated strong AMH expression in endometriosis tissues. Secondly, we demonstrated the ability of an anti‐AMH antibody, labelled with gadiolinium, to be directly detected by magnetic resonance in small endometriosis lesions (5 mm in diameter) in vivo in a mouse model. In conclusion, our data suggest that based on its expression pattern, AMH may serve to maintain physiological cellular homeostasis in different human tissues and organs. Moreover, it is strongly expressed in endometriosis lesions as a selective tissue specific contrast agent for in vivo detection of stromal endometriosis lesions. The potential significance of these findings could be further validated in a clinical setting. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-11-05T05:34:54.340738-05:
      DOI: 10.1002/jcp.24862
  • Pigment epithelium derived factor suppresses expression of Sost/sclerostin
           by osteocytes: Implication for its role in bone matrix mineralization
    • Authors: Feng Li; Na Song, Joyce Tombran‐Tink, Christopher Niyibizi
      Abstract: Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. Mechanisms by which PEDF regulates matrix mineralization remain unknown. We examined effect of exogenous PEDF on expression of osteoblastic and osteocytic related genes and proteins in mineralizing osteoblast culture. Mineralizing human osteoblasts supplemented with exogenous PEDF for 14 days deposited 47% more mineral than cells cultured without PEDF. Analysis of selected gene expression by cells in mineralizing cultures supplemented with exogenous PEDF showed reduction in expression of Sclerostin (Sost) by 70%, matrix extracellular phosphoglycoprotein (MEPE) by 75% and dentin matrix protein (DMP‐1) by 20% at day 14 of culture. Phosphate‐regulating gene with homologies to endopeptidases on the X chromosome (PHEX) expression was not affected. Western blotting and immunoprecipitation showed that sclerostin and MEPE synthesis by osteocytes were reduced by 50% and 60% respectively in mineralizing osteoblasts containing exogenous PEDF. Primary osteocytes exposed to PEDF also reduced synthesis of Sost/sclerostin by 50% within 24h. For osteoblastic genes, Bone sialoprotein (BSP) was expressed at 75% higher by day 7 in cultures containing exogenous PEDF while Col1A1 expression remained high at all‐time points. Total beta‐catenin was increased in mineralizing osteoblastic cells suggesting increased Wnt activity. Taken together, the data indicate that PEDF suppressed expression of factors that inhibit mineralization while enhancing those that promote mineralization. The findings also suggest that PEDF may regulate Sost expression by osteocytes leading to enhanced osteoblastic differentiation and increased matrix mineralization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-11-03T00:41:21.765712-05:
      DOI: 10.1002/jcp.24859
  • Protein Kinase C Inhibitor, GF109203X Attenuates Osteoclastogenesis, Bone
           Resorption and RANKL‐induced NF‐κB and NFAT activity
    • Authors: Jun Yao; Jia Li, Lin Zhou, Jianwen Cheng, Shek Man Chim, Ge Zhang, Julian M.W. Quinn, Jennifer Tickner, Jinmin Zhao, Jiake Xu
      Abstract: Osteolytic bone diseases are characterized by excessive osteoclast formation and activation. Protein kinase C (PKC)‐dependent pathways regulate cell growth, differentiation and apoptosis in many cellular systems, and have been implicated in cancer development and osteoclast formation. A number of PKC inhibitors with anti‐cancer properties have been developed, but whether they might also influence osteolysis (a common complication of bone invading cancers) is unclear. We studied the effects of the PKC inhibitor compound, GF109203X on osteoclast formation and activity, processes driven by receptor activator of NFκB ligand (RANKL). We found that GF109203X strongly and dose dependently suppresses osteoclastogenesis and osteoclast activity in RANKL‐treated primary mouse bone marrow cells. Consistent with this GF109203X reduced expression of key osteoclastic genes, including cathepsin K, calcitonin receptor, tartrate resistant acid phosphatase (TRAP) and the proton pump subunit V‐ATPase‐d2 in RANKL‐treated primary mouse bone marrow cells. Expression of these proteins is dependent upon RANKL‐induced NF‐κB and NFAT transcription factor actions; both were reduced in osteoclast progenitor populations by GF109203X treatment, notably NFATc1 levels. Furthermore, we showed that GF109203X inhibits RANKL‐induced calcium oscillation. Together, this study shows GF109203X may block osteoclast functions, suggesting that pharmacological blockade of PKC‐dependent pathways has therapeutic potential in osteolytic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-11-03T00:41:06.579636-05:
      DOI: 10.1002/jcp.24858
  • Glucose concentration and streptomycin alter in vitro muscle function and
    • Authors: Alastair Khodabukus; Keith Baar
      Abstract: Cell culture conditions can vary between laboratories and have been optimised for 2D cell culture. In this study, engineered muscle was cultured in 5.5 mM low glucose (LG) or 25 mM high glucose (HG) and in the absence or presence (+S) of streptomycin and the effect on C2C12 tissue‐engineered muscle function and metabolism was determined. Following 2 weeks differentiation, streptomycin (3‐fold) and LG (0.5‐fold) significantly decreased force generation. LG and/or streptomycin resulted in upward and leftward shifts in the force‐frequency curve and slowed time‐to‐peak tension and half‐relaxation time. Despite changes in contractile dynamics, no change in myosin isoform was detected. Instead, changes in troponin isoform, calcium sequestering proteins (CSQ and parvalbumin) and the calcium uptake protein SERCA predicted the changes in contractile dynamics. Culturing in LG and/or streptomycin resulted in increased fatigue resistance despite no change in the mitochondrial enzymes SDH, ATPsynthase and cytochrome C. However, LG resulted in increases in the β‐oxidation enzymes LCAD and VLCAD and the fatty acid transporter CPT‐1, indicative of a greater capacity for fat oxidation. In contrast, HG resulted in increased GLUT4 content and the glycolytic enzyme PFK, indicative of a more glycolytic phenotype. These data suggest that streptomycin has negative effects on force generation and that glucose can be used to shift engineered muscle phenotype via changes in calcium‐handling and metabolic proteins. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-30T15:18:36.324637-05:
      DOI: 10.1002/jcp.24857
  • TGFβ3 Regulates Periderm Removal through ΔNp63 in the Developing
    • Authors: Lihua Hu; Jingpeng Liu, Zhi Li, Ferhat Ozturk, Channabasavaiah Gurumurthy, Rose‐Anne Romano, Satrajit Sinha, Ali Nawshad
