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Journal Cover   Journal of Cellular Physiology
  [SJR: 1.948]   [H-I: 125]   [4 followers]  Follow
    
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
   Published by John Wiley and Sons Homepage  [1607 journals]
  • GRP78 / Dna K is a Target for Nexavar / Stivarga / Votrient in the
           Treatment of Human Malignancies, Viral Infections and Bacterial Diseases
    • Authors: Jane L. Roberts; Mehrad Tavallai, Aida Nourbakhsh, Abigail Fidanza, Tanya Cruz‐Luna, Elizabeth Smith, Paul Siembida, Pascale Plamondon, Kelly A. Cycon, Christopher D. Doern, Laurence Booth, Paul Dent
      Abstract: Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi‐kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug‐treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10 and cyclophilin A. Prolonged ‘rafenib / sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola / Hepatitis A and B viruses, respectively. Pre‐treatment with the ‘Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib / pazopanib and sildenafil was much more potent than sorafenib / pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever and Enterovirus 71 infection and reproduction. ‘Rafenib drugs / pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of ‘Rafenib drugs / pazopanib restored antibiotic sensitivity in pan‐antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-09T17:56:44.887878-05:
      DOI: 10.1002/jcp.25014
       
  • Development of an Experimental Animal Model for Lower Back Pain by
           Percutaneous Injury‐Induced Lumbar Facet Joint Osteoarthritis
    • Authors: Jae‐Sung Kim; Kasra Ahmadinia, Xin Li, John L Hamilton, Steven Andrews, Chris A. Haralampus, Guozhi Xiao, Hong‐Moon Sohn, Jae‐Won You, Yo‐Seob Seo, Gary S. Stein, Andre J Van Wijnen, Su‐Gwan Kim, Hee‐Jeong Im
      Abstract: We report generation and characterization of pain‐related behavior in a minimally‐invasive facet joint degeneration (FJD) animal model in rats. FJD was produced by a non‐open percutaneous puncture‐induced injury on the right lumbar FJs at three consecutive levels. Pressure hyperalgesia in the lower back was assessed by measuring the vocalization response to pressure from a force transducer. After hyperalgesia was established, pathological changes in lumbar FJs and alterations of intervertebral foramen size were assessed by histological and imaging analyses. To investigate treatment options for lumber FJ osteoarthritis‐induced pain, animals with established hyperalgesia were administered with analgesic drugs, such as morphine, a selective COX‐2 inhibitor, a non‐steroidal anti‐inflammatory drug (NSAID) (ketorolac), or pregabalin. Effects were assessed by behavioral pain responses. One week after percutaneous puncture‐induced injury of the lumbar FJs, ipsilateral primary pressure hyperalgesia developed and was maintained for at least 12 weeks without foraminal stenosis. Animals showed decreased spontaneous activity, but no secondary hyperalgesia in the hind paws. Histopathological and microfocus X‐ray computed tomography analyses demonstrated that the percutaneous puncture injury resulted in osteoarthritis‐like structural changes in the FJs cartilage and subchondral bone. Pressure hyperalgesia was completely reversed by morphine. The administration of celecoxib produced moderate pain reduction with no statistical significance while the administration of ketorolac and pregabalin produced no analgesic effect on FJ osteoarthritis‐induced back pain. Our animal model of non‐open percutanous puncture‐induced injury of the lumbar FJs in rats shows similar characteristics of low back pain produced by human facet arthropathy. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-09T17:56:26.532772-05:
      DOI: 10.1002/jcp.25015
       
  • Role of Contraction Duration in Inducing Fast‐To‐Slow
           Contractile and Metabolic Protein and Functional Changes in Engineered
           Muscle
    • Authors: Alastair Khodabukus; Leslie M. Baehr, Sue C. Bodine, Keith Baar
      Abstract: The role of factors such as frequency, contraction duration and active time in the adaptation to chronic low‐frequency electrical stimulation (CLFS) is widely disputed. In this study we explore the ability of contraction duration (0.6, 6, 60 and 600 secs) to induce a fast‐to‐slow shift in engineered muscle while using a stimulation frequency of 10Hz and keeping active time constant at 60%. We found that all contraction durations induced similar slowing of time‐to‐peak tension. Despite similar increases in total myosin heavy (MHC) levels with stimulation, increasing contraction duration resulted in progressive decreases in total fast myosin. With contraction durations of 60 and 600 secs, MHC IIx levels decreased and MHC IIa levels increased. All contraction durations resulted in fast‐to‐slow shifts in TnT and TnC but increased both fast and slow TnI levels. Half‐relaxation slowed to a greater extent with contraction durations of 60 and 600 secs despite similar changes in the calcium sequestering proteins calsequestrin and parvalbumin and the calcium uptake protein SERCA. All CLFS groups resulted in greater fatigue resistance than control. Similar increases in GLUT4, mitochondrial enzymes (SDH and ATPsynthase), the fatty acid transporter CPT‐1 and the metabolic regulators PGC‐1α and MEF2 were found with all contraction durations. However, the mitochondrial enzymes cytochrome C and citrate synthase were increased to greater levels with contraction durations of 60 and 600 seconds. These results demonstrate that contraction duration plays a pivotal role in dictating the level of CLFS‐induced contractile and metabolic adaptations in tissue engineered skeletal muscle. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-09T17:56:06.872202-05:
      DOI: 10.1002/jcp.24985
       
  • The Other Side of the RAAS ‐ Aldosterone Improves Migration of
           Cardiac Progenitor Cells
    • Authors: Stephanie Könemann; Kristin Wenzel, Sabine Ameling, Karina Grube, Elke Hammer, Raik Könemann, Rasmita Samal, Uwe Völker, Stephan B. Felix
      Abstract: Stem cell therapy is a promising new option for patients suffering from heart failure. Though many clinical studies show encouraging results, little is known about the signals which cause stem cells to home to diseased but not to healthy hearts. We hypothesized that aldosterone as one of the main players of heart failure functions as an attractant for progenitor cells and stimulates their migration. Stem cell antigen‐1 (Sca‐1) positive cells were isolated from the hearts of wild type FVB mice via magnetic cell sorting. The migration rate of the cells was determined using aldosterone as an attractant in a modified Boyden chamber (n = 5). Aldosterone led to a dose dependent increase in migration rate and this effect could be prevented by adding its blocker eplerenone. The mineralocorticoid receptor could be detected on Sca‐1+ cells via western blot and immunofluorescence. Therefore, aldosterone seems to play a role in stem cell migration and there the effect is most likely mediated by the mineralocorticoid receptor. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-08T17:47:27.86942-05:0
      DOI: 10.1002/jcp.25013
       
  • Effect of Diabetes Mellitus on Adipocyte‐Derived Stem Cells in Rat
    • Authors: Medet Jumabay; Jeremiah H. Moon, Huwate Yeerna, Kristina I. Boström
      Abstract: Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein (BMP)4 is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte‐derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co‐localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-08T17:47:10.011655-05:
      DOI: 10.1002/jcp.25012
       
  • Glycation of Wild‐Type Apomyoglobin Induces Formation of Highly
           Cytotoxic Species
    • Authors: Clara Iannuzzi; Vincenzo Carafa, Lucia Altucci, Gaetano Irace, Margherita Borriello, Roberto Vinciguerra, Ivana Sirangelo
      Abstract: Protein glycation is a non‐enzymatic, irreversible modification of protein amino groups by reactive carbonyl species leading to the formation of advanced glycation end products (AGEs). Several proteins implicated in neurodegenerative diseases have been found to be glycated in vivo and the extent of glycation is related to the pathologies of the patients. Although it is now accepted that there is a direct correlation between AGEs formation and the development of neurodegenerative diseases related to protein misfolding and amyloid aggregation, several questions still remain unanswered: whether glycation is the triggering event or just an additional factor acting on the aggregation pathway. We have recently shown that glycation of the amyloidogenic W7FW14F apomyoglobin mutant significantly accelerates the amyloid fibrils formation providing evidence that glycation actively participates to the process. In the present study, to test if glycation can be considered also a triggering factor in amyloidosis, we evaluated the ability of different glycation agents to induce amyloid aggregation in the soluble wild type apomyoglobin. Our results show that glycation covalently modifies apomyoglobin and induces conformational changes that lead to the formation of oligomeric species that are not implicated in amyloid aggregation. Thus, AGEs formation does not trigger amyloid aggregation in the wild type apomyoglobin but only induce the formation of soluble oligomeric species able to affect cell viability. The molecular bases of cell toxicity induced by AGEs formed upon glycation of wild type apomyoglobin have been also investigated. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-03T04:17:52.492318-05:
      DOI: 10.1002/jcp.25011
       
  • Lovastatin‐Mediated Changes in Human Tendon Cells
    • Authors: Maria Kuzma‐Kuzniarska; Hannah R. Cornell, Michael C. Moneke, Andrew J. Carr, Philippa A. Hulley
      Abstract: Statins are among the most widely prescribed drugs worldwide. Numerous studies have shown their beneficial effects in prevention of cardiovascular disease through cholesterol‐lowering and anti‐atherosclerotic properties. Although some statin patients may experience muscle‐related symptoms, severe side effects of statin therapy are rare, primarily due to extensive first‐pass metabolism in the liver. Skeletal muscles appear to be the main site of side effects, however recently some statin‐related adverse effects have been described in tendon. The mechanism behind these side effects remains unknown. This is the first study that explores tendon‐specific effects of statins in human primary tenocytes. The cells were cultured with different concentrations of lovastatin for up to 1 week. No changes in cell viability or morphology were observed in tenocytes incubated with therapeutic doses. Short‐term exposure to lovastatin concentrations outside the therapeutic range had no effect on tenocyte viability, however cell migration was reduced. Simvastatin and atorvastatin, two other drug family members, also reduced the migratory properties of the cells. Prolonged exposure to high concentrations of lovastatin induced changes in cytoskeleton leading to cell rounding and decreased levels of mRNA for matrix proteins, but increased BMP‐2 expression. Gap junctional communication was impaired but due to cell shape change and separation rather than direct gap junction inhibition. These effects were accompanied by inhibition of prenylation of Rap1a small GTPase. Collectively, we showed that statins in a dose‐dependent manner decrease migration of human tendon cells, alter their expression profile and impair the functional network, but do not inhibit gap junction function. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-02T02:04:27.795551-05:
      DOI: 10.1002/jcp.25010
       
  • Reflections on Cellular Physiology as a Catalytic Framework for Medicine
    • Authors: Arthur Perelman
      Abstract: These are the reflections of a retired physician who was introduced to cellular physiology in 1947 at the University of Pennsylvania. I majored in Zoology. I took the required major courses, which were interesting but not exciting. I was a premedical student at the time. Fortunately I was introduced to cellular physiology in 1947. The teacher was Professor Louis Heilbrunn, an amazing man dedicated to teaching and the love of his students. He had a number of graduate students at the time. The undergraduates were well integrated with these advanced students. We used his textbook of physiology and we were all proud of being his students. He gave us a new way of thinking about biology. It was exciting and we were treated as mature students. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-01T07:24:16.511292-05:
      DOI: 10.1002/jcp.25007
       
  • Increased Linear Bone Growth by GH in the Absence of SOCS2 is Independent
           of IGF‐1
    • Authors: R. Dobie; S.F. Ahmed, K.A. Staines, C. Pass, S. Jasim, V.E. MacRae, C. Farquharson
      Abstract: Growth hormone (GH) signalling is essential for postnatal linear bone growth, but the relative importance of GHs actions on the liver and/or growth plate cartilage remains unclear. The importance of liver‐derived insulin like‐growth factor‐1 (IGF‐1) for endochondral growth has recently been challenged. Here we investigate linear growth in Suppressor of Cytokine Signalling (SOCS2)‐2 knockout mice, which have enhanced growth despite normal systemic GH/IGF‐1 levels. Wild‐type embryonic ex vivo metatarsals failed to exhibit increased linear growth in response to GH, but displayed increased Socs2 transcript levels (p 
      PubDate: 2015-04-01T07:23:05.871481-05:
      DOI: 10.1002/jcp.25006
       
  • Immortalized Mouse Floxed Fam20c Dental Papillar Mesenchymal and
           Osteoblast Cell Lines Retain Their Primary Characteristics
    • Authors: Chao Liu; Xiaofang Wang, Hua Zhang, Xiaohua Xie, Peihong Liu, Ying Liu, Priyam H Jani, Yongbo Lu, Shuo Chen, Chunlin Qin
      Abstract: Fam20c is essential for the normal mineralization of dentin and bone. The generation of odontoblast and osteoblast cell lines carrying floxed Fam20c allele can offer valuable tools for the study of the roles of Fam20c in the mineralization of dentin and bone. The limited capability of the primary odontoblasts and osteoblasts to proliferate necessitates the development of odontoblast and osteoblast cell lines serving as substitutes for the study of differentiation and mineralization of the odontoblasts and osteoblasts. In this study, we established and characterized immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. The isolated primary mouse floxed Fam20c dental papilla mesenchymal cells and osteoblasts were immortalized by the infection of lentivirus containing Simian Virus 40 T‐antigen (SV40 T‐Ag). The immortalization of floxed Fam20c dental papilla mesenchymal cells and osteoblasts was verified by the long‐term passages and genomic integration of SV40 T‐Ag. The immortalized floxed Fam20c dental papilla mesenchymal and osteoblast cell lines not only proliferated at a high rate and retained the morphology of their primary counterparts, but also preserved the dentin and bone specific gene expression as the primary dental papilla mesenchymal cells and osteoblasts did. Consistently, the capability of the primary floxed Fam20c dental papilla mesenchymal cells and osteoblasts to mineralize was also inherited by the immortalized dental papilla mesenchymal and osteoblast cell lines. Thus, we have successfully generated the immortalized mouse floxed Fam20c dental papilla mesenchymal and osteoblast cell lines. This article is protected by copyright. All rights reserved
      PubDate: 2015-04-01T07:22:46.98116-05:0
      DOI: 10.1002/jcp.25008
       
  • TGF‐Beta Suppresses Ift88 Expression in Chondrocytic ATDC5 Cells
    • Authors: Makiri Kawasaki; Yoichi Ezura, Tadayoshi Hayata, Takuya Notomi, Yayoi Izu, Masaki Noda
      Abstract: IFT88 is an intraflagella transport protein, critical for the cilium and has been shown to be required for the maintenance of chondrocytes and cartilage. However, how IFT88 is controlled by cytokines that play a role in osteoarthritis is not well understood. Therefore, we examined the effects of TGF beta on the expression of IFT88. We used ATDC5 cells as chondrocytes and analyzed the effects of TGF beta on gene expression. TGF beta treatment suppresses the levels of Ift88 mRNA in a dose‐dependent manner starting from as low as 0.5 ng/ml and reaching the nadir at around 2 ng/ml. TGF beta treatment also suppresses the protein levels of Ift88. TGF beta suppression of Ift88 is still observed when the cells are cultured in the presence of a transcriptional inhibitor while the TGF beta suppression is weakened in the presence of a protein synthesis inhibitor, cycloheximide. TGF beta treatment suppresses the levels of Ift88 mRNA stability suggesting the presence of posttranscriptional regulation. TGF beta treatment reduces the number of cilia positive cells and suppresses average length of cilia. Knockdown of Ift88 by siRNA enhances TGF beta‐induced increase in type II collagen mRNA expression in ATDC5 cells revealing the suppressive role of Ift88 on TGF beta‐induced regulation of extracellular matrix protein expression. TGF beta also suppresses Ift88 mRNA expression in primary culture of chondrocytes. These data indicate that TGF beta regulates Ift88 gene expression at least in part via posttrascriptional manner. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-31T09:06:55.347463-05:
      DOI: 10.1002/jcp.25005
       
