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Journal Cover Journal of Cellular Physiology
  [SJR: 1.948]   [H-I: 125]   [4 followers]  Follow
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
   Published by John Wiley and Sons Homepage  [1598 journals]
  • Extracellular Matrix Stiffness Controls VEGF Signaling and Processing in
           Endothelial Cells
    • Authors: Kelsey D. Sack; Madelane Teran, Matthew A. Nugent
      Abstract: Vascular endothelial growth factor A (VEGF) drives endothelial cell maintenance and angiogenesis. Endothelial cell behavior is altered by the stiffness of the substrate the cells are attached to suggesting that VEGF activity might be influenced by the mechanical cellular environment. We hypothesized that extracellular matrix (ECM) stiffness modifies VEGF‐cell‐matrix tethering leading to altered VEGF processing and signaling. We analyzed VEGF binding, internalization, and signaling as a function of substrate stiffness in endothelial cells cultured on fibronectin (Fn) linked polyacrylamide gels. Cell produced extracellular matrices on the softest substrates were least capable of binding VEGF, but the cells exhibited enhanced VEGF internalization and signaling compared to cells on all other substrates. Inhibiting VEGF‐matrix binding with sucrose octasulfate decreased cell‐internalization of VEGF and, inversely, heparin pre‐treatment to enhance Fn‐matrix binding of VEGF increased cell‐internalization of VEGF regardless of matrix stiffness. β1 integrins, which connect cells to Fn, modulated VEGF uptake in a stiffness dependent fashion. Cells on hard surfaces showed decreased levels of activated β1 and inhibition of β1 integrin resulted in a greater proportional decrease in VEGF internalization than in cells on softer matrices. Extracellular matrix binding is necessary for VEGF internalization. Stiffness modifies the coordinated actions of VEGF‐matrix binding and β1 integrin binding/activation, which together are critical for VEGF internalization. This study provides insight into how the microenvironment may influence tissue regeneration and response to injury and disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-08T05:31:39.391953-05:
      DOI: 10.1002/jcp.25312
  • The Outflow Pathway: A Tissue with Morphological and Functional Unity
    • Abstract: The trabecular meshwork plays an important role in high‐tension glaucomas. Indeed, the TM is a true organ, through which the aqueous humor flows from the anterior chamber to Schlemm's canal. Until recently, the TM, which is constituted by endothelial‐like cells, was described as a kind of passive filter. In reality, it is much more. The cells delineating the structures of the collagen framework of the TM are endowed with a cytoskeleton, and are thus able to change their shape. These cells also have the ability to secrete the extracellular matrix, which expresses proteins and cytokines, and are capable of phagocytosis and autophagy. The cytoskeleton is attached to the nuclear membrane and can, in millionths of a second, send signals to the nucleus in order to alter the expression of genes in an attempt to adapt to biomechanical insult. Oxidative stress, as happens in aging, has a deleterious effect on the TM, leading eventually to cell decay, tissue malfunction, subclinical inflammation, changes in the extracellular matrix and cytoskeleton, altered motility, reduced outflow facility and (ultimately) increased IOP. TM failure is the most relevant factor in the cascade of events triggering apoptosis in the inner retinal layers, including ganglion cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-08T05:30:26.051956-05:
      DOI: 10.1002/jcp.25305
  • Osteoactivin Promotes Migration of Oral Squamous Cell Carcinomas
    • Authors: Oneida A. Arosarena; Raul A. dela Cadena, Michael F. Denny, Evan Bryant, Eric W. Barr, Ryan Thorpe, Fayez F. Safadi
      Abstract: Nearly 50% of patients with oral squamous cell carcinoma (OSCC) die of metastases or locoregional recurrence. Metastasis is mediated by cancer cell adhesion, migration and invasion. Osteoactivin (OA) overexpression plays a role in metastases in several malignancies. Objectives To determine how integrin interactions modulate OA‐induced OSCC cell migration; and to investigate OA effects on cell survival and proliferation. Materials and Methods We confirmed OA mRNA and protein overexpression in OSCC cell lines. We assessed OA's interactions with integrins using adhesion inhibition assays, fluorescent immunocytochemistry and co‐immunoprecipitation. We investigated OA‐mediated activation of mitogen‐activated protein kinases (MAPKs) and cell survival. Integrin inhibition effects on OA‐mediated cell migration were determined. We assessed effects of OA knock‐down on cell migration and proliferation. Results OA is overexpressed in OSCC cell lines, and serves as a migration‐promoting adhesion molecule. OA co‐localized with integrin subunits, and co‐immunoprecipitated with the subunits. Integrin blocking antibodies, especially those directed against the β1 subunit, inhibited cell adhesion (p = 0.03 for SCC15 cells). Adhesion to OA activated MAPKs in UMSCC14a cells and OA treatment promoted survival of SCC15 cells. Integrin‐neutralizing antibodies enhanced cell migration with OA in the extracellular matrix. OA knock‐down resulted in decreased proliferation of SCC15 and SCC25 cells, but did not inhibit cell migration. Conclusion OA in the extracellular matrix promotes OSCC cell adhesion and migration, and may be a novel target in the prevention of HNSCC spread. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-08T05:28:11.505983-05:
      DOI: 10.1002/jcp.25279
  • Identification of a hematopoietic cell dedifferentiation‐inducing
    • Abstract: It has long been realized that hematopoietic cells may have the capacity to trans‐differentiate into non‐lymphohematopoietic cells under specific conditions. However, the mechanisms and the factors for hematopoietic cell trans‐differentiation remain unknown. In an in vitro culture system, we found that using a conditioned medium from proliferating fibroblasts can induce a subset of hematopoietic cells to become adherent fibroblast‐like cells (FLCs). FLCs are not fibroblasts nor other mesenchymal stromal cells, based on their expression of type‐1 collagen and other stromal cell marker genes. To identify the active factors in the conditioned medium, we cultured fibroblasts in a serum‐free medium and collected it for further purification. Using the fractions from filter devices of different molecular weight cut‐offs, and ammonium sulfate precipitation collected from the medium, we found the active fraction is a protein. We then purified this fraction by using fast protein liquid chromatography (FPLC) and identified it by mass spectrometer as macrophage colony‐stimulating factor (M‐CSF). The mechanisms of M‐CSF‐inducing trans‐differentiation of hematopoietic cells seem to involve a tyrosine kinase signalling pathway and its known receptor. The FLCs express a number of stem cell markers including SSEA‐1 and ‐3, OCT3/4, NANOG and SOX2. Spontaneous and induced differentiation experiments confirmed that FLCs can be further differentiated into cell types of three germ layers. These data indicate that hematopoietic cells can be induced by M‐CSF to dedifferentiate to multipotent stem cells. This study also provides a simple method to generate multipotent stem cells for clinical applications. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-08T05:24:26.392863-05:
