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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]
  • 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
  • Neoadjuvant sequential docetaxel followed by high‐dose epirubicin in
           combination with cyclophosphamide administered concurrently with
    • Abstract: To report the results of the DECT trial, a phase II study of locally advanced or operable HER2‐positive breast cancer (BC) treated with taxanes and concurrent anthracyclines and trastuzumab. Eligible patients (stage IIA‐IIIB HER2‐positive BC),18‐75 years, normal organ functions, ECOG ≤1, and left ventricular ejection fraction (LVEF) ≥55% received 4 cycles of neoadjuvant docetaxel, 100 mg/m2 intravenously, plus trastuzumab 6 mg/kg (loading dose 8mg/kg) every 3 weeks, followed by four 3‐weekly cycles of epirubicin 120 mg/m2 and cyclophosphamide, 600 mg/m2, plus trastuzumab. Primary objective was pathologic complete response (pCR) rate, defined as ypT0/is ypN0 at definitive surgery. We enrolled 45 consecutive patients. All but 6 patients (13.3%) completed chemotherapy and all underwent surgery. pCR was observed in 28 patients (62.2%) overall and in 6 (66.7%) from the inflammatory subgroup. The classification and regression tree analysis showed a 100% pCR rate in patients with BMI≥25 and with hormone responsive disease. The median follow up was 46 months (8‐78). Four‐year recurrence‐free survival was 74.7% (95%CI, 58.2‐91.2). Seven patients (15.6%) recurred and one died. Treatment was well tolerated, with limiting toxicity being neutropenia. No clinical cardiotoxicity was observed. Six patients (13.4%) showed a transient LVEF decrease (
      PubDate: 2016-06-02T10:32:35.767155-05:
      DOI: 10.1002/jcp.25432
  • Microbial community metabolic modeling: A community data‐driven
           network reconstruction
    • Abstract: Metabolic network modeling of microbial communities provides an in‐depth understanding of community‐wide metabolic and regulatory processes. Compared to single organism analyses, community metabolic network modeling is more complex because it needs to account for interspecies interactions. To date, most approaches focus on reconstruction of high‐quality individual networks so that, when combined, they can predict community behaviors as a result of interspecies interactions. However, this conventional method becomes ineffective for communities whose members are not well characterized and cannot be experimentally interrogated in isolation. Here, we tested a new approach that uses community‐level data as a critical input for the network reconstruction process. This method focuses on directly predicting interspecies metabolic interactions in a community, when axenic information is insufficient. We validated our method through the case study of a bacterial photoautotroph‐heterotroph consortium that was used to provide data needed for a community‐level metabolic network reconstruction. Resulting simulations provided experimentally validated predictions of how a photoautotrophic cyanobacterium supports the growth of an obligate heterotrophic species by providing organic carbon and nitrogen sources. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:25:22.790673-05:
      DOI: 10.1002/jcp.25428
  • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:25:19.149981-05:
      DOI: 10.1002/jcp.25429
  • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:13:27.95026-05:0
      DOI: 10.1002/jcp.25426
  • 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. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:06:33.346872-05:
      DOI: 10.1002/jcp.25424
  • Sex differences in estrogen receptor α and β levels and
           activation status in LPS‐stimulated human macrophages
    • Authors: Ilaria Campesi; Maria Marino, Andrea Montella, Sara Pais, Flavia Franconi
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:06:28.352424-05:
      DOI: 10.1002/jcp.25425
  • Chrysophanol induces apoptosis of choriocarcinoma by through regulation of
           ROS and the AKT and ERK1/2 pathways
    • Authors: Whasun Lim; Changwon Yang, Fuller W. Bazer, Gwonhwa Song
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:03:58.124871-05:
      DOI: 10.1002/jcp.25423
  • Repression of primitive erythroid program is critical for the initiation
           of multi‐lineage hematopoiesis in mouse development
    • Authors: Toshiyuki Yamane; Chie Ito, Aya Washino, Kana Isono, Hidetoshi Yamazaki
      Abstract: Formation of the hematopoietic cells occurs in multiple steps. The first hematopoietic cells observed during ontogeny are primitive erythrocytes, which are produced in the early yolk sac within a limited temporal window. Multi‐lineage hematopoiesis, which supplies almost the entire repertoire of blood cell lineages, lags behind primitive erythropoiesis in the tissue. However, molecular mechanisms regulating sequential generation of primitive erythrocytes and multipotent hematopoietic progenitors in the yolk sac are largely unknown. In this study, the transcription factors involved in the development of hematopoietic cells were examined in purified progenitor cell populations from pluripotent stem cell cultures and from the yolk sac of developing embryos. We found that the earliest committed hematopoietic progenitors highly expressed Gata1, Scl/tal1, and Klf1 genes. Expression of these transcription factors, which is known to form a core erythroid transcriptional network, explained the prompt generation of primitive erythrocytes from these earliest progenitors. Importantly, the multipotent hematopoietic cells, which lack the differentiation potential into primitive erythroid cells, down‐regulated these genes during a transition from the earliest committed progenitors. In addition, we showed that Pu.1 is involved in the multipotent cell differentiation through the suppression of erythroid transcription program. We propose that these molecular mechanisms governed by transcription factors form sequential waves of primitive erythropoiesis and multi‐lineage hematopoiesis in the early yolk sac of developing embryos. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T09:57:07.656034-05:
      DOI: 10.1002/jcp.25422
  • Flavopiridol: An old drug with new perspectives' Implication for
           development of new drugs
    • Authors: Annamaria Cimini; Michele d'Angelo, Elisabetta Benedetti, Barbara D'Angelo, Giulio Laurenti, Andrea Antonosante, Loredana Cristiano, Antonella Di Mambro, Marcella Barbarino, Vanessa Castelli, Benedetta Cinque, Maria Grazia Cifone, Rodolfo Ippoliti, Francesca Pentimalli, Antonio Giordano
      Abstract: Glioblastoma, the most common brain tumor, is characterized by high proliferation rate, invasion, angiogenesis and chemo‐ and radio‐resistance. One of most remarkable feature of glioblastoma is the switch toward a glycolytic energetic metabolism that leads to high glucose uptake and consumption and a strong production of lactate. Activation of several oncogene pathways like Akt, c‐myc and ras, induces glycolysis and angiogenesis and acts to assure glycolysis prosecution, tumor proliferation and resistance to therapy. Therefore, the high glycolytic flux depends on the overexpression of glycolysis‐related genes resulting in an overproduction of pyruvate and lactate. Metabolism of glioblastoma thus represents a key issue for cancer research. Flavopiridol is a synthetic flavonoid that inhibits a wide range of Cyclin‐dependent kinase, that has been demonstrate to inactivate glycogen phosphorylase, decreasing glucose availability for glycolysis. In this work is reported the study of glucose metabolism upon flavopiridol treatment in the two different glioblastoma cell lines. The results obtained point towards an effect of flavopiridol in glycolytic cells, thus suggesting a possible new use of this compound or flavopiridol‐derived formulations in combination with anti‐proliferative agents in glioblastoma patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T09:52:49.630745-05:
      DOI: 10.1002/jcp.25421
  • Probing Micromechanical Properties of the Extracellular Matrix of Soft
           Tissues by Atomic Force Microscopy
    • Abstract: The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy (AFM) has unique capabilities to measure ECM mechanics at the scale at which cells probe the mechanical features of their microenvironment. Moreover, AFM measurements can be readily combined with bright field and fluorescence microscopy. Performing reliable mechanical measurements with AFM requires accurate calibration of the device and correct computation of the mechanical parameters. A suitable approach to isolate ECM mechanics from cell contribution is removing the cells by means of an effective decellularization process that preserves the composition, structure and mechanical properties of the ECM. AFM measurement of ECM micromechanics provides important insights into organ biofabrication, cell‐matrix mechanical crosstalk and disease‐induced tissue stiffness alterations. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T06:06:07.36028-05:0
      DOI: 10.1002/jcp.25420
  • How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy
    • Authors: Albert T. Gacerez; Benjamine Arellano, Charles L. Sentman
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T05:58:42.372515-05:
      DOI: 10.1002/jcp.25419
  • Milk‐Derived Nanoparticle Fraction Promotes the Formation of Small
           Osteoclasts But Reduces Bone Resorption
    • Authors: Marina C. Oliveira; Irene Di Ceglie, Onno J. Arntz, Wim B. van den Berg, Frank H.J. van den Hoogen, Adaliene V.M. Ferreira, Peter L.E.M. van Lent, Fons A.J. van de Loo
      Abstract: The general consensus is that milk promotes bone growth and density because is a source of calcium and contains components that enhance intestinal calcium uptake or directly affect bone metabolism. In this study we investigated the effect of bovine‐derived milk 100,000g pellet (P100), which contains nanoparticles (
      PubDate: 2016-06-02T05:51:51.12926-05:0
      DOI: 10.1002/jcp.25414
  • Targeting the breast cancer kinome
    • Authors: Samantha M. Miller; Daniel R. Goulet, Gary L. Johnson
      Abstract: Protein kinases are highly tractable targets for the treatment of many cancers including breast cancer, due to their essential role in tumor cell proliferation and survival. Sequencing of the breast cancer genome and transcriptome has defined breast cancer as a heterogeneous disease that is classified into five molecular subtypes: luminal A, luminal B, HER2‐enriched, basal‐like and claudin‐low. Each subtype displays a unique expression profile of protein kinases that can be targeted by small molecule kinase inhibitors or biologics. An understanding of genomic changes, including mutations or copy number variations, for specific protein kinases and dependencies on kinases across breast cancer subtypes is allowing for a more rational design of targeted breast cancer therapies. While specific kinase inhibitors have had success in the clinic, including the CDK4/6 inhibitor palbociclib in combination with aromatase inhibitors in luminal breast cancer, patients often become resistant to treatment. An understanding of the mechanisms allowing cells to bypass targeted kinase inhibition has led to the development of combination therapies that are more durable in pre‐clinical studies. However, the heterogeneity of resistance mechanisms and rapid adaptability of the kinome through feedback regulation greatly inhibit the long‐term efficacy of combination kinase inhibitor therapies. It is becoming apparent that epigenetic inhibitors such as HDAC and BET bromodomain inhibitors can block the transcriptional adaptability of tumor cells to kinase inhibitors and prevent the onset of resistance. Such novel combination therapies are currently showing promise in preclinical studies to markedly increase the durability of kinase inhibitors in breast cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-31T07:45:26.494401-05:
      DOI: 10.1002/jcp.25427
  • Epithelial‐to‐mesenchymal transition in
           paclitaxel‐resistant ovarian cancer cells is downregulated by
    • Authors: Vermont P Dia; Philipus Pangloli
      Abstract: Ovarian cancer (OVCA) is the deadliest of all gynecological cancers which is attributed to late presentation, persistence and development of chemoresistance. The objectives were to evaluate the association between OVCA paclitaxel‐resistance and epithelial‐to‐mesenchymal transition (EMT) and to determine the capability of luteolin to chemosensitize OVCA cells. X10 and X22 cells were 11.8–25.3‐fold and 7.8–8.6‐fold resistant to paclitaxel than 1AP cells. X10 and X22 cells exhibited a mesenchymal phenotype while 1AP has an epithelial characteristics. Furthermore, the expression of the epithelial marker E‐cadherin was downregulated while mesenchymal markers Vimentin and N‐cadherin were upregulated in X10 and X22 cells when compared to 1AP cells. Transcription factors Snail, Slug and Twist1 were upregulated in X10 cells while Twist1 was highly expressed in X22 cells. Luteolin treatment caused cytotoxicity being most potent to X10 OVCA cells. Treatment of non‐cytotoxic dose of luteolin at 15.625 µM chemosensitized X10 and X22 OVCA cells to paclitaxel as evidenced by reduced ED50 values from 11.8 to 0.2 µM and 8.6 to 3.6 µM for X10 and X22 cells, respectively. Moreover, luteolin treatment led to a more epithelial phenotype of X10 and X22 cells and modification of EMT markers indicating reversal of EMT. The mechanism involved is through reduction of phosphorylation of FAK and ERK leading to reduced nuclear translocation of p65. Our results highlight the significance of EMT in OVCA resistance to paclitaxel and warrant the investigation of luteolin as a potential therapeutic agent in chemoresistant OVCA. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-27T08:25:26.002642-05:
      DOI: 10.1002/jcp.25436
  • LPS induces hyper‐permeability of intestinal epithelial cells
    • Authors: Amir Bein; Alexander Zilbershtein, Michael Golosovsky, Dan Davidov, Betty Schwartz
      Abstract: Background Necrotizing Enterocolitis (NEC) is a severe inflammatory disorder leading to high morbidity and mortality rates. A growing body of evidence demonstrate the key role of the Toll like receptor 4 (TLR4) in NEC. This membranal receptor recognizes lipopolysaccharides (LPS) from the bacterial wall and triggers an inflammatory response. The aim of the present study was to elucidate the effect of LPS on paracellular permeability known to be severely affected in NEC. Methods IEC‐18 cells were treated with LPS and the effects on morphology, paracellular permeability and their associated gene and protein expressions were measured. Results LPS down regulated the expression of occludin and ZO‐1 mRNAs while up regulating Cdkn1a. In addition LPS caused a significant increase in paracellular permeability and epithelial barrier damage. Finally ZO‐1 protein was found to be spatially disarrayed in the intercellular junctions in response to LPS. Conclusion LPS adversely affected the functionality of the intestinal epithelial barrier suggesting a new mechanism by which bacterial infection may contribute to the development of NEC. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-26T09:11:37.61133-05:0