      Abstract: The periderm is a flat layer of epithelium created during embryonic development. During palatogenesis, the periderm forms a protective layer against premature adhesion of the oral epithelia, including the palate. However, the periderm must be removed in order for the medial edge epithelia (MEE) to properly adhere and form a palatal seam. Improper periderm removal results in a cleft palate. Although the timing of transforming growth factor β3 (TGFβ3) expression in the MEE coincides with periderm degeneration, its role in periderm desquamation is not known. Interestingly, murine models of knockout (‐/‐) TGFβ3, interferon regulatory factor 6 (IRF6) (‐/‐), and truncated p63 (ΔNp63) (‐/‐) are born with palatal clefts because of failure of the palatal shelves to adhere, suggesting that these genes regulate palatal epithelial differentiation. However, despite having similar phenotypes in null mouse models, no studies have analyzed the possible association between the TGFβ3 signaling cascade and the IRF6/ΔNp63 genes during palate development. Recent studies indicate that regulation of ΔNp63, which depends on IRF6, facilitates epithelial differentiation. We performed biochemical analysis, gene activity and protein expression assays with palatal sections of TGFβ3 (‐/‐), ΔNp63 (‐/‐), and wild‐type (WT) embryos, and primary MEE cells from WT palates to analyze the association between TGFβ3 and IRF6/ΔNp63. Our results suggest that periderm degeneration depends on functional TGFβ3 signaling to repress ΔNp63, thereby coordinating periderm desquamation. Cleft palate occurs in TGFβ3 (‐/‐) because of inadequate periderm removal that impedes palatal seam formation, while cleft palate occurs in ΔNp63 (‐/‐) palates because of premature fusion. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-30T15:14:43.751759-05:
      DOI: 10.1002/jcp.24856
  • Vitamin D Receptor and RXR in the Post‐Genomic Era
    • Authors: Mark D. Long; Lara E. Sucheston‐Campbell, Moray J. Campbell
      Pages: n/a - n/a
      Abstract: Following the elucidation of the human genome and components of the epigenome, it is timely to revisit what is known of vitamin D receptor (VDR) function. Early transcriptomic studies using microarray approaches focused on the protein coding mRNA that were regulated by the VDR, usually following treatment with ligand. These studies quickly established the approximate size and surprising diversity of the VDR transcriptome, revealing it to be highly heterogenous and cell type and time dependent. Investigators also considered VDR regulation of non‐protein coding RNA and again, cell and time dependency was observed. Attempts to integrate mRNA and miRNA regulation patterns are beginning to reveal patterns of co‐regulation and interaction that allow for greater control of mRNA expression, and the capacity to govern more complex cellular events. Alternative splicing in the trasncriptome has emerged as a critical process in transcriptional control and there is evidence of the VDR interacting with components of the splicesome. ChIP‐Seq approaches have proved to be pivotal to reveal the diversity of the VDR binding choices across cell types and following treatment, and have revealed that the majority of these are non‐canonical in nature. The underlying causes driving the diversity of VDR binding choices remain enigmatic. Finally, genetic variation has emerged as important to impact the transcription factor affinity towards genomic binding sites, and recently the impact of this on VDR function has begun to be considered. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:34:03.202427-05:
      DOI: 10.1002/jcp.24847
  • Regulation of Ribosomal Gene Expression in Cancer
    • Authors: Le Xuan Truong Nguyen; Aparna Raval, Jacqueline S. Garcia, Beverly S. Mitchell
      Pages: n/a - n/a
      Abstract: The ability of a cell to undergo malignant transformation is both associated with and dependent on a concomitant increase in protein synthesis due to increased cell division rates and biosynthetic activities. Protein synthesis, in turn, depends upon the synthesis of ribosomes and thus ultimately on the transcription of ribosomal RNA by RNA polymerase I that occurs in the nucleolus. Enlargement of nucleoli has long been considered a hallmark of the malignant cell, but it is only recently that the rate of synthesis of rRNA in the nucleolus has been recognized as both a critical regulator of cellular proliferation and a potential target for therapeutic intervention. As might be expected, the factors regulating rRNA synthesis are both numerous and complex. It is the objective of this review to highlight recent advances in understanding how rRNA synthesis is perturbed in transformed mammalian cells and to consider the impact of these findings on the development of new approaches to the treatment of malignancies. In‐depth analysis of the process of rRNA transcription itself may be found in several recently published reviews (Drygin et al., 2010, Annu Rev Pharmacol Toxicol 50:131–156; Bywater et al., 2013,Cancer Cell 22: 51–65; Hein et al., 2013,Trends Mol Med 19:643–654). J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:04:46.345249-05:
      DOI: 10.1002/jcp.24854
  • Short time tripterine treatment enhances endothelial progenitor cell
           function via heat shock protein 32
    • Authors: Chenhui Lu; Xiaoping Zhang, Denghai Zhang, Erli Pei, Jichong Xu, Tao Tang, Meng Ye, Georges Uzan, Kangkang Zhi, Maoquan Li
      Pages: n/a - n/a
      Abstract: Aims The dysfunction of endothelial progenitor cells (EPCs) limits their potential for the treatment of ischemia and atherosclerosis. Therefore, we investigated the effect of tripterine on EPC function and examined the underlying mechanisms. Methods and Results The effect of tripterine, an active component of Tripterygium wilfordii Hook, on the enhancement of EPC function and the efficiency of EPC transplantation was investigated in vitro and in vivo. Treatment of EPCs with tripterine at 2.5 µM for 4 h inhibited oxidized low‐density lipoprotein (ox‐LDL) induced ROS production, cell apoptosis, and cell senescence and improved the migration and tube formation capacities of EPCs treated with ox‐LDL (200 µg/ml). In vivo studies showed that tripterine conditioning of EPCs administered to ischemic foci improved blood perfusion and microvascular density in a mouse hindlimb ischemia model. Examination of the underlying mechanisms indicated that the effect of tripterine is mediated by the induction of heat shock protein 32 expression and the inhibition of JNK activation. Conclusion(s) The present results are of clinical significance because they suggest the potential of tripterine as a therapeutic agent to improve the efficacy of EPC transplantation for the treatment of ischemic diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:04:43.882389-05:
      DOI: 10.1002/jcp.24849
  • The Role of Epigenetic Mechanisms in Notch Signaling During Development
    • Authors: Ralf Schwanbeck
      Pages: n/a - n/a
