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Journal Cover Journal of Cellular Physiology
  [SJR: 1.948]   [H-I: 125]   [5 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
   Published by John Wiley and Sons Homepage  [1598 journals]
  • Effects of Conjugated Linoleic Acid Associated with Endurance Exercise on
           Muscle Fibres and Peroxisome Proliferator‐Activated Receptor γ
           Coactivator 1 α Isoforms
    • Authors: Rosario Barone; Claudia Sangiorgi, Antonella Marino Gammazza, Daniela D'Amico, Monica Salerno, Francesco Cappello, Cristoforo Pomara, Giovanni Zummo, Felicia Farina, Valentina Di Felice, Filippo Macaluso
      Abstract: Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator‐activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre‐type‐specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 µl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6‐week experimental period, whereas the placebo groups were gavaged with 35 µl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota‐Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2 and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre‐type‐specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre‐type‐specific hypertrophy induced by endurance exercise (of type I and IIb muscle fibres). This article is protected by copyright. All rights reserved
      PubDate: 2016-08-02T17:06:00.484007-05:
      DOI: 10.1002/jcp.25511
  • VIP Family Members Prevent Outer Blood Retinal Barrier Damage in a Model
           of Diabetic Macular Edema
    • Authors: Grazia Maugeri; Agata Grazia D'Amico, Caterina Gagliano, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata
      Abstract: Diabetic macular edema (DME), characterized by an increase of thickness in the eye macular area, is due to breakdown of the blood‐retinal barrier (BRB). Hypoxia plays a key role in the progression of this pathology by activating the hypoxia‐inducible factors. In the last years, various studies have put their attention on the role of pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in retinal dysfunction. However, until now, no study has investigated their protective role against the harmful combined effect of both hyperglycemia and hypoxia on outer BRB. Therefore, in the present study, we have analyzed the role of these peptides on permeability, restoration of tight junctions expression and inhibition of hyperglycemia/hypoxia‐induced apoptosis, in an experimental in vitro model of outer BRB. Our results have demonstrated that the peptides' treatment have restored the integrity of outer BRB induced by cell exposure to hyperglycemia/hypoxia. Their effect is mediated through the activation of phosphoinositide 3 kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling pathways. In conclusion, our study further clarifies the mechanism through which PACAP and VIP perform the beneficial effect on retinal damage induced by hyperglycemic/hypoxic insult, responsible of DME progression. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-02T17:05:58.282499-05:
      DOI: 10.1002/jcp.25510
  • Functional Effects of Cigarette Smoke‐Induced Changes in Airway
           Smooth Muscle Mitochondrial Morphology
    • Authors: Bharathi Aravamudan; Michael Thompson, Gary C. Sieck, Robert Vassallo, Christina M. Pabelick, Y.S. Prakash
      Abstract: Long‐term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). We previously showed that CS destabilizes the networked morphology of mitochondria in human ASM by regulating the expression of mitochondrial fission and fusion proteins via multiple signaling mechanisms. Emerging data link regulation of mitochondrial morphology to cellular structure and function. We hypothesized that CS‐induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling. Here, ASM cells were exposed to 1% cigarette smoke extract (CSE) for 48 hours to alter mitochondrial fission/fusion, or by inhibiting the fission protein Drp1 or the fusion protein Mfn2. Mitochondrial function was assessed via changes in bioenergetics or altered rates of proliferation and apoptosis. Our results indicate that both exposure to CS and inhibition of mitochondrial fission/ fusion proteins affect mitochondrial function (i.e., energy metabolism, proliferation and apoptosis) in ASM cells. In vivo, the airways in mice chronically exposed to CS are thickened and fibrotic, and the expression of proteins involved in mitochondrial function is dramatically altered in the ASM of these mice. We conclude that CS‐induced changes in mitochondrial morphology (fission/ fusion balance) correlate with mitochondrial function, and thus may control ASM proliferation, which plays a central role in airway health. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-30T15:00:30.808461-05:
      DOI: 10.1002/jcp.25508
  • Uric Acid Amplifies Aβ Amyloid Effects Involved in the Cognitive
           Dysfunction/Dementia: Evidences from an Experimental Model In Vitro
    • Authors: G Desideri; R Gentile, A Antonosante, E Benedetti, D Grassi, L Cristiano, A Manocchio, S Selli, R Ippoliti, C Ferri, Claudio Borghi, A Giordano, Annamaria Cimini
      Abstract: There is still a considerable debate concerning whether uric acid is neuroprotective or neurotoxic agent. To clarify this topic we tested the effects of uric acid on neuronal cells biology by using differentiated SHSY5Y neuroblastoma cells incubated with amyloid ß to reproduce an in vitro model of Alzheimer's disease. The incubation of cells with uric acid at the dose of 40 µM or higher significantly reduced cell viability and potentiated the proapoptotic effect of amyloid ß. Finally, uric acid enhanced the generation of 4‐hydroxynonenal and the expression of PPARβ/δ promoted by amyloid β, indicating a prooxidant effects. In conclusion, uric acid could exert a detrimental influence on neuronal cell biology being this influence further potentiated by concomitant exposure to neurotoxic stimuli. This effect is evident for uric acid concentrations close to those achievable in cerebrospinal fluid in presence of mild hyperuricemia thus suggesting a potential role of uric acid in pathophysiology of cognitive dysfunction. These effects are influenced by the concentrations of uric acid and by the presence of favouring conditions that commonly occur in neurodegenerative disorders and well as in the aging brain, including increased oxidative stress and exposure to amyloid β. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-30T14:55:25.58167-05:0
      DOI: 10.1002/jcp.25509
  • Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of
           Foetal Calf Serum
    • Authors: A. Karim; A. C. Hall
      Abstract: Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood however pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology and clustering of cultured bovine articular chondrocytes within stiff (2%w/v) and soft (0.2%w/v) 3‐dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; foetal calf serum (FCS) 1‐10%;380mOsm) for up to 7d. Chondrocytes were fluorescently‐labelled after 1, 3 and 7d with 5‐chloromethylfluorescein‐diacetate (CMFDA) and propidium iodide (PI) or 1,5‐bis{[2‐(di‐methylamino)ethyl]amino}‐4,8‐dihydroxyanthracene‐9,10‐dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology and clustering were quantified using VolocityTM software. In stiff gels after 7d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P 
      PubDate: 2016-07-29T16:35:23.286636-05:
      DOI: 10.1002/jcp.25507
  • Emerging Roles of Transforming Growth Factor β Signaling in Diabetic
    • Authors: Sarah E. Wheeler; Nam Y. Lee
      Abstract: Diabetic retinopathy (DR) is a serious complication of diabetes mellitus affecting about one third of diabetic adults. Despite its prevalence, treatment options are limited and often implemented only in the later stages of the disease. To date, the pathogenesis of DR has been extensively characterized in the context of elevated glucose, insulin, and VEGF signaling, although a growing number of other growth factors and molecules, including transforming growth factor‐β (TGF‐β) are being recognized as important contributors and/or therapeutic targets. Here, we review the complex roles of TGF‐β signaling in DR pathogenesis and progression. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-29T16:30:27.799001-05:
      DOI: 10.1002/jcp.25506
  • An Overview on Predictive Biomarkers of Testicular Germ Cell Tumors
    • Authors: Paolo Chieffi
      Abstract: Testicular germ cell tumors (TGCTs) are frequent solid malignant tumors and cause of death in men between 20–40 years of age. Genetic and environmental factors play an important role in the origin and development of TGCTs. Although the majority of TGCTs are responsive to chemotherapy, about 20% of patient presents incomplete response or tumors relapse. In addition, the current treatments cause acute toxicity and several chronic collateral effects, including sterility. The present mini‐review collectively summarize the most recent findings on the new discovered molecular biomarkers such as tyrosine kinases, HMGAs, Aurora B kinase, and GPR30 receptor predictive of TGCTs and as emerging new possible molecular targets for therapeutic strategies. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-29T10:10:23.798428-05:
      DOI: 10.1002/jcp.25482
  • Novel Instruments for the Implementation of Electrochemotherapy Protocols:
           From Bench Side to Veterinary Clinic
    • Authors: Enrico P. Spugnini; Stefano Fais, Tommaso Azzarito, Alfonso Baldi
      Abstract: Electrochemotherapy (ECT) is a medical strategy that allows an increased efficacy of chemotherapy agents after the application of permeabilizing electric pulses having appropriate characteristics (form, voltage, frequency). In the past ten years, the clinical efficacy of this therapeutic approach in several spontaneous models of tumors in animals has been shown. Moreover, some of the molecular and cellular mechanisms responsible for this phenomenon have been elucidated. Our group has been deeply involved in the development of new ECT protocols for companion animals, implementing the use of the technique as first line treatment, and evaluating different chemotherapy agents in laboratory animals as well as pets. This article summarizes the most important advances in veterinary ECT, including the development of novel equipment, therapeutic protocols and their translation to humans. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-28T06:35:31.771816-05:
      DOI: 10.1002/jcp.25505
  • Coumestrol Inhibits Proliferation and Migration of Prostate Cancer Cells
           by Regulating AKT, ERK1/2 and JNK MAPK Cell Signaling Cascades
    • Authors: Whasun Lim; Muhah Jeong, Fuller W. Bazer, Gwonhwa Song
      Abstract: Coumestrol is the one of the major phytoestrogens which is abundant in soybeans, legumes, brussel sprouts and spinach. The beneficial effects of coumestrol are well known in various biological processes including; neuroprotective effects on the nervous system, function of the female reproductive system, anti‐bacterial properties and anti‐cancer effects. Although the anti‐tumor activity of coumestrol has been demonstrated for ovarian, breast, lung and cervical cancers, little is known of its effects on prostate cancer. Therefore, in the present study, we investigated the chemotherapeutic effects of coumestrol on two prostate cancer cell lines, PC3 and LNCaP. Our results showed that coumestrol decreased proliferation and migration and induced apoptosis in both PC3 and LNCaP cells. Moreover, effects of coumestrol on cell signaling pathways were investigated and it increased phosphorylation of ERK1/2, JNK, P90RSK and P53 proteins in a dose‐ and time‐dependent manner whereas phosphorylation of AKT was reduced by coumestrol under the same conditions for culture of PC3 and LNCaP cells. In addition, mitochondrial dysfunction was induced by coumestrol as evidenced by a significant loss of mitochondrial membrane potential. Furthermore, cleavage of caspase‐3 and caspase‐9, the apoptotic proteins associated with mitochondria, also changed in response to coumestrol. Coumestrol also caused mitochondrial dysfunction resulting in an increase in ROS production in PC3 and LNCaP cells. These results suggest that coumestrol can inhibit progression of prostate cancer and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signaling pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-28T05:51:13.440339-05:
      DOI: 10.1002/jcp.25494
  • Tissue Inhibitor of Metalloproteinase 1 Influences Vascular Adaptations to
           Chronic Alterations in Blood Flow
    • Authors: Erin R. Mandel; Cassandra Uchida, Emmanuel Nwadozi, Armin Makki, Tara L. Haas
      Abstract: Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). We hypothesized that the loss of TIMP1 would enhance both ischemia and flow‐induced vascular remodeling by increasing MMP activity. TIMP1 deficient (Timp1−/−) and wild‐type (WT) C57BL/6 mice underwent unilateral femoral artery (FA) ligation or were treated with prazosin, an alpha‐1 adrenergic receptor antagonist, in order to investigate vascular remodeling to altered flow. Under basal conditions, Timp1−/− mice had reduced microvascular content as compared to WT mice. Furthermore, vascular remodeling was impaired in Timp1−/− mice. Timp1−/− mice displayed reduced blood flow recovery in response to FA ligation and no arteriogenic response to prazosin treatment. Timp1−/− mice failed to undergo angiogenesis in response to ischemia or prazosin, despite maintaining the capacity to increase VEGF‐A and eNOS mRNA. Vascular permeability was increased in muscles of Timp1−/− mice in response to both prazosin treatment and FA ligation, but this was not accompanied by greater MMP activity. This study highlights a previously undescribed integral role for TIMP1 in both vascular network maturation and adaptations to ischemia or alterations in flow. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-28T05:45:36.851462-05:
      DOI: 10.1002/jcp.25491
  • Aging Selectively Modulates Vitamin C Transporter Expression Patterns in
           the Kidney
    • Abstract: In the kidney, vitamin C is reabsorbed from the glomerular ultrafiltrate by sodium‐vitamin C cotransporter isoform 1 (SVCT1) located in the brush border membrane of the proximal tubules. Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging‐induced changes in the expression of vitamin C transporters in renal tissue. Aging induced significant morphological changes, such as periglomerular lymphocytic infiltrate and glomerular congestion, in the kidneys of SAMP8 mice, although no increase in collagen deposits was observed using 2‐photon microscopy analysis and second harmonic generation. The most characteristic histological alteration was the dilation of intracellular spaces in the basolateral region of proximal tubule epithelial cells. Furthermore, a combination of laser microdissection, qRT‐PCR and immunohistochemical analyses allowed us to determine that SVCT1 expression specifically increased in the proximal tubules from the outer strip of the outer medulla (segment S3) and cortex (segment S2) during aging and that these tubules also express GLUT1. We conclude that aging modulates vitamin C transporter expression and that renal over‐expression of SVCT1 enhances vitamin C reabsorption in aged animals that may synthesize less vitamin C. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T17:00:23.819469-05:
      DOI: 10.1002/jcp.25504
  • Late Endosomal Recycling of Open MHC‐I Conformers
    • Abstract: With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty Ld (eLd) molecules, an open form of murine MHC‐I allele, in fibroblast‐like cells. Pulse labeling of cell surface eLd with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC‐I conformers. Kinetic modeling, using in‐house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eLd demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eLd distributed into pre‐degradative multivesicular bodies (MVBs), these LE subsets were not a source for eLd recycling. The LE recycling of eLd did not require Rab7 membrane domains, as demonstrated by Rab7‐silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eLd in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T10:50:44.218757-05:
      DOI: 10.1002/jcp.25495
  • Nestin Expressed by Pre‐Existing Cardiomyocytes Recapitulated in
           Part an Embryonic Phenotype; Suppressive Role of p38 MAPK
    • Abstract: Nestin(+)‐cardiomyocytes were identified in the ischemically damaged human/rodent heart, albeit the cellular source and signaling events implicated in the appearance of the intermediate filament protein remained undefined. Expression of the enhanced green fluorescent protein (EGFP) driven by the second intron of the nestin gene identified a subpopulation of EGFP/nestin(+) cells that differentiated to a vascular phenotype in the peri‐infarct/infarct region of post‐MI mice albeit the transgene was not detected in nestin(+)‐cardiomyocytes. α‐MHC‐driven expression of the reporter mCherry was detected in troponin‐T(+)‐ and nestin(+)‐cardiomyocytes in the peri‐infarct/infarct region of post‐MI mice. However, the cell cycle re‐entry of nestin/mCherry(+)‐cardiomyocytes was not observed. Nestin staining was identified in a paucity of neonatal rat ventricular cardiomyocytes (NNVM). Exposure to phorbol 12, 13‐dibutyrate (PDBu) induced NNVM hypertrophy but did not promote nestin expression or Brdu incorporation. PDBu treatment of NNVMs phosphorylated p38 MAPK and HSP27 and HSP27 phosphorylation was abrogated by the p38 MAPK inhibitor SB203580. PDBu/SB203580 co‐treatment significantly increased the percentage of NNVMs that expressed nestin and incorporated Brdu. In the heart of embryonic 10.5 day mice, nestin was detected in cycling troponin‐T(+)‐cardiomyocytes. Nestin was also detected in embryonic rat ventricular cardiomyocytes and depletion of the intermediate filament protein attenuated cell cycle re‐entry. Thus, nestin expressed by pre‐existing cardiomyocytes following ischemic damage recapitulated in part an embryonic phenotype and may provide the requisite phenotype to initiate cell cycle re‐entry. However, the overt activation of the p38 MAPK pathway post‐MI may in part limit the appearance and inhibit the cell cycle re‐entry of nestin(+)‐cardiomyocytes. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T10:50:36.765274-05:
      DOI: 10.1002/jcp.25496
  • Neuronatin Protein in Health and Disease
    • Authors: Priyamvada M. Pitale; Wayne Howse, Marina Gorbatyuk
      Abstract: Neuronatin (NNAT) was first identified as a brain‐specific gene crucial for brain development. Over the years, NNAT has been studied in different developing and post‐developed tissues and organs. While NNAT manifests functional and structural similarities to the phospholamban gene, its physiological and pathological roles in healthy and diseased tissues have not been precisely identified. Ca2+ signaling, glucose transport, insulin secretion, and inflammation modulated at different pathological conditions have been proposed to be governed by NNAT. This review describes the current findings of cellular molecular pathways known to be modified concomitantly with an alteration in NNAT expression, and it highlights the need to conduct extensive investigation regarding the role of NNAT in health and disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T10:50:23.162398-05:
      DOI: 10.1002/jcp.25498
  • Tyrosine Residues Regulate Multiple Nuclear Functions of p54nrb
    • Authors: Ahn R. Lee; Wayne Hung, Ning Xie, Liangliang Liu, Leye He, Xuesen Dong
      Abstract: The non‐POU‐domain‐containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi‐functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site‐directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co‐immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein‐protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non‐specific binding affinity to anti‐phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co‐repression and RNA splicing in gene context‐dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co‐regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T10:45:26.08248-05:0
      DOI: 10.1002/jcp.25493
  • Glioblastoma Multiforme and Adult Neurogenesis in the
           Ventricular‐Subventricular Zone: A Review