      DOI: 10.1002/jcp.25435
  • Prostaglandin E2‐Dependent Phosphorylation of RAS Inhibition 1
           (RIN1) at Ser 291 and 292 Inhibits Transforming Growth
           Factor‐β‐Induced RAS Activation Pathway in Human Synovial
           Fibroblasts: Role in Cell Migration
    • Authors: Casimiro Gerarduzzi; QingWen He, Beibei Zhai, John Antoniou, John A. Di Battista
      Abstract: Prostaglandin E2 (PGE2)‐stimulated G‐protein coupled receptor (GPCR) activation inhibits pro‐fibrotic TGFβ‐dependent stimulation of human fibroblast to myofibroblast transition (FMT), though the precise molecular mechanisms are not fully understood. In the present study, we describe the PGE2‐dependent suppression and reversal of TGFβ‐induced events such as α‐sma expression, stress fiber formation, and Ras/Raf/ERK/MAPK pathway‐dependent activation of myofibroblast migration. In order to elucidate post ligand‐receptor signaling pathways, we identified a predominant PKA phosphorylation motif profile in human primary fibroblasts after treatment with exogenous PGE2 (EC50 30nM, Vmax 100nM), mimicked by the adenyl cyclase activator forskolin (EC50 5µM, Vmax 10µM). We used a global phosphoproteomic approach to identify a 2.5 fold difference in PGE2 induced phosphorylation of proteins containing the PKA motif. Deducing the signaling pathway of our migration data, we identified Ras inhibitor 1 (RIN1) as a substrate, whereby PGE2 induced its phosphorylation at Ser291 and at Ser292 by a 5.4‐ and 4.8‐fold increase, respectively. In a series of transient and stable over expression studies in HEK293T and HeLa cells using wild‐type (wt) and mutant RIN1 (Ser291/292Ala) or Ras constructs and siRNA knock‐down experiments, we showed that PGE2‐dependent phosphorylation of RIN1 resulted in the abrogation of TGFβ induced Ras/Raf signaling activation and subsequent downstream blockade of cellular migration, emphasizing the importance of such phosphosites in PGE2 suppression of wound closure. Over expression experiments in tandem with pull‐down assays indicated that specific Ser291/292 phosphorylation of RIN1 favoured binding to activated Ras. In principal, understanding PGE2‐GPCR activated signaling pathways mitigating TGFβ‐induced fibrosis may lead to more evidence‐based treatments against the disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-26T08:46:51.819046-05:
      DOI: 10.1002/jcp.25412
  • 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
  • Prototype of Multiplex Bead Assay for Quantification of Three Serum
           Biomarkers for In Vitro Diagnosis of Endometriosis
    • Authors: Pietro Giulio Signorile; Alfonso Baldi
      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. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-16T01:25:32.216622-05:
      DOI: 10.1002/jcp.25410
  • The Non‐Conventional Effects of Glucocorticoids in Cancer
    • Authors: Simra Azher; Omid Azami, Caterina Amato, Michael McCullough, Antonio Celentano, Nicola Cirillo
      Abstract: Synthetic corticosteroids are widely used for the treatment of a variety of diseases, including pre‐malignant and malignant conditions. In striking contrast, recent evidence suggests that corticosteroids can bear tumour‐promoting effects in solid tumours of epithelial origin. We have recently shown that epithelial tissues, including the mucosa of the oral cavity and the skin, are able to modulate the local concentration of active corticosteroids and to produce steroids de novo. This has important clinical and physiopathological implications, because tissue‐specific regulation of glucocorticoids plays a key role in the overall effect of these molecules. In the present review of the current English literature, performed using MEDLINE / PubMed / Ovid databases, we collected published evidence to demonstrate that corticosteroids induce effects that are more complex and controversial than previously acknowledged. Published studies clearly demonstrate that this class of molecules influences pathophysiological processes that are strictly related to malignancy, providing the rationale for further investigation. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-08T23:55:54.654891-05:
      DOI: 10.1002/jcp.25408
  • 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
  • SHP‐2 Phosphatase Prevents Colonic Inflammation by Controlling
           Secretory Cell Differentiation and Maintaining Host‐Microbiota
    • Abstract: Polymorphisms in the PTPN11 gene encoding for the tyrosine phosphatase SHP‐2 were described in patients with ulcerative colitis. We have recently demonstrated that mice with an intestinal epithelial cell‐specific deletion of SHP‐2 (SHP‐2IEC‐KO) develop severe colitis one month after birth. However, the mechanisms by which SHP‐2 deletion induces colonic inflammation remain to be elucidated. We generated SHP‐2IEC‐KO mice lacking Myd88 exclusively in the intestinal epithelium. The colonic phenotype was histologically analyzed and cell differentiation was determined by electron microscopy and lysozyme or Alcian blue staining. Microbiota composition was analyzed by 16S sequencing. Results show that innate defense genes including those specific to Paneth cells were strongly up‐regulated in SHP‐2‐deficient colons. Expansion of intermediate cells (common progenitors of the Goblet and Paneth cell lineages) was found in the colon of SHP‐2IEC‐KO mice while Goblet cell number was clearly diminished. These alterations in Goblet/intermediate cell ratio were noticed two weeks after birth, before the onset of inflammation and were associated with significant alterations in microbiota composition. Indeed, an increase in Enterobacteriaceae and a decrease in Firmicutes were observed in the colon of these mice, indicating that dysbiosis also occurred prior to inflammation. Importantly, loss of epithelial Myd88 expression inhibited colitis development in SHP‐2IEC‐KO mice, rescued Goblet/intermediate cell ratio and prevented NFκB hyperactivation and inflammation. These data indicate that SHP‐2 is functionally important for the maintenance of appropriate barrier function and host‐microbiota homeostasis in the large intestine. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-21T14:15:45.654798-05:
      DOI: 10.1002/jcp.25407
  • Epigenetics in Intestinal Epithelial Cell Renewal