  • Impairments of the Vascular KATP Channel Imposes Fatal Susceptibility to
           Experimental Diabetes Due to Multi‐Organ Injuries
    • Authors: Shan‐Shan Li; Ningren Cui, Yang Yang, Timothy C Trower, Yu‐Min Wei, Yang Wu, Shuang Zhang, Xin Jin, Chun Jiang
      Abstract: The vascular isoform of ATP‐sensitive K+ (KATP) channels regulates blood flow to all organs. The KATP channel is strongly inhibited by reactive oxygen and carbonyl species produced in diabetic tissue inflammation. To address how such channel inhibition impacts vascular regulation as well as tissue viability, we performed studies in experimental diabetic mice. Strikingly, we found that knockout of the Kcnj8 encoding Kir6.1 subunit (Kcnj8‐KO) caused mice to be fatally susceptible to diabetes. Organ perfusion studies suggested that the lack of this vascular K+ channel handicapped activity‐dependent vasodilation, leading to hypoperfusion, tissue hypoxia and multi‐organ failure. Morphologically, Kcnj8‐KO mice showed greater inflammatory cell infiltration, higher levels of expression of inflammation indicator proteins, more severe cell apoptosis and worse tissue disruptions. These were observed in the kidney, liver and heart under diabetic condition in parallel comparison to tissues from WT mice. Patch clamping and molecular studies showed that the KATP channel was S‐glutathionylated in experimental diabetes contributing to the inhibition of channel activity as well as the reduced arterial responses to vasodilators. These results suggest that the vascular KATP channel is organ‐protective in diabetic condition, and since the channel is suppressed by diabetic oxidative stress, therapeutical interventions to the maintenance of functional KATP channels may help to lower or prevent diabetic organ dysfunction. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-30T01:48:09.247148-05:
      DOI: 10.1002/jcp.25003
       
  • Hyperosmolarity‐Induced Down‐Regulation of Claudin‐2
           Mediated by Decrease in PKCβ‐Dependent GATA‐2 in MDCK
           Cells
    • Authors: Akira Ikari; Naoko Fujii, Shinya Hahakabe, Hisayoshi Hayashi, Masahiko Yamaguchi, Yasuhiro Yamazaki, Satoshi Endo, Toshiyuki Matsunaga, Junko Sugatani
      Abstract: Hyperosmolarity decreases claudin‐2 expression in renal tubular epithelial cells, but the molecular mechanism remains undefined. Here, we found that the hyperosmolarity‐induced decrease in claudin‐2 expression is inhibited by Go6983, a non‐selective protein kinase C (PKC) inhibitor, and PKCβ specific inhibitor in Madin‐Darby canine kidney II cells. Hyperosmolarity increased intracellular free Ca2+ concentration and phosphorylated PKCβ level, which were inhibited by RN‐1734, an antagonist of transient receptor potential vanilloid 4 channel. Phorbol 12‐myristate 13‐acetate, a PKC activator, decreased claudin‐2 expression. These results indicate hyperosmolarity decreases claudin‐2 expression mediated by the activation of RN‐1734‐sensitive channel and PKCβ. Hyperosmolarity decreased promoter activity of claudin‐2, which was inhibited by Go6983 and PKCβ inhibitor similar to those in real‐time PCR and Western blotting. The effect of hyperosmolarity on promoter activity was not observed in the construct of ‐469/‐6, a deletion mutant. Claudin‐2 has hyperosmolarity‐sensitive region in its promoter, which includes GATA binding site. Hyperosmolarity decreased the nuclear level of GATA‐2, which was inhibited by Go6983 and PKCβ inhibitor. Mutation of GATA binding site decreased the basal promoter activity and inhibited the effect of hyperosmolarity. In contrast, the hyperosmolarity‐induced decrease in reporter activity and claudin‐2 expression were rescued by over‐expression of wild type GATA‐2. Chromatin immunoprecipitation assay showed that GATA‐2 bound to promoter region of claudin‐2. These results suggest that hyperosmolarity decreases the expression level of claudin‐2 via a decrease in PKCβ‐dependent GATA‐2 transcriptional activity in renal tubular epithelial cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-30T01:46:57.244021-05:
      DOI: 10.1002/jcp.25004
       
  • Table of Contents: Volume 230, Number 7
    • PubDate: 2015-03-27T08:38:21.345328-05:
      DOI: 10.1002/jcp.24757
       
  • Journal of Cellular Physiology: Volume 230, Number 7, July 2015
    • Abstract: Cover: HSP60 expression at 60X magnification in a brain cancer cell treated for 6h with OSU‐03012 (also called AR‐12) and Viagra. See article by Booth et al. on pages 1661–1676.
      PubDate: 2015-03-27T08:38:21.306506-05:
      DOI: 10.1002/jcp.24756
       
  • Highlights: Volume 230, Number 7
    • PubDate: 2015-03-27T08:38:21.236731-05:
      DOI: 10.1002/jcp.24758
       
  • Editor's Choice
    • PubDate: 2015-03-27T08:38:16.369968-05:
      DOI: 10.1002/jcp.24984
       
  • The Role of Auxiliary Subunits for the Functional Diversity of
           Voltage‐Gated Calcium Channels
    • Authors: Marta Campiglio; Bernhard E. Flucher
      Abstract: Voltage‐gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore‐forming α1 subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice‐variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre‐existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macro‐molecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein‐protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein‐protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:58:40.644318-05:
      DOI: 10.1002/jcp.24998
       
  • Lactogenic Hormone Stimulation and Epigenetic Control of L‐Amino
           Acid Oxidase Expression in Lactating Mammary Glands
    • Authors: Kazuki Fujii; Haolin Zhang, Kento Usuda, Gen Watanabe, Kentaro Nagaoka
      Abstract: L‐amino acid oxidase (LAO), a classic flavoprotein, shows antibacterial activity by producing hydrogen peroxide. LAO exists in many tissues such as salivary gland, thymus, spleen, small intestine, and testis. In particular, LAO was highly expressed in mice milk and plays an important factor in innate immunity of mammary glands. However, the mechanism which LAO expression is regulated spatially and temporally in lactating mammary glands has been unclear. In this study, we showed the contribution of lactogenic hormone and epigenetic control on LAO gene expression. In monolayer of mammary epithelial cells, treatment of lactogenic hormone mixture, dexamethasone, insulin and prolactin, did not induce LAO mRNA expression and its promoter activity, even though one of milk protein β‐casein expression was stimulated. However, increase of LAO expression was observed when the cells were treated with lactogenic hormones in a 3‐dimensional culture. The results of chromatin immunoprecipitation analysis revealed that histone H3K18 acetylation increased and histone H3K27 tri‐methylation decreased with lactation, which is associated with a period of high LAO expression. Moreover, the treatment of histone methylation inhibitor (DZNep) as well as histone deacetylation inhibitor (Trichostatine A) induced LAO expression in monolayer of mammary cells. Taken together, this is the first demonstration showing that LAO expression is induced in cell culture, and stimulation of lactogenic hormone and change of histone modification are promising signals to show highly expression of LAO in lactating mammary glands. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:58:17.84328-05:0
      DOI: 10.1002/jcp.25000
       
  • Nuclear Translocation of Calpain‐2 Mediates Apoptosis of
           Hypertrophied Cardiomyocytes in Transverse Aortic Constriction Rat
    • Authors: Juan‐Juan Sheng; Hui Chang, Zhi‐Bin Yu
      Abstract: Apoptosis of cardiomyocytes plays an important role in the transition from cardiac hypertrophy to heart failure. Hypertrophied cardiomyocytes show enhanced susceptibility to apoptosis. Therefore, the aim of this study was to determine the susceptibility to apoptosis and its mechanism in hypertrophied cardiomyocytes using a rat model of transverse abdominal aortic constriction (TAC). Sixteen weeks of TAC showed compensatory and pathological hypertrophy in the left ventricle. TUNEL‐positive nuclei were significantly increased in TAC with angiotensin II (Ang II) treatment. Calpain inhibitor, PD150606, effectively inhibited Ang II‐induced apoptosis of hypertrophied cardiomyocytes. Ang II increased nuclear translocation of intracellular Ca2+ activated calpain‐2 in hypertrophied cardiomyocytes. Ang II enhanced the interaction between activated calpain‐2 and Ca2+/calmodulin‐dependent protein kinase II δB (CaMKIIδB), and promoted the degradation of CaMKIIδB by calpain‐2 in the nuclei of hypertrophied cardiomyocytes. Consequently, the depressed CaMKIIδB downregulated the expression of antiapoptotic Bcl‐2 leading to mitochondrial depolarization and release of cytochrome c led to apoptosis of hypertrophied cardiomyocytes. In conclusion, hypertrophied cardiomyocytes show increased susceptibility to apoptosis during Ang II stimulation via nuclear calpain‐2 and CaMKIIδB pathway. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:58:02.592822-05:
      DOI: 10.1002/jcp.24999
       
  • VEGF Enhances the Migration of MSCs in Neural Differentiation by
           Regulating Focal Adhesion Turnover
    • Authors: Huihui Wang; Xingkai Wang, Jing Qu, Qing Yue, Ya'nan Hu, Huanxiang Zhang
      Abstract: Mesenchymal stem cells (MSCs) hold great promise in neural regeneration, due to their intrinsic neuronal potential and migratory tropism to damaged nervous tissues. However, the chemotactic signals mediating the migration of MSCs remain poorly understood. Here, we investigated the regulatory roles for focal adhesion kinase (FAK) and Rac1 in vascular endothelial growth factor (VEGF)‐stimulated migration of MSCs in neural differentiation. We found that MSCs in various differentiation states show significant different chemotactic responses to VEGF and cells in 24‐h preinduction state possess the highest migration speed and efficiency. FAK, as the downstream signaling molecule, is involved in the VEGF‐induced migration by regulating the assembly and distribution of focal adhesions (FAs) and reorganization of F‐actin. The features of FAs and cytoskeletons and the ability of lamellipodia formation are closely related to the neural differentiation states of MSCs. VEGF promotes FA formation with an asymmetric distribution of FAs and induces the activation of Y397‐FAK and Y31/118‐paxillin of undifferentiated and 24‐h preinduced MSCs in a time‐dependent manner. Inhibition of FAK by PF‐228 or expressing FAK‐Y397F mutant impairs the dynamics of FAs in MSCs during VEGF‐induced migration. Furthermore, Rac1 regulates FA formation in a FAK‐dependent manner. Overexpression of constitutive activated mutants of Rac1 increases the number of FAs in undifferentiated and 24‐h preinduced MSCs, while VEGF‐induced increase of FA formation is decreased by inhibiting FAK by PF‐228. Collectively, these results demonstrate that FAK and Rac1 signalings coordinately regulate the dynamics of FAs during VEGF‐induced migration of MSCs in varying neural differentiation states. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:57:44.911676-05:
      DOI: 10.1002/jcp.24997
       
  • Determinants of Muscle and Bone Aging
    • Authors: E Curtis; A Litwic, C Cooper, E Dennison
      Abstract: Loss of bone and muscle with advancing age represent a huge threat to loss of independence in later life. “Osteoporosis represents a major public health problem through its association with fragility fractures, primarily of the hip, spine and distal forearm” (Moselhy et al., 2012). Sarcopenia, the age related loss of muscle mass and function, may add to fracture risk by increasing falls risk. In the context of muscle aging, it is important to remember that it is not just a decline in muscle mass which contributes to the deterioration of muscle function. Other factors underpinning muscle quality come into play, including muscle composition, aerobic capacity and metabolism, fatty infiltration, insulin resistance, fibrosis and neural activation. Genetic, developmental, endocrine and lifestyle factors, such as physical activity, smoking and poor diet have dual effects on both muscle and bone mass in later life and these will be reviewed here. These include poor nutrition, lack of physical activity and cigarette smoking, comorbidities or medication use. Recent work has highlighted a possible role for the early environment. Inflammaging is an exciting emerging research field that is likely to prove relevant to future work, including interventions designed to retard to reverse bone and muscle loss with age. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:57:24.088441-05:
      DOI: 10.1002/jcp.25001
       
  • Angiogenic Role of MMP‐2/9 Expressed on the Cell Surface of Early
           Endothelial Progenitor Cells/Myeloid Angiogenic Cells
    • Authors: Toshie Kanayasu‐Toyoda; Takeshi Tanaka, Akiko Ishii‐Watabe, Hiroko Kitagawa, Akifumi Matsuyama, Eriko Uchida, Teruhide Yamaguchi
      Abstract: Since the introduction of angiogenic cell therapy using early endothelial progenitor cells (EPCs), myeloid angiogenic cells (MACs) have been expected to be useful in treating ischemic diseases. In order to elucidate the angiogenic properties of MACs/EPCs, we clarified the characteristics of MACs as compared to M2 macrophages (Mϕs). Comparison of the gene expression profiles of MACs and late EPCs revealed that MACs expressed greater amounts of metalloproteinase (MMP)‐9. It should be noted that the profile of MMP‐2/9 expression on the cell surface of MACs was similar to that of M2 Mϕs, and that cell surface MMP‐2/9 might be an active form based on molecular size. In addition, the invasion of MACs was prohibited not only by MMP‐2/9 inhibitor, but also by the hyaluronidase treatment that caused the down‐regulation of MMP‐9 on the cell surface of MACs and inhibited their invasion activity. These results indicate that cell surface MMP‐2/9 plays an important role in the high invasion ability of MACs. The conditioned medium of both MACs and M2 Mϕs stimulated tube formation of endothelial cells in vitro. MACs caused an increase in vessel formation in in vivo models through the production of IL‐8. We propose that the role of MACs with cell surfaces expressing MMP‐2/9 is rapidly invading ischemic tissue. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-27T01:57:10.704862-05:
      DOI: 10.1002/jcp.25002
       
  • Loss of microRNA‐101 Promotes Epithelial to Mesenchymal Transition
           in Hepatocytes
    • Authors: Shuhua Zhao; Yuanyuan Zhang, Xiuxiu Zheng, Xiaolong Tu, Huanan Li, Jiangning Chen, Yuhui Zang, Junfeng Zhang
      Abstract: Epithelial‐to‐mesenchymal transition (EMT) has been implicated in embryonic development and various pathological events. However, the involvement of microRNA in the process of EMT remains to be fully defined in hepatocyte. ZEB1 is a well‐known transcriptional repressor of E‐cadherin and plays a major role in triggering EMT during organ fibrosis and cancer cell metastasis. Computational microRNA target predictions detect a conserved sequence matching to miR‐101 in the 3'UTR of ZEB1 mRNA. Our results confirm that miR‐101 suppresses ZEB1 expression by targeting the predicted site of ZEB1 3'UTR. Subsequent investigations show that miR‐101 is significantly downregulated in the cultured hepatocytes undergoing EMT and in the hepatocytes isolated from fibrotic liver. Along with the loss of miR‐101, the ZEB1 expression increases simultaneously in hepatocytes. In addition, miR‐101 levels in HCC cell lines are negatively associated with the ZEB1 productions and the metastatic potentials of tumor cells. Mechanistically, we demonstrate that miR‐101 significantly inhibits the TGF‐β1‐induced EMT in hepatocytes, whereas inhibition of miR‐101 promotes the EMT process as indicated by the changes of morphology, cell migration and the expression profiles of EMT markers. In the fibrotic liver, ectopic expression of miR‐101 can significantly downregulate ZEB1 in the hepatocyte and thereby reduces the mesenchymal marker expression. Moreover, miR‐101 significantly inhibits the proliferation and migration of HCC cell. Our results demonstrate that miR‐101 regulates HCC cell phenotype by upregulating the epithelial marker genes and suppressing the mesenchymal ones. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-25T02:51:19.937133-05:
      DOI: 10.1002/jcp.24995
       
  • Establishment of Four New Human Primary Cell Cultures from
           Chemo‐Naïve Italian Osteosarcoma Patients
    • Authors: Marcella Laschi; Giulia Bernardini, Michela Geminiani, Lorenzo Ghezzi, Loredana Amato, Daniela Braconi, Lia Millucci, Bruno Frediani, Adriano Spreafico, Alessandro Franchi, Domenico Campanacci, Rodolfo Capanna, Annalisa Santucci
      Abstract: Osteosarcoma (OS) is a primary highly malignant tumor of bone, affecting predominately adolescents and young adults between 10 and 20 years of age. OS is characterized by an extremely aggressive clinical course, with a rapid development of metastasis to the lung and distant bones. The relative rarity of the tumor contributes significantly to the difficulty in studying the molecular contributions to the disease process and thereby in the development of novel targeted therapeutic approaches for the cure of OS. Moreover, individual and ethnic differences in OS onset and in the responsiveness to irradiation and chemotherapy, makes restrictive and not effective the use of the most frequently studied OS commercial cell lines (SaOS‐2, U2OS, MG63). Actual experimental protocols should be applied on a variety of human primary cell cultures generated from OS patient tumors before the administration of a chemotherapeutic treatment. With this aim, we established four novel OS primary cell culture models based on chemo‐naïve osteoblastic, chondroblastic and small cell high grade OS tumors from adolescent patients of Italian origin. Several phenotypic features of these cells were biochemically characterized and compared with those of the original tumors, such as with those of established OS commercial cell lines and primary not transformed cells. The cultured cells appear to have retained the peculiar characteristics of the original tumors and could be used as new culture models for in vitro OS studies, aimed not only at a better understanding of the OS cellular origin and malignant progression but also at the identification of diagnostic markers and new therapeutic targets for OS. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-25T02:51:03.223684-05:
      DOI: 10.1002/jcp.24996
       