      Abstract: The Notch pathway is a highly conserved cell–cell communication pathway in metazoan involved in numerous processes during embryogenesis, development, and adult organisms. Ligand‐receptor interaction of Notch components on adjacent cells facilitates controlled sequential proteolytic cleavage resulting in the nuclear translocation of the intracellular domain of Notch (NICD). There it binds to the Notch effector protein RBP‐J, displaces a corepressor complex and enables the induction of target genes by recruitment of coactivators in a cell‐context dependent manner. Both, the gene‐specific repression and the context dependent activation require an intense communication with the underlying chromatin of the regulatory regions. Since the epigenetic landscape determines the function of the genome, processes like cell fate decision, differentiation, and self‐renewal depend on chromatin structure and its remodeling during development. In this review, structural features enabling the Notch pathway to read these epigenetic marks by proteins interacting with RBP‐J/Notch will be discussed. Furthermore, mechanisms of the Notch pathway to write and erase chromatin marks like histone acetylation and methylation are depicted as well as ATP‐dependent chromatin remodeling during the activation of target genes. An additional fine‐tuning of transcriptional regulation upon Notch activation seems to be controlled by the commitment of miRNAs. Since cells within an organism have to react to environmental changes, and developmental and differentiation cues in a proper manner, different signaling pathways have to crosstalk to each other. The chromatin status may represent one major platform to integrate these different pathways including the canonical Notch signaling. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:04:41.22321-05:0
      DOI: 10.1002/jcp.24851
  • Keeping Wnt Signalosome in Check by Vesicular Traffic
    • Authors: Qiang Feng; Nan Gao
      Pages: n/a - n/a
      Abstract: Wg/Wnts are paracrine and autocrine ligands that activate distinct signaling pathways while being internalized through surface receptors. In the field, contrasting views exist on whether, where, and how endocytosis may modulate Wnt signaling. We gather considerable amount of evidences to elaborate the point that signal‐receiving cells utilize distinct, flexible, and sophisticated vesicular trafficking mechanisms to keep Wnt signaling activity in check. Same molecules in a highly context‐dependent fashion serve as regulatory hub for various signaling purposes: amplification, maintenance, inhibition, and termination. Updates are provided for the regulatory mechanisms related to the three relevant cell surface complexes, Wnt‐Fzd‐LRP6, Dkk1‐Kremen‐LRP6, and R‐spondin‐LGR5‐RNF43, which potently influence Wnt signaling. We pay particular attentions to how cells achieve sustained and delicate control of Wnt signaling strength by employing comprehensive aspects of vesicular trafficking. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T14:03:37.979712-05:
      DOI: 10.1002/jcp.24853
  • Contractile and Metabolic Properties of Engineered Skeletal Muscle Derived
           From Slow and Fast Phenotype Mouse Muscle
    • Authors: Alastair Khodabukus; Keith Baar
      Pages: n/a - n/a
      Abstract: Satellite cells derived from fast and slow muscles have been shown to adopt contractile and metabolic properties of their parent muscle. Mouse muscle shows less distinctive fiber‐type profiles than rat or rabbit muscle. Therefore, in this study we sought to determine whether three‐dimensional muscle constructs engineered from slow soleus (SOL) and fast tibialis anterior (TA) from mice would adopt the contractile and metabolic properties of their parent muscle. Time‐to‐peak tension (TPT) and half‐relaxation time (1/2RT) was significantly slower in SOL constructs. In agreement with TPT, TA constructs contained significantly higher levels of fast myosin heavy chain (MHC) and fast troponin C, I, and T isoforms. Fast SERCA protein, both slow and fast calsequestrin isoforms and parvalbumin were found at higher levels in TA constructs. SOL constructs were more fatigue resistant and contained higher levels of the mitochondrial proteins SDH and ATP synthase and the fatty acid transporter CPT‐1. SOL constructs contained lower levels of the glycolytic enzyme phosphofructokinase but higher levels of the β‐oxidation enzymes LCAD and VLCAD suggesting greater fat oxidation. Despite no changes in PGC‐1α protein, SOL constructs contained higher levels of SIRT1 and PRC. TA constructs contained higher levels of the slow‐fiber program repressor SOX6 and the six transcriptional complex (STC) proteins Eya1and Six4 which may underlie the higher in fast‐fiber and lower slow‐fiber program proteins. Overall, we have found that muscles engineered from predominantly slow and fast mouse muscle retain contractile and metabolic properties of their native muscle. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T13:52:34.672339-05:
      DOI: 10.1002/jcp.24848
  • Chondroptosis in Alkaptonuric Cartilage
    • Authors: Lia Millucci; Giovanna Giorgetti, Cecilia Viti, Lorenzo Ghezzi, Silvia Gambassi, Daniela Braconi, Barbara Marzocchi, Alessandro Paffetti, Pietro Lupetti, Giulia Bernardini, Maurizio Orlandini, Annalisa Santucci
      Pages: n/a - n/a
      Abstract: Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above‐mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T13:52:27.162952-05:
      DOI: 10.1002/jcp.24850
  • Enhanced Phenotypic Alterations of Alveolar Type II Cells in Response to
           Aflatoxin G1‐Induced Lung Inflammation
    • Authors: Haitao Shen; Chunping Liu, Peilu Shao, Li Yi, Yuan Wang, Emily Mills Ko, Ziqiang Tian, Xin Zhao, Juan Wang, Lingxiao Xing, Xianghong Zhang
      Pages: n/a - n/a
      Abstract: Recently, we discovered that Aflatoxin G1 (AFG1) induces chronic lung inflammatory responses, which may contribute to lung tumorigenesis in Balb/C mice. The cancer cells originate from alveolar type II cells (AT‐II cells). The activated AT‐II cells express high levels of MHC‐II and COX‐2, may exhibit altered phenotypes, and likely inhibit antitumor immunity by triggering regulatory T cells (Tregs). However, the mechanism underlying phenotypic alterations of AT‐II cells caused by AFG1‐induced inflammation remains unknown. In this study, increased MHC‐II expression in alveolar epithelium was observed and associated with enhanced Treg infiltration in mouse lung tissues with AFG1‐induced inflammation. This provides a link between phenotypically altered AT‐II cells and Treg activity in the AFG1‐induced inflammatory microenvironment. AFG1‐activated AT‐II cells underwent phenotypic maturation since AFG1 upregulated MHC‐II expression on A549 cells and primary human AT‐II cells in vitro. However, mature AT‐II cells may exhibit insufficient antigen presentation, which is necessary to activate effector T cells, due to the absence of CD80 and CD86. Furthermore, we treated A549 cells with AFG1 and TNF‐α together to mimic an AFG1‐induced inflammatory response in vitro, and we found that TNF‐α and AFG1 coordinately enhanced MHC‐II, CD54, COX‐2, IL‐10, and TGF‐β expression levels in A549 cells compared to AFG1 alone. The phenotypic alterations of A549 cells in response to the combination of TNF‐α and AFG1 were mainly regulated by TNF‐α‐mediated induction of the NF‐κB pathway. Thus, enhanced phenotypic alterations of AT‐II cells were induced in response to AFG1‐induced inflammation. Thus, AT‐II cells are likely to suppress anti‐tumor immunity by triggering Treg activity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-21T13:52:14.909451-05:
      DOI: 10.1002/jcp.24852
  • PDE5 inhibitors enhance Celecoxib killing in multiple tumor types
    • Authors: Laurence Booth; Jane L. Roberts, Nichola Cruickshanks, Seyedmehrad Tavallai, Timothy Webb, Peter Samuel, Adam Conley, Brittany Binion, Harold F. Young, Andrew Poklepovic, Sarah Spiegel, Paul Dent
      Pages: n/a - n/a
      Abstract: The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L‐NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over‐expression of c‐FLIP‐s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-09T12:42:59.57235-05:0
      DOI: 10.1002/jcp.24843
  • Permanent culture of macrophages at physiological oxygen attenuates the
           antioxidant and immunomodulatory properties of dimethyl fumarate
    • Authors: Benjamin Haas; Sandra Chrusciel, Sarah Fayad‐Kobeissi, Jean‐Luc Dubois‐Randé, Francisco Azuaje, Jorge Boczkowski, Roberto Motterlini, Roberta Foresti
      Pages: n/a - n/a
      Abstract: We hypothesized that O2 tension influences the redox state and the immunomodulatory responses of inflammatory cells to dimethyl fumarate (DMF), an activator of the nuclear factor Nrf2 that controls antioxidant genes expression. This concept was investigated in macrophages permanently cultured at either physiological (5% O2) or atmospheric (20% O2) oxygen levels and then treated with DMF or challenged with lipopolysaccharide (LPS) to induce inflammation. RAW 264.7 macrophages cultured at 20% O2 exhibited a pro‐oxidant phenotype, reflected by a lower content of reduced glutathione, higher oxidized glutathione and increased production of reactive oxygen species when compared to macrophages continuously grown at 5% O2. At 20% O2, DMF induced a stronger antioxidant response compared to 5% O2 as evidenced by a higher expression of heme oxygenase‐1, NAD(P)H:quinone oxydoreductase‐1 and superoxide dismutase‐2. After challenge of macrophages with LPS, several pro‐inflammatory (iNOS, TNF‐α, MMP‐2, MMP‐9), anti‐inflammatory (arginase‐1, IL‐10) and pro‐angiogenic (VEGF‐A) mediators were evaluated in the presence or absence of DMF. All markers, with few interesting exceptions, were significantly reduced at 5% O2. This study brings new insights on the effects of O2 in the cellular adaptation to oxidative and inflammatory stimuli and highlights the importance of characterizing the effects of chemicals and drugs at physiologically relevant O2 tension. Our results demonstrate that the common practice of culturing cells at atmospheric O2 drives the endogenous cellular environment towards an oxidative stress phenotype, affecting inflammation and the expression of antioxidant pathways by exogenous modulators. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-09T12:42:44.639233-05:
      DOI: 10.1002/jcp.24844
  • Cyclooxygenase‐2 Regulates NLRP3 Inflammasome‐Derived
           IL‐1β Production
    • Authors: Kuo‐Feng Hua; Ju‐Ching Chou, Shuk‐Man Ka, Yu‐Ling Tasi, Ann Chen, Shih‐Hsiung Wu, Hsiao‐Wen Chiu, Wei‐Ting Wong, Yih‐Fuh Wang, Change‐Ling Tsai, Chen‐Lung Ho, Cheng‐Hsiu Lin
      Pages: n/a - n/a
      Abstract: The NLR family, pyrin domain‐containing 3 (NLRP3) inflammasome is a reactive oxygen species‐sensitive multiprotein complex that regulates IL‐1β maturation via caspase‐1. It also plays an important role in the pathogenesis of inflammation‐related disease. Cyclooxygenase‐2 (COX‐2) is induced by inflammatory stimuli and contributes to the pathogenesis of inflammation‐related diseases. However, there is currently little known about the relationship between COX‐2 and the NLRP3 inflammasome. Here, we describe a novel role for COX‐2 in regulating the activation of the NLRP3 inflammasome. NLRP3 inflammasome‐derived IL‐1β secretion and pyroptosis in macrophages were reduced by pharmaceutical inhibition or genetic knockdown of COX‐2. COX‐2 catalyzes the synthesis of prostaglandin E2 and increases IL‐1β secretion. Conversely, pharmaceutical inhibition or genetic knockdown of prostaglandin E2 receptor 3 reduced IL‐1β secretion. The underlying mechanisms for the COX‐2‐mediated increase in NLRP3 inflammasome activation were determined to be the following: (1) enhancement of lipopolysaccharide‐induced proIL‐1β and NLRP3 expression by increasing NF‐κB activation and (2) enhancement of the caspase‐1 activation by increasing damaged mitochondria, mitochondrial reactive oxygen species production and release of mitochondrial DNA into cytosol. Furthermore, inhibition of COX‐2 in mice in vivo with celecoxib reduced serum levels of IL‐1β and caspase‐1 activity in the spleen and liver in response to lipopolysaccharide (LPS) challenge. These findings provide new insights into how COX‐2 regulates the activation of the NLRP3 inflammasome and suggest that it may be a new potential therapeutic target in NLRP3 inflammasome‐related diseases. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T14:19:29.251235-05:
      DOI: 10.1002/jcp.24815
  • Effect of rMnSOD on survival signaling in pediatric high risk T‐cell
           acute lymphoblastic leukaemia
    • Authors: A Pica; A Di Santi, V D'angelo, A Iannotta, M Ramaglia, M Di Martino, Ml Pollio, A Schiattarella, A Borrelli, A Mancini, P Indolfi, F. Casale
      Pages: n/a - n/a
      Abstract: Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that defends against oxidative damage due to reactive oxygen species (ROS). A new isoform of MnSOD with cytotoxic activity was recently discovered in liposarcoma cells. Here, we tested the effectiveness of a recombinant form of this isoform (rMnSOD) on leukemic T cells, Jurkat cells, and normal lymphocytes. Our results confirm that leukemic T cells can internalize rMnSOD and that rMnSOD causes apoptosis of 99% of leukemic cells without showing toxic effects on healthy cells. Using light and electron microscopy, we determined that an rMnSOD concentration of 0.067 μM most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 μM rMnSOD resulted in high expression of the pro‐apoptotic protein Bax and low expression of the anti‐apoptotic protein Bcl‐2 in leukemia cells. Concerning signal transduction pathway no influence was observed after treatment except for Jurkat cells showing a slightly decreased expression of ERK phosphorylation. These results suggest that rMnSOD may be an effective and non‐toxic treatment option for T‐cell leukemia. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T11:36:06.767904-05:
      DOI: 10.1002/jcp.24840
  • Prostaglandin Dependent Control of an Endogenous Estrogen Receptor Agonist
           by Osteoblasts
    • Authors: Thomas L. McCarthy; Michael Centrella
      Pages: n/a - n/a