    • Abstract: A controlled balance between cell proliferation and differentiation is essential to maintain normal intestinal tissue renewal and physiology. Such regulation is powered by several intracellular pathways that are translated into the establishment of specific transcription programs, which influence intestinal cell fate along the crypt‐villus axis. One important check‐point in this process occurs in the transit amplifying zone of the intestinal crypts where different signalling pathways and transcription factors cooperate to manage cellular proliferation and differentiation, before secretory or absorptive cell lineage terminal differentiation. However, the importance of epigenetic modifications such as histone methylation and acetylation in the regulation of these processes is still incompletely understood. There have been recent advances in identifying the impact of histone modifications and chromatin remodelers on the proliferation and differentiation of normal intestinal crypt cells. In this review we discuss recent discoveries on the role of the cellular epigenome in intestinal cell fate, development and tissue renewal. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-07T16:25:48.709253-05:
      DOI: 10.1002/jcp.25401
  • 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
  • The Integrin‐Mediated ILK‐Parvin‐αPix Signalling
           Axis Controls Differentiation in Mammary Epithelial Cells
    • Authors: Nicholas Rooney; Pengbo Wang, Keith Brennan, Andrew P Gilmore, Charles H Streuli
      Abstract: Epithelial cell adhesion to the surrounding extracellular matrix is necessary for their proper behaviour and function. During pregnancy and lactation, mammary epithelial cells (MECs) receive signals from their interaction with laminin via β1‐integrin (β1‐itg) to establish apico‐basal polarity and to differentiate in response to prolactin. Downstream of β1‐itg, the scaffold protein Integrin Linked Kinase (ILK) has been identified as the key signal transducer that is required for both lactational differentiation and the establishment of apico‐basal polarity. ILK is an adaptor protein that forms the IPP complex with PINCH and Parvins, which are central to its adaptor functions. However, it is not known how ILK and its interacting partners control tissue‐specific gene expression. Expression of ILK mutants, which weaken the interaction between ILK and Parvin, revealed that Parvins have a role in mammary epithelial differentiation. This conclusion was supported by shRNA‐mediated knockdown of the Parvins. In addition, shRNA knockdown of the Parvin‐binding guanine nucleotide exchange factor αPix prevented prolactin‐induced differentiation. αPix depletion did not disrupt focal adhesions, MEC proliferation, or polarity. This suggests that αPix represents a differentiation‐specific bifurcation point in β1‐itg‐ILK adhesive signalling. In summary, this study has identified a new role for Parvin and αPix downstream of the integrin‐ILK signalling axis for MEC differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:41:06.985668-05:
      DOI: 10.1002/jcp.25390
  • DC‐STAMP: A Key Regulator in Osteoclast Differentiation
    • Abstract: Bone is a dynamic tissue that continuously undergoes remodeling with diverse functions including locomotion, hemopoiesis, and protection of internal organs. Osteoimmunology has recently become an emerging field of musculoskeletal research, which connects the bone and immune systems. Results from osteoimmunology studies provide a new concept that bone is not only an essential component of the musculoskeletal system, but it also participates in immune regulation. Many important factors involved in osteoimmunology regulation also participate in bone homeostasis. Bone homeostasis is achieved by a coordinated action between bone‐synthesizing osteoblasts and bone‐degrading osteoclasts. An imbalanced action between osteoblasts and osteoclasts often results in pathological bone diseases: osteoporosis is caused by an excessive osteoclast activity over that of osteoblasts, whereas osteopetrosis results from an increased osteoblast activity. This review focus on Dendritic Cell‐Specific Transmembrane Protein (DCSTAMP), an important protein currently considered as the master regulator of osteoclastogenesis. DC‐STAMP is required for cell‐cell fusion during osteoclast differentiation. Intriguingly, the frequency of circulating DCSTAMP+ cells is elevated during the pathogenesis of psoriatic diseases. Current data collectively suggest that DC‐STAMP also plays an imperative role in bone homeostasis by regulating the differentiation of both osteoclasts and osteoblasts. This article summarizes our current knowledge on DC‐STAMP by focusing on its interacting proteins, its regulation on osteoclastogenesis‐related genes, its possible involvement in immunoreceptor tyrosine‐based inhibitory motif (ITIM)‐mediated signaling cascade, and its potential of developing therapeutics for clinical applications. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-27T22:36:35.866617-05:
      DOI: 10.1002/jcp.25389
  • 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
  • Tissue Inhibitor of Metalloproteinase 1 Influences Vascular Adaptations to
           Chronic Alterations in Blood Flow
    • 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
  • Coumestrol Inhibits Proliferation and Migration of Prostate Cancer Cells
           by Regulating AKT, ERK1/2 and JNK MAPK Cell Signaling Cascades
    • 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
  • Tyrosine Residues Regulate Multiple Nuclear Functions of p54nrb
    • 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
  • Extracellular Proteasomes are Deficient in 19S Subunits as Revealed by
           iTRAQ Quantitative Proteomics
    • 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
  • 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
  • Neuronatin Protein in Health and Disease
    • 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
  • 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
  • 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
  • Erratum: Nanotopography Directs Mesenchymal Stem Cells to Osteoblast
           Lineage Through Regulation of microRNA‐SMAD‐BMP‐2
           Circuit. R. B. Kato, B. Roy, F. S. De Oliveira, E. P. Ferraz, P. T. De
           Oliveira, A. G. Kemper, M. Q. Hassan, A. L. Rosa, M. M. Beloti
  • Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,
           Differentiation, and Myotube Hypertrophy
    • Abstract: An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)2D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)2D3 and 25(OH)D3 affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D3 and 1,25(OH)2D3. We show that myoblasts not only responded to 1,25(OH)2D3, but also to the precursor 25(OH)D3 by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)2D3 as well as 25(OH)D3 stimulated VDR mRNA expression and in myotubes 1,25(OH)2D3 also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D3 metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)2D3 or 25(OH)D3 myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D3 to 24R,25(OH)2D3 which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2D3. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The
      Authors . Journal of Cellular Physiology Published by Wiley Periodicals, Inc. In differentiating C2C12 myoblasts and myotubes, 1,25(OH)2D3 as well as 25(OH)D3 stimulate VDR mRNA expression and in myotubes 1,25(OH)2D3 also stimulates MHC mRNA expression, however, this occurs without notable effects on expression of myogenic regulatory factors, the Akt/mTOR signaling pathway and myotube size. Interestingly, both differentiating myoblasts and myotubes express CYP27B1 and CYP24 mRNA and myotubes are able to synthesize 24R,25(OH)2D3 from 25(OH)D3. These data suggest that skeletal muscle is not only a direct target for vitamin D3 metabolites, but is also able to metabolize 25(OH)D3 and 1,25(OH)2D3.