      DOI: 10.1002/jcp.25239
  • Mesenchymal Stem Cells Ageing: Targeting the “Purinome” to
           Promote Osteogenic Differentiation and Bone Repair
    • Abstract: Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming cells. Such ability is compromised in elderly individuals resulting in bone disorders such as osteoporosis, also limiting their clinical usage for cell transplantation and bone tissue engineering strategies. In bone marrow niches, adenine and uracil nucleotides are important local regulators of osteogenic differentiation of MSCs. Nucleotides can be released to the extracellular milieu under both physiological and pathological conditions via (1) membrane cell damage, (2) vesicle exocytosis, (3) ATP‐binding cassette transporters, and/or (4) facilitated diffusion through maxi‐anion channels, hemichannels or ligand‐gated receptor pores. Nucleotides and their derivatives act via adenosine P1 (A1, A2A, A2B and A3) and nucleotide‐sensitive P2 purinoceptors comprising ionotropic P2X and G‐protein‐coupled P2Y receptors. Purinoceptors activation is terminated by membrane‐bound ecto‐nucleotidases and other ecto‐phosphatases, which rapidly hydrolyse extracellular nucleotides to their respective nucleoside 5'‐di‐ and mono‐phosphates, nucleosides and free phosphates or pyrophosphates. Current knowledge suggests that different players of the “purinome” cascade, namely nucleotide release sites, ecto‐nucleotidases and purinoceptors, orchestrate to fine‐tuning regulate the activity of MSCs in the bone microenvironment. Increasing studies, using osteoprogenitor cell lines, animal models and, more recently, non‐modified MSCs from postmenopausal women, raised the possibility to target chief components of the purinergic signaling pathway to regenerate the ability of aged MSCs to differentiate into functional osteoblasts. This review summarizes the main findings of those studies, prompting for novel therapeutic strategies to control ageing disorders where bone destruction exceeds bone formation, like osteoporosis, rheumatoid arthritis and fracture mal‐union. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-05T05:42:11.910789-05:
      DOI: 10.1002/jcp.25303
  • Expression of Ribosomal RNA and Protein Genes in Human Embryonic Stem
           Cells Is Associated with the Activating H3K4me3 Histone Mark
    • Authors: Sayyed K. Zaidi; Joseph R. Boyd, Rodrigo Grandy, Ricardo Medina, Jane B. Lian, Gary S. Stein, Janet L. Stein
      Abstract: Embryonic stem cells (ESCs) exhibit unrestricted and indefinite, but stringently controlled, proliferation and can differentiate into any lineage in the body. In the current study, we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs, we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. While many RPGs are expressed at levels that comparable to other human cell lines, a few RPGs also exhibit higher expression levels. In situ nuclear run‐on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome‐wide chromatin immunoprecipitation‐deep sequencing and bioinformatics approaches, we discovered that RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1, M and G2 phases of the cell cycle. Interestingly, the rDNA repeats are marked by the activating H3K4me3 only in the M phase, and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc, a known regulator of cell growth and proliferation, occupies both the rRNA genes and RPGs. Functionally, down‐regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together, our results show that expression of rRNA, which is regulated by the Myc pluripotency transcription factor, and of RPGs in hESCs is associated with the activating H3K4me3 modification. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-05T04:29:30.958349-05:
      DOI: 10.1002/jcp.25309
  • Fatty Acids and Breast Cancer: Make them on Site or have them Delivered
    • Authors: William B. Kinlaw; Paul W. Baures, Leslie E. Lupien, Wilson L. Davis, Nancy B. Kuemmerle
      Abstract: Brisk fatty acid (FA) production by cancer cells is accommodated by the Warburg effect. Most breast and other cancer cell types are addicted to fatty acids (FA), which they require for membrane phospholipid synthesis, signaling purposes, and energy production. Expression of the enzymes required for FA synthesis is closely linked to each of the major classes of signaling molecules that stimulate BC cell proliferation. This review focuses on the regulation of FA synthesis in BC cells, and the impact of FA, or the lack thereof, on the tumor cell phenotype. Given growing awareness of the impact of dietary fat and obesity on BC biology, we will also examine the less‐frequently considered notion that, in addition to de novo FA synthesis, the lipolytic uptake of preformed FA may also be an important mechanism of lipid acquisition. Indeed, it appears that cancer cells may exist at different points along a “lipogenic‐lipolytic axis”, and FA uptake could thwart attempts to exploit the strict requirement for FA focused solely on inhibition of de novo FA synthesis. Strategies for clinically targeting FA metabolism will be discussed, and the current status of the medicinal chemistry in this area will be assessed. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-04T12:41:43.136357-05:
      DOI: 10.1002/jcp.25332
  • Focal Adhesion Assembly Induces Phenotypic Changes and Dedifferentiation
           in Chondrocytes
    • Authors: Hyunjun Shin; Mi Nam Lee, Jin Seung Choung, Sanghee Kim, Byung Hyune Choi, Minsoo Noh, Jennifer H. Shin
      Abstract: The expansion of autologous chondrocytes in vitro is used to generate sufficient populations for cell‐based therapies. However, during monolayer culture, chondrocytes lose inherent characteristics and shift to fibroblast‐like cells as passage number increase. Here, we investigated passage‐dependent changes in cellular physiology, including cellular morphology, motility, and gene and protein expression, as well as the role of focal adhesion and cytoskeletal regulation in the dedifferentiation process. We found that the gene and protein expression levels of both the focal adhesion complex and small Rho GTPases are upregulated with increasing passage number and are closely linked to chondrocyte dedifferentiation. The inhibition of focal adhesion kinase (FAK) but not sm26all Rho GTPases induced the loss of fibroblastic traits and the recovery of collagen type II, aggrecan, and SOX9 expression levels in dedifferentiated chondrocytes. Based on these findings, we propose a strategy to suppress chondrogenic dedifferentiation by inhibiting the identified FAK or Src pathways while maintaining the expansion capability of chondrocytes in a 2D environment. These results highlight a potential therapeutic target for the treatment of skeletal diseases and the generation of cartilage in tissue‐engineering approaches. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-04T04:23:19.064156-05:
      DOI: 10.1002/jcp.25290
  • MicroRNA Regulation of Acute Lung Injury and Acute Respiratory Distress