      Abstract: Estrogen receptor (ER) activation has complex effects on bone cells, and loss of circulating estradiol adversely affects skeletal status in women. Hormone replacement therapy effectively circumvents bone loss after menopause, but enhances disease risk in other tissues. Here we show that prostaglandin E2 (PGE2) augments the activity of an osteoblast‐derived selective ER modulator, ObSERM. The stimulatory effect of PGE2 is replicated in part by either the PG receptor EP3 agonist 17‐phenyl trinor PGE2 or by the PG receptor FP agonist PGF2α. Whereas activation of the various PG receptors induces multiple downstream signals, the response to PGE2 was mimicked by activators of protein kinase C, and suppressed by inhibition of protein kinase C but not by inhibition of protein kinase A. Moreover, inhibition of nitric oxide synthesis and activation of the PTH and Wnt pathways increases ObSERM activity. Our studies therefore reveal that ObSERM activity is controlled in distinct ways and revise our understanding of ER activation within bone by agents or events associated with PG expression. They also predict ways to sustain or improve bone formation, fracture repair, and surgical healing without adding the risk of disease in other tissues where ER activation also has important biological functions. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T11:35:38.523439-05:
      DOI: 10.1002/jcp.24842
  • DNA‐PKcs deficiency inhibits glioblastoma cell‐derived
           angiogenesis after ionizing radiation
    • Authors: Yang Liu; Luwei Zhang, Yuanyuan Liu, Chao sun, Hong Zhang, Guoying Miao, Cui Xiao Di, Xin Zhou, Rong Zhou, Zhenhua Wang
      Pages: n/a - n/a
      Abstract: DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) plays a critical role in non‐homologous end‐joining repair of DNA double‐strand breaks (DSB) induced by ionizing radiation (IR). Little is known, however, regarding the relationship between DNA‐PKcs and IR‐induced angiogenesis; thus, in this study we aimed to further elucidate this relationship. Our findings revealed that lack of DNA‐PKcs expression or activity sensitized glioma cells to radiation due to the defective DNA DSB repairs and inhibition of phosphorylated AktSer473. Moreover, DNA‐PKcs deficiency apparently mitigated IR‐induced migration, invasion and tube formation of human microvascular endothelial cell (HMEC‐1) in conditioned media derived from irradiated DNA‐PKcs mutant M059J glioma cells or M059K glioma cells that have inhibited DNA‐PKcs kinase activity due to the specific inhibitor NU7026 or siRNA knockdown. Moreover, IR‐elevated vascular endothelial growth factor (VEGF) secretion was abrogated by DNA‐PKcs suppression. Supplemental VEGF antibody to irradiated‐conditioned media was negated enhanced cell motility with a concomitant decrease in phosphorylation of the FAK Try925 and SrcTry416. Furthermore, DNA‐PKcs suppression was markedly abrogated in IR‐induced transcription factor hypoxia inducible factor‐1α (HIF‐1α) accumulation, which is related to activation of VEGF transcription. These findings, taken together, demonstrate that depletion of DNA‐PKcs in glioblastoma cells at least partly suppressed IR‐inflicted migration, invasion and tube formation of HMEC‐1 cells, which may be associated with the reduced HIF‐1α level and VEGF secretion. Inhibition of DNA‐PKcs may be a promising therapeutic approach to enhance radio‐therapeutic efficacy for glioblastoma by hindering its angiogenesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T11:34:26.23108-05:0
      DOI: 10.1002/jcp.24841
  • Role of Methionine Adenosyltransferase α2 and β Phosphorylation
           and Stabilization in Human Hepatic Stellate Cell
    • Authors: Komal Ramani; Shant Donoyan, Maria Lauda Tomasi, Sunhee Park
      Pages: n/a - n/a
      Abstract: Myofibroblastic trans‐differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S‐adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up‐regulation during trans‐differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post‐translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans‐differentiation. Culture‐activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen‐activated protein kinase (MAPK) family members [ERK1/2, V‐Raf Murine Sarcoma Viral Oncogene Homolog B1 (B‐Raf), MEK]. Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B‐Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho‐sites destabilized them and prevented HSC trans‐differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans‐differentiation. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T09:36:32.892568-05:
      DOI: 10.1002/jcp.24839
  • Methods and Procedures in Adipose Stem Cells: State of the Art and
           Perspective For Translation Medicine
    • Authors: G.F. Nicoletti; F. De Francesco, F. D'Andrea, G.A. Ferraro
      Pages: n/a - n/a
      Abstract: Stem cells have potential in the retrieval and repair of injured tissue and renovation of organ function. To date, several studies have been carried out to elucidate how differentiation of stem cells can be used in regenerative medicine applications. Adipose tissue is an abundant and accessible source of stem cell, useful for regenerative therapeutic use. Adipose stem cells (ASCs) are favorable for future translational research and can be applied in many clinical settings. Adipose tissue repair has been recently adopted in clinical trials to prove that ASCs can be successfully used in patients. Variability in cell culture procedures (isolation, characterization and differentiation) may have an influence on the experimental outcome. In this report, we consider the selection mechanisms of ASCs using flow cytometry, cell culture, freezing/thawing, cell cycle evaluation, histochemistry/immunofluorescence and differentiation of ASCs. Both researchers and regulatory institutions should consider a new policy for GMP procedures and protocols, paying special attention to stem cell bio‐physiology, to facilitate more clinically oriented studies. ASCs show angiogenic properties, with prospects of repairing tissue damaged by radiotherapy, as well as possessing the ability to heal chronic wounds. They can also be useful in surgical practice. We focus on the potential clinical application of ASCs that are currently available regarding translational medicine and the methods and procedures for their isolation, differentiation and characterization. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T09:34:51.035164-05:
      DOI: 10.1002/jcp.24837
  • Pregnane X Receptor as the “Sensor and Effector” in Regulating
    • Authors: Xi Ma; Jingshu Chen, Yanan Tian
      Pages: n/a - n/a
      Abstract: The pregnane X receptor (PXR, NR1I2) is a ligand‐activated nuclear receptor which plays an essential role in organism's metabolic detoxification system by sensing the presence of xenobiotics and triggering detoxification responses. In addition to its role in xenobiotic metabolism, PXR has pleiotropic functions in regulating immune/inflammatory responses, cell proliferation, bile acid/cholesterol metabolism, glucose and lipid metabolism, steroid/endocrine homeostasis and bone metabolism. Recent research suggests that the PXR is required for maintaining healthy commensalism between microbiota and gut. Interestingly, the metabolites such as indole derivatives from commensal microbes serve as the ligands for the PXR in intestinal epithelium forming an intricate mutualistic interaction between host and microbiota. PXR‐regulated gene responses are controlled at epigenetic level by chromatin modifications, DNA methylation and noncoding RNA. Developmental alterations of the epigenome by exposure to the xenobiotics or diseases may produce persistent changes in PXR‐regulated physiological responses. These new areas of research promise to vastly increase our understanding of PXR‐regulated responses. In this review we highlight recent results on the epigenetic mechanisms for the PXR‐regulated gene expression and discuss the physiological significance of these findings. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-07T09:33:13.495063-05:
      DOI: 10.1002/jcp.24838
  • Gastroprotective Effects of L‐Lysine Salification of Ketoprofen in
           Ethanol‐Injured Gastric Mucosa
    • Authors: A. Cimini; L. Brandolini, R. Gentile, L. Cristiano, P. Menghini, A. Fidoamore, A. Antonosante, E. Benedetti, A. Giordano, M. Allegretti
      Pages: n/a - n/a
      Abstract: Ketoprofen L‐lysine salt (KLS), a NSAID, is widely used for its analgesic efficacy and tolerability. L‐lysine salification was reported to increase the solubility and the gastric absorption and tolerance of Ketoprofen. Since the management of NSAIDs gastrotoxicity still represents a major limitation in prolonged therapies, mainly when gastric lesions are present, this study investigated the gastro‐protective activity of L‐lysine by using a well‐established model of gastric mucosa injury, the ethanol‐gastric injury model. Several evidences show that the damaging action of ethanol could be attributed to the increase of ROS, which plays a key role in the increase of lipid peroxidation products, including malonyldialdehyde and 4‐hydroxy‐2‐nonenal. With the aim to unravel the mechanism of L‐lysine gastroprotection, cellular MDA levels and 4‐HNE protein adducts as markers of lipid peroxidation and a panel of key endogenous gastro‐protective proteins were assayed. The data obtained indicate a gastroprotective effect of L‐lysine on gastric mucosa integrity. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-06T22:53:18.120733-05:
      DOI: 10.1002/jcp.24809
  • Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in
           Dividing Human Cells
    • Authors: Anna Sobol; Paola Galluzzo, Shuang Liang, Brittany Rambo, Sylvia Skucha, Megan J. Weber, Sara Alani, Maurizio Bocchetta
      Pages: n/a - n/a
      Abstract: Hypoxic non‐small cell lung cancer (NSCLC) is dependent on Notch‐1 signaling for survival. Targeting Notch‐1 by means of γ‐secretase inhibitors (GSI) proved effective in killing hypoxic NSCLC. Post‐mortem analysis of GSI‐treated, NSCLC‐burdened mice suggested enhanced phosphorylation of 4E‐BP1 at threonines 37/46 in hypoxic tumor tissues. In vitro dissection of this phenomenon revealed that Amyloid Precursor Protein (APP) inhibition was responsible for a non‐canonical 4E‐BP1 phosphorylation pattern rearrangement‐a process, in part, mediated by APP regulation of the pseudophosphatase Styx. Upon APP depletion we observed modifications of eIF‐4F composition indicating increased recruitment of eIF‐4A to the mRNA cap. This phenomenon was supported by the observation that cells with depleted APP were partially resistant to silvestrol, an antibiotic that interferes with eIF‐4A assembly into eIF‐4F complexes. APP downregulation in dividing human cells increased the rate of global protein synthesis, both cap‐ and IRES‐dependent. Such an increase seemed independent of mTOR inhibition. After administration of Torin‐1, APP downregulation and Mechanistic Target of Rapamycin Complex 1 (mTORC‐1) inhibition affected 4E‐BP1 phosphorylation and global protein synthesis in opposite fashions. Additional investigations indicated that APP operates independently of mTORC‐1. Key phenomena described in this study were reversed by overexpression of the APP C‐terminal domain. The presented data suggest that APP may be a novel regulator of protein synthesis in dividing human cells, both cancerous and primary. Furthermore, APP appears to affect translation initiation using mechanisms seemingly dissimilar to mTORC‐1 regulation of cap‐dependent protein synthesis. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-06T01:57:08.048627-05:
      DOI: 10.1002/jcp.24835
  • Emerging Concept in DNA Methylation: Role of Transcription Factors in
           Shaping DNA Methylation Patterns
    • Authors: Claire Marchal; Benoit Miotto
      Pages: n/a - n/a
      Abstract: DNA methylation in mammals is a key epigenetic modification essential to normal genome regulation and development. DNA methylation patterns are established during early embryonic development, and subsequently maintained during cell divisions. Yet, discrete site‐specific de novo DNA methylation or DNA demethylation events play a fundamental role in a number of physiological and pathological contexts, leading to critical changes in the transcriptional status of genes such as differentiation, tumor suppressor or imprinted genes. How the DNA methylation machinery targets specific regions of the genome during early embryogenesis and in adult tissues remains poorly understood. Here, we report advances being made in the field with a particular emphasis on the implication of transcription factors in establishing and in editing DNA methylation profiles. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
      PubDate: 2014-10-06T01:56:48.790137-05:
      DOI: 10.1002/jcp.24836
  • Could lncRNAs be the Missing Links in Control of Mesenchymal Stem Cell
    • Authors: Coralee E. Tye; Jonathan A.R. Gordon, Lori A. Martin‐Buley, Janet L. Stein, Jane B. Lian, Gary S. Stein
      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.
      PubDate: 2014-09-25T00:21:51.876637-05:
      DOI: 10.1002/jcp.24834
  • LIGHT is a crucial mediator of airway remodeling
    • Authors: Jen‐Yu Hung; Shyh‐Ren Chiang, Ming‐Ju Tsai, Ying‐Ming Tsai, Inn‐Wen Chong, Jiunn‐Min Shieh, Ya‐Ling Hsu
      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.
      PubDate: 2014-09-24T00:46:50.134114-05:
      DOI: 10.1002/jcp.24832
  • Lkb1 deletion promotes ectopic lipid accumulation in muscle progenitor
           cells and mature muscles
    • Authors: Tizhong Shan; Pengpeng Zhang, Pengpeng Bi, Shihuan Kuang
      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.
      PubDate: 2014-09-24T00:44:13.870582-05:
      DOI: 10.1002/jcp.24831
  • UCP2‐related mitochondrial pathway participates in oroxylin
           A‐induced apoptosis in human colon cancer cells
    • Authors: Chen Qiao; Libin Wei, Qinsheng Dai, Yuxin Zhou, Qian Yin, Zhiyu Li, Yuanming Xiao, Qinglong Guo, Na Lu
      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.
      PubDate: 2014-09-24T00:44:07.232297-05:
      DOI: 10.1002/jcp.24833
  • Alpha‐synuclein as a Pathological Link between Chronic Traumatic
           Brain Injury and Parkinson's disease
    • Authors: Sandra A. Acosta; Naoki Tajiri, Ike de la Pena, Marina Bastawrous, Paul R. Sanberg, Yuji Kaneko, Cesar V. Borlongan
      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.