    • Abstract: Brain cancers account for less than 1,5% of all new cancer cases reported in the United States each year. Due to their invasive and heterogeneous nature, in addition to their resistance to multimodal treatments, these tumors are usually fatal. Gliomas, and in particular high‐grade astrocytomas such as glioblastoma multiforme (GBM), are the most common and lethal primary tumors of the central nervous system. The median survival of most patients is less than one year after application of multimodal therapies. The question is why are these cancers so injurious' And above all, how is it possible for a so carefully orchestrated area like the brain to develop such tumors' This brings us to the study of glioma stem cells, their specialized niches (perivascular and hypoxic), and the neurogenic phenomena that takes place within the adult ventricular‐subventricular zone: a structure that lies at the intersection between brain development and gliomagenesis. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-26T15:30:24.649733-05:
      DOI: 10.1002/jcp.25502
  • Role of Thrombin in the Pathogenesis of Central Nervous System
           Inflammatory Diseases
    • Authors: Safieh Ebrahimi; Najme Jaberi, Amir Avan, Mikhail Ryzhikov, Mohammad Reza Keramati, Mohammad Reza Parizadeh, Seyed Mahdi Hassanian
      Abstract: Thrombin initiates proinflammatory signaling responses through activation of protease‐activated receptors (PARs) in in vitro and in vivo systems. Proinflammatory signaling function of thrombin increases secretion of proinflammatory cytokines and chemokines, triggers vascular permeability, promotes leukocyte migration, and induces adhesion molecule expression. Thrombin as a potent signaling molecule is strongly implicated in a number of proinflammatory disorders including severe sepsis, cancer, cardiovascular disease, and of special interest in this review neurodegenerative disorders. This review summarizes the role of thrombin in the pathogenesis of central nervous system (CNS) inflammatory diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), promoting greater understanding and clinical management of these diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-26T15:30:21.373423-05:
      DOI: 10.1002/jcp.25501
  • Quantitative Analysis of Endocytic Recycling of Membrane Proteins by
           Monoclonal Antibody‐Based Recycling Assays
    • Abstract: In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin‐dependent endocytosis, represented by the transferrin receptor, and by clathrin‐independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin‐dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC‐conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti‐Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody‐capture protocols. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-26T15:25:23.719938-05:
      DOI: 10.1002/jcp.25503
  • Cytoskeleton Aberrations in Alkaptonuric Chondrocytes
    • Authors: Michela Geminiani; Silvia Gambassi, Lia Millucci, Pietro Lupetti, Giulia Collodel, Lucia Mazzi, Bruno Frediani, Daniela Braconi, Barbara Mazzocchi, Marcella Laschi, Giulia Bernardini, Annalisa Santucci
      Abstract: Alkaptonuria (AKU) is an ultra‐rare autosomal genetic disorder caused by a defect in the activity of the enzyme homogentisate 1,2‐dioxygenase (HGD) that leads to the accumulation of homogentisic acid (HGA) and its oxidized product, benzoquinone acetic acid (BQA), in the connective tissues causing a pigmentation called “ochronosis”. The consequent progressive formation of ochronotic aggregates generate a severe condition of oxidative stress and inflammation in all the affected areas. Experimental evidences have also proved the presence of serum amyloid A (SAA) in several AKU tissues and it allowed classifying AKU as a secondary amyloidosis. Although AKU is a multisystemic disease, the most affected system is the osteoarticular one and articular cartilage is the most damaged tissue. In this work, we have analyzed for the first time the cytoskeleton of AKU chondrocytes by means of immunofluorescence staining. We have shown the presence of SAA within AKU chondrocytes and finally we have demonstrated the co‐localization of SAA with three cytoskeletal proteins: actin, vimentin and β‐tubulin. Furthermore, in order to observe the ultrastructural features of AKU chondrocytes we have performed TEM analysis, focusing on the Golgi apparatus structure and, to demonstrate that pigmented areas in AKU cartilage are correspondent to areas of oxidation, 4‐HNE presence has been evaluated by means of immunofluorescence. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-25T14:55:30.2404-05:00
      DOI: 10.1002/jcp.25500
  • MiR‐145 Regulates Lipogenesis in Goat Mammary Cells via Targeting
           INSIG1 and Epigenetic Regulation of Lipid‐Related Genes
    • Authors: Hui Wang; Huaiping Shi, Jun Luo, Yongqing Yi, Dawei Yao, Xueying Zhang, Gongzhen Ma, Juan J. Loor
      Abstract: MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post‐transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid‐lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs and 821 pre‐miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR‐145) had significant differences in expression over the lactation cycle. Further studies revealed that miR‐145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR‐145. Overexpression of miR‐145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR‐145 impaired fatty acid synthesis. Inhibition of miR‐145 increased methylation levels of fatty acid synthase (FASN), stearoyl‐CoA desaturase 1 (SCD1), peroxisome proliferator‐activated receptor gamma (PPARG), and sterol regulatory element binding transcription factor 1 (SREBF1). Luciferase reporter assays confirmed that insulin induced gene 1 (INSIG1) is a direct target of miR‐145. These findings underscore the need for further studies to evaluate the potential for targeting miR‐145 for improving beneficial milk components in ruminant milk. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-22T16:10:27.305623-05:
      DOI: 10.1002/jcp.25499
  • BAG3 Protein Is Over‐ Expressed in Endometrioid Endometrial
    • Authors: Veronica Esposito; Carlo Baldi, Pio Zeppa, Michelina Festa, Luana Guerriero, Morena d'Avenia, Massimiliano Chetta, Fulvio Zullo, Vincenzo De Laurenzi, Maria Caterina Turco, Alessandra Rosati, Maurizio Guida
      Abstract: Endometrioid adenocarcinomas represent 80% of endometrial carcinomas1. Molecular features, including microsatellite instability, mutations of the PTEN, PIK3CA, K‐Ras and β‐catenin genes1 and dysregulations in sncRNAs (small non coding RNAs) are described for this disease. However, mechanisms and molecules that determine cell survival and response/resistance to therapy in different subtypes of this tumour are not fully clarified1. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-22T05:50:31.02862-05:0
      DOI: 10.1002/jcp.25489
  • 3‐Methylcholanthrene/Aryl‐Hydrocarbon Receptor–Mediated
           Hypertension through eNOS Inactivation
    • Abstract: Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)‐dependent pathway. We previously reported that 3‐methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3‐MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt‐mediated mechanism. The mechanism underlying the effects of 3‐MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3‐MC modified heat shock protein 90 (HSP90), caveolin‐1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA‐dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3‐MC reduced eNOS phosphorylation through the AhR/RhoA‐mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3‐MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin‐1. Immunofluorescence and western blot analysis revealed that 3‐MC reduced the amount of membrane‐bound activated eNOS, and a modified Griess assay revealed that 3‐MC concomitantly reduced NO production. However, simvastatin reduced 3‐MC–mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3‐MC. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-21T11:30:22.668827-05:
      DOI: 10.1002/jcp.25497
  • Atrogin‐1 Increases Smooth Muscle Contractility through Myocardin
    • Abstract: Atrogin‐1, an E3 ligase present in skeletal, cardiac and smooth muscle, down‐regulates myocardin protein during skeletal muscle differentiation. Myocardin, the master regulator of smooth muscle cell (SMC) differentiation, induces expression of smooth muscle marker genes through its association with serum response factor (SRF), which binds to the CArG box in the promoter. Myocardin undergoes ubiquitylation and proteasomal degradation. Evidence suggests that proteasomal degradation of myocardin is critical for myocardin to exert its transcriptional activity, but there is no report about the E3 ligase responsible for myocardin ubiquitylation and subsequent transactivation. Here, we showed that overexpression of atrogin‐1 increased contractility of cultured SMCs and mouse aortic tissues in organ culture. Overexpression of dominant‐negative myocardin attenuated the increase in SMC contractility induced by atrogin‐1. Atrogin‐1 overexpression increased expression of the SM contractile markers while downregulated expression of myocardin protein but not mRNA. Atrogin‐1 also ubiquitylated myocardin for proteasomal degradation in vascular SMCs. Deletion studies showed that atrogin‐1 directly interacted with myocardin through its amino acids 284‐345. Immunostaining studies showed nuclear localization of atrogin‐1, myocardin and the Rpt6 subunit of the 26S proteasome. Atrogin‐1 overexpression not only resulted in degradation of myocardin but also increased recruitment of RNA Polymerase II onto the promoters of myocardin target genes. In summary, our results have revealed the roles for atrogin‐1 in the regulation of smooth muscle contractility through enhancement of myocardin ubiquitylation/degradation and its transcriptional activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-21T11:20:36.573109-05:
      DOI: 10.1002/jcp.25485
  • Can Cholesterol Metabolism Modulation Affect Brain Function and
    • Authors: Veronica Cartocci; Michela Servadio, Viviana Trezza, Valentina Pallottini
      Abstract: Cholesterol is an important component for cell physiology. It regulates the fluidity of cell membranes and determines the physical and biochemical properties of proteins. In the central nervous system, cholesterol controls synapse formation and function and supports the saltatory conduction of action potential. In recent years, the role of cholesterol in the brain has caught the attention of several research groups since a breakdown of cholesterol metabolism has been associated with different neurodevelopmental and neurodegenerative diseases, and interestingly also with psychiatric conditions. The aim of this review is to summarize the current knowledge about the connection between cholesterol dysregulation and various neurologic and psychiatric disorders based on clinical and preclinical studies. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-20T12:06:03.33063-05:0
      DOI: 10.1002/jcp.25488
  • Vimentin as a Marker of Early Differentiating, Highly Motile Corneal
           Epithelial Cells
    • Abstract: Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial‐Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal‐like derivatives. Based on previous results showing that epithelial cells co‐express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co‐expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+/Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4‐5 layered epithelium, while there was a concomitant increase of both Pax‐6 expression and Krt+/Vim‐ cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co‐linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+/Krt+/ΔNp63α+/Pax‐6low/α6 integrin+ phenotype. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-20T12:01:04.868486-05:
      DOI: 10.1002/jcp.25487
  • Expression of P2 Purinergic Receptors in Mesenchymal Stem Cells and their
           Roles in Extracellular Nucleotide Regulation of Cell Functions
    • Abstract: Extracellular ATP and other nucleotides induce autocrine and/or paracrine purinergic signalling via activation of the P2 receptors on the cell surface, which represents one of the most common signalling mechanisms. Mesenchymal stem cells (MSC) are a type of multipotent adult stem cells that have many promising applications in regenerative medicine. There is increasing evidence to show that extracellular nucleotides regulate MSC functions and P2 receptor‐mediated purinergic signalling plays an important role in such functional regulation. P2 receptors comprise ligand‐gated ion channel P2X receptors and G‐protein‐coupled P2Y receptors. In this review, we provide an overview of the current understanding with respect to expression of the P2X and P2Y receptors in MSC and their roles in mediating extracellular nucleotide regulation of MSC proliferation, migration and differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-20T12:00:41.476616-05:
      DOI: 10.1002/jcp.25484
  • The β2‐Adrenoceptor Agonist Terbutaline Stimulates Angiogenesis
           via Akt and ERK Signaling
    • Authors: Stefanie Lemmens; Lauren Kusters, Annelies Bronckaers, Nathalie Geurts, Sven Hendrix
      Abstract: Angiogenesis is associated with changes in endothelial cell (EC) proliferation and tube formation, controlled by extracellular receptor‐activated kinase (ERK)/mitogen activated protein kinase (MAPK) and Akt signaling. Important regulators of these systems include hormones acting on G‐protein‐coupled receptors, such as beta 2‐adrenoceptors (β2‐ARs). In central nervous system (CNS) trauma, the importance of β2‐AR modulation has been highlighted, although the effects on revascularization remain unclear. Vascular protection and revascularization are, however, key to support regeneration. We have investigated the angiogenic capacity of the specific β2‐AR agonist terbutaline on ECs derived from the CNS, namely bEnd.3‐cells. As angiogenesis is a multistep process involving increased proliferation and tube formation of ECs, we investigated the effects of terbutaline on these processes. We show that terbutaline significantly induced bEnd.3 tube formation in a matrigel in vitro assay. Moreover, administration of specific inhibitors of ERK and Akt signaling both inhibited terbutaline‐induced tube formation. The proliferation rate of the ECs was not affected. In order to investigate the general effects of terbutaline in an organotypic system, we have used the chick chorioallantoic membrane (CAM)‐assay. Most importantly, terbutaline increased the number of blood vessels in this in ovo setting. Although we observed a positive trend, the systemic administration of terbutaline did not significantly improve the functional outcome, nor did it affect revascularization in our spinal cord injury model. In conclusion, these data indicate that terbutaline is promising to stimulate blood vessel formation, underscoring the importance of further research into the angiotherapeutic relevance of terbutaline and β2‐AR signaling after CNS‐trauma. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-20T11:50:35.977902-05:
      DOI: 10.1002/jcp.25483
  • Extracellular Proteasomes are Deficient in 19S Subunits as Revealed by
           iTRAQ Quantitative Proteomics
    • Authors: Anna S. Tsimokha; Julia J. Zaykova, Andrew Bottrill, Nikolai A. Barlev
      Abstract: Proteasome‐mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well‐documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ‐based quantitative proteomics (iTRAQ LC/MS‐MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. 62 of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP‐PIPs were associated with protein biosynthesis and, unlike CP‐PIPs, were under represented by chaperon/ATP‐binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-19T05:05:44.685377-05:
      DOI: 10.1002/jcp.25492
  • Erratum: ATP‐Sensitive Potassium Channel: A Novel Target for
           Protection Against UV‐Induced Human Skin Cell Damage by Cong Cao,
           Sarah Healey, Ashley Amaral, Avery Lee‐Couture, Shu Wan, Nicola
           Kouttab, Wenming Chu and Yinsheng Wan
    • PubDate: 2016-07-14T10:30:44.777224-05:
      DOI: 10.1002/jcp.25480
  • DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro
    • Authors: Li Duan; Yujie Liang, Bin Ma, Daming Wang, Wei Liu, Jianghong Huang, Jianyi Xiong, Liangquan Peng, Jielin Chen, Weimin Zhu, Daping Wang
      Abstract: DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full ‐scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome‐wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5‐azacytidine (5‐AzaC). This research revealed that the general methylation level of CpG was increased while the COL‐1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5‐AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL‐1A1 promoter was increased after 5‐AzaC treatment. The COL‐1A1 expression level was increased while that of SOX‐9 was decreased during the chondrocyte dedifferentiation. 5‐AzaC treatment up‐regulated the SOX‐9 expression while down‐regulated the COL‐1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage‐specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-12T16:26:00.893453-05:
      DOI: 10.1002/jcp.25486
  • Combined Multidimensional Microscopy as a Histopathology Imaging Tool
    • Authors: Gerald J. Shami; Delfine Cheng, Filip Braet
      Abstract: Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X‐ray micro‐computed tomography, bright‐field light microscopy and backscattered scanning electron microscopy, thus facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterise various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high‐resolution backscattered scanning electron microscopy, we were able to further characterise the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-09T09:15:35.773428-05:
      DOI: 10.1002/jcp.25470
  • Evidence for the Role of BAG3 in Mitochondrial Quality Control in
    • Authors: Farzaneh G. Tahrir; Tijana Knezevic, Manish K. Gupta, Jennifer Gordon, Joseph Y. Cheung, Arthur M. Feldman, Kamel Khalili
      Abstract: Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co‐chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates expression of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-06T06:50:22.924349-05:
      DOI: 10.1002/jcp.25476
  • Biochemical and Pathophysiological Premises to Positron Emission
           Tomography with Choline Radiotracers
    • Authors: Vincenzo Cuccurullo; Giuseppe Danilo Di Stasio, Laura Evangelista, Gabriella Castoria, Luigi Mansi
      Abstract: Choline is a quaternary ammonium base that represents an essential component of phospholipids and cell membranes. Malignant transformation is associated with an abnormal choline metabolism at a higher levels with respect to those exclusively due to cell multiplication. The use of Positron Emission Tomography/Computed Tomography (PET/CT) with radiocholine (RCH), labeled with 11C or 18F, is widely diffuse in oncology, with main reference to restaging of patients with prostate cancer. The enhanced concentration in neoplasm is based not only on the increasing growing rate, but also on more specific issues, such as the augmented uptake in malignant cells due to the up‐regulation of choline kinase. Furthermore the role of hypoxia in decreasing choline's uptake determine an in vivo concentration only in well oxygenated tumors, with a lower uptake when malignancy increases, i.e. in tumors positive at 18F‐Fluoro‐deoxyglucose. In this paper we have analyzed the most important issues related to the possible utilization of RCH in diagnostic imaging of human cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-06T06:46:59.668408-05:
      DOI: 10.1002/jcp.25478
  • The Effects of Acute and Chronic Exercise on Skeletal Muscle Proteome
    • Abstract: Skeletal muscle plasticity and its adaptation to exercise is a topic that is widely discussed and investigated due to its primary role in the field of exercise performance and health promotion. Repetitive muscle contraction through exercise stimuli leads to improved cardiovascular output and the regulation of endothelial dysfunction and metabolic disorders such as insulin resistance and obesity. Considerable improvements in proteomic tools and data analysis have broth some new perspectives in the study of the molecular mechanisms underlying skeletal muscle adaptation in response to physical activity. In this sense, this review updates the main relevant studies concerning muscle proteome adaptation to acute and chronic exercise, from aerobic to resistance training, as well as the proteomic profile of natural inbred high running capacity animal models. Also, some promising prospects in the muscle secretome field are presented, in order to better understand the role of physical activity in the release of extracellular microvesicles and myokines activity. Thus, the present review aims to update the fast‐growing exercise‐proteomic scenario, leading to some new perspectives about the molecular events under skeletal muscle plasticity in response to physical activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-06T06:46:56.937592-05:
      DOI: 10.1002/jcp.25477
  • Multifaceted Breast Cancer: The Molecular Connection with Obesity
    • Authors: Feola Antonia; Ricci Serena, Kouidhi Soumaya, Rizzo Antonietta, Penon Antonella, Formisano Pietro, Giordano Antonio, Di Carlo Angelina, Di Domenico Marina
      Abstract: Obesity is characterized by a disruption in energy balance regulation that results in an excess accumulation of body fat. Its increasing prevalence poses a major public health concern because it is a risk factor for a host of additional chronic conditions, including type 2 diabetes, hypertension, and cardiovascular disease. Obesity is increasingly recognized as a growing cause of cancer risk. In particular excessive adipose expansion during obesity causes adipose dysfunction and inflammation that can regulate tumor growth. In obesity, dysregulated systemic metabolism and inflammation induce hyperinsulinemia, hyperglycemia, dyslipidemia and enhance sex hormone production with increased secretion of proinflammatory adipokine that impact breast cancer development and progression. This review describes how adipose inflammation that characterizes obesity is responsible of microenvironment to promote cancer, and discuss how steroid hormones, that are essential for the maintenance of the normal development, growth and differentiation of the cells, influence the induction and progression of breast cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-01T05:36:16.572172-05:
      DOI: 10.1002/jcp.25475
  • An Innovative Assay for the Analysis of In Vitro Endothelial Remodeling:
           Experimental and Computational Evidence
    • Authors: Marco Scianna; Eleonora Bassino, Luca Munaron
      Abstract: The molecular and cellular mechanisms underlying vascular remodeling are currently investigated several experimental strategies which aim to mimic the complex environmental conditions found in vivo. Some of them focus on the tubulogenic activity of dispersed endothelial cell populations, while others evaluate vascular sprouting. Here we propose a new method to assess matrigel invasion starting from confluent or subconfluent monolayers of human microvascular ECs (HMVEC) seeded on different substrates. The experimental setting is also validated by an improved hybrid multiscale mathematical approach, which integrates a mesoscopic grid‐based cellular Potts model, that describes HMVEC phenomenology, with a continuous one, accounting for the kinetics of diffusing growth factors. Both experimental and theoretical approaches show that the endothelial potential to invade, migrate and organize in tubule structures is a function of selected environmental parameters. The present methodology is intended to be simple to use, standardized for rapid screening and suitable for mechanistic studies. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-30T10:30:30.870964-05:
      DOI: 10.1002/jcp.25468
  • microRNA‐30b Suppresses Epithelial‐Mesenchymal Transition and
           Metastasis of Hepatoma Cells
    • Authors: Xiaolin Sun; Shuhua Zhao, Huanan Li, Hanwen Chang, Zhen Huang, Zhi Ding, Lei Dong, Jiangning Chen, Yuhui Zang, Junfeng Zhang
      Abstract: Epithelial–mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis in HCC. Growing evidence indicates that upregulation of Snail, the major EMT inducer, significantly correlates with the metastasis and poor prognosis of HCC. Here, we investigate the underlying mechanism of miR‐30b in suppressing metastasis of hepatoma cells by targeting Snail. In this study, we found that miR‐30b was significantly downregulated and negatively associated with Snai1 production in HCC cell lines with higher metastatic potentials. Gain‐ and loss‐of‐function studies revealed that miR‐30b could dramatically inhibit in vitro HCC cell migration and invasion. In vivo orthotopic liver xenograft model further demonstrated that stable over‐expression of miR‐30b significantly repressed the local invasion and lung metastasis of hepatoma cells. Meanwhile, the restoration of miR‐30b expression suppressed the distant colonization of hepatoma cells. Both gain‐ and loss‐of‐function studies showed that miR‐30b suppressed the EMT of hepatoma cells as indicated by the morphology changes and deregulation of epithelial and mesenchymal markers. Using RNAi, we further investigated the role of Snai1 in HCC cell EMT and demonstrated that knockdown of Snai1 significantly inhibited the EMT and cancer cell metastasis. Additionally, miR‐30b exhibited inhibitory effects on HCC cell proliferation in vitro and in vivo. In conclusion, our findings highlight the significance of miR‐30b downregulation in HCC tumor metastasis and invasiveness, and implicate a new potential therapeutic target for HCC metastasis. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-30T10:25:27.648539-05:
      DOI: 10.1002/jcp.25466
  • Emerging from the Unknown: Structural and Functional Features of
           Agnoprotein of Polyomaviruses by A. Sami Saribas, Pascale Coric, Anahit
           Hamazaspyan, William Davis, Rachael Axman, Martyn K. White, Magid
           Abou‐Gharbia, Wayne Childers, Jon H. Condra, Serge Bouaziz and
           Mahmut Safak
    • PubDate: 2016-06-30T10:20:40.396414-05:
      DOI: 10.1002/jcp.25465
  • Parathyroid Hormone‐Related Protein Protects Osteoblastic Cells from
           Oxidative Stress by Activation of MKP1 Phosphatase
    • Abstract: Oxidative damage is an important contributor to the morphological and functional changes in osteoporotic bone. Aging increases the levels of reactive oxygen species (ROS) that cause oxidative stress and induce osteoblast apoptosis. ROS modify several signaling responses, including mitogen‐activated protein kinase (MAPK) activation, related to cell survival. Both parathyroid hormone (PTH) and its bone counterpart, PTH‐related protein (PTHrP), can regulate MAPK activation by modulating MAPK phosphatase‐1 (MKP1). Thus, we hypothesized that PTHrP might protect osteoblasts from ROS‐induced apoptosis by targeting MKP1. In osteoblastic MC3T3‐E1 and MG‐63 cells, H2O2 triggered p38, JNK, ERK and p66Shc phosphorylation and cell apoptosis. Meanwhile, PTHrP (1‐37) rapidly but transiently increased ERK and Akt phosphorylation without affecting p38, JNK or p66Shc activation. H2O2‐induced p38 and ERK phosphorylation and apoptosis were both decreased by pre‐treatment with specific kinase inhibitors or PTHrP (1‐37) in both osteoblastic cell types. These dephosphorylating and prosurvival actions of PTHrP (1‐37) were prevented by a phosphatase inhibitor cocktail, the phosphatase MKP1 inhibitor sanguinarine or a MKP1 siRNA. PTHrP (1‐37) promptly enhanced MKP1 protein and gene expression and MKP1‐dependent catalase activity in osteoblastic cells. Furthermore, exposure to PTHrP (1‐37) adsorbed in an implanted hydroxyapatite‐based ceramic into a tibial defect in aging rats increased MKP1 and catalase gene expression in the healing bone area. Our findings demonstrate that PTHrP counteracts the pro‐apoptotic actions of ROS by a mechanism dependent on MKP1‐induced dephosphorylation of MAPKs in osteoblasts. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-30T06:25:27.24047-05:0
      DOI: 10.1002/jcp.25473
  • Transcriptomic Analyses of Adipocyte Differentiation from Human
           Mesenchymal Stromal‐Cells (MSC)
    • Abstract: Adipogenesis is a physiological process required for fat‐tissue development, mainly involved in regulating the organism energetic‐state. Abnormal distribution‐changes and dysfunctions in such tissue are associated to different pathologies. Adipocytes are generated from progenitor cells, via a complex differentiating process not yet well understood. Therefore, we investigated differential mRNA and miRNA expression patterns of human mesenchymal stromal‐cells (MSC) induced and not induced to differentiate into adipocytes by next (second)‐generation sequencing. A total of 2,866 differentially‐expressed genes (101 encoding miRNA) were identified, with 705 (46 encoding miRNA) being upregulated in adipogenesis. They were related to different pathways, including PPARG, lipid, carbohydrate and energy metabolism, redox, membrane‐organelle biosynthesis and endocrine system. Downregulated genes were related to extracellular matrix and cell migration, proliferation and differentiation. Analyses of mRNA‐miRNA interaction showed that repressed miRNA‐encoding genes can act downregulating PPARG‐related genes; mostly the PPARG activator (PPARGC1A). Induced miRNA‐encoding genes regulate downregulated genes related to TGFB1. These results shed new light to understand adipose‐tissue differentiation and physiology, increasing our knowledge about pathologies like obesity, type‐2 diabetes and osteoporosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-28T04:17:05.839447-05:
      DOI: 10.1002/jcp.25472
  • Integrated Transcriptome Map Highlights Structural and Functional Aspects
           of the Normal Human Heart
    • Authors: Maria Caracausi; Allison Piovesan, Lorenza Vitale, Maria Chiara Pelleri
      Abstract: A systematic meta‐analysis of the available gene expression profiling datasets for the whole normal human heart generated a quantitative transcriptome reference map of this organ. TRAM (Transcriptome Mapper) software integrated 32 gene expression profile datasets from different sources returning a reference value of expression for each of the 43,360 known, mapped transcripts assayed by any of the experimental platforms used in this regard. Main findings include the visualization at the gene and chromosomal levels of the classical description of the basic histology and physiology of the heart, the identification of suitable housekeeping reference genes, the analysis of stoichiometry of gene products and the focusing on chromosome 21 genes, which are present in one excess copy in Down syndrome subjects, presenting cardiovascular defects in 30‐40% of cases. Independent in vitro validation showed an excellent correlation coefficient (r = 0.98) with the in silico data. Remarkably, heart/non‐cardiac tissues expression ratio may also be used to anticipate that effects of mutations will most probably affect or not the heart. The quantitative reference global portrait of gene expression in the whole normal human heart illustrates the structural and functional aspects of the whole organ and is a general model to understand the mechanisms underlying heart pathophysiology. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-27T06:55:22.777055-05:
      DOI: 10.1002/jcp.25471
  • SCD1 Alters Long‐Chain Fatty Acid (LCFA) Composition and its
           Expression is Directly Regulated by SREBP‐1 and PPARγ 1 in
           Dairy Goat Mammary Cells
    • Authors: Dawei Yao; Jun luo, Qiuya He, Hengbo Shi, Jun Li, Hui Wang, Huifen Xu, Zhi Chen, Yongqing Yi, Juan J. Loor
      Abstract: Stearoyl‐CoA desaturase 1 (SCD1) is a key enzyme for the synthesis of the monounsaturated fatty acids (MUFA) palmitoleic acid and oleic acid. In non‐ruminant species, SCD1 expression is known to be tightly regulated by a variety of transcription factors. Although the role of SCD1 and the transcriptional regulatory mechanism by SREBP‐1 and PPARs in other species is clear, changes in lipid metabolism related to SCD1 and via the regulation of SREBP‐1 or PPARG1 in ruminant mammary tissue remain largely unknown. Here, we demonstrated that SCD1 expression in goat mammary tissue is higher during lactation than the dry period. Overexpression of SCD1 increased the intracellular MUFA content and lipid accumulation, whereas SCD1 silencing resulted in a significant decrease in oleic acid concentration and triacylglycerol (TAG) accumulation. The overexpression of SREBF1 in goat mammary epithelial cells (GMEC) enhanced SCD1 expression and its promoter activity, but that effect was abolished when SREBF1 was silenced. Furthermore, deletion of sterol regulatory element (SRE) and the nuclear factor (NF‐Y) binding sites within a ‐1713∼ + 65‐base pair region of the SCD1 promoter completely abolished SREBP‐1‐induced SCD1 transcription. Otherwise, PPARG1 overexpression also stimulated the expression of SCD1 and its transcriptional activity directly via a PPAR response element (PPRE) in the SCD1 promoter. Together, these results indicate that SCD1 could markedly affect the fatty acid composition and rate of TAG synthesis through direct regulation via SREBP‐1 and PPARG1, hence, underscoring an important role of the enzyme and this transcription regulator in controlling mammary gland lipid synthesis in the goat. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-24T08:10:24.186014-05:
      DOI: 10.1002/jcp.25469
  • HDAC1, HDAC4 and HDAC9 Bind to PC3/Tis21/Btg2 and Are Required for its
           Inhibition of Cell Cycle Progression and Cyclin D1 Expression
    • Authors: Laura Micheli; Giorgio D'Andrea, Luca Leonardi, Felice Tirone
      Abstract: PC3/Tis21 is a transcriptional cofactor that inhibits proliferation in several cell types, including neural progenitors. Here we report that PC3/Tis21 associates with HDAC1, HDAC4 and HDAC9 in vivo, in fibroblast cells. Furthermore, when HDAC1, HDAC4 or HDAC9 are silenced in fibroblasts or in a line of cerebellar progenitor cells, the ability of PC3/Tis21 to inhibit proliferation is significantly reduced. Overexpression of HDAC1, HDAC4 or HDAC9 in fibroblasts and in cerebellar precursor cells synergizes with PC3/Tis21 in inhibiting the expression of cyclin D1, a cyclin selectively inhibited by PC3/Tis21. Conversely, the depletion of HDAC1 or HDAC4 (but not HDAC9) in fibroblasts and in cerebellar precursor cells significantly impairs the ability of PC3/Tis21 to inhibit cyclin D1 expression. An analysis of HDAC4 deletion mutants shows that both the amino‐ terminal moiety and the catalytic domain of HDAC4 associate to PC3/Tis21, but neither alone is sufficient to potentiate the inhibition of cyclin D1 by PC3/Tis21. As a whole, our findings indicate that PC3/Tis21 inhibits cell proliferation in a way dependent on the presence of HDACs, in fibroblasts as well as in neural cells. Considering that several reports have demonstrated that HDACs can act as transcriptional corepressors on the cyclin D1 promoter, our data suggest that the association of PC3/Tis21 to HDACs is functional to recruit them to target genes, such as cyclin D1, for repression of their expression. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-23T06:55:33.829497-05:
      DOI: 10.1002/jcp.25467
  • Further Support for ECM Control of Receptor Trafficking and Signaling
    • Authors: Lindsay Clegg; Feilim Mac Gabhann
      Abstract: Recently, Sack et al. presented an interesting, novel data set in JCP examining the effect of substrate stiffness on VEGF processing and signaling. The data represents a clear contribution to the field. However, the authors' conclusion that “extracellular matrix binding is essential for VEGF internalization” conflicts with other knowledge in the field, and is not supported by their data. Instead, their data demonstrates the effect of heparin addition and changing ECM stiffness on both VEGF binding to fibronectin and VEGF binding to endothelial receptors. This is consistent with other work showing that matrix‐ binding reduces VEGF‐VEGFR internalization, shifting downstream signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-20T09:45:37.567799-05:
      DOI: 10.1002/jcp.25464
  • DGKζ Downregulation Enhances Osteoclast Differentiation and Bone
           Resorption Activity Under Inflammatory Conditions
    • Authors: Kiyoshi Iwazaki; Toshiaki Tanaka, Yasukazu Hozumi, Masashi Okada, Rieko Tsuchiya, Ken Iseki, Matthew K. Topham, Kaneyuki Kawamae, Michiaki Takagi, Kaoru Goto
      Abstract: Bone homeostasis is maintained by a balance between resorption of the bone matrix and its replacement by new bone. Osteoclasts play a crucially important role in bone metabolism. They are responsible for bone resorption under pathophysiological conditions. Differentiation of these cells, which are derived from bone marrow cells, depends on receptor activator of NF‐κB ligand (RANKL). RANKL‐induced osteoclastogenesis is regulated by the phosphoinositide (PI) signaling pathway, in which diacylglycerol (DG) serves as a second messenger in signal transduction. In this study, we examined the functional implications of DG kinase (DGK), an enzyme family responsible for DG metabolism, for osteoclast differentiation and activity. Of DGKs, DGKζ is most abundantly expressed in osteoclast precursors such as bone marrow‐derived monocytes/macrophages. During osteoclast differentiation from precursor cells, DGKζ is downregulated at the protein level. In this regard, we found that DGKζ deletion enhances osteoclast differentiation and bone resorption activity under inflammatory conditions in an animal model of osteolysis. Furthermore, DGKζ deficiency upregulates RANKL expression in response to TNFα stimulation. Collectively, results suggest that DGKζ is silent under normal conditions, but it serves as a negative regulator in osteoclast function under inflammatory conditions. Downregulation of DGKζ might be one factor predisposing a person to osteolytic bone destruction in pathological conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-17T05:50:26.946779-05:
      DOI: 10.1002/jcp.25461
  • Pancreatic Endoderm‐Derived from Diabetic Patient‐Specific
           Induced Pluripotent Stem Cell Generates Glucose‐Responsive
           Insulin‐Secreting Cells
    • Authors: Bahareh Rajaei; Mehdi Shamsara, Masume Fakhr Taha, Mohammad Massumi, Mohammad Hossein Sanati
      Abstract: Human induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient‐ and disease‐specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin‐producing pancreatic β‐cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five‐step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose‐responsive insulin‐producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut‐tube endoderm, posterior foregut, pancreatic endoderm and endocrine precursor. Each stage of differentiation were characterized by stage‐specific markers. The produced cells exhibited many properties of functional β‐cells, including expression of critical β‐cells transcription factors, the potency to secrete C‐peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin‐secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs‐derived glucose‐responsive insulin‐secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-16T06:20:33.930205-05:
      DOI: 10.1002/jcp.25459
  • FGF23 Neutralizing Antibody Ameliorates Hypophosphatemia and Impaired FGF
           Receptor Signaling in Kidneys of HMWFGF2 Transgenic Mice
    • Authors: E. Du; L. Xiao, M. M. Hurley
      Abstract: High molecular weight FGF2 transgenic mice (HMWTg) phenocopy the Hyp mouse, homolog of human X‐linked hypophosphatemic rickets with phosphate wasting and abnormal fibroblast growth factor (FGF23), fibroblast growth factor receptor (FGFR), Klotho and mitogen activated protein kinases (MAPK) signaling in kidney. In this study, we assessed whether short term (24 hour) in vivo administration of FGF23 neutralizing antibody (FGF23Ab) could rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg male mice. Bone mineral density and bone mineral content in one‐month‐old HMWTg mice were significantly reduced compared with Control/VectorTg mice. Serum FGF23 was significantly increased in HMWTg compared with VectorTg. Serum phosphate was significantly reduced in HMWTg and was rescued by FGF23Ab. Serum parathyroid hormone (PTH) was significantly increased in HMWTg but was not reduced by FGF23Ab. 1, 25(OH)2 D was inappropriately normal in serum of HMWTg and was significantly increased in both Vector and HMWTg by FGF23Ab. Analysis of HMWTg kidneys revealed significantly increased mRNA expression of the FGF23 co‐receptor Klotho, transcription factor mRNAs for early growth response‐1 transcription factor (Egr‐1) and c‐fos were all significantly decreased by FGF23Ab. A significant reduction in the phosphate transporter Npt2a mRNA was also observed in HMWTg kidneys, which was increased by FGF23Ab. FGF23Ab reduced p‐FGFR1, p‐FGFR3, KLOTHO, p‐ERK1/2, C‐FOS and increased NPT2A protein in HMWTg kidneys. We conclude that FGF23 blockade rescued hypophosphatemia by regulating FGF23/FGFR downstream signaling in HMWTg kidneys. Furthermore, HMWFGF2 isoforms regulate PTH expression independent of FGF23/FGFR signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-16T06:15:25.784878-05:
      DOI: 10.1002/jcp.25458
  • Ethanol Extract of Cissus quadrangularis Enhances Osteoblast
           Differentiation and Mineralization of Murine Pre‐Osteoblastic
           MC3T3‐E1 Cells
    • Abstract: Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti‐osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre‐osteoblast cell line, MC3T3‐E1. Ethanolic extract of CQ (CQ‐E) was found to affect growth kinetics of MC3T3‐E1 cells in a dosage dependent manner. High concentrations of CQ‐E (more than 10 µg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non‐toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ‐E. CQ‐E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose‐dependent effect of CQ‐E with lower concentrations exhibiting anabolic and osteogenic properties. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:47.466708-05:
      DOI: 10.1002/jcp.25449
  • Transcriptome‐Based Analysis of Molecular Pathways for Clusterin
           Functions in Kidney Cells
    • Abstract: Clusterin (CLU) is a chaperone‐like protein and plays a protective role against renal ischemia‐reperfusion injury (IRI); however, the molecular pathways for its functions in the kidney are not fully understood. This study was designed to investigate CLU‐mediating pathways in kidney cells by using bioinformatics analysis. CLU null renal tubular epithelial cells (TECs) expressing human CLU cDNA (TEC‐CLUhCLU) or empty vector (TEC‐CLU‐/‐) were exposed to normoxia or hypoxia (1% O2). Transcriptome profiling with a significant 2‐fold change was performed using SurePrint G3 Mouse Gene Expression 8 × 60K microarray, and the signaling pathways was ranked by using Ingenuity pathway analysis. Here, we showed that compared to CLU null controls, ectopic expression of human CLU in CLU null kidney cells promoted cell growth but inhibited migration in normoxia, and enhanced cell survival in hypoxia. CLU expression affected expression of 3864 transcripts (1893 up‐regulated) in normoxia and 3670 transcripts (1925 up‐regulated) in hypoxia. CLU functions in normoxia were associated mostly with AKT2/PPP2R2B‐dependent PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling and as well with GSK3B‐mediated cell cycle progression. In addition to unfolded protein response (UPR) and/or endoplasmic reticulum (ER) stress, CLU‐enhanced cell survival in hypoxia was also associated with PIK3CD/MAPK1‐dependent PI3K/AKT, HIF‐α, PTEN, VEGF and ERK/MAPK signaling. In conclusion, our data showed that CLU functions in kidney cells were mainly mediated in a cascade manner by PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling, and specifically by activation of UPR/ER stress in hypoxia, providing new insights into the protective role of CLU in the kidney. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:40.582976-05:
      DOI: 10.1002/jcp.25415
  • MicroRNA‐590‐5p stabilizes Runx2 by targeting Smad7 during
           osteoblast differentiation
    • Authors: M. Vishal; S. Vimalraj, R. Ajeetha, M. Gokulnath, R. Keerthana, Z. He, N.C. Partridge, N. Selvamurugan
      Abstract: Mesenchymal stem cells (MSCs) are multipotent cells and their differentiation into the osteoblastic lineage is strictly controlled by several regulators, including microRNAs (miRNAs). Runx2 is a bone transcription factor required for osteoblast differentiation. Here, we used in silico analysis to identify a number of miRNAs that putatively target Runx2 and its co‐factors to mediate both positive and negative regulation of osteoblast differentiation. Among these miRNAs, miR‐590‐5p was selected and its expression was found to be increased during osteoblast differentiation. When mouse MSCs (mMSCs) were transiently transfected with a miR‐590‐5p mimic, we detected an increase in both calcium deposition and the mRNA expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP) and type I collagen genes. Smad7 was found to be among the putative target genes of miR‐590‐5p and its mRNA and protein expression decreased after miR‐590‐5p mimic transfection in human osteoblast‐like cells (MG63). Our analysis indicated that Runx2 was not a putative target of miR‐590‐5p. However, Runx2 protein, but not mRNA expression, increased after miR‐590‐5p mimic transfection in MG63 cells. Runx2 protein expression was increased with knockdown of Smad7 expression by Smad7 siRNA in these cells. We further identified that the 3′‐untranslated region of Smad7 was directly targeted by miR‐590‐5p; this was done using the luciferase reporter gene system. It is known that Smad7 inhibits osteoblast differentiation via Smurf2‐mediated Runx2 degradation. Hence, based on our results, we suggest that miR‐590‐5p promotes osteoblast differentiation by indirectly protecting and stabilizing the Runx2 protein by targeting Smad7 gene expression. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:34.725034-05:
      DOI: 10.1002/jcp.25434
  • Low‐Dose Farnesyltransferase Inhibitor Suppresses HIF‐1α
           and Snail Expression in Triple‐Negative Breast Cancer
           MDA‐MB‐231 Cells In Vitro
    • Abstract: The aggressiveness of triple‐negative breast cancer (TNBC), which lacks estrogen receptor, progesterone receptor and epidermal growth factor receptor 2 (HER2), represents a major challenge in breast cancer. Migratory and self‐renewal capabilities are integral components of invasion, metastasis and recurrence of TNBC. Elevated hypoxia‐inducible factor‐1α (HIF‐1α) expression is associated with aggressiveness of cancer. Nonetheless, how HIF‐1α expression is regulated and how HIF‐1α induces aggressive phenotype are not completely understood in TNBC. The cytotoxic effects of farnesyltransferase (FTase) inhibitors (FTIs) have been studied in cancer and leukemia cells. In contrast, the effect of FTIs on HIF‐1α expression has not yet been studied. Here, we show that clinically relevant low‐dose FTI, tipifarnib (300 nM), decreased HIF‐1α expression, migration and tumorsphere formation in human MDA‐MB‐231 TNBC cells under a normoxic condition. In contrast, the low‐dose FTIs did not inhibit cell growth and activity of the Ras pathway in MDA‐MB 231 cells. Tipifarnib‐induced decrease in HIF‐1α expression was associated with amelioration of the Warburg effect, hypermetabolic state, increases in Snail expression and ATP release, and suppressed E‐cadherin expression, major contributors to invasion, metastasis and recurrence of TBNC. These data suggest that FTIs may be capable of ameliorating the aggressive phenotype of TNBC by suppressing the HIF‐1α‐Snail pathway. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:28.989913-05:
      DOI: 10.1002/jcp.25411
  • Inhibition of mTOR Signaling Pathway Delays Follicle Formation in Mice
    • Authors: Jing Zhang; Wenwen Liu, Xinhui Sun, Feifei Kong, Ye Zhu, Yue Lei, Youqiang Su, Yiping Su, Jing Li
      Abstract: In mammalian ovaries, follicle assembly requires proper germ cell cyst breakdown and the invasion of somatic cells to encapsulate individual oocytes. Abnormalities in this process lead to a number of pathologies such as premature ovarian failure and infertility. As a conserved pathway regulating cell growth and metabolism in response to growth factors and nutrients, the roles of mTOR signaling in follicular development have been extensively studied in recent years. However, its functions during follicle formation remain unknown. In this study, the expression of p‐rpS6 (phospho‐ribosomal proteinS6), a downstream marker of mTORC1, showed dynamic changes in perinatal ovaries. When E18.5 ovaries, which mainly contained germ cell nests, were incubated with the mTOR inhibitors Rapamycin and Torin1 for 24 h, follicle assembly was delayed with differential somatic cell invasion into germ cell cyst among the groups. After transplanting treated or untreated ovaries into kidney capsules of recipient ovariectomized mice, follicular development was blocked in treated ovaries, as shown by fewer antral follicles and a higher proportion of primordial follicles. Further studies showed a significant decrease in somatic cell proliferation and the expression of marker genes related to follicular development (Kitl, Kit, Gdf9, Bmp15, Zp3, and Amhr2) in treated ovaries. Moreover, the addition of KITL, a growth factor that is mainly produced by pregranulosa cells during germ cell nest breakdown, rescued the extension of follicle formation induced by mTOR inhibitors. These results suggest that KITL functions downstream of mTOR in somatic cells to regulate their communication with oocytes during follicle formation. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T07:15:34.10707-05:0
      DOI: 10.1002/jcp.25456
  • Cholesterol and its Derivatives that Are Components of an Immune
           Stimulatory Drug Reversibly Inhibit Proteinase K
    • Authors: Namrata Singh; Debasish Bhattacharyya
      Abstract: Microorganisms express a variety of proteases that degrade many proteins of the host body and subvert host immune response. While elucidating the mechanism/s of an immune stimulatory drug that contains bile lipid, regulation of proteolytic activity was investigated. The drug and bile lipids both stabilize Proteinase K, an aggressive protease of fungal origin against auto‐digestion. Among the components of bile lipids, only cholesterol and its derivatives stabilize the enzyme. Biophysical evidences such as scattering of light, intrinsic and extrinsic fluorescence emission spectra, circular dichroism spectra, atomic force microscopy and transmission electron microscopy images indicated that cholesterol and its derivatives interact with Proteinase K. Inhibition kinetics using esterolysis of ATEE revealed non‐competitive inhibition by cholesterol. Surface Plasmon Resonance and mass spectrometric analysis indicated 1:1 stoichiometry of binding and with dissociation constant in the µM range. Further, the presence of four cholesterol recognition amino acid consensus motifs (CRAC motifs I‐IV) was identified in Proteinase K. Bioinformatics analysis revealed that the whole stretch of cholesterol interacts very well with the hydrophobic groove of motif II only among the four CRAC motifs. Variation of cholesterol content of HepG2 human liver carcinoma cells showed positive correlation with binding of fluorescence tagged Proteinase K. Under these conditions, binding of Proteinase K to the cells did not affect their morphology, viability and growth kinetics. Cell bound Proteinase K could be released by an excess of its substrate, thereby restoring reversibly its proteolytic activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T07:15:28.51616-05:0
      DOI: 10.1002/jcp.25457
  • Post‐Transcriptional Regulation of the Human Mu‐Opioid
           Receptor (MOR) by Morphine‐Induced RNA Binding Proteins hnRNP K and
    • Abstract: Expression of the mu‐opioid receptor (MOR) protein is controlled by extensive transcriptional and post‐transcriptional processing. MOR gene expression has previously been shown to be altered by a post‐transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)‐binding protein (hnRNP) K and poly(C)‐binding protein 1 (PCBP1) as post‐transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5‘‐untranslated region of human MOR mRNA. In addition, we also found that morphine‐induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine‐induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). This article is protected by copyright. All rights reserved
      PubDate: 2016-06-13T06:56:20.948968-05:
      DOI: 10.1002/jcp.25455
  • Affinity Selection of FGF2‐Binding Heparan Sulfates for Ex Vivo
           Expansion of Human Mesenchymal Stem Cells
    • Authors: Sampath Jeewantha Wijesinghe; Ling Ling, Sadasivam Murali, Hui Qing Yeong, Simon F.R. Hinkley, Susan M. Carnachan, Tracey J. Bell, Kunchithapadam Swaminathan, James H. Hui, Andre J. van Wijnen, Victor Nurcombe, Simon M. Cool
      Abstract: The future of human mesenchymal stem cells (hMSCs) as a successful cell therapy relies on bioprocessing strategies to improve the scalability of these cells without compromising their therapeutic ability. The culture‐expansion of hMSCs can be enhanced by supplementation with growth factors, particularly fibroblast growth factor 2 (FGF2). The biological activity of FGF2 is controlled through interactions with heparan sulfate (HS) that facilitates ligand‐receptor complex formation. We previously reported on an FGF2‐interacting HS variant (termed HS2) isolated in microgram amounts from embryonic tissue by anionic exchange chromatography that increased the proliferation and potency of hMSCs. Here we detail the isolation of gram amounts of an FGF2 affinity‐purified HS variant (HS8) using a scalable platform technology previously employed to generate HS variants with increased affinity for BMP‐2 or VEGF165. This process used a peptide sequence derived from the heparin‐binding domain of FGF2 as a substrate to affinity‐isolate HS8 from a commercially available source of porcine mucosal HS. Our data show that HS8 binds to FGF2 with higher affinity than to FGF1, FGF7, BMP2, PDGF‐BB, or VEGF165. Also, HS8 protects FGF2 from thermal destabilization and increases FGF signaling and hMSC proliferation through FGF receptor 1. Long‐term supplementation of cultures with HS8 increased both hMSC numbers and their colony‐forming efficiency without adversely affecting the expression of hMSC‐related surface antigens. This strategy further exemplifies the utility of affinity‐purifying HS variants against particular ligands important to the stem cell microenvironment and advocates for their addition as adjuvants for the culture‐expansion of hMSCs destined for cellular therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-13T06:56:16.469175-05:
      DOI: 10.1002/jcp.25454
  • Combination of Rapamycin and Resveratrol for Treatment of Bladder Cancer
    • Authors: Anya Alayev; Rachel S. Salamon, Naomi S. Schwartz, Adi Y. Berman, Sara L. Wiener, Marina K. Holz
      Abstract: Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin‐induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1‐/‐ MEF cells, and not in wild‐type MEFs. Similarly, resveratrol alone or in combination with rapamycin induced cell death in human bladder cancer cell lines. These data indicate that administration of resveratrol together with rapamycin may be a promising therapeutic option for treatment of bladder cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T10:01:48.493314-05:
      DOI: 10.1002/jcp.25443
  • Non‐Metabolic Role of PKM2 in Regulation of the HIV‐1 LTR
    • Authors: Satarupa Sen; Satish L. Deshmane, Rafal Kaminski, Shohreh Amini, Prasun K. Datta
      Abstract: Identification of cellular proteins, in addition to already known transcription factors such as NF‐κB, Sp1, C‐EBPβ, NFAT, ATF/CREB, and LEF‐1, which interact with the HIV‐1 LTR is critical in understanding the mechanism of HIV‐1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV‐1SF162 infection of monocyte/macrophages and reactivation of HIV‐1 in U1 cells, a macrophage model of latency. We observed that HIV‐1SF162 infection of monocyte/macrophages and reactivation of HIV‐1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA‐induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA‐induced U1 cells in comparison to PMA‐induced U937 cells. We focused on understanding the potential role of PKM2 in HIV‐1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA‐activated U1 and TZM‐ bl cells demonstrated the interaction of PKM2 with the HIV‐1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV‐1 LTR‐luciferase reporter in U937, U‐ 87 MG, and TZM‐bl cells. Using various truncated constructs of the HIV‐1 LTR, we mapped the region spanning ‐120 bp to ‐80 bp to be essential for PKM2‐mediated transactivation. This region contains the NF‐κB binding site and deletion of this site attenuated PKM2 mediated activation of HIV‐1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF‐κB. These observations demonstrate for the first time that PKM2 is a transcriptional co‐activator of HIV‐1 LTR. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T10:01:42.21665-05:0
      DOI: 10.1002/jcp.25445
  • Light Sheet Microscopy to Measure Protein Dynamics
    • Authors: Matthias Rieckher
      Abstract: Visualizing protein dynamics is the key to a quantitative understanding of molecular mechanisms in biological systems. Recent developments in fluorescent microscopy techniques allow for novel experimental approaches to study stochastic localization, distribution and movement of fluorescently labeled molecules in high resolution as they occur over time in the context of living cells and whole organisms. Particularly suitable for such studies is the application of light sheet microscopy, as it enables rapid in vivo imaging of a wide range and size of specimen, from whole organisms and organs, down to the dynamics of subcellular structures and single molecules. This article summarizes the principles of light sheet microscopy and its advantages over other optical imaging techniques, such as light microscopy and confocal microscopy, and highlights recent innovations that significantly enhance spatio‐temporal resolution. Also, this manuscript contains basic guidelines for the implementation of light sheet microscopy in the laboratory and presents thus far unpublished light sheet microscopy applications demonstrating the ability of this technology to measure protein dynamics in a whole‐organism context. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T01:31:54.123735-05:
      DOI: 10.1002/jcp.25451
  • Leucine‐Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast
           Requires Flotillin‐1 Mediated Endocytosis
    • Abstract: Basic, pre‐clinical and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine‐Rich Amelogenin Peptide ‐ LRAP), which is supposed to act as a signaling molecule in epithelial‐mesenchymal interactions. LRAP‐binding protein complexes from immortalized murine cementoblasts (OCCM‐30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin‐1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co‐precipitation and co‐localization assays as a protein‐binding partner for LRAP in OCCM‐30 cells. In addition, we found that exogenously added GST‐LRAP recombinant protein was internalized by OCCM‐30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1‐dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM‐30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp) and runt‐related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin‐assisted endocytosis, which suggests an involvement of LRAP in lipid‐raft‐dependent signaling pathways which are mediated by flotillin‐1. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:25:20.31246-05:0
      DOI: 10.1002/jcp.25453
  • Browning of White Fat: Novel Insight into Factors, Mechanisms and
    • Authors: Nevena Jeremic; Pankaj Chatuverdi, Suresh C. Tyagi
      Abstract: What is more interesting about brown adipose tissue (BAT) is its ability to provide thermogenesis, protection against obesity by clearing triglycerides and mitigating insulin resistance. White adipose tissue (WAT) on the other hand stores excess energy and secretes some endocrine factors like leptin for regulating satiety. For the last decade there has been an increasing interest in the browning of fat keeping in view its beneficial effects on metabolic disorders and protection in the form of perivascular fat. Obesity is one such metabolic disorder that leads to significant morbidity and mortality from obesity‐related disorders such as type 2 diabetes mellitus (T2D) and cardiovascular disease risk. Browning of white fat paves the way to restrict obesity and obesity related disorders. Although exercise has been the most common factor for fat browning, however there are other factors that involve: 1) beta aminoisobutyric acid (BAIBA); 2) gamma amino butyric acid (GABA); 3) PPARɣ agonists; 4) JAK inhibition; 5) IRISIN. In this review we propose two novel factors musclin and TFAM for fat browning. Musclin a myokine released from muscles during exercise activates PPARɣ which induces browning of WAT that has beneficial metabolic and cardiac effects. TFAM is a transcription factor that induces mitochondrial biogenesis. Since BAT is rich in mitochondria, higher expression of TFAM in WAT or TFAM treatment in WAT cells can induce browning of WAT. We propose that fat browning can be used as a therapeutic tool for metabolic disorders and cardiovascular diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:23:39.343408-05:
      DOI: 10.1002/jcp.25450
  • In Vitro and In Vivo Differentiation of Progenitor Stem Cells Obtained
           after Mechanical Digestion of Human Dental Pulp
    • Authors: Manuela Monti; Antonio Graziano, Silvana Rizzo, Cesare Perotti, Claudia Del Fante, Riccardo d'Aquino, CarloAlberto Redi, Ruggero Rodriguez y Baena
      Abstract: Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity. The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical‐grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate towards different cell types and to generate new bone in patients. We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio‐maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:23:35.810575-05:
      DOI: 10.1002/jcp.25452
  • Effects of sex and notch signaling on the osteocyte cell pool
    • Authors: Ernesto Canalis; Lauren Schilling, Stefano Zanotti
      Abstract: Osteocytes play a fundamental role in mechanotransduction and skeletal remodeling. Sex is a determinant of skeletal structure, and female C57BL/6J mice have increased osteoblast number in cancellous bone when compared to male mice. Activation of Notch in the skeleton causes profound cell‐context dependent changes in skeletal physiology. To determine the impact of sex and of Notch signaling on the osteocyte cell pool, we analyzed cancellous and cortical bone of 1 to 6 month old C57BL/6J or 129SvJ/C57BL/6J mice and determined the osteocyte number/area. There was an age‐dependent decline in osteocyte number in cancellous bone of male but not female mice, so that 6 month old female mice had a greater number of osteocytes than male littermates. Although differences between male and female mice were modest, female mice had ∼10‐15% greater number of osteocytes/area. RNA sequence analysis of osteocyte‐rich preparations did not reveal differences between sexes in the expression of genes known to influence bone homeostasis. Neither the activation of Notch1 nor the concomitant inactivation of Notch1 and Notch2 in Osterix (Sp7) or Dentin matrix protein 1 (Dmp1) expressing cells had a pronounced and consistent effect on cancellous or cortical bone osteocyte number in either sex. Moreover, inactivation of Notch1 and Notch2 in Dmp1 expressing cells did not influence the bone loss in a muscle immobilization model of skeletal unloading. In conclusion, cancellous bone osteocytes decline with age in male mice, cortical osteocytes are influenced by sex in younger mice, but osteocyte cell density is not affected substantially by Notch signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-07T11:50:40.061135-05:
      DOI: 10.1002/jcp.25433
  • Early Treatment with Enalapril and Later Renal Injury in Programmed Obese
           Adult Rats
    • Authors: Hyung Eun Yim; Kee Hwan Yoo, In Sun Bae, Young Sook Hong
      Abstract: Obesity‐related kidney disease should be prevented or retarded. We aimed to investigate whether early treatment with enalapril ameliorates later renal injury induced by early postnatal overnutrition. Three or ten male pups per mother were assigned to either the Obese or Lean group during the first 21 days of life. These pups were treated with enalapril (Obese enalapril, OE; Lean enalapril, LE) or vehicle (Obese control, OC; Lean control, LC) for 15–28 days. Body weight, blood pressure (BP), and renal alterations were determined at 3 months. Enalapril decreased body weight only in the Lean group at 3 months (P 
      PubDate: 2016-06-06T10:15:29.747504-05:
      DOI: 10.1002/jcp.25444
  • Gating Modulation of the Tumor‐Related Kv10.1 Channel by Mibefradil
    • Abstract: Several reports credit mibefradil with tumor suppressing properties arising from its known inhibition of Ca2+ currents. Given that mibefradil (Mb) is also known to inhibit K+ channels, we decided to study the interaction between this organic compound and the tumor‐related Kv10.1 channel. Here we report that Mb modulates the gating of Kv10.1. Mb induces an apparent inactivation from both open and early closed states where the channels dwell at hyperpolarized potentials. Additionally Mb accelerates the kinetics of current activation, in a manner that depends on initial conditions. Our observations suggest that Mb binds to the voltage sensor domain of Kv10.1 channels, thereby modifying the gating of the channels in a way that in some, but not all, aspects opposes to the gating effects exerted by divalent cations. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-03T06:58:46.738119-05:
      DOI: 10.1002/jcp.25448
  • T11TS Treatment Augments Apoptosis of Glioma Associated Brain Endothelial
           Cells, Hint towards Anti‐Angiogenic Action in Glioma
    • Authors: Debanjan Bhattacharya; Manoj Kumar Singh, Suhnrita Chaudhuri, Ankur Datta, Swapna Chaudhuri
      Abstract: Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11‐target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma associated brain endothelial cells. In the present study we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma associated brain endothelial cells. Annexin‐V/PI staining showed that T11TS treatment in glioma induced rats increases apoptosis of glioma associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC‐1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma associated brain endothelial cells. Flowcytometry, immunoblotting and in‐situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment upmodulated expression of p53, Bax, Fas, FasL and FADD in glioma associated endothelial cells and downregulated Bcl‐2 protein. T11TS therapy induced cytochrome‐c release into cytosol, activated caspase ‐9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas dependent extrinsic pathway. The pro‐apoptotic action of T11TS on glioma associated endothelial cells provides crucial insight into how T11TS exerts its anti‐angiogenic function in glioma. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-03T06:51:42.586441-05:
      DOI: 10.1002/jcp.25447
  • Cartilage‐specific and Cre‐dependent Nkx3.2 overexpression in
           vivo causes skeletal dwarfism by delaying cartilage hypertrophy
    • Abstract: Nkx3.2, the vertebrate homologue of Drosophila bagpipe, has been implicated as playing a role in chondrogenic differentiation. In brief, Nkx3.2 is initially expressed in chondrocyte precursor cells and, later during cartilage maturation, its expression is diminished in hypertrophic chondrocytes. In addition to Nkx3.2 expression analyses, previous studies using ex vivo chick embryo cultures and in vitro cell cultures have suggested that Nkx3.2 can suppress chondrocyte hypertrophy. However, it has never been demonstrated that Nkx3.2 functions in regulating chondrocyte hypertrophy during cartilage development in vivo. Here, we show that cartilage‐ specific and Cre‐dependent Nkx3.2 overexpression in mice results in significant postnatal dwarfism in endochondral skeletons, while intramembranous bones remain unaltered. Further, we observed significant delays in cartilage hypertrophy in conditional transgenic ciTg‐Nkx3.2 mice. Together, these findings confirm that Nkx3.2 is capable of controlling hypertrophic maturation of cartilage in vivo, and this regulation plays a significant role in endochondral ossification and longitudinal bone growth. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T12:28:17.938636-05:
      DOI: 10.1002/jcp.25446
  • Generation of neural crest‐like cells from human periodontal
           ligament cell‐derived induced pluripotent stem cells
    • Authors: Atsushi Tomokiyo; Kim Hynes, Jia Ng, Danijela Menicanin, Esther Camp, Agnes Arthur, Stan Gronthos, Peter Mark Bartold
      Abstract: Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue‐derived periodontal ligament (PDL) iPSC to differentiate into neural crest‐like cells (NCLC) relative to iPSC generated from a non‐neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC‐ and FF iPSC‐derived NCLC, which highly expressed HNK1. A high proportion of the HNK1‐positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1‐positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell‐associated markers than the PDL and FF HNK1‐negative populations. Interestingly, the HNK1‐positive cells derived from the PDL‐iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1‐positive cells derived from the FF‐iPSC. Our work suggests that HNK1‐enriched NCLC from neural crest tissue‐derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1‐low population and non‐neural crest iPSC‐derived NCLC. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:43:14.571204-05:
      DOI: 10.1002/jcp.25437
  • Gene and microRNA Expression Are Predictive of Tumor Response in Rectal
           Adenocarcinoma Patients Treated with Preoperative Chemoradiotherapy
    • Authors: Caterina Millino; Isacco Maretto, Beniamina Pacchioni, Maura Digito, Antonino De Paoli, Vincenzo Canzonieri, Edoardo D'Angelo, Marco Agostini, Flavio Rizzolio, Antonio Giordano, Andrea Barina, Senthilkumar Rajendran, Giovanni Esposito, Gerolamo Lanfranchi, Donato Nitti, Salvatore Pucciarelli
      Abstract: Preoperative chemoradiotherapy (pCRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). However, tumor response to pCRT is not uniform, and there are no effective predictive methods. This study investigated whether specific gene and miRNA expression are associated with tumor response to pCRT. Tissue biopsies were obtained from patients before pCRT and resection. Gene and miRNA expression were analyzed using a one‐color microarray technique that compares signatures between responders (R) and non‐responders (NR), as measured based on tumor regression grade. Two groups composed of 38 “exploration cohort” and 21 “validation cohort” LARC patients were considered for a total of 32 NR and 27 R patients. In the first cohort, using SAM Two Class analysis, 256 genes and 29 miRNAs that were differentially expressed between the NR and R patients were identified. The anti‐correlation analysis showed that the same 8 miRNA interacted with different networks of transcripts. The miR‐630 appeared only with the NR patients and was anti‐correlated with a single transcript: RAB5B. After PAM, the following 8 transcripts were strong predictors of tumor response: TMEM188, ITGA2, NRG, TRAM1, BCL2L13, MYO1B, KLF7 and GTSE1. Using this gene set, an unsupervised cluster analysis was applied to the validation cohort and correctly assigned the patients to the NR or R group with 85.7% accuracy, 90% sensitivity and 82% specificity. All three parameters reached 100% when both cohorts were considered together. In conclusion, gene and miRNA expression profiles may be helpful for predicting response to pCRT in LARC patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-25T17:40:42.055244-05:
      DOI: 10.1002/jcp.25441
  • Pin1, the Master Orchestrator of Bone Cell Differentiation
    • Abstract: Pin1 is an enzyme that specifically recognizes the peptide bond between phosphorylated serine or threonine (pS/pT‐P) and proline. This recognition causes a conformational change of its substrate, which further regulates downstream signaling. Pin1‐/‐ mice show developmental bone defects and reduced mineralization. Pin1 targets RUNX2 (Runt‐Related Transcription Factor 2), SMAD1/5, and β‐catenin in the FGF, BMP, and WNT pathways, respectively. Pin1 has multiple roles in the crosstalk between different anabolic bone signaling pathways. For example, it controls different aspects of osteoblastogenesis and increases the transcriptional activity of Runx2, both directly and indirectly. Pin1 also influences osteoclastogenesis at different stages by targeting PU.1 (Purine‐rich nucleic acid binding protein 1), C‐FOS, and DC‐STAMP. The phenotype of Pin1‐/‐ mice has led to the recent identification of multiple roles of Pin1 in different molecular pathways in bone cells. These roles suggest that Pin1 can be utilized as an efficient drug target in congenital and acquired bone diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-25T17:40:37.258658-05:
      DOI: 10.1002/jcp.25442
  • Association between aerobic exercise and rosiglitazone avoided the NAFLD
           and liver inflammation exacerbated in PPAR‐α knockout mice
    • Abstract: Nonalcoholic fatty liver disease (NAFLD) is one of the main liver diseases today, and may progress to steatohepatitis, cirrhosis and hepatocellular carcinoma. Some studies have shown the beneficial effects of aerobic exercise on reversing NAFLD. Aim: To verify whether chronic aerobic exercise improves the insulin resistance, liver inflammation and steatohepatitis caused by a high fat diet (HF) and whether PPARα is involved in these actions. Methods: C57BL6 wild type (WT) and PPAR‐α knockout (KO) mice were fed with a standard diet (SD) or HF during 12 weeks; the HF mice were trained on a treadmill during the last 8 weeks. Serum glucose and insulin tolerances, serum levels of aspartate aminotransferase, hepatic content of triacylglycerol, cytokines, gene expression and protein expression were evaluated in all animals. Results: Chronic exposure to HF diet increased triacylglycerol accumulation in the liver, leading to NAFLD, increased aminotransferase in the serum, increased peripheral insulin resistance, and higher adiposity index. Exercise reduced all these parameters in both animal genotypes. The liver lipid accumulation was not associated with inflammation; trained KO mice, however, presented a huge inflammatory response that was probably caused by a decrease in PPAR‐γ expression. Conclusion: We conclude that exercise improved the damage caused by a HF independently of PPARα, apparently by a peripheral fatty acid oxidation in the skeletal muscle. We also found that the absence of PPARα together with exercise leads to a decrease in PPAR‐γ and a huge inflammatory response. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-23T16:00:32.884989-05:
      DOI: 10.1002/jcp.25440
  • Ascorbic acid induces necrosis in human laryngeal squamous cell carcinoma
           via ROS, PKC, and calcium signaling
    • Abstract: Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N‐acetylcysteine blocked ascorbic acid–induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid–induced necrotic cell death was inhibited by Ro‐31‐8425 (PKC inhibitor) and BAPTA‐AM (cytosolic calcium–selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro‐31‐8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid‐treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-22T23:40:54.779976-05:
      DOI: 10.1002/jcp.25438
  • Intricate functions of matrix metalloproteinases in physiological and
           pathological conditions
    • Authors: Rahul Mittal; Amit P. Patel, Luca H. Debs, Desiree Nguyen, Kunal Patel, M'hamed Grati, Jeenu Mittal, Denise Yan, Prem Chapagain, Xue Zhong Liu
      Abstract: Matrix metalloproteinases (MMPs) are a diverse group of proteolytic enzymes and play an important role in the degradation and remodeling of the extracellular matrix (ECM). In normal physiological conditions, MMPs are usually minimally expressed. Despite their low expression, MMPs have been implicated in many cellular processes ranging from embryological development to apoptosis. The activity of MMPs is controlled at three different stages: (1) transcription, (2) zymogen activation, and (3) inhibition of active forms by tissue inhibitor metalloproteinases (TIMPs). They can collectively degrade any component of ECM and basement membrane, and their excessive activity has been linked to numerous pathologies mainly including, but not limited to, tumor evasion and metastasis. The lack of information about several MMPs and the steady stream of new discoveries suggest that there is much more to be studied in this field. In particular, there is a need for controlling their expression in disease states. Various studies over the past 30 years have found that each MMP has a specific mode of activation, action, and inhibition. Drugs specifically targeting individual MMPs could revolutionize the treatment of a great number of health conditions and tremendously reduce their burden. In this review article, we have summarized the recent advances in understanding the role of MMPs in physiological and pathological conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-17T16:25:35.052746-05:
      DOI: 10.1002/jcp.25430
  • Targeted Modification of Mitochondrial ROS Production Converts High
           Glucose‐Induced Cytotoxicity to Cytoprotection: Effects on
           Anesthetic Preconditioning
    • Authors: Filip Sedlic; Maria Muravyeva, Ana Sepac, Marija Sedlic, Anna Marie Williams, Meiying Yang, Xiaowen Bai, Zeljko J. Bosnjak
      Abstract: Contradictory reports on the effects of diabetes and hyperglycemia on myocardial infarction range from cytotoxicity to cytoprotection. The study was designed to investigate acute effects of high glucose‐driven changes in mitochondrial metabolism and osmolarity on adaptive mechanisms and resistance to oxidative stress of isolated rat cardiomyocytes. We examined the effects of high glucose on several parameters of mitochondrial bioenergetics, including changes in oxygen consumption, mitochondrial membrane potential and NAD(P)H fluorometry. Effects of high glucose on the endogenous cytoprotective mechanisms elicited by anesthetic preconditioning (APC) and the mediators of cell injury were also tested. These experiments included real‐time measurements of reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) opening in single cells by laser scanning fluorescence confocal microscopy, and cell survival assay. High glucose rapidly enhanced mitochondrial energy metabolism, observed by increase in NAD(P)H fluorescence intensity, oxygen consumption and mitochondrial membrane potential. This substantially elevated production of ROS, accelerated opening of the mPTP and decreased survival of cells exposed to oxidative stress. Abrogation of high glucose‐induced mitochondrial hyperpolarization with 2,4 dinitrophenol (DNP) significantly, but not completely, attenuated ROS production to a level similar to hyperosmotic mannitol control. DNP treatment reversed high glucose‐induced cytotoxicity to cytoprotection. Hyperosmotic mannitol treatment also induced cytoprotection. High glucose abrogated APC‐induced mitochondrial depolarization, delay in mPTP opening and cytoprotection. In conclusion, high glucose‐induced mitochondrial hyperpolarization abolishes APC and augments cell injury. Attenuation of high glucose‐induced ROS production by eliminating mitochondrial hyperpolarization protects cardiomyocytes. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-02T13:22:40.120836-05:
      DOI: 10.1002/jcp.25413
  • Differentiation of Keratinocytes Modulates Expression of Epidermal
           Elements of the Cutaneous Analog of
           Hypothalamus‐Pituitary‐Adrenal Axis
    • Abstract: It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus‐pituitary‐adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1‐3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from Day 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-06T17:02:14.046921-05:
      DOI: 10.1002/jcp.25400
  • Hypoxia Modulates the Swelling‐Activated Cl Current in Human
           Glioblastoma Cells: Role in Volume Regulation and Cell Survival
    • Authors: Luigi Sforna; Marta Cenciarini, Silvia Belia, Antonio Michelucci, Mauro Pessia, Fabio Franciolini, Luigi Catacuzzeno
      Abstract: The malignancy of glioblastoma multiform (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non‐tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling‐activated Cl current (ICl,swell). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia‐activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably the hypoxia‐induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell. Since a hypoxia‐induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia‐activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia‐induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-29T03:36:23.324328-05:
      DOI: 10.1002/jcp.25393
  • Quantification of Exosomes
    • Authors: E. H. Koritzinsky; J. M. Street, R. A. Star, P.S.T. Yuen