  • Regulation of MDM2 stability as a consequence of DNA damage
    • Authors: Jiaqi Li; Manabu Kurokawa
      Abstract: Cells in our body are constantly exposed to various stresses and threats to their genomic integrity. The tumor suppressor protein p53 plays a critical role in successful defense against these threats by inducing apoptotic cell death or cell cycle arrest. In unstressed conditions, p53 levels and activity must be kept low to prevent lethal activation of apoptotic and senescence pathways. However, upon DNA damage or other stressors, p53 is released from its inhibitory state to induce an array of apoptosis and cell cycle genes. Conversely, inactivation of p53 could promote unrestrained tumor proliferation and failure to appropriately undergo apoptotic cell death, which could, in turn, lead to carcinogenesis. The ubiquitin E3 ligase MDM2 is the most critical inhibitor of p53 that determines the cellular response to various p53‐activating agents, including DNA damage. MDM2 activity is controlled by post‐translational modifications, especially phosphorylation. However, accumulating evidence suggests that MDM2 is also regulated at the level of protein stability, which is controlled by the ubiquitin‐proteasome pathway. Here, we discuss how MDM2 can be regulated in response to DNA damage with particular focus on the regulation of MDM2 protein stability. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-24T17:18:28.279939-05:
      DOI: 10.1002/jcp.24994
       
  • The Role of Maternal Gestational Diabetes in Inducing Fetal Endothelial
           Dysfunction
    • Authors: Samar A Sultan; Wanting Liu, Yonghong Peng, W. Roberts, Donald Whitelaw, Anne M Graham
      Abstract: Gestational diabetes mellitus (GDM) is known to be associated with fetal endothelial dysfunction, however, the mechanisms are not fully understood. This study examines the effect of maternal diabetes on fetal endothelial function and gene expression under physiological glucose conditions (5mM). Human umbilical vein endothelial cell (HUVEC) isolated from diabetic mothers (d.HUVEC) grew more slowly than HUVEC isolated from healthy mothers (c.HUVEC) and had delayed doubling time despite increased levels of total vascular endothelial growth factor (VEGF) expression and protein production as determined by real time PCR and ELISA respectively. Using western blot, the levels of antiproliferative VEGF165b isoform were increased in d.HUVEC relative to c.HUVEC. Successful VEGF165b knockdown by small interfering RNA (siRNA) resulted in increased proliferation of d.HUVEC measured by MTT, compared with negative siRNA control, to similar levels measured in c.HUVEC. In addition, d.HUVEC generated excess levels of ROS as revealed by 2′,7′ Dichlorodihydrofluorescein Diacetate (DCFH‐DA) and Nitrotetrazolium blue (NBT). Using microarray, 102 genes were differentially overexpressed between d.HUVEC vs. c.HUVEC (>1.5 fold change; p 
      PubDate: 2015-03-23T05:03:44.550723-05:
      DOI: 10.1002/jcp.24993
       
  • The SWI/SNF ATPases are Required for Triple Negative Breast Cancer Cell
           Proliferation
    • Authors: Qiong Wu; Pasil Madany, Jacqueline Akech, Jason R. Dobson, Stephen Douthwright, Gillian Browne, Jennifer L. Colby, Georg E. Winter, James E. Bradner, Jitesh Pratap, Greenfield Sluder, Rohit Bhargava, Simion Chiosea, Andre J. van Wijnen, Janet L. Stein, Gary S. Stein, Jane B. Lian, Jeffrey A. Nickerson, Anthony N. Imbalzano
      Abstract: The Brahma (BRM) and Brahma‐related Gene 1 (BRG1) ATPases are highly conserved homologues that catalyze the chromatin remodeling functions of the multi‐subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in man cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-21T09:17:11.76436-05:0
      DOI: 10.1002/jcp.24991
       
  • TNFα post‐translationally targets ZnT2 to accumulate zinc in
           lysosomes
    • Authors: Stephen R. Hennigar; Shannon L. Kelleher
      Abstract: Mammary epithelial cells undergo widespread lysosomal‐mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor‐alpha (TNFα), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study was to determine how TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C‐terminus of ZnT2, allowing adaptor protein complex‐3 (AP‐3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wildtype (wt)‐ZnT2 from late endosomes (Pearson's coefficient=0.202±0.05 and 0.097±0.03; P
      PubDate: 2015-03-21T09:17:10.257474-05:
      DOI: 10.1002/jcp.24992
       
  • Cell cycle gene expression networks discovered using systems biology:
           Significance in carcinogenesis
    • Authors: RE Scott; PN Ghule, JL Stein, GS Stein
      Abstract: The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint by its association with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co‐vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty‐three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with p = e‐13 to e‐36. Cell cycle expression networks show species, sex and tissue variability and they are enriched in mRNA transcripts associated with mitosis many of which are associated with chromosomal instability. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-21T09:16:32.399557-05:
      DOI: 10.1002/jcp.24990
       
  • Runx1 is associated with breast cancer progression in MMTV‐PyMT
           transgenic mice and its depletion in vitro inhibits migration and invasion
           
    • Authors: Gillian Browne; Hanna Taipaleenmäki, Nicole M. Bishop, Sharath C. Madasu, Leslie M. Shaw, Andre J. van Wijnen, Janet L. Stein, Gary S. Stein, Jane B. Lian
      Abstract: Runx1 is a transcription factor essential for definitive hematopoiesis, and genetic abnormalities in Runx1 cause leukemia. Runx1 is functionally promiscuous and acts as either an oncogene or tumor suppressor gene in certain epithelial cancers. Recent evidence suggests that Runx1 is an important factor in breast cancer, however its role remains ambiguous. Here, we addressed whether Runx1 has a specific pathological role during breast cancer progression and show that Runx1 has an oncogenic function. We observed elevated Runx1 expression in a subset of human breast cancers. Furthermore, throughout the course of disease progression in a classical mouse model of breast cancer (i.e., the MMTV‐PyMT transgenic model), Runx1 expression increases in the primary site (mammary gland) and is further upregulated in tumors and distal lung metastatic lesions. Ex vivo studies using tumor epithelial cells derived from these mice express significantly higher levels of Runx1 than normal mammary epithelial cells. The tumor cells exhibit increased rates of migration and invasion, indicative of an aggressive cancer phenotype. Inhibition of Runx1 expression using RNA interference significantly abrogates these cancer‐relevant phenotypic characteristics. Importantly, our data establish that Runx1 contributes to murine mammary tumor development and malignancy and potentially represents a key disease‐promoting and prognostic factor in human breast cancer progression and metastasis. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-19T21:29:16.117121-05:
      DOI: 10.1002/jcp.24989
       
  • Upregulation of Runt‐Related Transcription Factor‐2 through
           CCAAT Enhancer Binding Protein‐Beta Signaling Pathway in Microglial
           BV‐2 Cells Exposed to ATP
    • Authors: Ryota Nakazato; Takeshi Takarada, Shinsuke Ikeno, Saki Nakamura, Takaya Kutsukake, Eiichi Hinoi, Yukio Yoneda
      Abstract: We have shown constitutive expression of the master regulator of osteoblastogenesis, runt‐related transcription factor‐2 (Runx2), by microglia cells outside bone. Here, we attempted to evaluate the pathological significance of Runx2 in microglial BV‐2 cells exposed to ATP at a high concentration. Marked upregulation of Runx2 transcript and protein expression was seen in cells exposed to 1 mM ATP for a period longer than 30 min without inducing cytotoxicity. The Runx2 upregulation by ATP was prevented by extracellular and intracellular Ca2+ chelators, while thapsigargin up‐regulated Runx2 expression alone without affecting the upregulation by ATP. A calmodulin antagonist prevented the upregulation by ATP, with calcineurin inhibitors being ineffective. Although ATP markedly increased nuclear levels of nuclear factor of activated T cell‐2 (NFAT2), Runx2 promoter activity was not simulated by the introduction of either NFAT1 or NFAT2, but facilitated by that of CCAAT enhancer binding protein‐α (C/EBPα), C/EBPβ and nuclear factor (erythroid‐derived 2)‐like‐2 (Nrf2). Exposure to ATP up‐regulated C/EBPβ and Nrf2, but not C/EBPα, expression, in addition to increasing nuclear levels of respective corresponding proteins. Runx2 upregulation by ATP was deteriorated by knockdown of C/EBPβ but not by that of Nrf2, however, while exposure to ATP up‐regulated matrix metalloproteinase‐13 (Mmp13) expression in a Runx2‐dependent manner. Overexpression of Runx2 up‐regulated Mmp13 expression with promoted incorporation of fluorescent beads into BV‐2 cells without ATP. These results suggest that extracellular ATP up‐regulates Runx2 expression through activation of the C/EBPβ signaling in a calmodulin‐dependent manner to play a pivotal role in phagocytosis in microglial BV‐2 cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-19T16:30:56.808946-05:
      DOI: 10.1002/jcp.24988
       
  • Upregulation of miR‐155 in CD4+ T Cells Promoted Th1 Bias in
           Patients with Unstable Angina
    • Authors: Yanmei Chen; Lang Li, Yongguang Lu, Qiang Su, Yuhan Sun, Yang Liu, Dong Yang
      Abstract: An imbalance between T helper 1 (Th1) and T helper 2 (Th2) cells has been reported to increase plaque instability in patients with unstable angina (UA). MicroRNAs play a vital role in the differentiation of CD4+ T cells. However, the role of microRNAs in regulation of Th1/Th2 balance in UA remains unclear. In this study, we aimed to elucidate microRNA expression profiles of circulating CD4+ T cells in UA and to explore the function of microRNAs in the Th1/Th2 balance. A total of 58 patients with UA and 31 control subjects without coronary artery disease were enrolled. Microarray analysis of the microRNA expression profiles of CD4+ T cells revealed that miR‐155 was the most significantly upregulated microRNA of the 451 differentially expressed microRNAs. The upregulation of miR‐155 expression was positively correlated with the percentage of Th1 cells and interferon‐gamma (IFN‐γ) levels in patients with UA. In addition, overexpression of miR‐155 in human circulating CD4+ T cells promoted Th1 differentiation. Further studies identified IFN‐γ receptor alpha chain (IFN‐γ Rα) mRNA as a direct and functional target of miR‐155. A luciferase reporter assay verified that miR‐155 directly targeted IFN‐γ Rα mRNA. Small‐interfering RNA‐mediated knockdown of IFN‐γ Rα mRNA showed effects similar to those of ectopic miR‐155 expression. Thus, our study indicated that upregulation of miR‐155 in circulating CD4+ T cells in patients with UA promoted a shift in the Th1/Th2 balance toward Th1 dominance by repressing IFN‐γ Rα, which may subsequently enhance plaque instability. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-11T05:57:13.457961-05:
      DOI: 10.1002/jcp.24987
       
  • Can Brown Fat Win the Battle against White Fat'
    • Authors: Sawsan Elattar; Ande Satyanarayana
      Abstract: A rapid growth in the overweight and obese population in the last few decades suggest that the current diet, exercise, awareness or drug strategies are still not effectively restraining the obesity epidemic. Obesity results from increased energy intake, and the body's energy balance shifts towards energy abundance. Therefore, current research is focused on developing new strategies aimed at increasing energy expenditure. As a result, brown adipose tissue (BAT) is receiving tremendous attention since the major function of BAT is to dissipate energy as heat. For example, mouse models that have increased BAT activity or increased numbers of brown‐like adipocytes within the white adipose tissue (WAT) are lean and protected from obesity. Alternatively, mouse models that lack BAT activity are more susceptible to age and diet‐induced obesity. However, a significant loss of BAT mass during the natural growth process in humans has created enormous challenges in effectively utilizing this tissue to increase energy expenditure. New strategies are primarily focused on expanding the BAT mass and/or activating the existing BAT. In this regard, recent finding that expression of early B cell factor‐2 (Ebf2) reprograms the white pre‐adipocytes into brown adipocytes is a significant break‐through in developing BAT‐mediated strategies to treat obesity. Here we review the major biological functions of WAT and BAT, which play critical but opposing roles in the energy spectrum, energy storage versus energy expenditure, and we evaluate whether activation and/or expansion of BAT is practically achievable to treat obesity in humans. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-11T05:47:58.157163-05:
      DOI: 10.1002/jcp.24986
       
  • Gap Junctions Enhance the Antiproliferative Effect of
           MicroRNA‐124‐3p in Glioblastoma Cells
    • Authors: Suzhi Zhang; Qin Wang, Lucy Liu, Xiaoting Hong, Yuan Zhang, Liang Tao
      Abstract: MicroRNA (miRNA) holds promise as a novel therapeutic tool for cancer treatment. However, the transfection efficiency of current delivery systems represents a bottleneck for clinical applications. Here, we demonstrate that gap junctions mediate an augmentative effect on the antiproliferation mediated by miR‐124‐3p in U87 and C6 glioblastoma cells. The functional inhibition of gap junctions using either siRNA or pharmacological inhibition eliminated the miR‐124‐3p‐mediated antiproliferation, whereas the enhancement of gap junctions with retinoic acid treatment augmented this miR‐124‐3p‐mediated antiproliferation. A similar effect was observed in glioblastoma xenograft models. More importantly, patch clamp and co‐culture assays demonstrated the transmission of miR‐124‐3p through gap junction channels into adjacent cells. In further exploring the impact of gap junction‐mediated transport of miR‐124‐3p on miR‐124‐3p target pathways, we found that miR‐124‐3p inhibited glioblastoma cell growth in part by decreasing the protein expression of cyclin‐dependent kinase 6, leading to cell cycle arrest at the G0/G1 phase; moreover, pharmacological regulation of gap junctions affected this cell cycle arrest. In conclusion, our results indicate that the “bystander” effects of functional gap junctions composed of connexin 43 enhance the antitumor effect of miR‐124‐3p in glioblastoma cells by transferring miR‐124‐3p to adjacent cells, thereby enhancing G0/G1 cell cycle arrest. These observations provide a new guiding strategy for the clinical application of miRNA therapy in tumor treatment. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-09T05:53:44.889884-05:
      DOI: 10.1002/jcp.24982
       
  • Cross‐Talk Between VEGF and BMP‐6 Pathways Accelerates
           Osteogenic Differentiation of Human Adipose‐Derived Stem Cells
    • Authors: Ching‐Ju Li; Vedavathi Madhu, Gary Balian, Abhijit S. Dighe, Quanjun Cui
      Abstract: Deficiency in vascular endothelial growth factor (VEGF) or bone morphogenetic proteins (BMPs) results in fracture non‐unions. Therefore, it is indispensable to comprehend the combined effect of VEGF and BMPs on the osteogenic differentiation of osteoprogenitor mesenchymal stem cells (MSCs) that are either naturally occurring at the fracture repair site or exogenously added to enhance the bone repair. We found that combination of VEGF and BMP‐6 enhanced COL1A2 expression, which, correlated with up‐regulated expression of osterix, Dlx5 and Msx2 in human adipose derived stem cells (hADSCs). Cross‐talk between VEGF and BMP‐6 pathways up‐regulated activation of p38 mitogen activated kinase (p38 MAPK) and inhibited activation of protein kinase B (PKB, also known as Akt) while phosphorylation of ‘mothers against decapentaplegic’ homologs 1/5/8 (Smads 1/5/8) and extracellular signal‐regulated kinases 1 and 2 (ERK 1/2) were not affected. Consistent with these findings, p38 inhibitor SB203580, or siRNA knock‐down of osterix, abrogated cross‐talk between the VEGF and BMP‐6 pathways and significantly reduced the observed up‐regulation of COL1A2. Nuclear translocation of the phosphorylated form of osterix was also inhibited by SB203580. While cross‐talk between the VEGF‐BMP‐6 pathways did not show an effect on the extent of mineralization, inhibition of any one of the three components that were up‐regulated through the cross‐talk i.e. osterix, Dlx5 and p38 activation, led to a complete inhibition of mineralization. Inhibition of PKB/Akt activation which is attenuated through the cross‐talk, significantly enhanced ALP gene expression. These observations imply that cross‐talk between the VEGF and BMP‐6 signaling pathways enhances osteogenic differentiation of MSCs. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-09T05:53:26.247591-05:
      DOI: 10.1002/jcp.24983
       