  • MMP‐8 Is Critical for Dexamethasone Therapy in Alkali‐Burned
           Corneas Under Dry Eye Conditions
    • Abstract: Our previous studies have shown that Dexamethasone (Dex) reduced the expression of matrix‐metalloproteinases (MMPs ‐1,‐3,‐9,‐13), IL‐1β and IL‐6, while it significantly increased MMP‐8 mRNA transcripts in a concomitant dry eye and corneal alkali burn murine model (CM). To investigate if MMP‐8 induction is responsible for some of the protective effects of Dex in CM, MMP‐8 knock out mice (MMP‐8KO) were subjected to the CM for 2 or 5 days and topically treated either with 2 μl of 0.1% Dexamethasone (Dex), or saline QID. A separate group of C57BL/6 mice were topically treated with Dex or BSS and received either 100 nM CAM12 (MMP‐8 inhibitor) or vehicle IP, QD. Here we demonstrate that topical Dex treated MMP‐8KO mice subjected to CM showed reduced corneal clarity, increased expression of inflammatory mediators (IL‐6, CXCL1, and MMP‐1 mRNA) and increased neutrophil infiltration at 2D and 5D compared to Dex treated WT mice. C57BL/6 mice topically treated with Dex and CAM12 IP recapitulated findings seen with MMP‐8KO mice. These results suggest that some of the anti‐inflammatory effects of Dex are mediated through increased MMP‐8 expression. J. Cell. Physiol. 231: 2506–2516, 2016. © 2016 Wiley Periodicals, Inc.
  • Radiation‐Induced RhoGDIβ Cleavage Leads to Perturbation of
           Cell Polarity: A Possible Link to Cancer Spreading
    • Abstract: The equilibrium between proliferation and apoptosis is tightly balanced to maintain tissue homeostasis in normal tissues and even in tumors. Achieving and maintaining such a balance is important for cancer regrowth and spreading after cytotoxic treatments. Caspase‐3 activation and tumor cell death following anticancer therapy as well as accompanying cell death pathways are well characterized, but their association to homeostasis of cancerous tissue and tumor progression remains poorly understood. Here we proposed a novel mechanism of cancer spreading induced by caspase‐3. RhoGDIβ, known as a direct cleavage substrate of caspase‐3, is overexpressed in many epithelial cancers. The N‐terminal‐truncated RhoGDIβ (ΔN‐RhoGDIβ) is accumulated in caspase‐3‐activated cells. Stable expression of ΔN‐RhoGDIβ in HeLa cells did not induce apoptosis, but impaired directional cell migration in a wound‐healing assay accompanied by a perturbed direction of cell division at the wound edge. Subcellular protein fractionation experiments revealed that ΔN‐RhoGDIβ but not wild‐type RhoGDIβ was present in the detergent‐soluble cytoplasmic and nuclear fractions and preferentially associated with Cdc42. Furthermore, Cdc42 activity was constitutively inhibited by stable expression of ΔN‐RhoGDIβ, resulting in increased radiation‐induced compensatory proliferation linking to RhoA activation. Thus, ΔN‐RhoGDIβ dominant‐negatively regulates Cdc42 activity and contributes to loss of polarity‐related functions. The caspase‐3‐cleaved RhoGDIβ is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage. J. Cell. Physiol. 231: 2493–2505, 2016. © 2016 Wiley Periodicals, Inc. The N‐terminal‐truncated RhoGDIβ (ΔN‐RhoGDIβ) is accumulated in caspase‐3‐activated cells. ΔN‐RhoGDIβ dominant‐negatively regulates Cdc42 activity and contributes to loss of polarity‐related functions. ΔN‐RhoGDIβ is a possible determinant to promote cancer spreading due to deregulation of directional organization of tumor cell population and inhibition of default equilibrium between proliferation and apoptosis after cytotoxic damage.
  • Deregulated E2F5/p38/SMAD3 Circuitry Reinforces the Pro‐Tumorigenic
           Switch of TGFβ Signaling in Prostate Cancer
    • Abstract: Transforming growth factor‐β signaling exerts divergent effects on normal and cancer cells, although mechanism underlying this differential behavior remains unclear. In this study, expression of 94 genes pertaining to the TGF‐β signaling pathway was compared between tumor and benign tissue samples from the human prostate gland to identify major discriminators driving prostate carcinogenesis. E2F5 was identified as one of the most deregulated genes in prostate cancer tissues, predominantly in samples with Gleason‐score 6. Expression of other deregulated components of TGF‐β signaling was examined by qRT‐PCR, Western blot, and immune‐staining. Function of E2F5 and p38 in prostate cancer was investigated using siRNA‐treatment of PC3 cell‐line followed by analyses of associated components and cell cycle. Observations revealed that E2F5 overexpression was accompanied by significantly higher phosphorylation of SMAD3 at Ser‐208 in the linker region (pSMAD3L) and p38 in tumor tissue. A striking difference in SMAD3 phosphorylation, marked by preponderance of pSMAD3L and pSMAD3C (Ser‐423 and 425) in tumor and benign tissues, respectively was noted. Co‐localization of E2F5 with pSMAD3L in the nuclei of tumor and PC3 cells indicated a functional interface between the proteins. Downregulation of E2F5 and p38 in PC3 cells resulted in marked reduction of phosphorylation of SMAD3 and perturbation of cell cycle with an arrest of cells in G1. Our findings unearthed that E2F5/p38 axis played a cardinal role in uncontrolled cellular proliferation in prostate cancer through pSMAD3L activation. It also underscores a strong potential for E2F5 to be incorporated as a tool in early detection of prostate cancer. J. Cell. Physiol. 231: 2482–2492, 2016. © 2016 Wiley Periodicals, Inc. Transforming growth factor‐β signaling exerts divergent effects on normal and cancer cells. Our study demonstrates that dysregulated E2F5/p38/pSMAD3L axis plays an important role in converting tumor‐suppressive TGFβ signaling into pro‐tumorigenic signaling.
  • Programmable Site‐Specific Nucleases for Targeted Genome Engineering
           in Higher Eukaryotes
    • Abstract: Recent advances in the targeted genome engineering enable molecular biologists to generate sequence specific modifications with greater efficiency and higher specificity in complex eukaryotic genomes. Programmable site‐specific DNA cleavage reagents and cellular DNA repair mechanisms have made this possible. These reagents have become powerful tools for delivering a site‐specific genomic double‐strand break (DSB) at the desired chromosomal locus, which produces sequence alterations through error‐prone non‐homologous end joining (NHEJ) resulting in gene inactivations/knockouts. Alternatively, the DSB can be repaired through homology‐directed repair (HDR) using a donor DNA template, which leads to the introduction of desired sequence modifications at the predetermined site. Here, we summarize the role of three classes of nucleases; zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system in achieving targeted genome modifications. Further, we discuss the progress towards the applications of programmable site‐specific nucleases (SSNs) in treating human diseases and other biological applications in economically important higher eukaryotic organisms such as plants and livestock. J. Cell. Physiol. 231: 2380–2392, 2016. © 2016 Wiley Periodicals, Inc. In this review article, we discuss the properties of programmable site‐specific nucleases and their biomedical and biological applications in higher eukaryotic organisms.