      Abstract: Exosomes are released by cells as self‐contained vesicles with an intact lipid bilayer that encapsulates a small portion of the parent cell. Exosomes have been studied widely as information‐rich sources of potential biomarkers that can reveal cellular physiology. We suggest that quantification is essential to understand basic biological relationships between exosomes and their parent cells and hence the underlying interpretation of exosome signals. The number of methods for quantifying exosomes has expanded as interest in exosomes has increased. However, a consensus on proper quantification has not developed, making each study difficult to compare to another. Overcoming this ad hoc approach will require widely available standards that have been adequately characterized, and multiple comparative studies across platforms. We outline the current status of these technical approaches and our view of how they can become more coherent. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:35:45.891505-05:
      DOI: 10.1002/jcp.25387
  • Stiffness of Intact Endothelial Cells from Fresh Aortic Bifurcations of
           Atherosclerotic Rabbits ‐ Atomic Force Microscopic Study
    • Authors: Kozaburo Hayashi; Michitaka Higaki
      Abstract: Stiffness of intact endothelial cells (ECs) in the abdominal aorta (AA) and in the medial and lateral wall of the common iliac artery (CIA(Medial) and CIA(Lateral), respectively), which were freshly obtained from cholesterol‐fed rabbits, were measured with an atomic force microscopic indentation method. In the areas away from atherosclerotic plaques (Off‐plaque), ECs were significantly stiffer in CIA(Medial) than in the other two locations; this result was similar to that from normal diet‐fed animals. On the other hand, there were no significant differences in the stiffness of ECs located on atherosclerotic plaques (On‐plaque) among the three sites; the stiffness was equal to those in “Off‐plaque” wall of CIA(Lateral) and AA. Moreover, the stiffness of ECs covering plaques decreased with the progression of atherosclerosis. The precise quantification of the stiffness of vascular ECs would provide a better understanding of cellular remodeling and adaptation in atherosclerosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:43:15.558698-05:
      DOI: 10.1002/jcp.25379
  • Generation of Human Lens Epithelial‐Like Cells from
           Patient‐Specific Induced Pluripotent Stem Cells
    • Authors: Dan Li; Xiaodi Qiu, Jin Yang, Tianjin Liu, Yi Luo, Yi Lu
      Abstract: Cataractogenesis begins from the dynamic lens epithelial cells (LECs) and adjacent fiber cells. LECs derived from cell lines cannot maintain the crystalline expression as the primary LECs. The current study aimed to efficiently generate large numbers of human LECs from patient‐specific induced pluripotent stem cells (iPSCs). Anterior lens capsules were collected from cataract surgery and were used to culture primary hLECs. iPSCs were induced from these primary hLECs by lentiviral transduction of Oct4, Sox2, Klf4 and c‐Myc. Then, the generated iPSCs were re‐differentiated into hLECs by the 3‐step addition of defined factor combinations (Noggin, BMP4/7, bFGF and EGF) modified from an established method. During the re‐differentiation process, colonies of interest were isolated using a glass picking tool and cloning cylinders based on the colony morphology. After two steps of isolation, populations of LEC‐like cells (LLCs) were generated and identified by the expression of lens marker genes by qPCR, western blot and immunofluorescence staining. The study introduced a modified protocol to isolate LLCs from iPSCs by defined factors in a short time frame. This technique could be useful for mechanistic studies of lens‐related diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-15T02:56:45.612403-05:
      DOI: 10.1002/jcp.25374
  • Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis
           via the PI3K/AKT and ERK1/2 MAPK Pathways
    • Authors: Whasun Lim; Sunwoo Park, Fuller W. Bazer, Gwonhwa Song
      Abstract: Apigenin is a flavonoid found in parsley, onions, oranges, tea, chamomile, wheat and sprouts. It has a variety of biological properties including anti‐oxidant, anti‐mutagenic, anti‐carcinogenic, anti‐inflammatory, anti‐proliferative and anti‐spasmodic effects. Based on epidemiological and case‐control studies, apigenin is regarded as a novel chemotherapeutic agent against various cancer types. However, little is known about the effects of apigenin on choriocarcinoma cells. Therefore, we investigated the anti‐cancer effects of apigenin on choriocarcinoma cells (JAR and JEG3) in the present study. Apigenin reduced viability and migratory properties, increased apoptosis, and suppressed mitochondrial membrane potential in both the JAR and JEG3 cells. In addition, apigenin predominantly decreased phosphorylation of AKT, P70RSK and S6 whereas the phosphorylation of ERK1/2 and P90RSK was increased by apigenin treatment of JAR and JEG3 cells in a dose‐dependent manner. Moreover, treatment of JAR and JEG3 cells with both apigenin and pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126) revealed synergistic anti‐proliferative effects. Collectively, these results indicated that the apigenin is an invaluable chemopreventive agent that inhibits progression and metastasis of choriocarcinoma cells through regulation of PI3K/AKT and ERK1/2 MAPK signal transduction mechanism. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-11T02:43:28.687733-05:
      DOI: 10.1002/jcp.25372
  • Alternative Start Codon Connects eIF5A to Mitochondria
    • Abstract: Eukaryotic translation initiation factor 5A (eIF5A), a protein containing the amino acid residue hypusine required for its activity, is involved in a number of physiological and pathological cellular processes. In humans, several EIF5A1 transcript variants encode the canonical eIF5A1 isoform B, whereas the hitherto uncharacterized variant A is expected to code for a hypothetical eIF5A1 isoform, referred to as isoform A, which has an additional N‐terminal extension. Herein, we validate the existence of eIF5A1 isoform A and its production from transcript variant A. In fact, variant A was shown to encode both eIF5A1 isoforms A and B. Mutagenic assays revealed different efficiencies in the start codons present in variant A, contributing to the production of isoform B at higher levels than isoform A. Immunoblotting and mass spectrometric analyses showed that isoform A can undergo hypusination and acetylation at specific lysine residues, as observed for isoform B. Examination of the N‐terminal extension suggested that it might confer mitochondrial targeting. Correspondingly, we found that isoform A, but not isoform B, co‐purified with mitochondria when the proteins were overproduced. These findings suggest that eIF5A1 isoform A has a role in mitochondrial function. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-08T06:02:49.087117-05:
      DOI: 10.1002/jcp.25370
  • Mitochondria and Mitochondrial ROS in Cancer: Novel Targets for Anticancer
    • Authors: Yuhui Yang; Svetlana Karakhanova, Werner Hartwig, Jan G. D'Haese, Pavel P. Philippov, Jens Werner, Alexandr V. Bazhin
      Abstract: Mitochondria are indispensable for energy metabolism, apoptosis regulation and cell signaling. Mitochondria in malignant cells differ structurally and functionally from those in normal cells and participate actively in metabolic reprogramming. Mitochondria in cancer cells are characterized by reactive oxygen species (ROS) overproduction, which promotes cancer development by inducing genomic instability, modifying gene expression and participating in signaling pathways. Mitochondrial and nuclear DNA mutations caused by oxidative damage that impair the oxidative phosphorylation process will result in further mitochondrial ROS production, completing the “vicious cycle” between mitochondria, ROS, genomic instability and cancer development. The multiple essential roles of mitochondria have been utilized for designing novel mitochondria‐targeted anticancer agents. Selective drug delivery to mitochondria helps to increase specificity and reduce toxicity of these agents. In order to reduce mitochondrial ROS production, mitochondria‐targeted antioxidants can specifically accumulate in mitochondria by affiliating to a lipophilic penetrating cation and prevent mitochondria from oxidative damage. In consistence with the oncogenic role of ROS, mitochondria‐targeted antioxidants are found to be effective in cancer prevention and anticancer therapy. A better understanding of the role played by mitochondria in cancer development will help to reveal more therapeutic targets, and will help to increase the activity and selectivity of mitochondria‐targeted anticancer drugs. In this review we summarized the impact of mitochondria on cancer and gave summary about the possibilities to target mitochondria for anticancer therapies. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-19T20:43:18.622087-05:
      DOI: 10.1002/jcp.25349
  • Characterization of Notch Signaling During Osteogenic Differentiation in
           Human Osteosarcoma Cell Line MG63
    • Abstract: Osteogenic differentiation is a multi‐step process controlled by a complex molecular framework. Notch is an evolutionarily conserved intercellular signaling pathway playing a prominent role in cell fate and differentiation, although the mechanisms by which this pathway regulates osteogenesis remain controversial. This study aimed to investigate, in vitro, the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. Cells were cultured in basal condition (control) and in osteoinductive medium (OM). Notch inhibitors were also added in OM to block Notch pathway. During osteogenic differentiation, early (alkaline phosphatase activity and collagen type I) and late osteogenic markers (osteocalcin levels and matrix mineralization), as well as the gene expression of the main osteogenic transcription factors (Runx2, Osterix, and Dlx5) increased. Time dependent changes in the expression of specific Notch receptors were identified in OM versus control with a significant reduction in the expression of Notch1 and Notch3 receptors in the early phase of differentiation, and an increase of Notch2 and Notch4 receptors in the late phase. Among Notch nuclear target genes, Hey1 expression was significantly higher in OM than control, while Hes5 expression decreased. Osteogenic markers were reduced and Hey1 was significantly inhibited by Notch inhibitors, suggesting a role for Notch through the canonical pathway. In conclusion, Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4, and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3, and Hes5, maintaining an undifferentiated status. J. Cell. Physiol. 231: 2652–2663, 2016. © 2016 Wiley Periodicals, Inc. This study investigated in vitro the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. During osteogenic differentiation, early and late osteogenic markers, as well as the gene expression of the main osteogenic transcription factors increased. Time dependent changes in the expression of specific Notch receptors and nuclear target genes were also identified. Our results showed that Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4, and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3, and Hes5, maintaining an undifferentiated status.
  • Phospholipidomic Profile Variation on THP‐1 Cells Exposed to Skin or
           Respiratory Sensitizers and Respiratory Irritant
    • Abstract: Occupational exposure to low molecular weight reactive chemicals often leads to development of allergic reactions such as allergic contact dermatitis and respiratory allergies. Further insights into the interaction of these chemicals with physiopathological relevant cellular models might provide the foundations for novel non‐animal approaches to safety assessment. In this work we used the human THP‐1 cell line to determine phospholipidome changes induced by the skin sensitizer 1‐fluoro‐2,4‐dinitrobenzene (DNFB), the respiratory allergen hexamethylene diisocyanate (HDI), and the irritant methyl salicylate (MESA). We detected that these chemicals differently induce lipid peroxidation and modulate THP‐1 IL‐1β, IL‐12B, IL‐8, CD86, and HMOX1 transcription. Decreased phosphatidylethanolamine content was detected in cells exposed to MESA, while profound alterations in the relative abundance of cardiolipin species were observed in cells exposed to DNFB. All chemicals tested induced a decrease in the relative abundance of plasmanyl phosphatidylcholine species PC (O‐16:0e/18:1) and phosphatidylinositol species PI (34:1), while increasing PI (38:4). An increased abundance of oleic acid was observed in the phospholipids of cells exposed to DNFB while a decreased abundance of palmitic acid was detected in cells treated with MESA or DNFB. We conclude that both specific and common alterations at phospholipidome levels are triggered by the different chemicals, while not allowing a complete distinction between them using a Canonical Analysis of Principal Coordinates (CAP). The common effects observed at phospholipids level with all the chemicals tested might be related to unspecific cell cytotoxic mechanisms that nevertheless may contribute to the elicitation of specific immune responses. J. Cell. Physiol. 231: 2639–2651, 2016. © 2016 Wiley Periodicals, Inc. The skin sensitizer 1‐fluoro‐2,4‐dinitrobenzene (DNFB), the respiratory allergen hexamethylene diisocyanate (HDI), and the irritant methyl salicylate (MESA) trigger both specific and common alterations at THP‐1 phospholipidome level. To some extent these alterations are dependent on the oxidative capacity of the chemicals and may be important in shaping THP‐1 cells’ maturation.
  • Quercetin Partially Preserves Development of Osteoblast Phenotype in Fetal
           Rat Calvaria Cells in an Oxidative Stress Environment
    • Abstract: Studies are needed to improve understanding of the osteoblast antioxidant response, and the balance between oxidative homeostasis and osteoblast differentiation. The flavonol quercetin aglycone (QRC) up‐regulates the osteoblast antioxidant response in vitro without suppressing osteoblast phenotype, suggesting that QRC may preserve osteoblast phenotypic development in cells subsequently exposed to oxidative stress, which suppresses osteoblast differentiation. The aims of this study were to assess the extent that QRC pretreatment preserved development of the osteoblast phenotype in cells subsequently cultured with hydrogen peroxide, an oxidative stressor, and to characterize alterations in the osteoblast antioxidant response and in key antioxidant signaling pathways. We hypothesized that pretreatment with QRC would preserve phenotypic development after hydrogen peroxide treatment, suppress the hydrogen peroxide‐induced antioxidant response, and that the antioxidant response would involve alterations in Nrf2 and ERK1/2 signaling. Results showed that treating fetal rat calvarial osteoblasts for 4 days (D5‐9) with 300 μM hydrogen peroxide resulted in fewer alkaline phosphatase‐positive cells and mineralized nodules, altered cell morphology, and significantly lower osteoblast phenotypic gene expression (P 
  • Atp2c2 Is Transcribed From a Unique Transcriptional Start Site in Mouse
           Pancreatic Acinar Cells
    • Abstract: Proper regulation of cytosolic Ca2+ is critical for pancreatic acinar cell function. Disruptions in normal Ca2+ concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca2+ homeostasis by promoting rapid Ca2+ movement. Determining how expression of Ca2+ modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas‐specific isoform for Secretory Pathway Ca2+ ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA‐seq), chromatin immunoprecipitation for epigenetic modifications and promoter‐reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2’s 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca2+. RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca2+ levels. Combined, these results suggest SPCA2C affects Ca2+ homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca2+ ATPases. J. Cell. Physiol. 231: 2768–2778, 2016. © 2016 Wiley Periodicals, Inc. A novel isoform for SPCA2 (termed SPAC2C) is generated from a unique transcriptional start site within pancreatic acinar cells. Unlike other Ca2+ ATPases, which lower cytosolic Ca2+, SPCA2C increases cytosolic Ca2+, suggesting a novel function for this protein. This is important as SPCA2C is down‐regulated during acinar cell stress and may reflect an effort by these cells to minimize damage associated with dysregulated Ca2+ levels.
  • Kynurenine Modulates MMP‐1 and Type‐I Collagen Expression Via
           Aryl Hydrocarbon Receptor Activation in Dermal Fibroblasts
    • Abstract: Dermal fibrosis is characterized by a high deposition of extracellular matrix (ECM) and tissue cellularity. Unfortunately all means of treating this condition are unsatisfactory. We have previously reported the anti‐fibrotic effects of Kynurenine (Kyn), a tryptophan metabolite, in fibrotic rabbit ear model. Here, we report the mechanism by which Kyn modulates the expression of key ECM components in dermal fibroblasts. The results showed that Kyn activates aryl hydrocarbon receptor (AHR) nuclear translocation and up‐regulates cytochrome‐P450 (CYP1A‐1) expression, the AHR target gene. A specific AHR antagonist, 6,2′,4′‐trimethoxyflavone, inhibited the Kyn‐dependent modulation of CYP1A‐1, MMP‐1, and type‐I collagen expression. Establishing the anti‐fibrogenic effect of Kyn and its mechanism of action, we then developed nano‐fibrous Kyn slow‐releasing dressings and examined their anti‐fibrotic efficacy in vitro and in a rat model. Our results showed the feasibility of incorporating Kyn into PVA/PLGA nanofibers, prolonging the Kyn release up to 4 days tested. Application of medicated‐dressings significantly improved the dermal fibrosis indicated by MMP‐1 induction, alpha‐smooth muscle actin and type‐I collagen suppression, and reduced tissue cellularity, T‐cells and myofibroblasts. This study clarifies the mechanism by which Kyn modulates ECM expression and reports the development of a new slow‐releasing anti‐fibrogenic dressing. J. Cell. Physiol. 231: 2749–2760, 2016. © 2016 Wiley Periodicals, Inc. Kynurenine modulates the expression of collagen and collagenase (MMP‐1) via aryl hydrocarbon receptor signaling pathway. The medicated nanofibrous wound dressings, developed in this study, can be used as an effective slow releasing drug delivery system in order to prevent the post‐burn and pots‐surgical hypertrophic scar formation.
  • TNF‐α Induces Caspase‐1 Activation Independently of
           Simultaneously Induced NLRP3 in 3T3‐L1 Cells
    • Abstract: The intracellular cysteine protease caspase‐1 is critically involved in obesity‐induced inflammation in adipose tissue. A substantial body of evidence from immune cells, such as macrophages, has shown that caspase‐1 activation depends largely on a protein complex, called the NLRP3 inflammasome, which consists of the NOD‐like receptor (NLR) family protein NLRP3, the adaptor protein ASC, and caspase‐1 itself. However, it is not fully understood how caspase‐1 activation is regulated within adipocytes upon inflammatory stimuli. In this study, we show that TNF‐α‐induced activation of caspase‐1 is accompanied by robust induction of NLRP3 in 3T3‐L1 adipocytes but that caspase‐1 activation may not depend on the NLRP3 inflammasome. Treatment of 3T3‐L1 cells with TNF‐α induced mRNA expression and activation of caspase‐1. Although the basal expression of NLRP3 and ASC was undetectable in unstimulated cells, TNF‐α strongly induced NLRP3 expression but did not induce ASC expression. Interestingly, inhibitors of the ERK MAP kinase pathway strongly suppressed NLRP3 expression but did not suppress the expression and activation of caspase‐1 induced by TNF‐α, suggesting that NLRP3 is dispensable for TNF‐α‐induced caspase‐1 activation. Moreover, we did not detect the basal and TNF‐α‐induced expression of other NLR proteins (NLRP1a, NLRP1b, and NLRC4), which do not necessarily require ASC for caspase‐1 activation. These results suggest that TNF‐α induces caspase‐1 activation in an inflammasome‐independent manner in 3T3‐L1 cells and that the ERK‐dependent expression of NLRP3 may play a role independently of its canonical role as a component of inflammasomes. J. Cell. Physiol. 231: 2761–2767, 2016. © 2016 Wiley Periodicals, Inc. In this study, we propose that TNF‐α induces caspase‐1 activation in an inflammasome‐independent manner in 3T3‐L1 cells and that the ERK‐dependent expression of NLRP3 may play a role independently of its canonical role as a component of inflammasomes.