  • Inhibition of PKC‐induced COX‐2 and IL‐8 Expression in
           Human Breast Cancer Cells by Glucosamine
    • Authors: Wan‐Yu Chou; Kun‐Han Chuang, David Sun, Yu‐Hsiu Lee, Pu‐Hong Kao, Yen‐Yu Lin, Hsei‐Wei Wang, Yuh‐Lin Wu
      Abstract: Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase‐2 (COX‐2) and interleukin‐8 (IL‐8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti‐inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC‐regulation of COX‐2/IL‐8 expression and the associated impact on breast cancer. In MCF‐7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX‐2 and IL‐8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E2 (PGE2) and IL‐8. Glucosamine is able to promote COX‐2 protein degradation in a calpain‐dependent manner and IL‐8 protein degradation in calpain‐dependent and proteasome‐dependent manners. The MAPK and NF‐κB pathways are involved in PKC‐induced COX‐2 expression, but only the NF‐κB pathway is involved in PKC‐induced IL‐8 expression. Glucosamine attenuates PKC‐mediated IκBα phosphorylation, nuclear NF‐κB translocation, and NF‐κB reporter activation. Both PGE2 and IL‐8 promote cell proliferation and IL‐8 induces cell migration; thus glucosamine appears to suppress PKC‐induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX‐2 and IL‐8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF‐κB signaling pathway, resulting in an inhibition of breast cancer cell growth. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-09T00:21:52.1074-05:00
      DOI: 10.1002/jcp.24955
       
  • Oral squamous cancer cell exploits hnRNP A1 to regulate cell cycle and
           proliferation
    • Authors: Cheng Yu; Jihua Guo, Yu Liu, Jun Jia, Rong Jia, Mingwen Fan
      Abstract: Oral squamous cell carcinoma (OSCC) is a common human malignant tumor with high mortality. So far, the molecular pathogenesis of OSCC remains largely unclear. Heterogeneous nuclear ribonucleoprotein (HnRNP) A1 is an important multi‐function splicing factor and closely related to tumorigenesis. HnRNP A1 is overexpressed in various tumors, and promotes aerobic glycolysis and elongation of telomere, but the function of hnRNP A1 in cell cycle and proliferation remains unclear. We found that hnRNP A1 was overexpressed in OSCC tissues, and was required for the growth of OSCC cells. Moreover, hnRNP A1 was highly expressed in the G2/M cell cycle phase. Knockdown of hnRNP A1 induced G2/M arrest. DNA microarray assay result showed that hnRNP A1 regulated the expression of a number of target genes associated with G2/M phase. Moreover, hnRNP A1 controlled the alternative splicing of CDK2 exon 5. These findings suggested that hnRNP A1 plays key roles in the regulation of cell cycle progression and pathogenesis of OSCC. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-09T00:21:11.929479-05:
      DOI: 10.1002/jcp.24956
       
  • β‐arrestin2 contributes to cell viability and proliferation via
           the down‐regulation of FOXO1 in castration‐resistant prostate
           cancer
    • Authors: Xiaolu Duan; Zhenzhen Kong, Yang Liu, Zhiwen Zeng, Shujue Li, Wenqi Wu, Weidong Ji, Bicheng Yang, Zhijian Zhao, Guohua Zeng
      Abstract: β‐arrestin2 has been identified to act as a corepressor of androgen receptor (AR) signaling by binding to AR and serving as a scaffold to affect the activity and expression of AR in androgen‐dependent prostate cancer cells; however, little is known regarding its role in castration‐resistant prostate cancer (CRPC) progression. Here, our data demonstrated that β‐arrestin2 contributes to the cell viability and proliferation of CRPC via the down‐regulation of FOXO1 activity and expression. Mechanistically, in addition to its requirement for FOXO1 phosphorylation induced by IGF‐1, β‐arrestin2 could inhibit FOXO1 activity in an Akt‐independent manner and delay FOXO1 dephosphorylation through the inhibition of PP2A phosphatase activity and the attenuation of the interaction between FOXO1 and PP2A. Furthermore, β‐arrestin2 could down‐regulate FOXO1 expression via ubiquitylation and proteasomal degradation. Together, our results identified a novel role for β‐arrestin2 in the modulation of the CRPC progress through FOXO1. Thus, the characterization of β‐arrestin2 may represent an alternative therapeutic target for CRPC treatment. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-09T00:20:38.20275-05:0
      DOI: 10.1002/jcp.24963
       
  • p14ARF Prevents Proliferation of Aneuploid Cells by Inducing
           p53‐Dependent Apoptosis
    • Authors: Lorena Veneziano; Viviana Barra, Laura Lentini, Sergio Spatafora, Aldo Di Leonardo
      Abstract: Weakening the Spindle Assembly Checkpoint by reduced expression of its components induces chromosome instability and aneuploidy that are hallmarks of cancer cells. The tumor suppressor p14ARF is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14ARF is involved in the maintenance of aneuploid cells in primary human cells suggesting that it could be part of a pathway controlling their proliferation. To investigate further this aspect, p14ARF was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. p14ARF re‐expression reduced the number of aneuploid cells in MAD2 post‐transcriptionally silenced cells. Also aberrant mitoses, frequently displayed in MAD2‐depleted cells, were decreased when p14ARF was expressed at the same time. In addition p14ARF ectopic expression in MAD2‐depleted cells induced apoptosis associated with increased p53 protein levels. Conversely, p14ARF ectopic expression did not induce apoptosis in HCT116 p53KO cells. Collectively, our results suggest that the tumor suppressor p14ARF may have an important role in counteracting proliferation of aneuploid cells by activating p53‐dependent apoptosis. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-08T22:21:10.033109-05:
      DOI: 10.1002/jcp.24976
       
  • Paracrine Factors Secreted by MSCs Promote Astrocyte Survival Associated
           with GFAP Downregulation After Ischemic Stroke Via p38 MAPK and JNK
    • Authors: Weiyi Huang; Bingke Lv, Huijun Zeng, Dandan Shi, Yi Liu, Fanfan Chen, Feng Li, Xinghui Liu, Rong Zhu, Lei Yu, Xiaodan Jiang
      Abstract: Astrocytes are critical for ischemic stroke, and understanding their role in mesenchymal stem cell (MSC)‐mediated protection against ischemic injury is important. The paracrine capacity of MSCs has been proposed as the principal mechanism contributing to the protection and repair of brain tissue. In the present study, an in vitro oxygen–glucose deprivation (OGD) model was used to mimic ischemic injury. OGD‐induced astrocytes were reperfused with MSC‐conditioned medium (MSC‐CM) or co‐cultured with MSCs for 24 h to create an environment abundant in paracrine factors. The results indicated that both situations could protect astrocytes from apoptosis, increase cell metabolic activity, and reduce glial fibrillary acidic protein (GFAP) overexpression; however, the effects of co‐culturing with MSCs were more positive. Paracrine factors suppressed the activation of p38 MAPK, JNK, and their downstream targets p53 and STAT1. Inhibition of p38 MAPK, JNK, p53 and STAT1 attenuated astrocyte injury and/or GFAP upregulation. Activation of p38 MAPK and JNK suppressed the beneficial effects of paracrine factors, resulting in decreased survival and GFAP overexpression. These results suggest that paracrine factors inhibit p38 MAPK and JNK, and most likely by regulating their downstream targets, p53 and STAT1, to promote astrocyte survival associated with GFAP downregulation after ischemic stroke in vitro. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-05T06:59:39.56217-05:0
      DOI: 10.1002/jcp.24981
       
  • Roles for Histone Acetylation in Regulation of Telomere Elongation and
           Two‐Cell State in Mouse ES Cells
    • Authors: Jiameng Dan; Jiao Yang, Yifei Liu, Andrew Xiao, Lin Liu
      Abstract: Mammalian telomeres and subtelomeres are marked by heterochromatic epigenetic modifications, including repressive DNA methylation and histone methylation (e.g. H3K9me3 and H4K20me3). Loss of these epigenetic marks results in increased rates of telomere recombination and elongation. Other than these repressive epigenetic marks, telomeric and subtelomeric H3 and H4 are underacetylated. Yet, whether histone acetylation also regulates telomere length has not been directly addressed. We thought to test the effects of histone acetylation levels on telomere length using histone deacetylase (HDAC) inhibitor (sodium butyrate, NaB) that mediates histone hyperacetylation and histone acetyltransferase (HAT) inhibitor (C646) that mediates histone hypoacetylation. We show that histone hyperacetylation dramatically elongates telomeres in wild‐type ES cells, and only slightly elongates telomeres in Terc‐/‐ ES cells, suggesting that Terc is involved in histone acetylation‐induced telomere elongation. In contrast, histone hypoacetylation shortens telomeres in both wild‐type and Terc‐/‐ ES cells. Additionally, histone hyperacetylation activates 2‐cell (2C) specific genes including Zscan4, which is involved in telomere recombination and elongation, whereas histone hypoacetylation represses Zscan4 and 2C genes. These data suggest that histone acetylation levels affect the heterochromatic state at telomeres and subtelomeres, and regulate gene expression at subtelomeres, linking histone acetylation to telomere length maintenance. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-05T06:38:35.334387-05:
      DOI: 10.1002/jcp.24980
       
  • Phosphorylation of Runx2, Induced by Cyclic Mechanical Tension Via ERK1/2
           Pathway, Contributes to Osteodifferentiation of Human Periodontal Ligament
           Fibroblasts
    • Authors: Dapeng Ren; Chunling Wang, Xiao Yuan, Fulan Wei, Lihua Hu, Shuangyan Yang
      Abstract: Occlusal force is an important stimulus for maintaining periodontal homeostasis. This is attributed to the quality of human periodontal ligament fibroblasts (hPDLFs) that could transfer occlusal force into biological signals modulating osteoblst differentiation. However, few studies investigated the mechanism of occlusal force‐induced osteodifferentiation of hPDLFs. In our study, we used the cyclic mechanical tension (CMT) at 10% elongation with 0.5HZ to mimic occlusal force, and explored its effects on osteogenesis of hPDLFs. Firstly, elevated expressions of several osteoblast marker genes (Runx2, ATF4, SP7, OCN and BSP), as well as activated ERK1/2 pathway were detected during CMT loading for 1h, 3h, 6h, 12h, 18h and 24h. To gain further insight into how CMT contributed to those effects, we focused on the classic ERK1/2‐Runx2 pathway by inhibiting ERK1/2 and overexpressing Runx2. Our results reflected that Runx2 overexpression alone could induce osteodifferentiation of hPDLFs. Meanwhile, CMT loading could intensify while combined ERK1/2 blockage could weaken this process. Furthermore, we found that CMT promoted Runx2 transcription and phosphorylation via ERK1/2; protein level of phospho‐Runx2 (p‐Runx2), rather than Runx2, was in parallel with mRNA expressions of SP7, OCN and BSP. Taken together, our study proved that p‐Runx2, elevated by CMT via ERK1/2 pathway, is the predominate factor in promoting osteoblast differentiation of hPDLFs. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-05T00:34:02.893174-05:
      DOI: 10.1002/jcp.24972
       
  • EGF Suppresses the Initiation and Drives the Reversion of
           TGF‐β1‐Induced Transition in Hepatic Oval Cells Showing
           the Plasticity of Progenitor Cells
    • Authors: Ping Wang; Ai‐Ting Yang, Min Cong, Tian‐Hui Liu, Dong Zhang, Jian Huang, Xiao‐Fei Tong, Sheng‐Tao Zhu, Yong Xu, Shu‐Zhen Tang, Bao‐En Wang, Hong Ma, Ji‐Dong Jia, Hong You
      Abstract: Transforming growth factor‐β1 (TGF‐β1) induces hepatic progenitors to tumor initiating cells through epithelial‐mesenchymal transition (EMT), thus raising an important drawback for stem cell‐based therapy. How to block and reverse TGF‐β1‐induced transition is crucial for progenitors' clinical application and carcinogenic prevention. Rat adult hepatic progenitors, hepatic oval cells, experienced E‐cadherin to N‐cadherin switch and changed to α‐smooth muscle actin (α‐SMA) positive cells after TGF‐β1 incubation, indicating EMT. When TGF‐β1 plus EGF were co‐administrated to these cells, EGF dose‐dependently suppressed the cadherin switch and α‐SMA expression. Interestingly, if EGF was applied to TGF‐β1‐pretreated cells, the cells that have experienced EMT could return to their epithelial phenotype. Abruption of EGF receptor revealed that EGF exerts its blockage and reversal effects through phosphorylation of ERK1/2 and Akt. These findings suggest an important attribute of EGF on opposing and reversing TGF‐β1 effects, indicating the plasticity of hepatic progenitors. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-05T00:23:08.846357-05:
      DOI: 10.1002/jcp.24962
       
  • Antidepressant effects of exercise: a role for the
           adiponectin‐PGC‐1α‐kynurenine triad'
    • Authors: Fabian Sanchis‐Gomar; Carme Pérez Quilis, Alejandro Lucia
      Abstract: It is well‐recognized that exercise improves mental health, e.g., by decreasing depressive behaviors, improving hippocampal‐dependent learning and neurogenesis, and increasing dendritic plasticity. Yet how exercise influences the brain at the molecular level is not clearly understood. Yau et al recently reported that the antidepressant effects of physical exercise are mainly mediated by adiponectin, an adipocyte‐secreted hormone (‘adipocytokine’) with neuroprotective effects at the central nervous system level (Yau et al., 2014). This article is protected by copyright. All rights reserved
      PubDate: 2015-03-04T13:27:20.841098-05:
      DOI: 10.1002/jcp.24964
       
  • Genetic architecture of sexual dimorphism in humans
    • Authors: Nichole Rigby; Rob J. Kulathinal
      Abstract: Males and females differ across a broad spectrum of morphological, physiological, and behavioral characters. In fact, sexually dimorphic traits typically contribute the largest component of phenotypic variance in most taxa that use sex to reproduce. However, we know very little about the mechanisms that maintain these dimorphic states and how these sexually dimorphic traits evolve. Here, we review our current knowledge of the underlying genetic basis of sexual dimorphism in humans. First, we briefly review the etiology of sex differences starting from sex determination's initial switch early in embryogenesis. We then survey recent sex‐biased transcriptomic expression literature in order to provide additional insight into the landscape of sex‐biased gene expression in both gonadal and non‐gonadal tissues: from overall prevalence to tissue specificity to conservation across species. Finally, we discuss implications of sex‐biased genetic architecture to human health and disease in light of the National Institute of Health's recently proposed initiative to promote study samples from both sexes. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-04T13:26:26.847538-05:
      DOI: 10.1002/jcp.24979
       
  • OSU‐03012 and Viagra Treatment Inhibits the Activity of Multiple
           Chaperone Proteins and Disrupts the Blood Brain Barrier: Implications for
           Anti‐Cancer Therapies
    • Authors: Laurence Booth; Jane L. Roberts, Mehrad Tavallai, Aida Nourbakhsh, John Chuckalovcak, Jori Carter, Andrew Poklepovic, Paul Dent
      Abstract: We examined the interaction between OSU‐03012 (also called AR‐12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone GRP78/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU‐03012 to kill stem‐like GBM cells. Treatment of cells with OSU‐03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK‐eIF2α‐ATF4‐CHOP signaling and was blocked by GRP78 over‐expression. In vivo OSU‐03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU‐03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over‐expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU‐03012/sildenafil treated mouse we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU‐03012/sildenafil lethality. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-03T18:29:32.931709-05:
      DOI: 10.1002/jcp.24977
       
  • In Vitro Evaluation of Different Methods of Handling Human Liposuction
           Aspirate and Their Effect on Adipocytes and Adipose Derived Stem Cells
    • Authors: Paola Palumbo; Gianfranca Miconi, Benedetta Cinque, Cristina La Torre, Francesca Lombardi, Giovanni Zoccali, Gino Orsini, Pietro Leocata, Maurizio Giuliani, Maria Grazia Cifone
      Abstract: Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue‐derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10‐20‐30 min) with the centrifugation technique at different speeds (90‐400‐1500xg). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, “middle layer” and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105 + , CD90 + , CD45‐) were analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500xg. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400xg are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400xg centrifugation could be the recommended method when the necessary instrumentation is available. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-03T18:29:16.641805-05:
      DOI: 10.1002/jcp.24965
       