      PubDate: 2014-09-24T00:43:55.605155-05:
      DOI: 10.1002/jcp.24830
  • 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
    • 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
  • 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
    • 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
    • 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
  • The Role of Notch Receptors in Transcriptional Regulation
    • Abstract: Notch signaling has pleiotropic context‐specific functions that have essential roles in many processes, including embryonic development and maintenance and homeostasis of adult tissues. Aberrant Notch signaling (both hyper‐ and hypoactive) is implicated in a number of human developmental disorders and many cancers. Notch receptor signaling is mediated by tightly regulated proteolytic cleavages that lead to the assembly of a nuclear Notch transcription complex, which drives the expression of downstream target genes and thereby executes Notch's functions. Thus, understanding regulation of gene expression by Notch is central to deciphering how Notch carries out its many activities. Here, we summarize the recent findings pertaining to the complex interplay between the Notch transcriptional complex and interacting factors involved in transcriptional regulation, including co‐activators, cooperating transcription factors, and chromatin regulators, and discuss emerging data pertaining to the role of Notch‐regulated noncoding RNAs in transcription. This article is protected by copyright. All rights reserved
  • Thymoquinone Restores Radiation‐Induced TGF‐β Expression
           and Abrogates EMT in Chemoradiotherapy of Breast Cancer Cells
    • 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‐β), a key regulator in epithelial–mesenchymal transdifferentiation and radio‐resistance. In this viewpoint, we employed thymoquinone as a radiosensitizer to investigate its migration and invasion 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 αV 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 downregulated whereas mesenchymal markers like integrin αV, MMP9, and MMP2 were upregulated by irradiation treatment; however thymoquinone pre‐sensitization has reverted the expression of these proteins back 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. 230: 620–629, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Inhibition of Metalloproteinase Activity in FANCA Is Linked to Altered
           Oxygen Metabolism
    • 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 patients. 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. 230: 603–609, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • The Differential Palmitoylation States of N‐Ras and H‐Ras
           Determine Their Distinct Golgi Subcompartment Localizations
    • 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 N‐Ras) 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. 230: 610–619, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Modulation of Mast Cell Proliferative and Inflammatory Responses by
           Leukotriene D4 and Stem Cell Factor Signaling Interactions
    • 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. 230: 595–602, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • PLCβ1a and PLCβ1b Selective Regulation and Cyclin D3 Modulation
           Reduced by Kinamycin F During K562 Cell Differentiation
    • Abstract: Here we report that both PLCβ1a and PLCβ1b 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 PLCβ1 isozymes even though the results point out that the effect of the drug is mainly directed toward the expression of the PLCβ1a isoform. We have identified a different role for the two isozymes as regulators of K562 differentiation process induced by kinamycin F. The overexpression of PLCβ1b induced an increase in γ‐globin expression even in the absence of kinamycin F. Moreover during K562 differentiation, cyclin D3 level is regulated by PLCβ1 signaling pathway. Namely the amplification of the expression of the PLCβ1a, but not of PLCβ1b, 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 PLCβ1b is mainly present in the nucleus in respect to PLCβ1a. Our data indicate that the amplification of PLCβ1a expression, following treatment with kinamycin F, confers a real advantage to K562 cells viability and protects cells themselves from apoptosis. J. Cell. Physiol. 230: 587–594, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Signaling Pathways Involved in Megakaryocyte‐Mediated Proliferation
           of Osteoblast Lineage Cells
    • 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 (Lennert et al., 1975; Thiele et al., 1999; 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. 230: 578–586, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Blocking the Expression of Both Bone Sialoprotein (BSP) and Osteopontin
           (OPN) Impairs the Anabolic Action of PTH in Mouse Calvaria Bone
    • 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 5× higher in −/− versus +/+ 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. 230: 568–577, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • LPS Promote the Odontoblastic Differentiation of Human Dental Pulp Stem
           Cells via MAPK Signaling Pathway
    • 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. 230: 554–561, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Detection of Phosphorylated Insulin Receptor in Colorectal Adenoma and
           Adenocarcinoma: Implications for Prognosis and Clinical Outcome
    • 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 
  • Fibronectin Peptides as Potential Regulators of Hepatic Fibrosis Through
           Apoptosis of Hepatic Stellate Cells
    • Abstract: The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX‐2 based on oligonucleosomal DNA fragmentation, caspase‐3 and ‐9 activation, Bcl‐2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease‐9 (MMP‐9) expression in human monocytes. J. Cell. Physiol. 230: 546–553, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • MicroRNA‐195 Chemosensitizes Colon Cancer Cells to the
           Chemotherapeutic Drug Doxorubicin by Targeting the First Binding Site of
           BCL2L2 mRNA
    • Abstract: The mechanisms underlying doxorubicin (Dox) resistance in colon cancer cells are not fully understood. MicroRNA (miRNA) play important roles in tumorigenesis and drug resistance. However, the relationship between miRNA and Dox resistance in colon cancer cells has not been previously explored. In this study, we utilized microRNA array and real‐time PCR to verify that miR‐127, miR‐195, miR‐22, miR‐137 were significantly down‐regulated, while miR‐21, miR‐592 were up‐regulated in both HT29/DOX and LOVO/DOX cell lines. In vitro cell viability assay showed that knockdown of miR‐195 in HT29 and LOVO cells caused a marked inhibition of Dox‐induced cytotoxicity. Moreover, we explored that miR‐195 is involved in repression of BCL2L2 expression through targeting its 3′‐untranslated region, especially the first binding site within its mRNA. Furthermore, down‐regulation of miR‐195 conferred DOX resistance in parental cells and reduced cell apoptosis activity, while over‐expression of miR‐195 sensitized resistant cells to DOX and enhanced cell apoptosis activity, all of which can be partly rescued by BCL2L2 siRNA and cDNA expression. These results may have implications for therapeutic strategies aiming to overcome colon cancer cell resistance to Dox. J. Cell. Physiol. 230: 535–545, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Journal of Cellular Physiology: Volume 230, Number 3, March 2015
    • Abstract: Cover: Mechanisms of lncRNA function. See article by Tye et al. on pages 526–534.