    • Authors: Subbiah Rajasekaran; Dhamotharan Pattarayan, P. Rajaguru, P. S. Sudhakar Gandhi, Rajesh K. Thimmulappa
      Abstract: The acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI) is a very common condition associated with critically ill patients, which causes substantial morbidity and mortality worldwide. Despite decades of research, effective therapeutic strategies for clinical ALI/ARDS are not available. In recent years, microRNAs (miRNAs), small non‐coding molecules have emerged as a major area of biomedical research as they post‐transcriptionally regulate gene expression in diverse biological and pathological processes, including ALI/ARDS. In this context, this present review summarizes a large body of evidence implicating miRNAs and their target molecules in ALI/ARDS originating largely from studies using animal and cell culture model systems of ALI/ARDS. We have also focused on the involvement of miRNAs in macrophage polarization, which play a critical role in regulating the pathogenesis of ALI/ARDS. Finally, the possible future directions that might lead to novel therapeutic strategies for the treatment of ALI/ARDS are also reviewed. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-04T04:19:00.353386-05:
      DOI: 10.1002/jcp.25316
  • Differential Neuronal Plasticity of Dental Pulp Stem Cells from Exfoliated
           Deciduous and Permanent Teeth Towards Dopaminergic Neurons
    • Authors: Debanjana Majumdar; Mohammad Kanafi, Ramesh Bhonde, Pawan Gupta, Indrani Datta
      Abstract: Based on early occurrence in chronological‐age, stem‐cells from human exfoliated deciduous teeth (SHED) has been reported to possess better differentiation‐potential towards certain cell‐lineage in comparison to stem‐cells from adult teeth (DPSCs). Whether this same property between them extends for the yield of functional central nervous system neurons is still not evaluated. Hence we aim to assess the neuronal plasticity of SHED in comparison to DPSCs towards dopaminergic‐neurons and further, if the difference is reflected in a differential expression of sonic‐hedgehog (SHH)‐receptors and basal‐expressions of tyrosine‐hydroxylase [TH; through cAMP levels]. Human SHED and DPSCs were exposed to midbrain‐cues [SHH, fibroblast growth‐factor8 and basic fibroblast growth‐factor], and their molecular, immunophenotypical and functional characterization was performed at different time‐points of induction. Though SHED and DPSCs spontaneously expressed early‐neuronal and neural‐crest marker in their naïve state, only SHED expressed a high basal‐expression of TH. The upregulation of dopaminergic transcription‐factors Nurr1, Engrailed1 and Pitx3 was more pronounced in DPSCs. The yield of TH‐expressing cells decreased from 49.8% to 32.16% in SHED while it increased from 8.09% to 77.47% in DPSCs. Dopamine release and intracellular‐Ca2+ influx upon stimulation (KCl & ATP) was higher in induced DPSCs. Significantly lower‐expression of SHH‐receptors was noted in naïve SHED than DPSCs, which may explain the differential neuronal plasticity. In addition, unlike DPSCs, SHED showed a down‐regulation of cyclic adenosine‐monophosphate (cAMP) upon exposure to SHH; possibly another contributor to the lesser differentiation‐potential. Our data clearly demonstrates for the first time that DPSCs possess superior neuronal plasticity towards dopaminergic‐neurons than SHED; influenced by higher SHH‐receptor and lower basal TH expression. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-04T02:12:11.785108-05:
      DOI: 10.1002/jcp.25314
  • Conversion of Prostate Adenocarcinoma to Small Cell Carcinoma‐Like
           by Reprogramming
    • Abstract: The lineage relationship between prostate adenocarcinoma and small cell carcinoma was studied by using the LuCaP family of xenografts established from primary neoplasm to metastasis. Expression of four stem cell transcription factor (TF) genes, LIN28A, NANOG, POU5F1, SOX2, were analyzed in the LuCaP lines. These genes, when force expressed in differentiated cells, can reprogram the recipients into stem‐like induced pluripotent stem (iPS) cells. Most LuCaP lines expressed POU5F1, while LuCaP 145.1, representative of small cell carcinoma, expressed all four. Through transcriptome database query, many small cell carcinoma genes were also found in stem cells. To test the hypothesis that prostate cancer progression from “differentiated” adenocarcinoma to “undifferentiated” small cell carcinoma could involve re‐expression of stem cell genes, the four TF genes were transfected via lentiviral vectors into five adenocarcinoma LuCaP lines – 70CR, 73CR, 86.2, 92, 105CR – as done in iPS cell reprogramming. The resultant cells from these five transfections displayed a morphology of small size and dark appearing unlike the parentals. Transcriptome analysis of LuCaP 70CR* (* to denote transfected progeny) revealed a unique gene expression close to that of LuCaP 145.1. In a prostate principal components analysis space based on cell‐type transcriptomes, the different LuCaP transcriptome datapoints were aligned to suggest a possible ordered sequence of expression changes from the differentiated luminal‐like adenocarcinoma cell types to the less differentiated, more stem‐like small cell carcinoma types and LuCaP 70CR*. Prostate cancer progression can thus be molecularly characterized by loss of differentiation with re‐expression of stem cell genes. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-04T02:11:09.728444-05:
      DOI: 10.1002/jcp.25313
  • Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound
           Healing Process
    • Authors: Cristina La Torre; Benedetta Cinque, Francesca Lombardi, Gianfranca Miconi, Paola Palumbo, Zoran Evtoski, Giuseppe Placidi, Donatella Fanini, Anna Maria Cimini, Elisabetta Benedetti, Maurizio Giuliani, Maria Grazia Cifone
      Abstract: An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S‐nitroso‐N‐acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analysed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0‐28 h) and the wound area was analysed through software included in Matlab®. As early as 15 minutes, SNAP induced significant cytoskeletal remodelling, as shown by immunostaining (phalloidin‐labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki‐67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP‐induced migration and proliferation were antagonized by the NO‐sensitive GC inhibitor 1H‐[1,2,4]oxadiazolo[‐4,3‐a]quinoxalin‐1‐one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP‐induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR‐beta antagonist (GSK0660) may interfere with NO‐mediated wound healing process. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-03T13:51:13.810184-05:
      DOI: 10.1002/jcp.25331
  • Ubiquitin‐Related Roles of β‐Arrestins in Endocytic
           Trafficking and Signal Transduction