  • Transcription Factor Tfe3 Directly Regulates Pgc‐1Alpha in Muscle
    • Authors: Nunciada Salma; Jun S. Song, Zolt Arany, David E. Fisher
      Abstract: The microphthalmia (MiT) family of transcription factors is an important mediator of metabolism. Family members Mitf and Tfeb directly regulate the expression of the master regulator of metabolism, peroxisome‐proliferator activated receptor gamma coactivator‐1 alpha (Pgc‐1alpha), in melanomas and in the liver, respectively. Pgc‐1alpha is enriched in tissues with high oxidative capacity and plays an important role in the regulation of mitochondrial biogenesis and cellular metabolism. In skeletal muscle, Pgc‐1alpha affects many aspects of muscle functionally such as endurance, fiber‐type switching and insulin sensitivity. Tfe3 also regulates muscle metabolic genes that enhance insulin sensitivity in skeletal muscle. Tfe3 has not yet been shown to regulate Pgc‐1alpha expression. Our results reported here show that Tfe3 directly regulates Pgc‐1alpha expression in myotubes. Tfe3 ectopic expression induces Pgc‐1alpha, and Tfe3 silencing suppresses Pgc‐1alpha expression. This regulation is direct, as shown by Tfe3's binding to E‐boxes on the Pgc‐1alpha proximal promoter. We conclude that Tfe3 is a critical transcription factor that regulates Pgc‐1alpha gene expression in myotubes. Since Pgc‐1alpha coactivates numerous biological programs in diverse tissues, the regulation of its expression by upstream transcription factors such Tfe3 implies potential opportunities for the treatment of diseases where modulation of Pgc‐1alpha expression may have important clinical outcomes. This article is protected by copyright. All rights reserved
      PubDate: 2015-03-03T18:29:00.613669-05:
      DOI: 10.1002/jcp.24978
       
  • A SMYD3 Small‐Molecule Inhibitor Impairing Cancer Cell Growth
    • Authors: Alessia Peserico; Aldo Germani, Paola Sanese, Armenio Jourge Barbosa, Valeria Di Virgilio, Raffaella Fittipaldi, Edoardo Fabini, Carlo Bertucci, Greta Varchi, Mary Pat Moyer, Giuseppina Caretti, Alberto Del Rio, Cristiano Simone
      Abstract: SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi‐mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI‐121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 levels. Of note, the extent of cell growth inhibition by BCI‐121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-28T06:02:14.012173-05:
      DOI: 10.1002/jcp.24975
       
  • In vivo Sodium Tungstate Treatment Prevents E‐Cadherin Loss Induced
           by Diabetic Serum in HK‐2 Cell Line
    • Authors: Romina Bertinat; Pamela Silva, Elizabeth Mann, Xuhang Li, Francisco Nualart, Alejandro J. Yáñez
      Abstract: Diabetic nephropathy (DN) is characterized by interstitial inflammation and fibrosis, which is the result of chronic accumulation of extracellular matrix produced by activated fibroblasts in the renal tubulointerstitium. Renal proximal tubular epithelial cells (PTECs), through the process of epithelial‐to‐mesenchymal transition (EMT), are the source of fibroblasts within the interstitial space, and loss of E‐cadherin has shown to be one of the earliest steps in this event. Here, we studied the effect of the anti‐diabetic agent sodium tungstate (NaW) in the loss of E‐cadherin induced by transforming growth factor (TGF) β‐1, the best‐characterized in vitro EMT promoter, and serum from untreated or NaW‐treated diabetic rats in HK‐2 cell line, a model of human kidney PTEC. Our results showed that both TGFβ‐1 and serum from diabetic rat induced a similar reduction in E‐cadherin expression. However, E‐cadherin loss induced by TGFβ‐1 was not reversed by NaW, whereas sera from NaW‐treated rats were able to protect HK‐2 cells. Searching for soluble mediators of NaW effect, we compared secretion of TGFβ isoforms and vascular endothelial growth factor (VEGF)‐A, which have opposite actions on EMT. 1mM NaW alone reduced secretion of both TGFβ‐1 and ‐2, and stimulated secretion of VEGF‐A after 48 h. However, these patterns of secretion were not observed after diabetic rat serum treatment, suggesting that protection from E‐cadherin loss by serum from NaW‐treated diabetic rats originates from an indirect rather than a direct effect of this salt on HK‐2 cells, via a mechanism independent of TGFβ and VEGF‐A functions. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-28T06:01:42.497004-05:
      DOI: 10.1002/jcp.24974
       
  • A New Medical Device Rigeneracons Allows to Obtain Viable
           Micro‐Grafts from Mechanical Disaggregation of Human Tissues
    • Authors: Letizia Trovato; Manuela Monti, Claudia del Fante, Marila Cervio, Milla Lampinen, Lucia Ambrosio, Carlo Alberto Redi, Cesare Perotti, Esko Kankuri, Gennaro Ambrosio, Ruggero Rodriguez y Baena, Giuseppe Pirozzi, Antonio Graziano
      Abstract: Autologous graft is considered the gold standard of graft materials, however, this approach is still limited due to both small amount of tissue that can be collected and to reduced cell viability of cells that can be obtained. The aim of this preliminary study was to demonstrate the efficacy of an innovative medical device called Rigeneracons® (CE certified Class I) to provide autologous micro‐grafts immediately available to be used in the clinical practice. Moreover, Rigeneracons® is an instrument able to create micro‐grafts enriched of progenitors cells which maintain their regenerative and differentiation potential. We reported preliminary data about viability cell of samples derived from different kind of human tissues, such as periosteum, cardiac atrial appendage biopsy and lateral rectus muscle of eyeball and disaggregated by Rigeneracons®. In all cases we observed that micro‐grafts obtained by Rigeneracons® displayed high cell viability. Furthermore, by cell characterization of periosteum samples, we also evidenced an high positivity to mesenchymal cell markers, suggesting an optimal regenerative potential. This article is protected by copyright. All rights reserved
      PubDate: 2015-02-28T06:01:27.847462-05:
      DOI: 10.1002/jcp.24973
       
  • Quantifying Heterogeneity and Dynamics of Clonal Fitness in Response to
           Perturbation
    • Authors: Peter Frick; Bishal Paudel, Darren Tyson, Vito Quaranta
      First page: 1403
      Abstract: The heterogeneous dynamics of clonal lineages within a cell population, in aggregate, shape both normal and pathological biological processes. Studies of clonality typically relate the fitness of clones to their relative abundance, thus requiring long‐term experiments and limiting conclusions about the heterogeneity of clonal fitness in response to perturbation. We present, for the first time, a method that enables a dynamic, global picture of clonal fitness within a mammalian cell population. This novel assay allows facile comparison of the structure of clonal fitness in a cell population across many perturbations. By utilizing high‐throughput imaging, our methodology provides ample statistical power to define clonal fitness dynamically and to visualize the structure of perturbation‐induced clonal fitness within a cell population. We envision that this technique will be a powerful tool to investigate heterogeneity in biological processes involving cell proliferation, including development and drug response. This article is protected by copyright. All rights reserved
      PubDate: 2015-01-20T06:26:35.650196-05:
      DOI: 10.1002/jcp.24888
       
  • Role of the unfolded protein response, GRP78 and GRP94 in organ
           homeostasis
    • Authors: Genyuan Zhu; Amy S. Lee
      First page: 1413
      Abstract: The endoplasmic reticulum (ER) is a cellular organelle where secretory and membrane proteins, as well as lipids, are synthesized and modified. When cells are subjected to ER stress, an adaptive mechanism referred to as the Unfolded Protein Response (UPR) is triggered to allow the cells to restore homeostasis. Evidence has accumulated that the UPR pathways provide specialized and unique roles in diverse development and metabolic processes. The glucose regulated proteins (GRPs) are traditionally regarded as ER proteins with chaperone and calcium binding properties. The GRPs are constitutively expressed at basal levels in all organs, and as stress‐inducible ER chaperones, they are major players in protein folding, assembly and degradation. This conventional concept is augmented by recent discoveries that GRPs can be actively translocated to other cellular locations such as the cell surface, where they assume novel functions that regulate signaling, proliferation, apoptosis and immunity. Recent construction and characterization of mouse models where the gene encoding for the UPR components and the GRPs is genetically altered provide new insights on the physiological contribution of these proteins in vivo. This review highlights recent progress towards the understanding of the role of the UPR and two major GRPs (GRP78 and GRP94) in regulating homeostasis of organs arising from the endoderm, mesoderm and ectoderm. GRP78 and GRP94 exhibit shared and unique functions, and in specific organs their depletion elicits adaptive responses with physiological consequences. J. Cell. Physiol. 230: 1413–1420, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:13.215307-05:
      DOI: 10.1002/jcp.24923
       
  • A hyaluronic acid‐based compound inhibits fibroblast senescence
           induced by oxidative stress in vitro and prevents oral mucositis in vivo
    • Authors: Nicola Cirillo; Antonio Vicidomini, Michael McCullough, Antonio Gambardella, Yazan Hassona, Stephen S. Prime, Giuseppe Colella
      First page: 1421
      Abstract: Virtually all patients receiving radio‐ and chemotherapy for cancer develop oral mucositis, a severe and highly debilitating condition. The onset of mucositis is thought to involve the production of reactive oxygen species (ROS) in the submucosa. Here we investigated a possible protective effect of a commercial formulation of hyaluronic acid (HA) enriched with amino acids (Mucosamin®) against the damage induced by oxidative stress both in vitro and in vivo. Transient exposure of normal human oral fibroblasts to hydrogen peroxide (H2O2) led to irreversible senescence, as demonstrated by sustained increase in the levels of p16INK4A and SA‐βGal. Conditioned media from senescent fibroblasts induced detrimental effects on keratinocytes, as shown by reduced metabolic activity and migration capability. Pre‐treatment with Mucosamin® prevented H2O2‐induced, but not TGF‐β‐induced, fibroblast senescence with a concomitant reduction of fibroblast‐induced loss of keratinocyte vitality and functional activity. Finally, data from a case‐series of patients undergoing radio/chemotherapy strongly suggested that prophylactic use of the hyaluronic acid‐based compound in the form of a spray may be effective in preventing the onset of oral mucositis. J. Cell. Physiol. 230: 1421–1429, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:23.709299-05:
      DOI: 10.1002/jcp.24908
       
  • Interleukin‐17 induces CC chemokine receptor 6 expression and cell
           migration in colorectal cancer cells
    • Authors: Chih‐Chien Chin; Cheng‐Nan Chen, Hsing‐Chun Kuo, Chung‐Sheng Shi, Meng Chiao Hsieh, Yi‐Hung Kuo, Shui‐Yi Tung, Kam‐Fai Lee, Wen‐Shih Huang
      First page: 1430
      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. 230: 1430–1437, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:15.877763-05:
      DOI: 10.1002/jcp.24796
       
  • The anti‐spasticity drug baclofen alleviates collagen‐induced
           arthritis and regulates dendritic cells
    • Authors: Shichao Huang; Jianxin Mao, Bin Wei, Gang Pei
      First page: 1438
      Abstract: Baclofen is used clinically as a drug that treats spasticity, which is a syndrome characterized by excessive contraction of the muscles and hyperflexia in the central nervous system (CNS), by activating GABAB receptors (GABABRs). Baclofen was recently reported to desensitize chemokine receptors and to suppress inflammation through the activation of GABABRs. GABABRs are expressed in various immune cells, but the functions of these receptors in autoimmune diseases remain largely unknown. In this study, we investigated the effects of baclofen in murine collagen‐induced arthritis (CIA). Oral administration of baclofen alleviated the clinical development of CIA, with a reduced number of IL‐17‐producing T helper 17 (TH17) cells. In addition, baclofen treatment suppressed dendritic cell (DC)‐primed TH17 cell differentiation by reducing the production of IL‐6 by DCs in vitro. Furthermore, the pharmacological and genetic blockade of GABABRs in DCs weakened the effects of baclofen, indicating that GABABRs are the molecular targets of baclofen on DCs. Thus, our findings revealed a potential role for baclofen in the treatment of CIA, as well as a previously unknown signaling pathway that regulates DC function. J. Cell. Physiol. 230: 1438–1447, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:20.043311-05:
      DOI: 10.1002/jcp.24884
       
  • FADD phosphorylation impaired islet morphology and function
    • Authors: Chun Yao; Hongqin Zhuang, Wei Cheng, Yan Lin, Pan Du, Bingya Yang, Xiaofeng Huang, Sheng Chen, Qingang Hu, Zi‐Chun Hua
      First page: 1448
      Abstract: Previous studies have indicated that Fas‐FasL pathway and its downstream caspase‐8 can regulate islet mass and insulin secretion. As a classical adaptor in Fas‐FasL signaling, Fas‐associated death domain‐containing protein (FADD) takes part in many non‐apoptosis processes regulated by its phosphorylation. However, its role in islets has not been evaluated to date. Here, through comparative proteomics and bioinformatic analysis on FADD phosphorylated (FADD‐D) and wild‐type (WT) MEFs, we found three proteins involved in islet differentiation and function were dysregulated due to FADD phosphorylation. The mouse model of FADD‐D, which mimics constitutive phosphorylated FADD expression in mice, was further analyzed to address this issue. We confirmed the proteomic results by immunohistological analyses on pancreatic islets. In addition, we found that FADD‐D mice displayed decreased islet area, and the glucose stimulated insulin secretion (GSIS) of FADD‐D islets was impaired. These data suggest a novel role of FADD in islet development and insulin secretion. J. Cell. Physiol. 230: 1448–1456, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:15.310152-05:
      DOI: 10.1002/jcp.24885
       
  • Generation of tumor‐specific cytotoxic T‐lymphocytes from the
           peripheral blood of colorectal cancer patients for adoptive T‐cell
           transfer
    • Authors: Silvia Carluccio; Serena Delbue, Lucia Signorini, Elisabetta Setola, Anna Bagliani, Alberto Della Valle, Andrea Galli, Pasquale Ferrante, Marco Bregni
      First page: 1457
      Abstract: This study designs a strategy for an adoptive cellular therapy (ACT) protocol based on the ex‐vivo selection of autologous peripheral blood‐derived CD8‐enriched T‐cells, stimulated with dendritic cells (DCs) that had been pulsed with apoptotic tumor cells to generate cytotoxic T lymphocytes (CTLs) with anti‐tumor activity. Seventy‐eight colorectal cancer (CRC) patients were enrolled in this study. Tumor tissues and peripheral blood (PB) were obtained at surgery. Tissues were mechanically dissociated and cultured to obtain a primary tumor cell line from each patient. DCs were derived from peripheral blood mononuclear cells (PBMCs) using magnetic positive selection of CD14+ monocytes. Anti‐tumor CTLs were elicited in co‐/micro‐cultures using DCs as antigen‐presenting cells, autologous apoptotic tumor cells as a source of antigens, and CD8+ T lymphocytes as effectors. Interferon‐γ (IFN‐γ) secretion was assessed by ELISpot assays to evaluate the activation of the CTLs against the autologous tumor cells. Primary tumor cell lines were obtained from 20 of 78 patients (25.6%). DCs were generated from 26 patients, and of them, corresponding tumor cell lines were derived from six patients. ELISpot results showed that significant IFN‐γ secretion was detected after different numbers of stimulations for two patients, whereas weak secretion was observed for three patients. Despite difficulties due to contamination of several primary tumor cell lines with gut intestinal flora, the results suggest that the generation of tumor‐specific CTLs is feasible from patients with CRC, and could be useful for supporting an ACT approach in CRC. J. Cell. Physiol. 230: 1457–1465, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:14.568188-05:
      DOI: 10.1002/jcp.24886
       
  • Angiotensin receptor I stimulates osteoprogenitor proliferation through
           TGFβ‐mediated signaling
    • Authors: Francesca Querques; Bruno Cantilena, Carmine Cozzolino, Maria Teresa Esposito, Fabiana Passaro, Silvia Parisi, Barbara Lombardo, Tommaso Russo, Lucio Pastore
      First page: 1466
      Abstract: Clinical studies of large human populations and pharmacological interventions in rodent models have recently suggested that anti‐hypertensive drugs that target angiotensin II (Ang II) activity may also reduce loss of bone mineral density. Here, we identified in a genetic screening the Ang II type I receptor (AT1R) as a potential determinant of osteogenic differentiation and, implicitly, bone formation. Silencing of AT1R expression by RNA interference severely impaired the maturation of a multipotent mesenchymal cell line (W20–17) along the osteoblastic lineage. The same effect was also observed after the addition of the AT1R antagonist losartan but not the AT2R inhibitor PD123,319. Additional cell culture assays traced the time of greatest losartan action to the early stages of W20–17 differentiation, namely during cell proliferation. Indeed, addition of Ang II increased proliferation of differentiating W20–17 and primary mesenchymal stem cells and this stimulation was reversed by losartan treatment. Cells treated with losartan also displayed an appreciable decrease of activated (phosphorylated)‐Smad2/3 proteins. Moreover, Ang II treatment elevated endogenous transforming growth factor β (TGFβ) expression considerably and in an AT1R‐dependent manner. Finally, exogenous TGFβ was able to restore high proliferative activity to W20–17 cells that were treated with both Ang II and losartan. Collectively, these results suggest a novel mechanism of Ang II action in bone metabolism that is mediated by TGFβ and targets proliferation of osteoblast progenitors. J. Cell. Physiol. 230: 1466–1474, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:24.390181-05:
      DOI: 10.1002/jcp.24887
       