  • Oncofetal Epigenetic Bivalency in Breast Cancer Cells: H3K4 and H3K27
           Tri‐Methylation as a Biomarker for Phenotypic Plasticity
    • Abstract: Alterations in the epigenetic landscape are fundamental drivers of aberrant gene expression that contribute to cancer progression and pathology. Understanding specific modes of epigenetic regulation can be used to identify novel biomarkers or targets for therapeutic intervention to clinically treat solid tumors and leukemias. The bivalent marking of gene promoters by H3K4me3 and H3K27me3 is a primary mechanism to poise genes for expression in pluripotent embryonic stem cells (ESC). In this study we interrogated three well‐established mammary cell lines to model epigenetic programming observed among breast cancer subtypes. Evidence is provided for a distinct bivalent signature, activating and repressive histone marks co‐residing at the same gene promoter, in the MCF7 (ESR/PGR+) luminal breast cancer cell line. We identified a subset of genes, enriched for developmental pathways that regulate cellular phenotype and signaling, and partially recapitulate the bivalent character observed in ESC. We validated the biological relevance of this “oncofetal epigenetic” signature using data from ESR/PGR+ tumor samples from breast cancer patients. This signature of oncofetal epigenetic control is an informative biomarker and may provide novel therapeutic targets, selective for both recurring and treatment‐resistant cancers. J. Cell. Physiol. 231: 2474–2481, 2016. © 2016 Wiley Periodicals, Inc.
  • Priming Endothelial Cells With a Melanoma‐Derived Extracellular
           Matrix Triggers the Activation of αvβ3/VEGFR2 Axis
    • Abstract: The unique composition of tumor‐produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM‐ECM), ECM produced by a melanoma cell line (MV3‐ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin‐C and laminin and a lower expression of fibronectin. When cultured directly on MV3‐ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3‐ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK‐Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma‐ECM triggers integrin‐dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up‐regulate angiogenesis. J. Cell. Physiol. 231: 2464–2473, 2016. © 2016 Wiley Periodicals, Inc.
  • Skin Metabolite, Farnesyl Pyrophosphate, Regulates Epidermal Response to
           Inflammation, Oxidative Stress, and Migration
    • Abstract: Skin produces cholesterol and a wide array of sterols and non‐sterol mevalonate metabolites, including isoprenoid derivative farnesyl pyrophosphate (FPP). To characterize FPP action in epidermis, we generated transcriptional profiles of primary human keratinocytes treated with zaragozic acid (ZGA), a squalene synthase inhibitor that blocks conversion of FPP to squalene resulting in endogenous accumulation of FPP. The elevated levels of intracellular FPP resulted in regulation of epidermal differentiation and adherens junction signaling, insulin growth factor (IGF) signaling, oxidative stress response and interferon (IFN) signaling. Immunosuppressive properties of FPP were evidenced by STAT‐1 downregulation and prominent suppression of its nuclear translocation by IFNγ. Furthermore, FPP profoundly downregulated genes involved in epidermal differentiation of keratinocytes in vitro and in human skin ex vivo. Elevated levels of FPP resulted in induction of cytoprotective transcriptional factor Nrf2 and its target genes. We have previously shown that FPP functions as ligand for the glucocorticoid receptor (GR), one of the major regulator of epidermal homeostasis. Comparative microarray analyses show significant but not complete overlap between FPP and glucocorticoid regulated genes, suggesting that FPP may have wider transcriptional impact. This was further supported by co‐transfection and chromatin immunoprecipitation experiments where we show that upon binding to GR, FPP recruits β‐catenin and, unlike glucocorticoids, recruits co‐repressor GRIP1 to suppress keratin 6 gene. These findings have many clinical implications related to epidermal lipid metabolism, response to glucocorticoid therapy as well as pleiotropic effects of cholesterol lowering therapeutics, statins. J. Cell. Physiol. 231: 2452–2463, 2016. © 2016 Wiley Periodicals, Inc.
  • A Novel Biological Role of α‐Mangostin in Modulating
           Inflammatory Response Through the Activation of SIRT‐1 Signaling
    • Abstract: Several studies have shown that xanthones obtained from Garcinia Mangostana (GM) have remarkable biological activities. α‐mangostin (α‐MG) is the main constituent of the fruit hull of the GM. Several findings have suggested that SIRT‐1, a nuclear histone deacetylase, could influence cellular function by the inhibition of NF‐kB signaling. ROS can inhibit SIRT‐1 activity by initiating oxidative modifications on its cysteine residues, and suppression of SIRT‐1 enhances the NF‐κB signaling resulting in inflammatory responses. The goals of the present study were to evaluate the quantity of α‐MG in the methanolic extract of GM (Vithagroup Spa) and to investigate the activity of this xanthone in U937 cell line and in human monocytes from responsive to inflammatory insult analyzing the possible changes on the activation of SIRT‐1 protein via NF‐Kb. Cells were treated with the methanolic extract of GM and/or LPS. The chromatographic separation of α‐MG was performed by an HPLC analysis. EX 527, a specific SIRT‐1 inhibitor, was used to determine if SIRT‐1/NfkB signaling pathway might be involved in α‐MG action on cells. Our results show that α‐MG inhibits p65 acetylation and down‐regulates the pro‐inflammatory gene products as COX‐2, iNOS via SIRT‐1 activation. Cells treated with EX 527 showed an up‐regulation of NFkB acetylation and an over expression of inducible enzymes and their product of catalysis (NO and PGE2). These results suggest that α‐MG may be useful for the development of alternative pharmacological strategies aimed at reducing the inflammatory process. J. Cell. Physiol. 231: 2439–2451, 2016. © 2016 Wiley Periodicals, Inc.
  • The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares,
           Fusion, and Fenestrations
    • Abstract: In eukaryotes, the nuclear envelope (NE) physically separates nuclear components and activities from rest of the cell. The NE also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the NE is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the NE of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and, expands asymmetrically during mitotic delay. Moreover, its NE is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast NE lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast NE have uncovered interesting, and likely conserved, processes that contribute to NE dynamics. In particular, we discuss the processes that drive and enable NE expansion and the dramatic changes in the NE that lead to extensions and fenestrations. J. Cell. Physiol. 231: 2353–2360, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA
  • Current Controversies in the Management of Myeloma Bone Disease
    • Abstract: Recent significant advances in the treatment of multiple myeloma have resulted in an improvement in median overall survival from 4.6 years, for patients diagnosed between 2001 and 2005, to 6.1 years, for those diagnosed between 2006 and 2010 (Kumar et al., 2014). However, myeloma bone lesions persist in the absence of active disease and continue to be frequent and significant causes of patient morbidity and contribute to mortality. While bisphosphonate therapy in combination with anti‐myeloma therapy remains the cornerstone of skeletal disease management in myeloma, open questions regarding the optimal management of patients with myeloma bone disease remain. This article will address when to initiate and stop bone‐targeted therapy in patients with monoclonal gammopathies, duration of bisphosphonate treatment in the era of more effective anti‐myeloma treatment, the role of bone resorption markers in determining the dosing schedule for anti‐resorptive therapy, risks and benefits of long term anti‐resorptive therapy, and whether anti‐resorptive therapies should be stopped to enhance the potential anabolic effects of proteasome antagonists and other anabolic agents. J. Cell. Physiol. 231: 2374–2379, 2016. © 2016 Wiley Periodicals, Inc.