  • E‐Cadherin Facilitates Protein Kinase D1 Activation and Subcellular
    • Abstract: Protein kinase D 1 (PKD1) is a serine/threonine kinase implicated in the regulation of diverse cellular functions including cell growth, differentiation, adhesion and motility. The current model for PKD1 activation involves diacylglycerol (DAG) binding to the C1 domain of PKD1 which results in the translocation of PKD1 to subcellular membranes where PKD1 is phosphorylated and activated by protein kinase C (PKC). In this study, we have identified a novel regulation of PKD1 activation. The epithelial cell membrane protein E‐cadherin physically binds to PKD1 which leads to a subcellular redistribution of PKD1. Furthermore, artificial targeting of PKD1 to the membrane leads to PKD1 activation in a PKC‐independent manner, indicating that membrane attachment is sufficient enough to activate PKD1. The presence of E‐cadherin dynamically regulates PKD1 activation by Bryostatin 1, a potent activator of PKD1, and its substrate phosphorylation specificity, implying a loss of E‐cadherin during cancer metastasis could cause the re‐distribution PKD1 and re‐wiring of PKD1 signaling for distinct functions. The knocking down of PKD1 in lung epithelial cell line A549 results in an epithelial to mesenchymal transition with changes in biomarker expression, cell migration and drug resistance. These results extend our previous understanding of PKD1 regulation and E‐cadherin signaling functions and may help to explain the diversified functions of PKD1 in various cells. J. Cell. Physiol. 231: 2741–2748, 2016. © 2016 Wiley Periodicals, Inc. E‐cadherin intracellular domain directly binds to PKD1 C1 domain. This interesction regulates PKD1 subcellular localization and activation.
  • Stimulatory Effects of Coumestrol on Embryonic and Fetal Development
           Through AKT and ERK1/2 MAPK Signal Transduction
    • Abstract: Successful establishment of pregnancy is required for fetal‐maternal interactions regulating implantation, embryonic development and placentation. A uterine environment with insufficient growth factors and nutrients increases the incidence of intrauterine growth restriction (IUGR) leading to an impaired uterine environment. In the present study, we demonstrated the effects of the phytoestrogen coumestrol on conceptus development in the pig that is regarded as an excellent biomedical animal model for research on IUGR. Results of this study indicated that coumestrol induced migration of porcine trophectoderm (pTr) cells in a concentration‐dependent manner. In response to coumestrol, the phosphorylation of AKT, P70S6K, S6, ERK1/2 MAPK, and P90RSK proteins were activated in pTr cells and ERK1/2 MAPK and P90RSK phosphorylation was prolonged for a longer period than for the other proteins. To identify the signal transduction pathway induced by coumestrol, pharmacological inhibitors U0126 (an ERK1/2 inhibitor) and LY294002 (a PI3K inhibitor) were used to pretreat pTr cells. The results showed that coumestrol‐induced phosphorylation of ERK1/2 MAPK and P90RSK was blocked by U0126. In addition, the increased phosphorylation in response to coumestrol was completely inhibited following pre‐treatment incubation of pTr cells in the presence of LY294002 and U0126. Furthermore, these two inhibitors suppressed the ability of coumestrol to induce migration of pTr cells. Collectively, these findings suggest that coumestrol affects embryonic development through activation of the PI3K/AKT and ERK1/2 MAPK cell signal transduction pathways and improvement in the uterine environment through coumestrol supplementation may provide beneficial effects of enhancing embryonic and fetal survival and development. J. Cell. Physiol. 231: 2733–2740, 2016. © 2016 Wiley Periodicals, Inc. Coumestrol stimulates migration of trophectoderm cells which is required for elongation and development of porcine conceptuses. Coumestrol affects embryonic development through activation of the PI3K/AKT and ERK1/2 MAPK cell signal transduction pathways.
  • Prototype of Multiplex Bead Assay for Quantification of Three Serum
           Biomarkers for In Vitro Diagnosis of Endometriosis
    • Abstract: Endometriosis is a very common disease, affecting 10% of women in the reproductive age. To date, a significant delay between onset of the symptoms and definitive diagnosis is caused by the lack of a reliable non‐invasive diagnostic test. Recently, the potential value as diagnostic markers for endometriosis of three proteins (Zn‐alpha2‐glycoprotein, serum albumin, and complement C3 precursor), has been showed. In this article, we have defined the experimental conditions for the development of a multiplex bead array assay for rapid and simultaneous quantification of these three biomarkers in the serum of patients with endometriosis. Finally, pivotal experiments on a small cohort of patients have confirmed the diagnostic value of this assay. J. Cell. Physiol. 231: 2622–2627, 2016. © 2016 Wiley Periodicals, Inc. To date, a significant delay between onset of the symptoms and definitive diagnosis is caused by the lack of a reliable non‐invasive diagnostic test. We have defined the experimental conditions and the diagnostic value of a multiplex bead array assay for rapid and simultaneous quantification of three biomarkers (Zn‐alpha2‐glycoprotein, serum albumin and complement C3 precursor) in the serum of patients with endometriosis.
  • Properties of Adult Lung Stem and Progenitor Cells
    • Abstract: The last decade has seen significant progress in understanding the organisation of regenerative cells in the adult lung. Cell‐lineage tracing and in vitro clonogenic assays have enabled the identification and characterisation of endogenous lung epithelial stem and progenitor cells. Selective lung injury models, and genetically engineered mice have revealed highly conserved gene networks, factors, signalling pathways, and cellular interactions important in maintaining lung homeostasis and regulating lung regeneration and repair following injury. This review describes the current models of lung epithelial stem and progenitor cell organisation in adult mice, and the impediments encountered in translational studies aiming to identify and characterise their human homologs. J. Cell. Physiol. 231: 2582–2589, 2016. © 2016 Wiley Periodicals, Inc. This review describes the current models of lung epithelial stem and progenitor cell organisation in adult mice, and the impediments encountered in translational studies aiming to identify and characterise their human homologs.
  • How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy
    • Abstract: Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor‐ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590–2598, 2016. © 2016 Wiley Periodicals, Inc. Chimeric antigen receptor (CAR) T cells are able to eliminate late stage hematological cancers and have a great potential to treat many types of cancer. This review summarizes how the design of CARs alters their function and T cell responses.
  • l‐glutamine Improves Skeletal Muscle Cell Differentiation and
           Prevents Myotube Atrophy After Cytokine (TNF‐α) Stress Via
           Reduced p38 MAPK Signal Transduction
    • Abstract: Tumour Necrosis Factor‐Alpha (TNF‐α) is chronically elevated in conditions where skeletal muscle loss occurs. As l‐glutamine can dampen the effects of inflamed environments, we investigated the role of l‐glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF‐α (20 ng · ml−1) ± l‐glutamine (20 mM). TNF‐α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf‐I, and Igfbp5. Furthermore, when administered to mature myotubes, TNF‐α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf‐I, Igfbp2, and glutamine synthetase and parallel increases in Fox03, Cfos, p53, and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non‐significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF‐α. Importantly, 20 mM l‐glutamine reduced p38 MAPK activity in TNF‐α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. l‐glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf‐Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and restored atrophic gene expression of Fox03 back to baseline in TNF‐α conditions. In conclusion, l‐glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. l‐glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. J. Cell. Physiol. 9999: 231: 2720–2732, 2016. © 2016 Wiley Periodicals, Inc. l‐glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed cytokine environment (TNF‐α) via regulation of p38 MAPK singaling. l‐Glutamine supplementation evoked reductions in p38 MAPK following extensive increases after TNF‐α administration. This was associated with downstream regulation of gene transcription associated with growth and survival including increased; Myogenin, Igf‐Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and reduced atrophic gene expression of Fox03 back to baseline in TNF‐α conditions. l‐glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing.
  • Cover Image, Volume 231, Number 12, December 2016
    • Abstract: Cover: The cover image, by Sabine Mai et al., is based on the Research Article XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of Telomeres in Tumor Cells,
      DOI : 10.1002/jcp.25378.
  • Table of Contents, Editor's Choice, Highlights
  • CRISPR‐Cas‐Assisted Multiplexing (CAM): Simple Same‐Day
           Multi‐Locus Engineering in Yeast
    • Abstract: Demands on the industrial and academic yeast strain engineer have increased significantly in the era of synthetic biology. Installing complex biosynthetic pathways and combining point mutations are tedious and time‐consuming using traditional methods. With multiplex engineering tools, these tasks can be completed in a single step, typically achieving up to sixfold compression in strain engineering timelines. To capitalize on this potential, a variety of yeast CRISPR‐Cas methods have been developed, differing largely in how the guide RNA (gRNA) reagents that direct the Cas9 nuclease are delivered. However, in nearly all reported protocols, the time savings of multiplexing is offset by multiple days of cloning to prepare the required reagents. Here, we discuss the advantages and opportunities of CRISPR‐Cas‐assisted multiplexing (CAM), a same‐day, cloning‐free method for multi‐locus engineering in yeast. J. Cell. Physiol. 231: 2563–2569, 2016. © 2016 Wiley Periodicals, Inc. Installing complex biosynthetic pathways and combining point mutations in yeast are tedious and time‐consuming using traditional methods. Here, we discuss the advantages and opportunities of CRISPR‐Cas‐assisted multiplexing (CAM), a same‐day, cloning‐free method for multi‐locus engineering.
  • XPO1 Inhibition Preferentially Disrupts the 3D Nuclear Organization of
           Telomeres in Tumor Cells
    • Abstract: Previous work has shown that the three‐dimensional (3D) nuclear organization of telomeres is altered in cancer cells and the degree of alterations coincides with aggressiveness of disease. Nuclear pores are essential for spatial genome organization and gene regulation and XPO1 (exportin 1/CRM1) is the key nuclear export protein. The Selective Inhibitor of Nuclear Export (SINE) compounds developed by Karyopharm Therapeutics (KPT‐185, KPT‐330/selinexor, and KPT‐8602) inhibit XPO1 nuclear export function. In this study, we investigated whether XPO1 inhibition has downstream effects on the 3D nuclear organization of the genome. This was assessed by measuring the 3D telomeric architecture of normal and tumor cells in vitro and ex vivo. Our data demonstrate for the first time a rapid and preferential disruption of the 3D nuclear organization of telomeres in tumor cell lines and in primary cells ex vivo derived from treatment‐naïve newly diagnosed multiple myeloma patients. Normal primary cells in culture as well as healthy lymphocyte control cells from the same patients were minimally affected. Using both lymphoid and non‐lymphoid tumor cell lines, we found that the downstream effects on the 3D nuclear telomere structure are independent of tumor type. We conclude that the 3D nuclear organization of telomeres is a sensitive indicator of cellular response when treated with XPO1 inhibitors. J. Cell. Physiol. 231: 2711–2719, 2016. © 2016 Wiley Periodicals, Inc. This study shows that XPO1 inhibition directly disrupts the 3D nuclear telomere organization of tumor cell lines and of primary tumor cells (multiple myeloma) from treatment naive patients. Normal cells are only minimally afffected.
  • Serum From Advanced Heart Failure Patients Promotes Angiogenic Sprouting
           and Affects the Notch Pathway in Human Endothelial Cells
    • Abstract: It is unknown whether components present in heart failure (HF) patients’ serum provide an angiogenic stimulus. We sought to determine whether serum from HF patients affects angiogenesis and its major modulator, the Notch pathway, in human umbilical vein endothelial cells (HUVECs). In cells treated with serum from healthy subjects or from patients at different HF stage we determined: (1) Sprouting angiogenesis, by measuring cells network (closed tubes) in collagen gel. (2) Protein levels of Notch receptors 1, 2, 4, and ligands Jagged1, Delta‐like4. We found a higher number of closed tubes in HUVECs treated with advanced HF patients serum in comparison with cells treated with serum from mild HF patients or controls. Furthermore, as indicated by the reduction of the active form of Notch4 (N4IC) and of Jagged1, advanced HF patients serum inhibited Notch signalling in HUVECs in comparison with mild HF patients’ serum and controls. The circulating levels of NT‐proBNP (N‐terminal of the pro‐hormone brain natriuretic peptide), a marker for the detection and evalutation of HF, were positively correlated with the number of closed tubes (r = 0.485) and negatively with Notch4IC and Jagged1 levels in sera‐treated cells (r = −0.526 and r = −0.604, respectively). In conclusion, we found that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in HUVECs. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it. J. Cell. Physiol. 231: 2700–2710, 2016. © 2016 Wiley Periodicals, Inc. It is unknown whether components present in heart failure (HF) patients’ serum provide an angiogenic stimulus. We show that sera from advanced HF patients promote sprouting angiogenesis and dysregulate Notch signaling in human umbilical vein endothelial cells. Our study provides in vitro evidence of an angiogenic stimulus arising during HF progression and suggests a role for the Notch pathway in it.
  • Role of PTHrP(1‐34) Pulse Frequency Versus Pulse Duration to Enhance
           Mesenchymal Stromal Cell Chondrogenesis
    • Abstract: Generation of phenotypically stable, articular chondrocytes from mesenchymal stromal cells (MSCs) is still an unaccomplished task, with formation of abundant, hyaline extracellular matrix, and avoidance of hypertrophy being prime challenges. We recently demonstrated that parathyroid hormone‐related protein (PTHrP) is a promising factor to direct chondrogenesis of MSCs towards an articular phenotype, since intermittent PTHrP application stimulated cartilage matrix production and reduced undesired hypertrophy. We here investigated the role of frequency, pulse duration, total exposure time, and underlying mechanisms in order to unlock the full potential of PTHrP actions. Human MSC subjected to in vitro chondrogenesis for six weeks were exposed to 2.5 nM PTHrP(1‐34) pulses from days 7 to 42. Application frequency was increased from three times weekly (3 × 6 h/week) to daily maintaining either the duration of individual pulses (6 h/day) or total exposure time (18 h/week; 2.6 h/day). Daily PTHrP treatment significantly increased extracellular matrix deposition regardless of pulse duration and suppressed alkaline‐phosphatase activity by 87%. High total exposure time significantly reduced cell proliferation at day 14. Pulse duration was critically important to significantly reduce IHH expression, but irrelevant for PTHrP‐induced suppression of the hypertrophic markers MEF2C and IBSP. COL10A1, RUNX2, and MMP13 expression remained unaltered. Decreased IGFBP‐2, ‐3, and ‐6 expression suggested modulated IGF‐I availability in PTHrP groups, while drop of SOX9 protein levels during the PTHrP‐pulse may delay chondroblast formation and hypertrophy. Overall, the significantly optimized timing of PTHrP‐pulses demonstrated a vast potential to enhance chondrogenesis of MSC and suppress hypertrophy possibly via superior balancing of IGF‐ and SOX9‐related mechanisms. J. Cell. Physiol. 231: 2673–2681, 2016. © 2016 Wiley Periodicals, Inc. Daily PTHrP treatment significantly increased extracellular matrix deposition regardless of pulse duration and suppressed alkaline‐phosphatase activity by 87%. Pulse duration was critically important to significantly reduce IHH expression, but irrelevant for PTHrP‐induced suppression of the hypertrophic markers MEF2C and IBSP. Decreased IGFBP‐2 and ‐6 expression suggested modulated IGF‐I availability in PTHrP groups while drop of SOX9 protein levels during the PTHrP‐pulse may delay chondroblast formation and hypertrophy. Overall, the significantly optimized timing of PTHrP‐pulses demonstrated a vast potential to enhance chondrogenesis of MSC and suppress hypertrophy possibly via superior balancing of IGF‐ and SOX9‐related mechanisms.
  • The Androgen Receptor Regulates PPARγ Expression and Activity in
           Human Prostate Cancer Cells
    • Abstract: The peroxisome proliferator activated receptor gamma (PPARγ) is a ligand‐activated transcription factor that regulates growth and differentiation within normal prostate and prostate cancers. However the factors that control PPARγ within the prostate cancers have not been characterized. The goal of this study was to examine whether the androgen receptor (AR) regulates PPARγ expression and function within human prostate cancer cells. qRT‐PCR and Western blot analyses revealed nanomolar concentrations of the AR agonist dihydrotestosterone (DHT) decrease PPARγ mRNA and protein within the castration‐resistant, AR‐positive C4‐2 and VCaP human prostate cancer cell lines. The AR antagonists bicalutamide and enzalutamide blocked the ability of DHT to reduce PPARγ levels. In addition, siRNA mediated knockdown of AR increased PPARγ protein levels and ligand‐induced PPARγ transcriptional activity within the C4‐2 cell line. Furthermore, proteasome inhibitors that interfere with AR function increased the level of basal PPARγ and prevented the DHT‐mediated suppression of PPARγ. These data suggest that AR normally functions to suppress PPARγ expression within AR‐positive prostate cancer cells. To determine whether increases in AR protein would influence PPARγ expression and activity, we used lipofectamine‐based transfections to overexpress AR within the AR‐null PC‐3 cells. The addition of AR to PC‐3 cells did not significantly alter PPARγ protein levels. However, the ability of the PPARγ ligand rosiglitazone to induce activation of a PPARγ‐driven luciferase reporter and induce expression of FABP4 was suppressed in AR‐positive PC‐3 cells. Together, these data indicate AR serves as a key modulator of PPARγ expression and function within prostate tumors. J. Cell. Physiol. 231: 2664–2672, 2016. © 2016 Wiley Periodicals, Inc. The goal of this study was to determine whether the androgen receptor (AR) signaling pathway influences the expression and function of PPAR gamma within human prostate cancer cells. Our results demonstrate that the presence of AR suppresses PPAR gamma protein levels, and inhibits the ability of PPAR gamma agonists to regulate transcription and suppress prostate cancer cell proliferation.