  • GABARAP is a determinant of apoptosis in growth‐arrested chicken
           embryo fibroblasts
    • Authors: Scott Maynard; Romita Ghosh, Ying Wu, Shi Yan, Tetsuaki Miyake, Mark Gagliardi, Karen Rethoret, P‐A. Bédard
      First page: 1475
      Abstract: Nutrient depletion triggers a series of adaptive processes as part of the unfolded protein response or UPR. These processes reduce stress to the endoplasmic reticulum by enhancing its protein folding capacity or ability to promote the degradation of dysfunctional proteins. Failure to restore ER homeostasis causes the activation of lethal pathways. The expression of a dominant negative mutant of C/EBPβ (Δ184‐C/EBPβ) alters this balance in chicken embryo fibroblasts (CEF). As a result, CEF display enhanced survival upon prolonged nutrient depletion. Starved Δ184‐C/EBPβ–expressing CEF display pronounced features of autophagy characterized by the appearance of large vesicles containing amorphous material, the formation of smaller double‐membrane vesicles (autophagosomes) and processing of LC3 and GABARAP. However, there were marked differences in the expression and processing of these proteins. In both normal and Δ184‐C/EBPβ expressing CEF, the lipidated form of LC3 (form II) accumulated during starvation but was detectable even when cells were actively dividing in complete medium. In contrast, GABARAP expression and lipidation were strongly stimulated in response to starvation. Inhibition of LC3 expression by RNA interference led to apoptosis in normal CEF even in the absence of starvation but stable and near complete repression of GABARAP was tolerated. Moreover, the inhibition of GABARAP enhanced CEF survival and abolished the expression of the pro‐apoptotic CHOP factor in conditions of starvation, suggesting a reduced level of ER stress. Therefore, GABARAP is a determinant of apoptosis in CEF subjected to prolonged nutrient depletion. J. Cell. Physiol. 230: 1475–1488, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:19.319957-05:
      DOI: 10.1002/jcp.24889
       
  • 15‐Lipoxygenase Promotes Chronic Hypoxia‐Induced Phenotype
           Changes of PASMCs Via Positive Feedback‐Loop of BMP4
    • Authors: Xiufeng Yu; Liuping Wei, Ping Lu, Tingting Shen, Xia Liu, Tingting Li, Bo Zhang, Hao Yu, Daling Zhu
      First page: 1489
      Abstract: Our laboratory has previously demonstrated that 15‐lipoxygenase (15‐LO)/15‐hydroxyeicosatetr‐aenoic acid (15‐HETE) is involved in hypoxic pulmonary arterial hypertension (PAH). Phenotypical alterations of vascular smooth muscle cells are considered to be an important stage in the development of PAH, whereas the underlying mechanisms and signaling systems are still unclear. Here, we determined the contribution of 15‐LO/15‐HETE signaling in the hypoxia–induced phenotype changes of pulmonary arterial smooth muscle cells (PASMCs). To accomplish this, cellular and molecular changes in pulmonary vascular remodeling were detected in PAH patients and rats exposed to hypoxia. We found that the hypoxia‐induced alterations in PASMCs phenotypes were reversed by the inhibition of 15‐LO/15‐HETE or inhibition of BMP4/BMPRI. Hypoxia‐induced 15‐LO1/2 expression in rat PASMCs was significantly abolished by small interfering RNA targeted at BMP4. Meanwhile, BMP4/BMPRI‐15‐LO/15‐HETE had a positive feedback mechanism. Furthermore, ERK and p38MAPK act as the downstream of the 15‐LO/15‐HETE‐BMP4/BMPRI signaling. Our results suggest that chronic hypoxia promotes phenotypical alterations of PASMCs due to the interaction between 15‐LO/15‐HETE and BMP4/BMPRI. Our study reveals a novel mechanism of hypoxia‐induced pulmonary vascular remodeling and suggested new therapeutic strategies for the targeting of 15‐LO/15‐HETE and BMP4/BMPRI in PAH treatment. J. Cell. Physiol. 230: 1489–1502, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:09.359907-05:
      DOI: 10.1002/jcp.24893
       
  • Intrinsic resistance triggered under acid loading within normal esophageal
           epithelial cells: NHE1‐ and ROS‐mediated survival
    • Authors: Sun Young Park; Yeon Joo Lee, Eun Jeong Cho, Chang Yell Shin, Uy Dong Sohn
      First page: 1503
      Abstract: The transition to a pathological phenotype such as Barrett's esophagus occurs via induction of resistance upon repeated contact with gastric refluxate in esophagus. This study examined the molecular changes within normal esophageal epithelial cells (EECs) under short‐term acid loading and the role of these changes in defensive resistance against acidic cytotoxicity. After primary cultured EECs were exposed to pH 4‐acidified medium (AM4), cell viability was determined by the MTT assay. Reactive oxygen species (ROS) and NAD(P)H oxidase (NOX) activity were measured. Activation of the mitogen‐activated protein kinases (MAPKs) MEK/ERK1/2, p38 and JNK; phosphoinositol‐3‐kinase (PI3K)/Akt, and nuclear factor‐kappa B (NF‐κB) were detected by Western blot analysis or immunofluorescence staining. AM4 incubation induced intracellular ROS generation accompanied by increase in NOX activity, which was further increased by Na+/H+ exchange‐1 (NHE1)‐dependent inhibition but was prevented by inhibition of NOX or mitochondria complex I. AM4 also induced phosphorylation of MEK/ERK1/2, p38 MAPK, PI3K/Akt, and nuclear translocation of NF‐κB, and all these effects, except for p38 MAPK phosphorylation, were abolished by inhibition of ROS. ROS‐dependent PI3K/Akt activation, which mediates NF‐κB nuclear translocation, was inhibited by protein tyrosine kinase (PTK) inhibitors and NHE1‐specific inhibitor. All inhibitors of NHE, ROS, PTK, PI3K, or NF‐κB further decreased AM4‐induced cell viability. Acid loading in the presence of NHE1‐dependent protection induced ROS generation by activating NOX and mitochondria complex I, which stimulated PTK/PI3K/Akt/NF‐κB‐dependent survival in EEC. Our data indicate that normal EEC initially respond to acid loading through intrinsic survival activation. J. Cell. Physiol. 230: 1503–1514, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:12.464056-05:
      DOI: 10.1002/jcp.24896
       
  • Exploration of molecular pathways mediating electric field‐directed
           schwann cell migration by RNA‐seq
    • Authors: Li Yao; Yongchao Li, Jennifer Knapp, Peter Smith
      First page: 1515
      Abstract: In peripheral nervous systems, Schwann cells wrap around axons of motor and sensory neurons to form the myelin sheath. Following spinal cord injury, Schwann cells regenerate and migrate to the lesion and are involved in the spinal cord regeneration process. Transplantation of Schwann cells into injured neural tissue results in enhanced spinal axonal regeneration. Effective directional migration of Schwann cells is critical in the neural regeneration process. In this study, we report that Schwann cells migrate anodally in an applied electric field (EF). The directedness and displacement of anodal migration increased significantly when the strength of the EF increased from 50 mV/mm to 200 mV/mm. The EF did not significantly affect the cell migration speed. To explore the genes and signaling pathways that regulate cell migration in EFs, we performed a comparative analysis of differential gene expression between cells stimulated with an EF (100 mV/mm) and those without using next‐generation RNA sequencing, verified by RT‐qPCR. Based on the cut‐off criteria (FC > 1.2, q 
      PubDate: 2015-03-27T08:38:24.933258-05:
      DOI: 10.1002/jcp.24897
       
  • Biosafety evidence for human dedifferentiated adipocytes
    • Authors: Antonella Poloni; Giulia Maurizi, Domenico Mattiucci, Elena Busilacchi, Stefania Mancini, Giancarlo Discepoli, Augusto Amici, Massimo Falconi, Saverio Cinti, Pietro Leoni
      First page: 1525
      Abstract: Mature adipocytes have shown dynamic plasticity to be converted into fibroblast‐like and lipid‐free cells. After the dedifferentiation process, these cells re‐entered the cell cycle and acquired a high proliferation potential, becoming a valid source of stem cells. However, many aspects of the cellular biosafety about dedifferentiated fat cells remained unclear. This study aimed to elucidate their potential susceptibility to malignant transformation and to ascertain the safety of these cells for clinical use. To evaluate the genomic stability of dedifferentiated adipocytes, telomere length, hTERT gene transcription, the capacity of these cells to grow in an anchorage‐independent manner and the presence of DNA damage by single cell gel electrophoresis assay were studied. Spontaneous chromosomal alterations were excluded by cytogenetic analysis and the expression level of c‐myc and p53, tumor associated genes, were assessed, evaluating also p53 loss of function mutations. Despite the high proliferation capacity of dedifferentiated adipocytes, these cells showed stable telomere length compared with mature adipocytes, no hTERT transcriptions and consequently no telomerase activity, suggesting that both transformation and senescence were avoided. A constant expression level of c‐myc and p53, the inability of dedifferentiated adipocytes to grow in an anchorage‐independent manner, the absence of DNA damage suggested the safety of these cells. Moreover, a normal karyotype was preserved throughout the dedifferentiation process. Data in vivo showed that dedifferentiated adipocytes analyzed for tumorigenicity did not develop tumors. In conclusion, our data indicated that dedifferentiated adipocytes could be a relatively easily accessible resource for cell therapy and regenerative medicine. J. Cell. Physiol. 230: 1525–1533, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:26.295811-05:
      DOI: 10.1002/jcp.24898
       
  • Impaired expression of HIF‐2α induces compensatory expression
           of HIF‐1α for the recovery from anemia
    • Authors: Ikki Tsuboi; Toshiharu Yamashita, Masumi Nagano, Kenichi Kimura, Georgina To'a Salazar, Osamu Ohneda
      First page: 1534
      Abstract: Erythropoiesis is strongly influenced by the interactions between stromal cells and erythroid progenitors, as well as by a key regulatory factor, erythropoietin (EPO). We previously generated mice with a knockdown mutation of Hif‐2α (referred to as kd/kd) and found that these kd/kd mice exhibited normocytic anemia, even though the EPO expression was not severely affected. However, the VCAM‐1 expression in spleen endothelial cells (EC), which is regulated by HIF‐2α, was impaired, resulting in defective erythroid maturation. A deficiency of HIF‐2α clearly led to pancytopenia. However, the critical level of HIF‐2α required for erythropoiesis has not yet been elucidated. In this study, we generated HIF‐2α knockdown/knockout heterozygous mice (kd/null). Strikingly, anemia was observed in the kd/null mice, but the red blood cell indices were significantly improved compared to those of kd/kd mice. In the spleens of kd/null mice, higher HIF‐1α activity and expansion of the red pulp area were observed compared to those of kd/kd mice. Importantly, EC isolated from kd/null spleens showed high expression of VEGF receptors, FLK‐1 and FLT‐1, which are regulated by HIF‐1α instead of HIF‐2α under hypoxic conditions. We also found higher expression of phosphorylated ERK and higher proliferative activity in the EC isolated from kd/null mice compared to those from kd/kd mice. While the HIF‐2α expression was diminished, HIF‐1α bound to the HRE region in the promoters of genes that are normally regulated by HIF‐2α. These results suggest that there is a compensatory pathway involving HIF‐1α that regulates the expression of some HIF‐2α target genes. J. Cell. Physiol. 230: 1534–1548, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:25.664062-05:
      DOI: 10.1002/jcp.24899
       
  • Lipidomic approach towards deciphering anandamide effects in rat decidual
           cell
    • Authors: Marta Almada; M. Rosário Domingues, M. Luísa Dória, Bruno M. Fonseca, Natércia A. Teixeira, Georgina Correia‐da‐Silva
      First page: 1549
      Abstract: Altered phospholipid (PL) metabolism has been associated with pregnancy disorders. Moreover, lipid molecules such as endocannabinoids (eCBs) and prostaglandins (PGs) are important mediators of reproductive events. In humans, abnormal decidualization has been linked with unexplained infertility, miscarriage and endometrial pathologies. Anandamide (AEA), the major eCB, induces apoptosis in rat decidual cells. In this study, the PL profile of rat decidual cells was characterized by a Mass spectrometry (MS) based lipidomic approach. Furthermore, we analyzed a possible correlation between changes in PL of rat decidual cells' membrane and AEA‐induced apoptosis. We found an increase in phosphatidylserine and a reduction of cardiolipin and phophatidylinositol relative contents. In addition, we observed an increase in the content of alkyl(alkenyl) acylPL, plasmalogens, and of long chain fatty acids especially with high degrees of unsaturation, as well as an increase in lipid hydroperoxides in treated cells. These findings provide novel insights on deregulation of lipid metabolism by anandamide, which may display further implications in decidualization process. J. Cell. Physiol. 230: 1549–1557, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:17.644887-05:
      DOI: 10.1002/jcp.24901
       
  • TGF‐β1 up‐regulates connexin43 expression: A potential
           mechanism for human trophoblast cell differentiation
    • Authors: Jung‐Chien Cheng; Hsun‐Ming Chang, Lanlan Fang, Ying‐Pu Sun, Peter C.K. Leung
      First page: 1558
      Abstract: Connexin43 (Cx43)‐mediated gap junctional intercellular communication (GJIC) are required for human trophoblast differentiation. To date, whether Cx43 mediates TGF‐β1‐induced trophoblast differentiation has not been determined. We showed that treatment with TGF‐β1 increased Cx43 expression and GJIC in HTR‐8/SVneo human trophoblast cells. In addition, Smad and ERK1/2 signaling pathways were involved in TGF‐β1‐induced up‐regulation of Cx43. Moreover, TGF‐β1 increased the expression of the syncytiotrophoblast marker, β‐hCG. Importantly, knockdown of Cx43 abolished the TGF‐β1‐induced up‐regulation of β‐hCG. Furthermore, overexpression of Cx43 up‐regulated β‐hCG expression. These results provide evidence that Cx43 and GJIC activity are up‐regulated by TGF‐β1 in human trophoblast cells, which subsequently contributes to TGF‐β1‐induced trophoblast differentiation. J. Cell. Physiol. 230: 1558–1566, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:18.308861-05:
      DOI: 10.1002/jcp.24902
       
  • Resveratrol inhibits NLRP3 inflammasome activation by preserving
           mitochondrial integrity and augmenting autophagy
    • Authors: Ya‐Ping Chang; Shuk‐Man Ka, Wan‐Han Hsu, Ann Chen, Louis Kuoping Chao, Chai‐Ching Lin, Cho‐Chen Hsieh, Ming‐Cheng Chen, Huan‐Wen Chiu, Chen‐Lung Ho, Yi‐Chich Chiu, May‐Lan Liu, Kuo‐Feng Hua
      First page: 1567
      Abstract: The NLRP3 inflammasome is a caspase‐1‐containing multi‐protein complex that controls the release of IL‐1β and plays important roles in the development of inflammatory disease. Here, we report that resveratrol, a polyphenolic compound naturally produced by plants, inhibits NLRP3 inflammasome‐derived IL‐1β secretion and pyroptosis in macrophages. Resveratrol inhibits the activation step of the NLRP3 inflammasome by suppressing mitochondrial damage. Resveratrol also induces autophagy by activating p38, and macrophages treated with an autophagy inhibitor are resistant to the suppressive effects of resveratrol. In addition, resveratrol administration mitigates glomerular proliferation, glomerular sclerosis, and glomerular inflammation in a mouse model of progressive IgA nephropathy. These findings were associated with decreased renal mononuclear leukocyte infiltration, reduced renal superoxide anion levels, and inhibited renal NLRP3 inflammasome activation. Our data indicate that resveratrol suppresses NLRP3 inflammasome activation by preserving mitochondrial integrity and by augmenting autophagy. J. Cell. Physiol. 230: 1567–1579, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:10.182992-05:
      DOI: 10.1002/jcp.24903
       