  • Inheritance of Cytosine Methylation
    • Abstract: There are numerous examples of parental transgenerational inheritance that is epigenetic. The informational molecules include RNA, chromatin modifications, and cytosine methylation. With advances in DNA sequencing technologies, the molecular and epigenetic mechanisms mediating these effects are now starting to be uncovered. This mini‐review will highlight some of the examples of epigenetic inheritance, the establishment of cytosine methylation in sperm, and recent genomic studies linking sperm cytosine methylation to epigenetic effects on offspring. A recent paper examining changes in diet and sperm cytosine methylation from pools of eight animals each, found differences between a normal diet, a high fat diet, and a low protein diet. However, epivariation between individuals within a group was greater than the differences between groups obscuring any potential methylation changes linked to diet. Learning more about epivariation may help unravel the mechanisms that regulate cytosine methylation. In addition, other experimental and genetic systems may also produce more dramatic changes in the sperm methylome, making it easier to unravel potential transgenerational phenomena. J. Cell. Physiol. 231: 2346–2352, 2016. © 2016 Wiley Periodicals, Inc.
  • Laser Direct‐Write Onto Live Tissues: A Novel Model for Studying
           Cancer Cell Migration
    • Abstract: Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. Recently, our laboratories have combined a novel tissue culture model and laser direct‐write, a form of bioprinting, to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Herein, we highlight this new model as a tool for quantifying cancer cell motility and effects on angiogenesis and lymphangiogenesis in an intact network that matches the complexity of a real tissue. Application of our proposed methodology offers an innovative ex vivo tissue perspective for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion. J. Cell. Physiol. 231: 2333–2338, 2016. © 2016 Wiley Periodicals, Inc. Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. In this article, we highlight a new model that combines laser direct‐write, a form of bioprinting, with a tissue culture model to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Application of our proposed methodology offers an innovative ex vivo tool for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion.
  • Lipid‐Targeting Peptide Probes for Extracellular Vesicles
    • Abstract: Extracellular vesicles released from cells are under intense investigation for their roles in cell–cell communication and cancer progression. However, individual vesicles have been difficult to probe as their small size renders them invisible by conventional light microscopy. However, as a consequence of their small size these vesicles possess highly curved lipid membranes that offer an unconventional target for curvature‐sensing probes. In this article, we present a strategy for using peptide‐based biosensors to detect highly curved membranes and the negatively charged membrane lipid phosphatidylserine, we delineate several assays used to validate curvature‐ and lipid‐targeting mechanisms, and we explore potential applications in probing extracellular vesicles released from sources such as apoptotic cells, cancer cells, or activated platelets. J. Cell. Physiol. 231: 2327–2332, 2016. © 2016 Wiley Periodicals, Inc.
  • The Cholinergic Signaling Responsible for the Expression of a
           Memory‐Related Protein in Primary Rat Cortical Neurons
    • Abstract: Cholinergic dysfunction in the brain is closely related to cognitive impairment including memory loss. In addition to the degeneration of basal forebrain cholinergic neurons, deficits in the cholinergic receptor signaling may also play an important role. In the present study, to examine the cholinergic signaling pathways responsible for the induction of a memory‐related postsynaptic protein, a cholinergic agonist carbachol was used to induce the expression of activity‐regulated cytoskeleton associated protein (Arc) in primary rat cortical neurons. After pretreating neurons with various antagonists or inhibitors, the levels of carbachol‐induced Arc protein expression were detected by Western blot analysis. The results show that carbachol induces Arc protein expression mainly through activating M1 acetylcholine receptors and the downstream phospholipase C pathway, which may lead to the activation of the MAPK/ERK signaling pathway. Importantly, carbachol‐mediated M2 receptor activation exerts negative effects on Arc protein expression and thus counteracts the enhanced effects of M1 activation. Furthermore, it is suggested for the first time that M1‐mediated enhancement of N‐methyl‐D‐aspartate receptor (NMDAR) responses, leading to Ca2+ entry through NMDARs, contributes to carbachol‐induced Arc protein expression. These findings reveal a more complete cholinergic signaling that is responsible for carbachol‐induced Arc protein expression, and thus provide more information for developing treatments that can modulate cholinergic signaling and consequently alleviate cognitive impairment. J. Cell. Physiol. 231: 2428–2438, 2016. © 2016 Wiley Periodicals, Inc. Cholinergic agonist carbachol induces the expression of a memory‐related protein, Arc, mainly through activating M1 acetylcholine receptors and the downstream phospholipase C pathway, leading to the activation of the MAPK/ERK signaling pathway. In addition, M1‐mediated enhancement of N‐methyl‐D‐aspartate receptor (NMDAR) responses, leading to Ca2+ entry through NMDARs, contributes to carbachol‐induced Arc protein expression.
  • iPSCs, a Future Tool for Therapeutic Intervention in Mitochondrial
           Disorders: Pros and Cons
    • Abstract: Mitochondrial disorders, although individually are rare, taken together constitute a big group of diseases that share a defect in the oxidative phosphorylation system. Up to now, the development of therapies for these diseases is very slow and ineffective due in part to the lack of appropriate disease models. Therefore, there is an urgent need for the discovery of new therapeutic interventions. Regarding this, the generation of induced pluripotent stem cells (iPSCs) has opened new expectations in the regenerative medicine field. However, special cares and considerations must be taken into account previous to a replacement therapy. J. Cell. Physiol. 231: 2317–2318, 2016. © 2016 Wiley Periodicals, Inc.