    • Abstract: The non‐visual arrestins, β‐arrestin1 and β‐arrestin2 were originally identified as proteins that bind to seven‐transmembrane receptors (7TMRs, also called G protein‐coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors and non‐receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non‐receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β‐ arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β‐arrestins in protein ubiquitination is evolutionarily conserved. β‐ arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β‐arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β‐arrestin plays a critical role in stabilizing β‐arrestin‐7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin‐ dependent regulation of 7TMRs as well as β‐arrestins and the potential role of reversible ubiquitination as a ‘biological switch’ in signal transduction. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-03T03:46:58.859088-05:
      DOI: 10.1002/jcp.25317
  • First Evidence of DAAM1 Localization during the Post‐Natal
           Development of Rat Testis and in Mammalian Sperm
    • Authors: Paolo Pariante; Raffaele Dotolo, Massimo Venditti, Diana Ferrara, Aldo Donizetti, Francesco Aniello, Sergio Minucci
      Abstract: Dishevelled‐associated activator of morphogenesis 1 (DAAM1) is a formin‐family protein involved in nucleation of unbranched actin filaments and in cytoskeletal organization through Wnt‐Dishevelled PCP pathway, which participates in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. While its role has been investigated during development and in somatic cells, its potential association with the germinal compartment and reproduction is still unexplored. In this work, we assessed the possible association of DAAM1 with the morphogenesis of rat testis. We studied its expression and profiled its localization versus actin and tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, DAAM1 shares its localization with actin in Sertoli cells, gonocytes and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, while only actin is detectable at the forming blood‐testis barrier. DAAM1, then, follows the development of the acrosome system throughout spermiogenesis, and it is finally retained inside the cytoplasmic droplet in mature gametes, as corroborated by additional immunolocalization data on both rat and human sperm. Unlike the DAAM1, actin keeps its localization in Sertoli cells, and tubulin is associated with their protruding cytoplasm during the process. Our data support, for the first time, the hypothesis of a role for DAAM1 in cytoskeletal organization during Mammalian testis morphogenesis and gamete progression, while also hinting at its possible investigation as a morphological marker of germ cell and sperm physiology. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T08:06:20.256924-05:
      DOI: 10.1002/jcp.25330
  • Emerging from the Unknown: Structural and Functional Features of
           Agnoprotein of Polyomaviruses
    • Abstract: Agnoprotein is an important regulatory protein of polyomaviruses, including JCV, BKV and SV40. In the absence of its expression, these viruses are unable to sustain their productive life cycle. It is a highly basic phosphoprotein that localizes mostly to the perinuclear area of infected cells, although a small amount of the protein is also found in nucleus. Much has been learned about the structure and function of this important regulatory protein in recent years. It forms highly stable dimers/oligomers in vitro and in vivo through its Leu/Ile/Phe‐rich domain. Structural NMR studies revealed that this domain adopts an alpha‐helix conformation and plays a critical role in the stability of the protein. It associates with cellular proteins, including YB‐1, p53, Ku70, FEZ1, HP1α, PP2A, AP‐3, PCNA and α‐SNAP; and viral proteins, including small t antigen, large T antigen, HIV‐1 Tat, and JCV VP1; and significantly contributes the viral transcription and replication. This review summarizes the recent advances in the structural and functional properties of this important regulatory protein. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T08:06:14.347529-05:
      DOI: 10.1002/jcp.25329
  • The Spectrum of E2F in Liver Disease – Mediated Regulation in
           Biology and Cancer
    • Authors: Justin T. Huntington; Xing Tang, Lindsey N. Kent, Carl R. Schmidt, Gustavo Leone
      Abstract: Uncoordinated cell growth is one of the fundamental concepts in carcinogenesis and occurs secondary to dysregulation of the cell cycle. The E2Fs are a large family of transcription factors and are key regulators of the cell cycle. The activation of E2Fs is intimately regulated by retinoblastoma 1 (RB1). The RB pathway has been implicated in almost every human malignancy. Recently there have been exciting developments in the E2F field using animal models to better understand the role of E2Fs in vivo. Genetic mouse models have proven essential in implicating E2Fs in hepatocellular carcinoma (HCC) and liver disease. In this review, the general structure and function of E2Fs as well as the role for E2Fs in the development of HCC and liver disease is evaluated. Specifically what is known about E2Fs in human disease is explored in depth and future directions are discussed. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T05:18:42.335798-05:
      DOI: 10.1002/jcp.25242
  • Interactions Between Fatty Acid Transport Proteins, Genes that Encode for
           Them, and Exercise: A Systematic Review
    • Authors: Avindra F. Jayewardene; Yorgi Mavros, Anneliese Reeves, Dale P. Hancock, Tom Gwinn, Kieron B. Rooney
      Abstract: Long‐chain fatty acid (LCFA) movement into skeletal muscle involves a highly mediated process in which lipid rafts are utilised in the cellular membrane, involving numerous putative plasma membrane‐associated LCFA transport proteins. The process of LCFA uptake and oxidation is of particular metabolic significance both at rest and during light to moderate exercise. A comprehensive systematic search of electronic databases was undertaken to investigate whether exercise alters protein and / or gene expression of putative LCFA transport proteins. There were 31 studies meeting all eligibility criteria, of these 13 utilised an acute exercise protocol and 18 examined chronic exercise adaptations. 17 involved a study design incorporating an exercise stimulus, while the remaining 14 incorporated an exercise and diet stimulus. Divergent data relating to acute exercise, as well as prolonged exercise training (≥3 weeks), on protein content (PC) response was identified for proteins CD36, FABPpm and CAV1. Messenger ribonucleic acid (mRNA) data did not always correspond to functional PC, supporting previous suggestions of a disconnect due to potentially limiting factors post gene expression. The large array of study designs, cohorts and primary dependent variables within the studies included in the present review elucidate the complexity of the interaction between exercise and LCFA transport proteins. Summary of the results in the present review validate the need for further targeted investigation within this topic, and provide an important information base for such research. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T05:18:02.208666-05:
      DOI: 10.1002/jcp.25281
  • Evidence Based Theory for Integrated Genome Regulation of Ontogeny –
           an Unprecedented Role of Nuclear FGFR1 Signaling
    • Authors: Michal K. Stachowiak; Ewa K. Stachowiak