  • TNF‐related weak inducer of apoptosis (TWEAK) regulates junctional
           proteins in tubular epithelial cells via canonical NF‐κB
           pathway and ERK activation
    • Authors: Sergio Berzal; Cristian González‐Guerrero, Sandra Rayego‐Mateos, Álvaro Ucero, Carlos Ocaña‐Salceda, Jesús Egido, Alberto Ortiz, Marta Ruiz‐Ortega, Adrián M. Ramos
      First page: 1580
      Abstract: The tubular epithelium may be intrinsically involved in promoting kidney injury by junctional instability, epithelial‐mesenchymal transition (EMT) and extracellular matrix remodelling. In this work, we investigated whether the pleiotropic and proinflammatory cytokine tumor necrosis factor‐like weak inducer of apoptosis (TWEAK), could be able to disturb junctional protein expression and to induce EMT of tubular cells. In cultured murine proximal tubular cells TWEAK induced phenotypic changes that were accompanied by F‐actin redistribution, loss of epithelial adherent (E‐cadherin, Cadherin‐16, β‐catenin) and tight junction (ZO‐1) proteins, and re‐expression of the mesenchymal protein Vimentin. The transcriptional repressors Snail and HNF1β were also modulated by TWEAK. In a murine model of obstructive renal pathology, TWEAK expression correlated with the appearance of the mesenchymal marker αSMA in kidney tubular cells. Mechanistically, the epithelial changes induced by TWEAK, including loss of epithelial integrity and EMT, via Fn14 were TGF‐β1 independent, but mediated by several intracellular signaling systems, including the canonical NF‐κB, ERK activation and the vitamin D receptor modulation. These results highlight potential contributions of TWEAK‐induced inflammatory mechanisms that could unveil new pathogenic effects of TWEAK starting tubulointerstitial damage and fibrosis. J. Cell. Physiol. 230: 1580–1593, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:10.837519-05:
      DOI: 10.1002/jcp.24905
       
  • Wild‐type and specific mutant androgen receptor mediates
           transcription via 17β‐estradiol in sex hormone‐sensitive
           cancer cells
    • Authors: Takao Susa; Reina Ikaga, Takashi Kajitani, Masayoshi Iizuka, Hiroko Okinaga, Mimi Tamamori‐Adachi, Tomoki Okazaki
      First page: 1594
      Abstract: We previously encountered regulatory processes wherein dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone‐related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR), in breast cancer MCF‐7 cells. Here, we investigated whether such aberrant ligand‐nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed large amounts of AR at negligible levels of ERα/β or progesterone receptor. Both suppression of PTHrP and activation of prostate‐specific antigen genes were observed after independent administration of 17β‐estradiol (E2), DHT, or R5020. Consistent with the notion that the LNCaP AR lost its ligand specificity due to a mutation (Thr‐Ala877), experiments with siRNA targeting the respective NR revealed that the AR monopolized the role of the mediator of shared hormone‐dependent regulation, which was invariably associated with nuclear translocation of this mutant AR. Microarray analysis of gene regulation by DHT, E2, or R5020 disclosed that more than half of the genes downstream of the AR (Thr‐Ala877) overlapped in the LNCaP cells. Of particular interest, we realized that the AR (wild‐type [wt]) and AR (Thr‐Ala877) were equally responsible for the E2‐AR interactions. Fluorescence microscopy experiments demonstrated that both EGFP‐AR (wt) and EGFP‐AR (Thr‐Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Furthermore, reporter assays revealed that some other cancer cells exhibited aberrant E2‐AR (wt) signaling similar to that in the LNCaP cells. We herein postulate the presence of entangled interactions between wt AR and E2 in certain hormone‐sensitive cancer cells. J. Cell. Physiol. 230: 1594–1606, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:21.440484-05:
      DOI: 10.1002/jcp.24906
       
  • Effect of NAD on PARP‐mediated insulin sensitivity in oleic acid
           treated hepatocytes
    • Authors: Jing Pang; Ju Cui, Huan Gong, Chao Xi, Tie‐mei Zhang
      First page: 1607
      Abstract: High serum free fatty acids levels are associated with the development of insulin resistance in type 2 diabetes; however, the precise mechanisms underlying this lipid toxicity are unclear. To investigate whether PARP1 activation and NAD depletion are involved in the impairment of insulin sensitivity associated with lipotoxicity, HepG2 cells were cultured with 500 μM oleic acid for 48 h. Oleic acid‐treated cells exhibited increased ROS generation, lipid accumulation and PARP1 activation. Treatment with the PARP1 inhibitor PJ34 and transfection with PARP1 small interfering RNA both prevented the oleic acid‐induced impairment of the insulin signaling pathway. Furthermore, treatment with PJ34 reversed the oleic acid‐induced decrease in intracellular NAD concentration, while exogenous NAD protected cells against oleic acid‐induced insulin insensitivity. Combined NAD and PJ34 administration did not enhance the effects obtained by treatment with either NAD or PJ34 alone. Interestingly, when cells were treated with the SIRT1 inhibitor EX527, the protective effects of PJ34 and NAD treatment were diminished. Taken together, these data suggest that NAD depletion by PARP1 activation is essential for the modulation of insulin sensitivity in oleic acid‐induced lipotoxicity. J. Cell. Physiol. 230: 1607–1613, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:17.294672-05:
      DOI: 10.1002/jcp.24907
       
  • Human olfactory bulb neural stem cells mitigate movement disorders in a
           rat model of Parkinson's disease
    • Authors: Hany E.S. Marei; Samah Lashen, Amany Farag, Asmaa Althani, Nahla Afifi, Abd‐Elmaksoud A, Shaymaa Rezk, Roberto Pallini, Patrizia Casalbore, Carlo Cenciarelli
      First page: 1614
      Abstract: Parkinson's disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. Neural stem cells (NSCs) are multipotent stem cells that are capable of differentiating into different neuronal and glial elements. The production of DA neurons from NSCs could potentially alleviate behavioral deficits in Parkinsonian patients; timely intervention with NSCs might provide a therapeutic strategy for PD. We have isolated and generated highly enriched cultures of neural stem/progenitor cells from the human olfactory bulb (OB). If NSCs can be obtained from OB, it would alleviate ethical concerns associated with the use of embryonic tissue, and provide an easily accessible cell source that would preclude the need for invasive brain surgery. Following isolation and culture, olfactory bulb neural stem cells (OBNSCs) were genetically engineered to express hNGF and GFP. The hNFG‐GFP‐OBNSCs were transplanted into the striatum of 6‐hydroxydopamin (6‐OHDA) Parkinsonian rats. The grafted cells survived in the lesion environment for more than eight weeks after implantation with no tumor formation. The grafted cells differentiated in vivo into oligodendrocyte‐like (25 ± 2.88%), neuron‐like (52.63 ± 4.16%), and astrocyte ‐like (22.36 ± 1.56%) lineages, which we differentiated based on morphological and immunohistochemical criteria. Transplanted rats exhibited a significant partial correction in stepping and placing in non‐pharmacological behavioral tests, pole and rotarod tests. Taken together, our data encourage further investigations of the possible use of OBNSCs as a promising cell‐based therapeutic strategy for Parkinson's disease. J. Cell. Physiol. 230: 1614–1629, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:11.564767-05:
      DOI: 10.1002/jcp.24909
       
  • miR‐1470 mediates lapatinib induced p27 upregulation by targeting
           c‐jun
    • Authors: Weiwei Nie; Wei Song, Wenwen Zhang, Yanru Wang, Aiyu Zhu, Jiaqing Shao, Xiaoxiang Guan
      First page: 1630
      Abstract: Our previous study indicated that lapatinib induces p27‐dependent G1 arrest through both transcriptional and post‐translational mechanisms. Using miRNA microarray technology and quantitative RT‐PCR, we further investigated the potential miRNAs that involved in p27 upregulation and Her‐2 signaling pathway alteration with lapatinib treatment. A subset of 7 miRNAs was significantly affected in both 0.5 µM and 2.0 µM and 24 h and 48 h lapatinib treatment. Among them, only miR‐1470, miR‐126, and miR‐1208 were identified in the Her‐2 pathway after KEGG pathway analysis. However, luciferase reporter assay confirmed that miR‐1470 directly recognized the 3′‐untranslated region of c‐jun transcripts, which was consistent with TargetScan analysis. miR‐1470 significantly decreased c‐jun expression, thus miR‐1470 may repressc‐jun activation of cyclinD1 expression, and consequently promoted the upregulation of p27, a key molecule in the cell cycle arrest. Taken together, the present study provided the first evidences that miR‐1470 mediated lapatinib induced p27 upregulation by targeting c‐jun. J. Cell. Physiol. 230: 1630–1639, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:20.545758-05:
      DOI: 10.1002/jcp.24910
       
  • Cannabinoid receptor 1 but not 2 mediates macrophage phagocytosis by
           G(α)i/o/RhoA/ROCK signaling pathway
    • Authors: Ping Mai; Lei Tian, Le Yang, Lin Wang, Lin Yang, Liying Li
      First page: 1640
      Abstract: Phagocytosis is critical to macrophages linking innate and adaptive immune reaction. Cannabinoid receptor 1 (CB1) and 2 (CB2) mediate immune modulation. However, the role of cannabinoid receptors in macrophage phagocytosis is undefined. In this study, we found that two murine macrophage lines (J774A.1 and RAW264.7) and peripheral blood macrophages all expressed CB1 and CB2 by immunofluorescence‐staining, real time RT‐PCR and Western blot. Macrophage phagocytic activity was determined by quantifying fluorescent intensity of the engulfed BioParticles or fluorescence‐activated cell sorting. mAEA (CB1 agonist) enhanced phagocytosis of macrophages, but JWH133 (CB2 agonist) had no influence. Pharmacological or genetic ablation of CB1 inhibited mAEA‐enhanced phagocytosis, while CB2 had no such effects. Meanwhile, activation of CB1 increased GTP‐bounding active form of small GTPase RhoA, but not Rac1 or Cdc42. AM281 (CB1 antagonist) and pertussis toxin (PTX, G(α) i/o protein inhibitor) decreased GTP‐bound RhoA protein level with mAEA. In addition, PTX, C3 Transferase (RhoA inhibitor) or Y27632 (Rho‐associated kinase ROCK inhibitor) attenuated CB1‐mediated phagocytosis. These results confirm that activation of CB1 regulates macrophage phagocytosis through G(α) i/o/RhoA/ROCK signaling pathway. Moreover, activation of CB1 induced significant up‐regulation of CB1 expression by real time RT‐PCR and Western blot analysis, but not CB2. It indicated the existence of a positive feedback between CB1 activation and CB1 expression. The up‐regulation of CB1 was RhoA‐independent but it may contribute to maintaining high phagocytic activity of macrophages for a longer time. In conclusion, CB1 mediates macrophage phagocytosis by G(α) i/o/RhoA/ROCK signal axis. These data further underline the role of CB1 in macrophage phagocytic process. J. Cell. Physiol. 230: 1640–1650, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:16.840451-05:
      DOI: 10.1002/jcp.24911
       
  • Ubiquitin‐specific protease 22 (USP22) positively regulates RCAN1
           protein levels through RCAN1 de‐ubiquitination
    • Authors: Ahyoung Hong; Ji Eun Lee, KWANG CHUL Chung
      First page: 1651
      Abstract: Protein ubiquitination can be reversed by de‐ubiquitinating enzymes (DUBs), which are classified into two main classes, cysteine proteases and metalloproteases. Cysteine proteases include ubiquitin‐specific proteases (USPs) and ubiquitin C‐terminal hydrolases. USP22 is a USP family member and a component of the mammalian Spt‐Ada‐Gcn5 acetyltransferase transcriptional coactivating complex. Regulator of calcineurin 1 (RCAN1; also known as DSCR1 or MCIP1) functions as an endogenous inhibitor of calcineurin signaling. In the present study, we have identified a novel interaction between USP22 and RCAN1 (RCAN1–1S) in the mammalian cells. In addition, the overexpression of USP22 caused the increase of RCAN1 protein stability. USP22 antagonized the actions of FBW7, NEDD4–2, and β‐TrCP E3 ligase on RCAN1 and promoted RCAN1 de‐ubiquitination. Moreover, we found that RCAN1 was bound to USP22 in basal conditions, and interferon‐α (IFN‐α) treatment caused the dissociation of RCAN1 from USP22, which subsequently triggered RCAN1 ubiquitination and proteasome degradation. Taken together, these results suggest that USP22 positively regulates RCAN1 levels, which would consequently affect diverse RCAN1‐linked cellular processes, such as the inflammatory process involving the release of IFN‐α. J. Cell. Physiol. 230: 1651–1660, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:22.170112-05:
      DOI: 10.1002/jcp.24917
       
  • GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease
    • Authors: Laurence Booth; Jane L. Roberts, Devin R. Cash, Seyedmehrad Tavallai, Sophonie Jean, Abigail Fidanza, Tanya Cruz‐Luna, Paul Siembiba, Kelly A. Cycon, Cynthia N. Cornelissen, Paul Dent
      First page: 1661
      Abstract: The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU‐03012 (AR‐12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70, HSP90, GRP94, GRP58, HSP27, HSP40 and HSP60. OSU‐03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola/Marburg/Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively. Pre‐treatment with OSU‐03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya, Mumps, Measles, Rubella, RSV, CMV, and Influenza viruses. OSU‐03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi‐drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU‐03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU‐03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus, Dna K and bacterial phosphodiesterases are novel antibiotic targets, and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. J. Cell. Physiol. 230: 1661–1676, 2015. © 2014 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
      PubDate: 2015-03-27T08:38:22.831938-05:
      DOI: 10.1002/jcp.24919
       
  • Functional role of the KCa3.1 potassium channel in synovial fibroblasts
           from rheumatoid arthritis patients
    • Authors: Kristin Friebel; Roland Schönherr, Raimund W. Kinne, Elke Kunisch
      First page: 1677
      Abstract: Rheumatoid arthritis synovial fibroblasts (RA‐SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. New druggable targets in RA‐SFs would therefore be of high therapeutic interest. The present study shows that the intermediate‐conductance, calcium‐activated potassium channel KCa3.1 (KCNN4) is expressed at the mRNA and protein level in RA‐SFs, is functionally active, and has a regulatory impact on cell proliferation and secretion of pro‐inflammatory and pro‐destructive mediators. Whole‐cell patch‐clamp recordings identified KCa3.1 as the dominant potassium channel in the physiologically relevant membrane voltage range below 0 mV. Stimulation with transforming growth factor β1 (TGF‐β1) significantly increased transcription, translation, and channel function of KCa3.1. Inhibition of KCa3.1 by the selective, pore‐blocking inhibitor TRAM‐34, (and, in part, by siRNA) significantly reduced cell proliferation, as well as expression and secretion of pro‐inflammatory factors (IL‐6, IL‐8, and MCP1) and the tissue‐destructive protease MMP3. These effects were observed in non‐stimulated and/or TGF‐β1‐stimulated RA‐SFs. Since small molecule‐based interference with KCa3.1 is principally well tolerated in clinical settings, further evaluation of channel blockers in models of rheumatoid arthritis may be a promising approach to identify new pharmacological targets and develop new therapeutic strategies for this debilitating disease. J. Cell. Physiol. 230: 1677–1688, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:13.94927-05:0
      DOI: 10.1002/jcp.24924
       