  • Translocation of Endogenous Danger Signal HMGB1 From Nucleus to Membrane
           Microvesicles in Macrophages
    • Abstract: High mobility group box 1 (HMGB1) is a nuclear protein that can be released from activated or dead cells. Extracellular HMGB1 can serve as a “danger signal” and novel cytokine that mediates sterile inflammation. In addition to its soluble form, extracellular HMGB1 can also be carried by membrane microvesicles. However, the cellular mechanisms responsible for nuclear HMGB1 translocation to the plasma membrane and release onto membrane microvesicles have not been investigated. Tobacco smoking is a major cause of sterile inflammation in many diseases. Smoking also increases blood levels of HMGB1. In this study, we found that exposure of macrophages to tobacco smoke extract (TSE) stimulated HMGB1 expression, redistribution, and release into the extracellular milieu both as a soluble molecule and, surprisingly, as a microvesicle‐associated form (TSE‐MV). Inhibition of chromosome region maintenance‐1 (CRM1), a nuclear exporter, attenuated TSE‐induced HMGB1 redistribution from the nucleus to the cytoplasm, and then its release on TSE‐MVs. Our study demonstrates a novel mechanism for the translocation of nuclear HMGB1 to the plasma membrane, and then its release in a microvesicle‐associated form. J. Cell. Physiol. 231: 2319–2326, 2016. © 2016 Wiley Periodicals, Inc.
  • Table of Contents, Editor's Choice, Highlights
  • KCa3.1−/− Mice Do Not Develop CIA: Regulatory Role for KCa3.1
           in Autoimmune Arthritis
  • Are Wnts Retrogradely Transported to the ER'
    • Abstract: A recent report showed that Drosophila miR‐307a initiates endoplasmic reticulum (ER) stress in wingless (wg)‐expressing cells by suppression of the evolutionarily conserve Wnt secretion factor Wntless (Wls). Interestingly, the authors noted that wg has a putative C‐terminal dilysine motif (KKVY), which is required for its apparent retrograde Golgi‐to‐ER transport. Wls suppression resulted in ER stress, which was phenocopied by several other manipulations that impaired wg secretion in flies, as well as Wnt5a secretion in mammalian cells. The authors surmised that their data “reveals a previously unknown Golgi‐to‐ER retrograde route of wg, and elucidates a correlation between Wnt secretion and ER stress.” However, there are obvious caveats to this interpretation, as ER stress resulting from Wnt secretion impairment could be readily explained by its inability to leave the ER, and not resulting from Golgi‐to‐ER retrograde transport. J. Cell. Physiol. 231: 2315–2316, 2016. © 2016 Wiley Periodicals, Inc.
  • Cover Image, Volume 231, Number 11, November 2016
    • Abstract: The cover image, by Hyun‐Seob Song et al., is based on the Article Microbial Community Metabolic Modeling: A Community Data‐Driven Network Reconstruction,
      DOI : 10.1002/jcp.25428. Confocal micrograph depicting a metabolically coupled microbial consortium composed of cyanobacterium Thermosynechococcus elongatus (red) supporting heterotrophic bacterium, Meiothermus ruber (green). Scientists at PNNL and ANL proposed a new approach for microbial community metabolic modeling using this phototroph‐heterotroph co‐culture as a model system to study how microorganisms living in communities coordinate their metabolisms in response to partnership. Imaged by PNNL scientist, William B. Chrisler.
  • Annexin A1 Is a Physiological Modulator of Neutrophil Maturation and
           Recirculation Acting on the CXCR4/CXCL12 Pathway
    • Abstract: Neutrophil production and traffic in the body compartments is finely controlled, and the strong evidences support the role of CXCL12/CXCR4 pathway on neutrophil trafficking to and from the bone marrow (BM). We recently showed that the glucocorticoid‐regulated protein, Annexin A1 (AnxA1) modulates neutrophil homeostasis and here we address the effects of AnxA1 on steady‐state neutrophil maturation and trafficking. For this purpose, AnxA1−/− and Balb/C wild‐type mice (WT) were donors of BM granulocytes and mesenchymal stem cells and blood neutrophils. In vivo treatments with the pharmacological AnxA1 mimetic peptide (Ac2‐26) or the formyl peptide receptor (FPR) antagonist (Boc‐2) were used to elucidate the pathway of AnxA1 action, and with the cytosolic glucocorticoid antagonist receptor RU 38486. Accelerated maturation of BM granulocytes was detected in AnxA1−/− and Boc2‐treated WT mice, and was reversed by treatment with Ac2‐26 in AnxA1−/− mice. AnxA1 and FPR2 were constitutively expressed in bone marrow granulocytes, and their expressions were reduced by treatment with RU38486. Higher numbers of CXCR4+ neutrophils were detected in the circulation of AnxA1−/− or Boc2‐treated WT mice, and values were rescued in Ac2‐26‐treated AnxA1−/− mice. Although circulating neutrophils of AnxA1−/− animals were CXCR4+, they presented reduced CXCL12‐induced chemotaxis. Moreover, levels of CXCL12 were reduced in the bone marrow perfusate and in the mesenchymal stem cell supernatant from AnxA1−/− mice, and in vivo and in vitro CXCL12 expression was re‐established after Ac2‐26 treatment. Collectively, these data highlight AnxA1 as a novel determinant of neutrophil maturation and the mechanisms behind blood neutrophil homing to BM via the CXCL12/CXCR4 pathway. J. Cell. Physiol. 231: 2418–2427, 2016. © 2016 Wiley Periodicals, Inc.
  • The Impact of Epigenetics on Mesenchymal Stem Cell Biology
    • Abstract: Changes in epigenetic marks are known to be important regulatory factors in stem cell fate determination and differentiation. In the past years, the investigation of the epigenetic regulation of stem cell biology has largely focused on embryonic stem cells (ESCs). Contrarily, less is known about the epigenetic control of gene expression during differentiation of adult stem cells (AdSCs). Among AdSCs, mesenchymal stem cells (MSCs) are the most investigated stem cell population because of their enormous potential for therapeutic applications in regenerative medicine and tissue engineering. In this review, we analyze the main studies addressing the epigenetic changes in MSC landscape during in vitro cultivation and replicative senescence, as well as follow osteocyte, chondrocyte, and adipocyte differentiation. In these studies, histone acetylation, DNA methylation, and miRNA expression are among the most investigated phenomena. We describe also epigenetic changes that are associated with in vitro MSC trans‐differentiation. Although at the at initial stage, the epigenetics of MSCs promise to have profound implications for stem cell basic and applied research. J. Cell. Physiol. 231: 2393–2401, 2016. © 2016 Wiley Periodicals, Inc. Epigenetic marks are known to be important regulatory factors in stem cell fate determination and differentiation. In this paper, we reviewed studies on epigenetic changes that govern mesenchymal stem cell biology.
  • Can Cholesterol Metabolism Modulation Affect Brain Function and
    • 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
  • 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
  • BAG3 Protein Is Over‐ Expressed in Endometrioid Endometrial
    • 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
  • An Overview on Predictive Biomarkers of Testicular Germ Cell Tumors
    • 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
  • DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro
    • 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
  • 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
  • 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
  • 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
  • The β2‐Adrenoceptor Agonist Terbutaline Stimulates Angiogenesis
           via Akt and ERK Signaling
    • 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
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