  • Chrysophanol Induces Apoptosis of Choriocarcinoma Through Regulation of
           ROS and the AKT and ERK1/2 Pathways
    • Abstract: Chrysophanol is an anthraquinone compound, mainly isolated from rhubarb, with anti‐cancer effects on some types of cancer cells. However, effects of chrysophanol on human choriocarcinoma cells are not known. Therefore, the objective of this study was to determine effects of chrysophanol on choriocarcinoma cells (JAR and JEG‐3) and identify signal transduction cascades activated by chrysophanol. Results of present study, showed that chrysophanol decreased cell viability and induced apoptosis of JEG‐3, but not JAR cells, in a dose‐dependent manner. Chrysophanol also increased oxidative stress in JEG‐3 cells by inducing ROS generation followed by mitochondrial dysfunction including depolarization of mitochondrial inner membrane potential. Western blot analysis revealed that ERK1/2, P90RSK, AKT, and P70S6K were increased significantly in JEG‐3 cells by chrysophanol. Next, we investigated chrysophanol‐mediated effects on proliferation of JEG‐3 cells using pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126). Inhibition of AKT and ERK1/2 prevented chrysophanol‐induced stimulation of proliferation of JEG‐3 cells. In addition, the phosphorylation of AKT and ERK1/2 was suppressed by LY294002 and U0126 in JEG‐3 cells treated with chrysophanol, whereas, the AKT protein was activated by pre‐treatment of JEG‐3 cells with U0126. Furthermore, we compared therapeutic effects of chrysophanol with cisplatin and paclitaxel which are conventional salvage regimens for choriocarcinoma. Our results verified that chrysophanol has synergistic effects with traditional therapy to increase apoptosis of JEG‐3 cells. Collectively, these results indicate that chrysophanol is a potential effective chometherapeutic agent for treatment of choriocarcinoma therapy, and minimizing side effects of conventional treatment regimens. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc. Results of the present study indicated that chrysophanol has anti‐cancer effects by reducing cell viability and inducing apoptosis by increasing ROS production and decreasing mitochondrial membrane potential through the ERK1/2 and AKT signaling cascades. Moreover, chrysophanol is a potential effective chometherapeutic agent for treatment of choriocarcinoma therapy when combined with cisplatin and paclitaxel and minimizing side effects of conventional treatment regimens.
  • Anti‐Diabetic Agent Sodium Tungstate Induces the Secretion of
           Pro‐ and Anti‐Inflammatory Cytokines by Human Kidney Cells
    • Abstract: Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti‐diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type‐2‐diabetic (D‐RPTEC) and non‐diabetic (N‐RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development, and repair, all processes that are dysregulated during DKD. In basal conditions, D‐RPTEC cells secreted higher levels of prototypical pro‐inflammatory IL‐6, IL‐8, and MCP‐1 than N‐RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL‐6, IL‐8, and MCP‐1 secretion in both N‐ and D‐RPTEC, together with lower levels of IL‐1 RA, IL‐4, IL‐10, and GM‐CSF, suggesting that it may contribute to the extent of renal damage/repair during DKD. Besides, NaW induced the accumulation of IκBα, the main inhibitor protein of one major pathway involved in cytokine production, suggesting further anti‐inflammatory effect in the long‐term. A better understanding of the mechanisms involved in the interplay between the anti‐diabetic and immunomodulatory properties of NaW will facilitate future studies about its clinical relevance. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc. Sodium tungstate (NaW) exerts anti‐diabetic activities. We further show that NaW induces IL‐6, IL‐8, and MCP‐1 secretion, together with lower levels of IL1‐RA, IL‐4, IL‐10, GM‐CSF in renal proximal tubule epithelial cells from diabetic and non‐diabetic subjects, suggesting that it may contribute to the extent of renal damage/repair of diabetic kidney.
  • Sex Differences in Estrogen Receptor α and β Levels and
           Activation Status in LPS‐Stimulated Human Macrophages
    • Abstract: Immune function, inflammation, and atherosclerosis display sex differences and are influenced by 17β‐estradiol through estrogen receptors subtypes ERα and ERβ. Male tissues express active ERs, but their possible involvement in inflammation in males has never been assessed. Macrophages express both ERα and ERβ and offer the opportunity to evaluate the role of ER levels and activation in inflammation. We assessed the ability of lipopolysaccharide (LPS) to modulate, in a sex‐specific way, the expression and the activation status of ERα and ERβ in blood monocytes‐derived macrophages (MDMs) from men and women. MDMs were incubated with 100 ng/ml LPS for 24 h and used to evaluate ERα, ERβ, P‐ERα, p38, and P‐p38 expression by Western Blotting. In basal conditions, ERα and ERβ were significantly higher in female MDMs than in male MDMs. LPS up‐regulated ERα and ERα phosphorylation in both sexes, with a significantly higher effect observed in male MDMs, and down‐regulated ERβ level only in female MDMs. p38 and P‐p38 proteins, indicative of ERβ activity, did not show sex differences both in basal conditions and after LPS treatment. Finally, ERα/ERβ and P‐ERα/ERα ratios were significantly higher in male MDMs than in female ones. Our data indicate, for the first time, that LPS affects ERα but not ERβ activation status. We identify a significant role of ERα in LPS‐mediated inflammatory responses in MDMs, which represents an initial step in understanding the influence of sex in the relationship between LPS and ERα. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 Wiley Periodicals, Inc.
  • Animal Models of Congenital Cardiomyopathies Associated With Mutations in
           Z‐Line Proteins
    • Abstract: The cardiac Z‐line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. The Z‐line is important for efficient force generation and transmission as well as the maintenance of structural stability and integrity. Furthermore, it is a nodal point for intracellular signaling, in particular mechanosensing and mechanotransduction. Mutations in various genes encoding Z‐line proteins have been associated with different cardiomyopathies, including dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction, and mutations even within the same gene can cause widely different pathologies. Animal models have contributed to a great advancement in the understanding of the physiological function of Z‐line proteins and the pathways leading from mutations in Z‐line proteins to cardiomyopathy, although genotype–phenotype prediction remains a great challenge. This review presents an overview of the currently available animal models for Z‐line and Z‐line associated proteins involved in human cardiomyopathies with special emphasis on knock‐in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z‐line proteins. Pros and cons of mouse models will be discussed and a future outlook will be given. J. Cell. Physiol. 9999: 1–15, 2016. © 2016 Wiley Periodicals, Inc. The cardiac Z‐line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. This review presents an overview of the currently available animal models for Z‐line and Z‐line associated proteins involved in human cardiomyopathies with special emphasis on knock‐in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z‐line proteins.
  • Ginkgolic Acid Inhibits Invasion and Migration and
           TGF‐β‐Induced EMT of Lung Cancer Cells Through
           PI3K/Akt/mTOR Inactivation
    • Abstract: Epithelial‐to‐mesenchymal transition (EMT) is a critical cellular phenomenon regulating tumor metastases. In the present study, we investigated whether ginkgolic acid can affect EMT in lung cancer cells and the related underlying mechanism(s) of its actions. We found that ginkgolic acid C15:1 (GA C15:1) inhibited cell proliferation, invasion, and migration in both A549 and H1299 lung cancer cells. GA C15:1 also suppressed the expression of EMT related genes (Fibronectin, Vimentin, N‐cadherin, MMP‐9, MMP‐2, Twist and Snail) and suppressed TGF‐β‐induced EMT as assessed by reduced expression of mesenchymal markers (Fibronectin, Vimentin, N‐cadherin), MMP‐9, MMP‐2, Twist and Snail. However, GA C15:1 did not affect the expression of various epithelial marker proteins (Occludin and E‐cadherin) in both A549 and H1299 cells. TGF‐β‐induced morphologic changes from epithelial to mesenchymal cells and induction of invasion and migration were reversed by GA C15:1. Finally, GA C15:1 not only abrogated basal PI3K/Akt/mTOR signaling cascade, but also reduced TGF‐β‐induced phosphorylation of PI3K/Akt/mTOR pathway in lung cancer cells. Overall, these findings suggest that GA C15:1 suppresses lung cancer invasion and migration through the inhibition of PI3K/Akt/mTOR signaling pathway and provide a source of potential therapeutic compounds to control the metastatic dissemination of tumor cells. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc. GA C15:1 also suppressed the expression of EMT related genes. GA C15:1 suppressed TGF‐beta‐induced mesenchymal markers, MMP‐9, MMP‐2, Twist and Snail. GA C15:1 suppresses metastatic lung cancer through inhibition of PI3K/Akt/mTOR activation.
  • Unique Regulatory Mechanisms for the Human Embryonic Stem Cell Cycle
    • Abstract: The cell cycle in pluripotent human embryonic stem cells is governed by unique mechanisms that support unrestricted proliferation and competency for endodermal, mesodermal and ectodermal differentiation. The abbreviated G1 period with retention of uncompromised fidelity for genetic and epigenetic mechanisms operative in control of proliferation support competency for expansion of the pluripotent cell population that is fundamental for initial stages of development. Regulatory events during the G1 period of the pluripotent cell cycle are decisive for the transition from pluripotency to lineage commitment. Recent findings indicate that a G2 cell cycle pause is present in both endodermal and mesodermal lineage cells, and is obligatory for differentiation to endoderm. This article is protected by copyright. All rights reserved
  • Pathophysiology of the Desmo‐Adhesome
    • Abstract: Advances in our understanding of desmosomal diseases have provided a clear demonstration of the key role played by desmosomes in tissue and organ physiology, highlighting the importance of their dynamic and finely regulated structure. In this context, non‐desmosomal regulatory molecules have acquired increasing relevance in the study of this organelle resulting in extending the desmosomal interactome, named the “desmo‐adhesome”. Spatiotemporal changes in the expression and regulation of the desmo‐adhesome underlie a number of genetic, infectious, autoimmune, and malignant conditions. The aim of the present article was to examine the structural and functional relationship of the desmosome, by providing a comprehensive, yet focused overview of the constituents targeted in human disease. The inclusion of the novel regulatory network in the desmo‐adhesome pathophysiology opens new avenues to a deeper understanding of desmosomal diseases, potentially unveiling pathogenic mechanisms waiting to be explored. This article is protected by copyright. All rights reserved
  • Chemoresistance of Lung and Breast Cancer Cells Growing Under Prolonged
           Periods of Serum Starvation
    • Abstract: The efficacy of chemotherapy is hindered by both tumor heterogeneity and acquired or intrinsic multi‐ drug resistance caused by the contribution of multidrug resistance proteins and stemness‐associated prosurvival markers. Therefore, targeting multi‐drug resistant cells would be much more effective against cancer. In this study, we characterized the chemoresistance properties of adherent (anchorage‐ dependent) lung H460 and breast MCF‐7 cancer cells growing under prolonged periods of serum starvation (PPSS). We found that under PPSS, both cell lines were highly resistant to Paclitaxel, Colchicine, Hydroxyurea, Obatoclax, Wortmannin and LY294002. Levels of several proteins associated with increased stemness such as Sox2, MDR1, ABCG2 and Bcl‐2 were found to be elevated in H460 cells but not in MCF‐7 cells. While pharmacological inhibition of either MDR1, ABCG2, Bcl‐2 with Verapamil, Sorafenib or Obatoclax respectively decreased the levels of their target proteins under routine culture conditions as expected, such inhibition did not reverse PX resistance in PPSS conditions. Paradoxically, treatment with inhibitors in serum‐starved conditions produced an elevation of their respective target proteins. In addition, we found that Digitoxin, an FDA approved drug that decrease the viability of cancer cells growing under PPSS, downregulates the expression of Sox2, MDR1, phospho‐ AKT, Wnt5a/b and β‐catenin. Our data suggests that PPSS‐induced chemoresistance is the result of extensive rewiring of intracellular signaling networks and that multi‐resistance can be effectively overcome by simultaneously targeting multiple targets of the rewired network. Furthermore, our PPSS model provides a simple and useful tool to screen drugs for their ability to target multiple pathways of cancer resistance. This article is protected by copyright. All rights reserved
  • Store Operated Ca2+ Entry in Oocytes Modulates the Dynamics of
           IP3‐Dependent Ca2+ Release from Oscillatory to Tonic
    • Abstract: Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal and amplitude features. Here we investigate the role of Store‐Operated Ca2+ Entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release is coupled to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3R) results in a pump‐leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER‐plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3Rs at distant sites, a process we refer to as ‘Ca2+ teleporting’. Therefore, sustained SOCE activation in the presence of an IP3‐dependent ‘leak’ pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. This article is protected by copyright. All rights reserved
  • DJ‐1/Park7 Sensitive Na+/H+ Exchanger 1 (NHE1) in CD4+ T Cells
    • Abstract: DJ‐1/Park7 is a redox‐sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ‐1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na+/H+ exchanger 1 (NHE1). ROS formation in CD4+ T cells plays a decisive role in regulating inflammatory responses. In the present study we explored whether DJ‐1 is expressed in CD4+ T cells and affects ROS production as well as NHE1 in those cells. To this end, DJ‐1 and NHE1 transcript and protein levels were quantified by qRT‐PCR and Western blotting respectively, intracellular pH (pHi) utilizing bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' –dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ‐1 was expressed in CD4+ T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4+ T cells from DJ‐1 deficient mice than in CD4+ T cells from wild type mice. Antioxidant N‐acetyl‐cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ‐1 deficient CD4+ T cells, and blunted the difference between DJ‐1‐/‐ and DJ‐1+/+ CD4+ T cells, an observation pointing to a role of ROS in the up‐regulation of NHE1 in DJ‐1‐/‐ CD4+ T cells. In conclusion, DJ‐1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4+ T cells. This article is protected by copyright. All rights reserved
  • Dedicator of Cytokinesis 2 in Cell Signaling Regulation and Disease
    • Abstract: Dedicator of cytokinesis 2 (DOCK2) is a CDM family protein containing DOCK homology region (DHR) ‐1 and DHR‐2, Src‐homology 3 (SH3) domain, and C‐terminal polybasic amino acid cluster. The CDM family consists of 11 mammalian members and is classified into four subfamilies, the DOCK‐A, ‐B, ‐C, and ‐D. DOCK2 is a member of DOCK‐A subfamily and an atypical guanine exchange factor regulating the loading of GTP to activate Rac. It is primarily found in peripheral blood, spleen, and thymus and mainly expressed in lymphocytes and macrophages of various organs. DOCK2 is also expressed in microglial in brain and is induced in neointima smooth muscle following vascular injury. Functionally, DOCK2 is involved in cell motility, polarity, adhesion, proliferation, and apoptosis. It is essential for lymphocyte migration and activation as well as neutrophil chemotaxis. DOCK2 also regulates the differentiation of natural killer T cells, type 2 T helper cells, and plasmacytoid dendritic cells. In addition, it is important for the growth of B cell lymphoma and prostate cancer cells. Deletion of DOCK2 enables long‐term cardiac allograft survival. Moreover, DOCK2 is associated with the Alzheimer Disease, HIV development, and the early‐onset of invasive infections. Recently, we found that DOCK2 plays a critical role in SMC phenotypic modulation and vascular remodeling. In this review, we will briefly summarize recent advancement of DOCK2 function. This article is protected by copyright. All rights reserved
  • Neurotransmitters: The Critical Modulators Regulating Gut‐Brain Axis
    • Abstract: Neurotransmitters including catecholamines and serotonin play a crucial role in maintaining homeostasis in the human body. Studies on these neurotransmitters mainly revolved around their role in the “fight or flight” response, transmitting signals across a chemical synapse and modulating blood flow throughout the body. However, recent research has demonstrated that neurotransmitters can play a significant role in the gastrointestinal (GI) physiology. Norepinephrine (NE), epinephrine (E), dopamine (DA), and serotonin have recently been a topic of interest because of their roles in the gut physiology and their potential roles in gastrointestinal and central nervous system pathophysiology. These neurotransmitters are able to regulate and control not only blood flow, but also affect gut motility, nutrient absorption, gastrointestinal innate immune system, and the microbiome. Furthermore, in pathological states such as inflammatory bowel disease (IBD) and Parkinson's disease, the levels of these neurotransmitters are dysregulated, therefore causing a variety of gastrointestinal symptoms. Research in this field has shown that exogenous manipulation of catecholamine serum concentrations can help in decreasing symptomology and/or disease progression. In this review article, we discuss the current state‐of‐the‐art research and literature regarding the role of neurotransmitters in regulation of normal gastrointestinal physiology, their impact on several disease processes, and novel work focused on the use of exogenous hormones and/or psychotropic medications to improve disease symptomology. This article is protected by copyright. All rights reserved
  • MAP Kinase‐Dependent RUNX2 Phosphorylation Is Necessary for
           Epigenetic Modification of Chromatin During Osteoblast Differentiation
    • Abstract: RUNX2, an essential transcription factor for osteoblast differentiation and bone formation, is activated by ERK/MAP kinase‐dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2‐binding regions of Bglap2 and Ibsp. Growth of MC3T3‐E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin‐bound P‐ERK, P‐RUNX2, p300 and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di‐methylation, another gene activation‐associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono‐, di‐, and tri‐methylation, in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation‐dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2‐deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non‐phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin‐associated P‐ERK, p300 and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P‐RUNX2‐dependent process involving epigenetic modifications of specific promoter regions. This article is protected by copyright. All rights reserved
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