  • Activation Of Nfatc2 in osteoblasts causes osteopenia
    • Authors: Stefano Zanotti; Ernesto Canalis
      First page: 1689
      Abstract: Nuclear factor of activated T‐cells (Nfat) c1 to c4 are transcription factors that play an undisputable role in osteoclastogenesis. However, Nfat function in osteoblastic cells is controversial. Constitutive activation of Nfatc1 and c2 in osteoblasts suppresses cell function, although the study of Nfat in vivo has yielded conflicting results. To establish the consequences of Nfatc2 activation in osteoblasts, we generated transgenic mice where a 3.6 kb fragment of the collagen type I α1 promoter directs expression of a constitutively active Nfatc2 mutant (Col3.6‐Nfatc2). The skeletal phenotype of Col3.6‐Nfatc2 mice of both sexes and of sex‐matched littermate controls was investigated by microcomputed tomography and histomorphometry. Col3.6‐ Nfatc2 mice were born at the expected Mendelian ratio and appeared normal. Nfatc2 expression was confirmed in parietal bones from 1 and 3 month old transgenic mice. One month old Col3.6‐Nfatc2 female mice exhibited cancellous bone compartment osteopenia secondary to a 30% reduction in bone formation. In contrast, cancellous femoral bone volume and bone formation were not altered in male transgenics, whereas osteoblast number was higher, suggesting incomplete osteoblast maturation. Indices of bone resorption were not affected in either sex. At 3 months of age, the skeletal phenotype evolved; and Col3.6‐Nfatc2 male mice exhibited vertebral osteopenia, whereas femoral cancellous bone was not affected in either sex. Nfatc2 activation in osteoblasts had no impact on cortical bone structure. Nfatc2 activation inhibited alkaline phosphatase activity and mineralized nodule formation in bone marrow stromal cell cultures. In conclusion, Nfatc2 activation in osteoblasts inhibits bone formation and causes cancellous bone osteopenia. J. Cell. Physiol. 230: 1689–1695, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company
      PubDate: 2015-03-27T08:38:18.790288-05:
      DOI: 10.1002/jcp.24928
       
  • The Immunomodulatory and Therapeutic Effects of Mesenchymal Stromal Cells
           for Acute Lung Injury and Sepsis
    • Abstract: It is increasingly recognized that immunomodulation represents an important mechanism underlying the benefits of many stem cell therapies, rather than the classical paradigm of transdifferentiation and cell replacement. In the former paradigm, the beneficial effects of cell therapy result from paracrine mechanism(s) and/or cell‐cell interaction as opposed to direct engraftment and repair of diseased tissue and/or dysfunctional organs. Depending on the cell type used, components of the secretome, including microRNA (miRNA) and extracellular vesicles, may be able to either activate or suppress the immune system even without direct immune cell contact. Mesenchymal stromal cells (MSCs), also referred to as mesenchymal stem cells, are found not only in the bone marrow, but in a wide variety of organs and tissues. In addition to any direct stem cell activities, MSCs were the first stem cells recognized to modulate immune response, and therefore they will be the focus of this review. Specifically, MSCs appear to be able to effectively attenuate acute and protracted inflammation via interactions with components of both innate and adaptive immune systems. To date, this capacity has been exploited in a large number of pre‐clinical studies and MSC immunomodulatory therapy has been attempted with various degrees of success in a relatively large number of clinical trials. Here we will explore the various mechanism employed by MSCs to effect immunosuppression as well as review the current status of its use to treat excessive inflammation in the context of acute lung injury and sepsis in both pre‐clinical and clinical settings. This article is protected by copyright. All rights reserved
       
  • TRPC1 Regulates Calcium‐Activated Chloride Channels in Salivary
           Gland Cells
    • Abstract: Calcium‐activated chloride channel (CaCC) plays an important role in modulating epithelial secretion. It has been suggested that in salivary tissues, sustained fluid secretion is dependent on Ca2+ influx that activates ion channels such as CaCC to initiate Cl‐ efflux. However direct evidence as well as the molecular identity of the Ca2+ channel responsible for activating CaCC in salivary tissues is not yet identified. Here we provide evidence that in human salivary cells, an outward rectifying Cl‐ current was activated by increasing [Ca2+]i, which was inhibited by the addition of pharmacological agents niflumic acid (NFA), an antagonist of CaCC, or T16Ainh‐A01, a specific TMEM16a inhibitor. Addition of thapsigargin (Tg), that induces store‐depletion and activates TRPC1‐mediated Ca2+ entry, potentiated the Cl‐ current, which was inhibited by the addition of a non‐specific TRPC channel blocker SKF96365 or removal of external Ca2+. Stimulation with Tg also increased plasma membrane expression of TMEM16a protein, which was also dependent on Ca2+ entry. Importantly, in salivary cells, TRPC1 silencing, but not that of TRPC3, inhibited CaCC especially upon store depletion. Moreover, primary acinar cells isolated from submandibular gland also showed outward rectifying Cl‐ currents upon increasing [Ca2+]i. These Cl‐ currents were again potentiated with the addition of Tg, but inhibited in the presence of T16Ainh‐A01. Finally, acinar cells isolated from the submandibular glands of TRPC1 knockout mice showed significant inhibition of the outward Cl‐ currents without decreasing TMEM16a expression. Together the data suggests that Ca2+ entry via the TRPC1 channels is essential for the activation of CaCC. This article is protected by copyright. All rights reserved
       
  • Melanoma Treatments: Advances and Mechanisms
    • Abstract: Advances in the understanding of the molecular pathogenesis of melanoma and in cancer immunology have led to the rational design and recent clinical implementation of a variety of novel therapies for metastatic melanoma. BRAF and MEK inhibitors that target the MAPK pathway have high rates of clinical response in BRAF‐mutant melanoma. Therapies that modulate the immune response to melanoma, including monoclonal antibodies that interfere with pathways that inhibit T‐cell function, have been shown to be effective in melanoma. Lessons learned from these encouraging results are driving the development of new treatments for melanoma and other cancers. This review will focus on the science and clinical findings related to targeted therapies that inhibit BRAF or MEK as well as the immunotherapies that block the CTLA‐4 or PD‐1 pathways. Other experimental and combinatorial therapeutic approaches will also be discussed. This article is protected by copyright. All rights reserved
       
  • Functional Characterization of Nupr1L, a novelp53‐Regulated Isoform
           of the High Mobility Group (HMG)‐Related Protumoral Protein Nupr1
    • Abstract: We have previously demonstrated a crucial role of NUPR1 in tumor development and progression. In this work we report the functional characterization of a novel Nupr1‐like isoform (NUPR1L) and its functional interaction with the protumoral factor NUPR1. Through the use of primary sequence analysis, threading, and homology‐based molecular modeling as well as expression and immunolocalization, studies reveal that NUPR1L displays properties which are similar to member of the HMG‐like family of chromatin regulators including its ability to translocate to the cell nucleus and bind to DNA. Analysis of the NUPR1L promoter showed the presence of two p53‐response elements at positions ‐37 and ‐7 respectively. Experiments using reporter assays combined with site‐directed mutagenesis and using cells with controllable p53 expression demonstrate that both of these sequences are responsible for the regulation of NUPR1L expression by p53. Congruently, NUPR1L gene expression is activated in response to DNA damage induced by Oxaliplatin treatment or cell cycle arrest induced by serum starvation, two well‐validated methods to achieve p53 activation. Interestingly, expression of NUPR1L down‐regulates the expression of NUPR1, its closely related protumoral isoform, by a mechanism that involves the inhibition of its promoter activity. At the cellular level, overexpression of NUPR1L induces G1 cell cycle arrest and a decrease in their cell viability, an effect that is mediated, at least in part, by down‐regulating NUPR1 expression. Combined these experiments constitute the first functional characterization of NUPR1L as a new p53‐induced gene which negatively regulates the protumoral factor NUPR1. This article is protected by copyright. All rights reserved
       
  • Myeloid Zinc Finger 1 (MZF‐1) Regulates Expression of the CCN2/CTGF
           and CCN3/NOV Genes in the Hematopoietic Compartment
    • Abstract: Connective Tissue Growth Factor (CCN2/CTGF) and Nephroblastoma Overexpressed (CCN3/NOV) execute key functions within the hematopoietic compartment. Both are abundant in the bone marrow stroma, which is a niche for hematopoiesis and supports marrow function. Roles for 1,25‐dihydroxyvitamin D3 (calcitriol) and all‐trans retinoic acid in the bone marrow have also been elucidated. Interestingly, some of the annotated roles of these vitamins overlap with established functions of CCN2 and CCN3. Yet, no factor has been identified that unifies these observations. In this study, we report the regulation of the CTGF and NOV genes by Myeloid Zinc Finger‐1 (MZF‐1), a hematopoietic transcription factor. We show the interaction of MZF‐1 with the CTGF and NOV promoters in several cell types. Up‐regulation of MZF‐1 via calcitriol and vitamin A induces expression of CTGF and NOV, implicating a role for these vitamins in the functions of these two genes. Lastly, knockdown of MZF1 reduces levels of CTGF and NOV. Collectively, our results argue that MZF‐1 regulates the CTGF and NOV genes in the hematopoietic compartment, and may be involved in their respective functions in the stroma. This article is protected by copyright. All rights reserved
       
  • Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation in
           Vivo and Osteoprogenitor Differentiation ex Vivo
    • Abstract: Initial identification of osteoactivin (OA)/glycoprotein non‐melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased 3‐fold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post‐fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV‐promoter (OA‐Tg). Western blot analysis showed increased OA/gpnmb in OA‐Tg osteoblasts, compared to wild‐type (WT). In OA‐Tg mouse femurs versus WT littermates, micro‐CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA‐Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over‐expressed in osteoclasts in OA‐Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX‐1 and RANK‐L, and decreased osteoclast numbers in OA‐Tg, compared to WT mice, indicating decreased bone remodeling in OA‐Tg mice. The proliferation rate of OA‐Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA‐Tg osteoprogenitors. Quantitative RT‐PCR analysis showed increased TGF‐β1 and TGF‐β receptors I and II expression in OA‐Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. This article is protected by copyright. All rights reserved
       
  • Treg(s) in Cancer: Friends or Foe'
    • Abstract: Immune escape is a hallmark of cancer. Regulatory T cells (Treg) have been described to maintain peripheral tolerance. The role of Treg in cancer is ambiguous, as they are central inhibitory regulators in solid tumors, whereas during inflammation‐driven tumorigenesis they prevent cancer initiation by restraining inflammation. As a consequence, under conditions with chronic inflammation that may initiate malignant transformation, application rather than depletion of Treg may be helpful. In solid tumors, however, the success story of immune‐activating antibodies targeting checkpoint molecules of T cell activation fuels the hope that Treg inactivation or depletion may additionally boost anti‐tumor immune response. In this review we summarize important aspects on the dual role of Treg in cancer to provide a rationale for future Treg targeting attempts. This article is protected by copyright. All rights reserved
       
  • Para‐Cresyl Sulfate Acutely Impairs Vascular Reactivity and Induces
           Vascular Remodeling
    • Abstract: Chronic kidney disease (CKD) is characterized by vascular remodeling and the retention of uremic toxins, several of which are independently associated with the high cardiovascular mortality rate in CKD patients. Whether the association between these uremic toxins and cardiovascular mortality is due to induction of vascular dysfunction and resulting vascular remodeling remains to be determined. This study evaluates the effects of para‐cresyl sulfate (PCS), a newly identified uremic toxin, on vascular function and remodeling. PCS acutely induced oxidative stress in both endothelial and vascular smooth muscle cells, with a maximal effect at 0.15 mM, corresponding to the mean ‘uremic’ concentration found in dialysis patients. PCS significantly increased within 30 min phenylephrine‐induced contraction of mouse thoracic aorta, through direct activation of rho‐kinase, independently of oxidative stress induction, as demonstrated by the capacity of rho‐kinase inhibitor Y‐27632 to abolish this effect. After exposure of the aorta to PCS for 48 h, we observed inward eutrophic remodeling, a hallmark of uremic vasculopathy characterized by a reduction of the area of both lumen and media, with unchanged media/lumen ratio. In conclusion, elevated PCS concentrations such as those observed in CKD patients, by promoting both vascular dysfunction and vascular remodeling, may contribute to the development of hypertension and to cardiovascular mortality in CKD. This article is protected by copyright. All rights reserved
       
  • Anti Proliferative and Pro Apoptotic Effects of Flavonoid Quercetin Are
           Mediated by CB1 Receptor in Human Colon Cancer Cell Lines
    • Abstract: Quercetin, the major constituent of flavonoid and widely present in fruits and vegetables, is an attractive compound for cancer prevention due to its beneficial anti proliferative effects, showing a crucial role in the regulation of apoptosis and cell cycle signaling. In vitro studies have demonstrated that quercetin specifically influences colon cancer cell proliferation. Our experiments, using human colon adenocarcinoma cells, confirmed the anti proliferative effect of quercetin and gave intriguing new insight in to the knowledge of the mechanisms involved. We observed a significant increase in the expression of the endocannabinoids receptor (CB1‐R) after quercetin treatment. CB1‐R can be considered an estrogen responsive receptor and quercetin, having a structure similar to that of the estrogens, can interact with CB1‐R leading to the regulation of cell growth. In order to clarify the contribution of the CB1‐R to the quercetin action, we investigated some of the principal molecular pathways that are inhibited or activated by this natural compound. In particular we detected the inhibition of the major survival signals like the PI3K/Akt/mTOR and an induction of the pro apoptotic JNK/JUN pathways. Interestingly, the metabolism of β‐catenin was modified by flavonoid both directly and through activated CB1‐R. In all the experiments done, the quercetin action has proven to be reinforced by anandamide (Met‐F‐AEA), a CB1‐R agonist, and partially counteracted by SR141716, a CB1‐R antagonist. These findings open new perspectives for anticancer therapeutic strategies. This article is protected by copyright. All rights reserved
       
  • Regulation of Osteoblast Migration Involving Receptor Activator of Nuclear
           Factor (NF)‐Kappa B (RANK) Signaling
    • Abstract: Bone remodeling requires osteoclast activation, resorption and reversal, prior to osteoblast migration into the bone pit. The Receptor Activator of NF‐κB (RANK) signaling pathway plays an important role in bone remodeling. Two components of the RANK signaling pathway, RANK Ligand (RANKL) and the decoy receptor Osteoprotegerin (OPG), are expressed predominantly on the surface of osteoblasts, while RANK is principally expressed on the surface of osteoclasts. However, RANK has also been reported to be expressed on the surface of osteoblasts and osteosarcoma tumor cells. Treatment with soluble RANKL (sRANKL) of both normal osteoblasts and osteosarcoma tumor cells activated phosphorylation of ERK, p38MAPK, Akt and p65NF‐κB. However, modified Boyden chamber assays and wound repair assays showed differential response to sRANKL‐induced chemotactic migration in normal osteoblasts and osteosarcoma tumor cells. In contrast to previously published results, both normal osteoblasts and osteosarcoma tumor cells responded to sRANKL‐induced chemotactic migration but the normal osteoblasts did so only in the presence of an ERK pathway inhibitor. For both normal and tumor cells, the chemotactic response could be blocked by inhibiting the PI3K/Akt or p65NF‐κB pathway. Response to sRANKL in normal and tumor cells suggests a role for RANK/ERK‐mediated signaling in normal osteoblasts chemotactic migration during bone remodeling that is altered or lost during osteosarcoma tumorigenesis. This article is protected by copyright. All rights reserved
       
  • Ephrin Reverse Signaling Mediates Palatal Fusion and
           Epithelial‐to‐Mesenchymal Transition Independently of
           Tgfß3
    • Abstract: The mammalian secondary palate forms from shelves of epithelia‐covered mesenchyme that meet at midline and fuse. The midline epithelial seam (MES) is thought to degrade by apoptosis, epithelial‐to‐mesenchymal transition (EMT), or both. Failure to degrade the MES blocks fusion and causes cleft palate. It was previously thought that transforming growth factor ß3 (Tgfß3) is required to initiate fusion. Members of the Eph tyrosine kinase receptor family and their membrane‐bound ephrin ligands are expressed on the MES. We demonstrated that treatment of mouse palates with recombinant EphB2/Fc to activate ephrin reverse signaling (where the ephrin acts as a receptor and transduces signals from its cytodomain) was sufficient to cause mouse palatal fusion when Tgfß3 signaling was blocked by an antibody against Tgfß3 or by an inhibitor of the TgfßrI serine/threonine receptor kinase. Cultured palatal epithelial cells traded their expression of epithelial cell markers for that of mesenchymal cells and became motile after treatment with EphB2/Fc. They concurrently increased their expression of the EMT‐associated transcription factors Snail, Sip1, and Twist1. EphB2/Fc did not cause apoptosis in these cells. These data reveal that ephrin reverse signaling directs palatal fusion in mammals through a mechanism that involves EMT but not apoptosis and activates a gene expression program not previously associated with reverse signaling. This article is protected by copyright. All rights reserved
       
  • The Role of Mitochondrial Derived Peptides (MDPs) in Metabolism
    • Abstract: The term “mitokines” refers to signals derived from mitochondria that have an impact on other cells or tissues (Durieux et al., 2011). Rather than being simply a set of DNA composed by 37 genes, the mitochondrial DNA (mtDNA) is quite complex and includes small RNAs (Mercer et al., 2011). Mitochondrial‐derived peptides (MDPs) are encoded by functional short open reading frames (sORFs) in the mtDNA.
       
 
 
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