      Abstract: Genetic experiments have positioned the fgfr1 gene at the top of the gene hierarchy that governs gastrulation, as well as the subsequent development of the major body axes, nervous system, muscles, and bones, by affecting downstream genes that control the cell cycle, pluripotency and differentiation, as well as microRNAs. Studies show that this regulation is executed by a single protein, the nuclear isoform of FGFR1 (nFGFR1), which integrates signals from development‐initiating factors such as Retinoic Acid (RA), and operates at the interface of genomic and epigenomic information. nFGFR1 cooperates with a multitude of Transcriptional Factors (TFs), and targets thousands of genes encoding for mRNAs, as well as miRNAs in top ontogenic networks. nFGFR1 binds to the promoters of ancient proto‐oncogenes and tumor suppressor genes, in addition to binding to metazoan morphogens that delineate body axes, and construct the nervous system, as well as mesodermal and endodermal tissues. The discovery of pan‐ontogenic gene programming by Integrative Nuclear FGFR1 Signaling (INFS) impacts our understanding of ontogeny, as well as developmental pathologies, and holds new promise for reconstructive medicine, and cancer therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T05:17:32.182356-05:
      DOI: 10.1002/jcp.25298
  • Prolonged Survival of Transplanted Osteoblastic Cells Does Not Directly
           Accelerate the Healing of Calvarial Bone Defects
    • Authors: Megumi Kitami; Masaru Kaku, Juan Marcelo Rosales Rocabado, Takako Ida, Nami Akiba, Katsumi Uoshima
      Abstract: Considering the increased interest in cell‐based bone regeneration, it is necessary to reveal the fate of transplanted cells and their substantive roles in bone regeneration. The aim of this study was to analyze the fate of transplanted cells and the effect of osteogenic cell transplantation on calvarial bone defect healing. An anti‐apoptotic protein, heat shock protein (HSP) 27, was overexpressed in osteoblasts. Then, the treated osteoblasts were transplanted to calvarial bone defect and their fate was analyzed to evaluate the significance of transplanted cell survival. Transient overexpression of Hsp27 rescued MC3T3‐E1 osteoblastic cells from H2O2‐induced apoptosis without affecting osteoblastic differentiation in culture. Transplantation of Hsp27‐overexpressing cells, encapsulated in collagen gel, showed higher proliferative activity and fewer apoptotic cells in comparison with control cells. After 4‐week of transplantation, both control cell‐ and Hsp27 overexpressed cell‐transplanted groups showed significantly higher new bone formation in comparison with cell‐free gel‐transplantation group. Interestingly, the prolonged survival of transplanted osteoblastic cells by Hsp27 did not provide additional effect on bone healing. The transplanted cells in collagen gel survived for up to 4‐week but did not differentiate into bone‐forming osteoblasts. In conclusion, cell‐containing collagen gel accelerated calvarial bone defect healing in comparison with cell‐free collagen gel. However, prolonged survival of transplanted cells by Hsp27 overexpression did not provide additional effect. These results strongly indicate that cell transplantation‐based bone regeneration cannot be explained only by the increment of osteogenic cells. Further studies are needed to elucidate the practical roles of transplanted cells that will potentiate successful bone regeneration. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T05:16:31.552021-05:
      DOI: 10.1002/jcp.25302
  • New Treatment of Medullary and Papillary Human Thyroid Cancer: Biological
           Effects of Hyaluronic Acid Hydrogel Loaded With Quercetin Alone or in
           Combination to an Inhibitor of Aurora Kinase
    • Authors: Vincenzo Quagliariello; Emilia Armenia, Caterina Aurilio, Francesco Rosso, Ottavia Clemente, Gabriele de Sena, Manlio Barbarisi, Alfonso Barbarisi
      Abstract: The aim of this paper is based on the use of a hyaluronic acid hydrogel of Quercetin tested alone and in combination to an inhibitor of Aurora Kinase type A and B (SNS‐314) on human medullary and papillary thyroid cancer cells. Biological investigations were focused on the cellular uptake of the hydrogel, cell viability, antioxidant and cytokines secretion studies. Quercetin delivered from hydrogel show a time and CD44 dependent interaction with both cell lines with significant anti‐inflammatory effects. Combination of Quercetin and SNS‐314 leads to a synergistic cytotoxic effect on medullary TT and papillary BCPAP cell lines with a significant reduction of the IC50 value. These results, highlights the importance of synergistic effect of the hyaluronic acid hydrogel of Quercetin with SNS‐314 in the regulation of human thyroid cancer cell proliferation and emphasize the anti‐tumor activity of these molecules. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T05:15:48.523024-05:
      DOI: 10.1002/jcp.25283
  • Eriodictyol Inhibits RANKL‐Induced Osteoclast Formation and Function
           via Inhibition of NFATc1 Activity
    • Authors: Fangming Song; Lin Zhou, Jinmin Zhao, Qian Liu, Mingli Yang, Renxiang Tan, Jun Xu, Ge Zhang, Julian M.W. Quinn, Jennifer Tickner, Yuanjiao Huang, Jiake Xu
      Abstract: RANKL induces differentiation and function of osteoclasts through triggering multiple signaling cascades, including NF‐κB, MAPK, and Ca2+‐dependent signals, which induce and activate critical transcription factor NFATc1. Targeting these signaling cascades may serve as an effective therapy against osteoclast‐related diseases. Here, by screening a panel of natural plant extracts with known anti‐inflammatory, anti‐tumor or anti‐oxidant properties for possible anti‐osteoclastogenic activities we identified Eriodictyol. This flavanone potently suppressed RANKL‐induced osteoclastogenesis and bone resorption in a dose‐dependent manner without detectable cytotoxicity, suppressing RANKL‐induced NF‐κB, MAPK and Ca2+ signaling pathways. Eriodictyol also strongly inhibited RANKL‐induction of c‐Fos levels (a critical component of AP‐1 transcription factor required by osteoclasts) and subsequent activation of NFATc1, concomitant with reduced expression of osteoclast specific genes including cathepsin K (Ctsk), V‐ATPase‐d2 subunit, and tartrate resistant acid phosphatase (TRAcP/Acp5). Taken together, these data provide evidence that Eriodictyol could be useful for the prevention and treatment of osteolytic disorders associated with abnormally increased osteoclast formation and function. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T05:05:37.384833-05:
      DOI: 10.1002/jcp.25304
  • Magnesium Supplementation Potentiates Neural Stem Cell Proliferation in
           the Hippocampus of Young and Aged Mice
    • Authors: Shanshan Jia; Yunpeng Liu, Yang Shi, Yihe Ma, Yixin Hu, Meiyan Wang, Xue Li