  • Table of Contents: Volume 230, Number 3
  • Editor's Choice
  • Highlights: Volume 230, Number 3
  • LncRNAs: New Players in Gliomas, With Special Emphasis on the Interaction
           of lncRNAs With EZH2
    • Abstract: Gliomas are the most common primary malignancy in the brain, accounting for 50–60%. Despite all the efforts of cytoreductive surgery in combination with intense chemoradiotherapy, glioma remains an incurable disease. Recent studies have shown that long noncoding RNAs (lncRNAs) are involved in the pathology of gliomas. LncRNAs are involved in many cellular processes, such as angiogenesis, invasion, cell proliferation, and apoptosis. In this review we focus on the dysregulation of lncRNAs in gliomas. We also address that epigenetic modification such as DNA methylation and microRNAs interact with lncRNAs in gliomas. In addition, the interaction of lncRNAs with signaling pathways in gliomas is discussed systematically, with particular emphasis on the interaction of lncRNAs with EZH2. Such approaches provide valuable insights into the potential future applications of lncRNAs in the treatment of gliomas. J. Cell. Physiol. 230: 496–503, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Acetylcholine Inhibits Tumor Necrosis Factor α Activated Endoplasmic
           Reticulum Apoptotic Pathway via EGFR‐PI3K Signaling in
    • 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.
  • Interleukin‐17 induces CC chemokine receptor 6 expression and cell
           migration in colorectal cancer cells
    • 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.
  • CRM197 in combination with shRNA interference of VCAM‐1 displays
           enhanced inhibitory effects on human glioblastoma cells
    • 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.
  • High levels of GPR30 protein in human testicular carcinoma in situ and
           seminomas correlate with low levels of estrogen receptor‐beta and
           indicate a switch in estrogen responsiveness
    • Abstract: The G protein‐coupled estrogen receptor (GPR30) is suggested to be involved in non‐nuclear estrogen signalling and is expressed in a variety of hormone dependent cancer entities. It is well established that oestrogens are involved in pathological germ cell proliferation including testicular germ cell tumours. This study was performed to further elucidate the role of this receptor and the possible correlation with the estrogen receptor β in human testicular carcinoma in situ (CIS), seminomas and in GC1 and TCam‐2 germ cell lines; in addition, a Tissue Micro‐Array was built using the most representative areas from 25 cases of human testicular seminomas and 20 cases of CIS. The expression of ERβ and GPR30 were observed by using Western blot analysis in combination with immunocytochemistry and immunofluorescence analyses. Here, we show that down regulation of ERβ associates with GPR30 over‐expression both in human testicular CIS and seminomas. In addition, we show that 17β‐oestradiol induces the ERK1/2 activation and increases c‐Fos expression through GPR30 associated with ERβ down‐regulation in TCam‐2 cell line. The present results suggest that exposure to oestrogens or oestrogen‐mimics, in some as of yet undefined manner, diminishes the ERβ‐mediated growth restraint in CIS and in human testicular seminoma, probably due to ERβ down‐regulation associated to GPR30 increased expression indicating that GPR30 could be a potential therapeutic target to design specific inhibitors. This article is protected by copyright. All rights reserved
  • Methotrexate‐Induced Bone Marrow Adiposity Is Mitigated by Folinic
           Acid Supplementation Through the Regulation of Wnt/β‐Catenin
    • 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 0.75 mg/kg MTX once daily for 5 days, receiving saline or 0.75 mg/kg FA 6 h after MTX. FA rescue alleviated the MTX‐induced bone marrow adiposity, 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. 230: 648–656, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Pin1 Plays a Critical Role as a Molecular Switch in Canonical BMP
    • 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. 230: 640–647, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Palmitate Induces ER Stress and Autophagy in H9c2 Cells: Implications for
           Apoptosis and Adiponectin Resistance
    • 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 (100 μM) 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 palmitate‐induced 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. 230: 630–639, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • In type 2 diabetes mellitus glycated albumin alters macrophage gene
           expression impairing ABCA1‐mediated cholesterol efflux
    • Abstract: Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE‐albumin induces oxidative stress, which is linked to a reduction in ABCA‐1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2‐HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C‐ or DM2‐HSA to measure the 14C‐cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA‐1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real‐time RT‐PCR. An increased mean mass was observed in DM2‐HSA compared with C‐HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3, and HDL2 was impaired in DM2‐HSA‐treated cells, which was related to greater intracellular lipid accumulation. DM2‐HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA‐1 protein content decreased. Compared with C‐HAS‐treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2‐HSA treatment. Stearoyl‐Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2‐HSA. The level of glycation that occurs in vivo in DM2‐HSA‐treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2. This article is protected by copyright. All rights reserved
  • Anti‐inflammatory/tissue repair macrophages enhance the
           cartilage‐forming capacity of human bone marrow‐derived
           mesenchymal stromal cells
    • Abstract: Macrophages are key players in healing processes. However little is known on their capacity to modulate the differentiation potential of mesenchymal stem/stromal cells (MSC). Here we investigated whether macrophages (Mf) with, respectively, pro‐inflammatory and tissue‐remodeling traits differentially modulate chondrogenesis of bone marrow derived‐MSC (BM‐MSC). We demonstrated that coculture in collagen scaffolds of BM‐MSC with Mf derived from monocytes polarized with M‐CSF (M‐Mf), but not with GM‐CSF (GM‐Mf) resulted in significantly higher glycosaminoglycan (GAG) content than what would be expected from an equal number of BM‐MSC alone (defined as chondro‐induction). Moreover, type II collagen was expressed at significantly higher levels in BM‐MSC/M‐Mf as compared to BM‐MSC/GM‐Mf constructs, while type X collagen expression was unaffected. In order to understand the possible cellular mechanism accounting for chondro‐induction, developing monoculture and coculture tissues were digested and the properties of the isolated BM‐MSC analysed. We observed that as compared to monocultures, in coculture with M‐Mf, BM‐MSC decreased less markedly in number and exhibited higher clonogenic and chondrogenic capacity. Despite their chondro‐inductive effect in vitro, M‐Mf did not modulate the cartilage tissue maturation in subcutaneous pockets of nude mice, as evidenced by similar accumulation of type X collagen and calcified tissue. Our results demonstrate that coculture of BM‐MSC with M‐Mf results in synergistic cartilage tissue formation in vitro. Such effect seems to result from the survival of BM‐MSC with high chondrogenic capacity. Studies in an orthotopic in vivo model are necessary to assess the clinical relevance of our findings in the context of cartilage repair. This article is protected by copyright. All rights reserved
  • MSCs Modified With ACE2 Restore Endothelial Function Following LPS
           Challenge by Inhibiting the Activation of RAS
    • 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, treatments that 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 lipopolysaccharide (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. 230: 691–701, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Roles of SATB2 in Site‐Specific Stemness, Autophagy and Senescence
           of Bone Marrow Mesenchymal Stem Cells
    • 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. 230: 680–690, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • Influence of Nuclear Blebs and Micronuclei Status on the Growth Kinetics
           of Human Mesenchymal Stem Cells
    • 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. 230: 657–666, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
  • The Cooperation of CREB and NFAT Is Required for PTHrP‐Induced RANKL
           Expression in Mouse Osteoblastic Cells
    • 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. 230: 667–679, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
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