      Abstract: In the adult brain, neural stem cells (NSCs) can self‐renew and generate all neural lineage types, and they persist in the sub‐granular zone (SGZ) of the hippocampus and the sub‐ventricular zone (SVZ) of the cortex. Here, we show that dietary‐supplemented magnesium regulates the NSC pool in the adult hippocampus. We found that administration of both short‐ and long‐term regimens of magnesium‐L‐threonate (MgT), a magnesium ion supplement, increased the number of hippocampal NSCs. We demonstrated that in young mice, dietary supplementation with MgT significantly enhanced NSC proliferation in the SGZ. Importantly, in aged mice that underwent long‐term (12‐month) supplementation with MgT, MgT did not deplete the hippocampal NSC reservoir but rather curtailed the age‐associated decline in NSC proliferation. We further established an association between extracellular magnesium concentrations and NSC self‐renewal in vitro by demonstrating that elevated Mg2+ concentrations can maintain or increase the number of cultured hippocampal NSCs. Our study also suggests key signaling pathways for cell growth and proliferation as candidates of Mg2+'s target of action on NSC self‐renewal. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T05:04:15.234901-05:
      DOI: 10.1002/jcp.25306
  • The Involvement of Parasympathetic and Sympathetic Nerve in the
           Inflammatory Reflex
    • Abstract: Production of inflammatory cytokines plays important roles in the response against tissue injury and in host defense. Alterations in the production of inflammatory cytokines may cause local or systemic inflammatory imbalance, culminating in organ failure or lethal systemic inflammation. The cholinergic anti‐inflammatory pathway has been implicated as an important mechanism to regulate inflammation of targeted tissue. In this review, we discuss important advances, conflicting and controversial findings regarding the involvement of parasympathetic vagus and sympathetic splenic nerve through acetylcholine (ACh) release and α7 nicotinic acetylcholine receptor (nAChRα7) activation in the spleen. In addition, we address the involvement of cholinergic control of inflammation in other organs innerved by the vagus nerve such as gut, liver, kidney and lung, and independent of parasympathetic innervations such as skin and skeletal muscle. Then, other structures and mechanisms independent of vagus or splenic nerve may be involved in this process, such as local cells and motor neurons producing ACh. Altogether, the convergence of these findings may contribute to current anti‐inflammatory strategies involving selective drug‐targeting and electrical nerve stimulation. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T05:01:53.676523-05:
      DOI: 10.1002/jcp.25307
  • Reprogramming for Cardiac Regeneration – Strategies for Innovation
    • Abstract: It is well‐known that the human myocardium has a low capacity for self‐regeneration. This fact is especially important after acute myocardial infarction with subsequent heart failure and adverse tissue remodeling. New potential strategies have recently emerged for treating heart diseases, such as the possibility of generating large quantities of cardiomyocytes through genetic iPSC reprogramming, transdifferentiation for in vitro disease modeling, in vivo therapies or telomerase gene reactivation. Approaches based on these techniques may represent the new horizon in cardiology with an appropriate 180‐degree turn perspective. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T04:59:57.74836-05:0
      DOI: 10.1002/jcp.25311
  • Metabolic Modification in Gastrointestinal Cancer Stem Cells:
           Characteristics and Therapeutic Approaches
    • Authors: Angela Maria Di Francesco; Amelia Toesca, Carlo Cenciarelli, Antonio Giordano, Antonio Gasbarrini, Maria Ausiliatrice Puglisi
      Abstract: Currently, there is much interest in the characterization of metabolic profiling of cancer stem cells (CSCs), a small subset of tumour cells with self‐renewal capacity. Indeed, ever‐growing evidence indicate that metabolism and stemness are highly intertwined processes in tumour tissue. In this review, we analyze the potential metabolic targeting strategies for eradicating CSCs that could help to develop a more effective therapeutic approach for gastrointestinal cancers. Indeed, the successful elimination of a tumour requires an anticancer therapy that affects both cancer cells and CSCs. The observation that gastrointestinal CSCs possess higher inducible nitric oxide sinthase (iNOS) expression, lower reactive oxygen species (ROS) production and a different metabolism respect to no‐CSCs tumour cells has paved the way to develop drugs targeting CSC specific signaling. In particular, several studies have highlighted that metformin, aldehyde dehydrogenase 1 and iNOS inhibitors selectively suppressed CSC growth and that combinatorial therapy of them with standard chemotherapeutic drugs had a synergistic effect resulting in reduced tumour burden and delayed tumour recurrence. Thus, the possibility of combining specific CSC metabolism inhibitors with existing therapeutic approaches could have profound anticancer effects, changing the conventional treatment approaches to gastrointestinal cancers. This article is protected by copyright. All rights reserved
      PubDate: 2016-02-02T04:58:40.772889-05:
      DOI: 10.1002/jcp.25318
  • Corticosteroid‐Induced MKP‐1 Represses Pro‐Inflammatory
           Cytokine Secretion by Enhancing Activity of Tristetraprolin (TTP) in ASM
    • Authors: Pavan Prabhala; Kristin Bunge, Qi Ge, Alaina J. Ammit
      Abstract: Exaggerated cytokine secretion drives pathogenesis of a number of chronic inflammatory diseases, including asthma. Anti‐inflammatory pharmacotherapies, including corticosteroids, are front‐line therapies and although they have proven clinical utility, the molecular mechanisms responsible for their actions are not fully understood. The corticosteroid‐inducible gene, mitogen‐activated protein kinase (MAPK) phosphatase 1 (MKP‐1, DUSP1) has emerged as a key molecule responsible for the repressive effects of steroids. MKP‐1 is known to deactivate p38 MAPK phosphorylation and can control the expression and activity of the mRNA destabilizing protein ‐ tristetraprolin (TTP). But whether corticosteroid‐induced MKP‐1 acts via p38 MAPK‐mediated modulation of TTP function in a pivotal airway cell type, airway smooth muscle (ASM), was unknown. While pretreatment of ASM cells with the corticosteroid dexamethasone (preventative protocol) is known to reduce ASM synthetic function in vitro, the impact of adding dexamethasone after stimulation (therapeutic protocol) had not been explored. Whether dexamethasone modulates TTP in a p38 MAPK‐dependent manner in this cell type was also unknown. We address this herein and utilize an in vitro model of asthmatic inflammation where ASM cells were stimulated with the pro‐asthmatic cytokine tumor necrosis factor (TNF) and the impact of adding dexamethasone 1 hr after stimulation assessed. IL‐6 mRNA expression and protein secretion was significantly repressed by dexamethasone acting in a temporally distinct manner to increase MKP‐1, deactivate p38 MAPK and modulate TTP phosphorylation status. In this way, dexamethasone‐induced MKP‐1 acts via p38 MAPK to switch on the mRNA destabilizing function of TTP to repress pro‐inflammatory cytokine secretion from ASM cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-30T06:47:04.182305-05:
      DOI: 10.1002/jcp.25327
  • Bee Venom Accelerates Wound Healing in Diabetic Mice by Suppressing
           Activating Transcription Factor‐3 (ATF‐3) and Inducible Nitric
           Oxide Synthase (iNOS)‐Mediated Oxidative Stress and Recruiting Bone
           Marrow‐Derived Endothelial Progenitor Cells
    • Authors: Gamal Badr; Wael N. Hozzein, Badr M. Badr, Ahmad Al Ghamdi, Heba M. Saad Eldien, Olivier Garraud
      Abstract: Multiple mechanisms contribute to impaired diabetic wound healing including impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Bee venom (BV) has been used as an anti‐inflammatory agent for the treatment of several diseases. Nevertheless, the effect of BV on the healing of diabetic wounds has not been studied. Therefore, in this study, we investigated the impact of BV on diabetic wound closure in a type I diabetic mouse model. Three experimental groups were used: group 1, non‐diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice treated with BV. We found that the diabetic mice exhibited delayed wound closure characterized by a significant decrease in collagen production and prolonged elevation of inflammatory cytokines levels in wounded tissue compared to control non‐diabetic mice. Additionally, wounded tissue in diabetic mice revealed aberrantly up‐regulated expression of ATF‐3 and iNOS followed by a marked elevation in free radical levels. Impaired diabetic wound healing was also characterized by a significant elevation in caspase‐3, ‐8 and ‐9 activity and a marked reduction in the expression of TGF‐β and VEGF, which led to decreased neovascularization and angiogenesis of the injured tissue by impairing EPC mobilization. Interestingly, BV treatment significantly enhanced wound closure in diabetic mice by increasing collagen production and restoring the levels of inflammatory cytokines, free radical, TGF‐β and VEGF. Most importantly, BV‐treated diabetic mice exhibited mobilized long‐lived EPCs by inhibiting caspase activity in the wounded tissue. Our findings reveal the molecular mechanisms underlying improved diabetic wound healing and closure following BV treatment. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-30T06:46:42.007725-05:
      DOI: 10.1002/jcp.25328
  • Let‐7d miRNA Shows Both Antioncogenic and Oncogenic Functions in
           Osteosarcoma‐Derived 3AB‐OS Cancer Stem Cells
    • Abstract: Osteosarcoma (OS), an aggressive highly invasive and metastatic bone‐malignancy, shows therapy resistance and recurrence, two features that likely depend on cancer stem cells (CSCs), which hold both self‐renewing and malignant potential. So, effective anticancer therapies against OS should specifically target and destroy CSCs. We previously found that the let‐7d microRNA was downregulated in the 3AB‐OS‐CSCs, derived from the human OS‐MG63 cells. Here, we aimed to assess whether let‐7d modulation affected tumorigenic and stemness properties of these OS‐CSCs. We found that let‐7d‐overexpression reduced cell proliferation, by decreasing CCND2 and E2F2 cell‐cycle‐activators and increasing p21 and p27 CDK‐inhibitors. Let‐7d also decreased sarcosphere‐and‐colony forming ability, two features associated with self‐renewing, and it reduced the expression of stemness genes, including Oct3/4, Sox2, Nanog, Lin28B and HMGA2. Moreover, let‐7d induced mesenchymal‐to‐epithelial‐transition, as shown by both N‐Cadherin‐E‐cadherin‐switch and decrease in vimentin. Surprisingly, such switch was accompanied by enhanced migratory/invasive capacities, with a strong increase in MMP9, CXCR4 and VersicanV1. Let‐7d‐ overexpression also reduced cell sensitivity to apoptosis induced by both serum‐starvation and various chemotherapy drugs, concomitant with decrease in caspase‐3 and increase in BCL2 expression. Our data suggest that let‐7d in 3AB‐OS‐CSCs could induce plastic‐transitions from CSCs‐to‐non‐CSCs and vice‐versa. To our knowledge this is the first study to comprehensively examine the expression and functions of let‐7d in OS‐CSCs. By showing that let‐7d has both tumor suppressor and oncogenic functions in this context, our findings suggest that, before prospecting new therapeutic strategies based on let‐7d modulation, it is urgent to better define its multiple functions. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-28T04:42:07.921937-05:
      DOI: 10.1002/jcp.25291
  • Signaling and Gene Regulatory Networks Governing Definitive Endoderm
           Derivation from Pluripotent Stem Cells
    • Authors: Abdulshakour Mohammadnia; Moein Yaqubi, Farzaneh Pourasgari, Eric Neely, Hossein Fallahi, Mohammad Massumi
      Abstract: The generation of definitive endoderm (DE) from Pluripotent Stem Cells (PSCs) is a fundamental stage in the formation of highly organized visceral organs, such as the liver and pancreas. Currently, there is a need for a comprehensive study that illustrates the involvement of different signaling pathways and their interactions in the derivation of DE cells from PSCs. This study aimed to identify signaling pathways that have the greatest influence on DE formation using analyses of transcriptional profiles, protein‐protein interactions, protein‐DNA interactions, and protein localization data. Using this approach, signaling networks involved in DE formation were constructed using systems biology and data mining tools, and the validity of the predicted networks was confirmed experimentally by measuring the mRNA levels of hub genes in several PSCs‐derived DE cell lines. Based on our analyses, seven signaling pathways including the BMP, ERK1‐ERK2, FGF, TGF‐beta, MAPK, Wnt and PIP signaling pathways and their interactions, were found to play a role in the derivation of DE cells from PSCs. Lastly, the core gene regulatory network governing this differentiation process was constructed. The results of this study could improve our understanding surrounding the efficient generation of DE cells for the regeneration of visceral organs. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-28T04:40:25.91172-05:0
      DOI: 10.1002/jcp.25308
  • Improved Mitochondrial and Methylglyoxal‐Related Metabolisms Support
           Hyperproliferation Induced by 50 Hz Magnetic Field in Neuroblastoma
    • Authors: Stefano Falone; Silvano Santini, Silvia di Loreto, Valeria Cordone, Marta Grannonico, Patrizia Cesare, Marisa Cacchio, Fernanda Amicarelli
      Abstract: Extremely low frequency magnetic fields (ELF‐MF) are common environmental agents that are suspected to promote later stages of tumorigenesis, especially in brain‐derived malignancies. Even though ELF magnetic fields have been previously linked to increased proliferation in neuroblastoma cells, no previous work has studied whether ELF‐MF exposure may change key biomolecular features, such as anti‐glycative defence and energy re‐programming, both of which are currently considered as crucial factors involved in the phenotype and progression of many malignancies. Our study investigated whether the hyperproliferation that is induced in SH‐SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field is supported by an improved defense towards methylglyoxal (MG), which is an endogenous cancer‐static and glycating α‐oxoaldehyde, and by rewiring of energy metabolism. Our findings show that not only the ELF magnetic field interfered with the biology of neuron‐derived malignant cells, by de‐differentiating further the cellular phenotype and by increasing the proliferative activity, but also triggered cytoprotective mechanisms through the enhancement of the defense against MG, along with a more efficient management of metabolic energy, presumably to support the rapid cell outgrowth. Intriguingly, we also revealed that the MF‐induced bioeffects took place after an initial imbalance of the cellular homeostasis, which most likely created a transient unstable milieu. The biochemical pathways and molecular targets revealed in this research could be exploited for future approaches aimed at limiting or suppressing the deleterious effects of ELF magnetic fields. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-28T04:40:03.365209-05:
      DOI: 10.1002/jcp.25310
  • Zebrafish as a Model for the Study of Chaperonopathies
    • Authors: Gianfranco Bellipanni; Francesco Cappello, Federica Scalia, Everly Conway de Macario, Alberto JL Macario, Antonio Giordano
      Abstract: There is considerable information on the clinical manifestations and mode of inheritance for many genetic chaperonopathies but little is known on the molecular mechanisms underlying the cell and tissue abnormalities that characterize them. This scarcity of knowledge is mostly due to the lack of appropriate animal models that mimic closely the human molecular, cellular, and histological characteristics. In this article we introduce zebrafish as a suitable model to study molecular and cellular mechanisms pertaining to human chaperonopathies. Genetic chaperonopathies manifest themselves from very early in life so it is necessary to examine the impact of mutant chaperone genes during development, starting with fertilization and proceeding throughout the entire ontogenetic process. Zebrafish is amenable to such developmental analysis as well as studies during adulthood. In addition, the zebrafish genome contains a wide range of genes encoding proteins similar to those that form the chaperoning system of humans. This, together with the availability of techniques for genetic manipulations and for examination of all stages of development, makes zebrafish the organism of choice for the analysis of the molecular features and pathogenic mechanisms pertaining to human chaperonopathies. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-27T06:46:27.446181-05:
      DOI: 10.1002/jcp.25319
  • Polyglucosan Molecules Induce Mitochondrial Impairment and Apoptosis in
           Germ Cells Without Affecting the Integrity and Functionality of Sertoli
    • Abstract: Glycogen is the main storage form of glucose; however, the accumulation of glycogen‐like glucose polymers can lead to degeneration and cellular death. Previously, we reported that the accumulation of glycogen in testis of transgenic animals overexpressing a constitutively active form of glycogen synthase enhances the apoptosis of pre‐meiotic male germ cells and a complete disorganization of the seminiferous tubules. Here we sought to further identify the effects of glycogen storage in cells from the seminiferous tubules and the mechanism behind the pro‐apoptotic activity induced by its accumulation. Using an in vitro culture of Sertoli cells (line 42GPA9) and spermatocyte‐like cells (line GC‐1) expressing a superactive form of glycogen synthase or the Protein Targeting to Glycogen (PTG), we found that glycogen synthesized in both cell lines is poorly branched. In addition, the immunodetection of key molecules of apoptotic events suggests that cellular death induced by polyglucosan molecules affects GC‐1 cells, but not 42GPA9 cells by mitochondrial impairment and activation of an intrinsic apoptotic pathway. Furthermore, we analyzed the effects of glycogen deposition during the establishment of an in vitro blood‐testis barrier. The results using a non‐permeable fluorescent molecule showed that, in conditions of over‐synthesis of glycogen, 42GPA9 cells do not lose their capacity to generate an impermeable barrier and the levels of connexin43, occludin, and ZO1 proteins were not affected. These results suggest that the accumulation of polyglucosan molecules has a selective effect—triggered by the intrinsic activation of the apoptotic pathway—in germ cells without directly affecting Sertoli cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-21T06:54:39.640951-05:
      DOI: 10.1002/jcp.25315
  • The Lineage Specification of Mesenchymal Stem Cells Is Directed by the
           Rate of Fluid Shear Stress
    • Authors: Juan Lu; Yijuan Fan, Xiaoyuan Gong, Xin Zhou, Caixia Yi, Yinxing Zhang, Jun Pan
      Abstract: The effective regulation of fluid shear stress (FSS) on the lineage specification of mesenchymal stem cells (MSCs) remains to be addressed. We hypothesized that when MSCs are recruited to musculoskeletal system following stimulation, their differentiation into osteogenic or chondrogenic cells is directed by the rate of FSS (ΔSS) through modulation of the mechanosensitive, cation‐selective channels (MSCCs), intracellular calcium levels and F‐actin. To this end, MSCs were exposed to laminar FSS linearly increased from 0 to 10 dyn/cm2 in 0, 2 or 20 min and maintained at 10 dyn/cm2 for a total of 20 min (termed as ΔSS 0‐0', 0‐2' and 0‐20', respectively, representing more physiological (0‐0') and non‐physiological (0‐2' and 0‐20') ΔSS treatments). Our results showed 0‐0' facilitated MSC differentiation towards chondrogenic and not osteogenic phenotype, by promoting moderate intracellular calcium concentration ([Ca2+]i) increase from the calcium channels with the exception of MSCCs or intracellular calcium stores, and F‐actin organization. In contrast, 0‐2' promoted MSCs towards osteogenic and not chondrogenic phenotype, by inducing significant [Ca2+]i increase mainly from the MSCCs, and F‐actin assembly. However, 0‐20' elicited the modest osteogenic and chondrogenic phenotypes, as it induced the lowest [Ca2+]i increase mainly from MSCCs, and F‐actin assembly. Our results suggest that compared to the more physiological ΔSS, the non‐physiological ΔSS favors [Ca2+]i influx from MSCCs. An appropriate non‐physiological ΔSS (0‐2') even elicits a large [Ca2+]i influx from the MSCCs that reverses the lineage specification of MSCs, providing validation for the high mechanosensitivity of MSCs and guidance for training osteoporosis and osteoarthritis patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-15T11:14:31.808751-05:
      DOI: 10.1002/jcp.25278
  • Genetic Polymorphism of CHRM2 in COPD: Clinical Significance and
           Therapeutic Implications
    • Authors: Emanuela Cherubini; Maria Cristina Esposito, Davide Scozzi, Fabrizio Terzo, Giorgia Amira Osman, Salvatore Mariotta, Rita Mancini, Pierdonato Bruno, Alberto Ricci
      Abstract: Chronic Obstructive Pulmonary Disease (COPD) is a common preventable and treatable disease, characterized by persistent airflow limitation not fully reversible. However, a number of patients with COPD respond to bronchodilator agents. Some studies have shown polymorphisms in the b2‐adrenergic (ADRb2) and muscarinic M2 and M3 receptors (CHRM) that may participate in the modulation of the receptor responses. This study was designed to investigate the existence and the role of adrenergic and muscarinic receptor polymorphisms and their functional impact in COPD. Eighty‐two patients with COPD and 17 healthy smokers were recruited and screened for ADRb2 (T164I and R175R), for CHRM2 (rs1824024) and for CHRM3 (–513C/A and –492C/T). Among the polymorphisms studied our results was not able to demonstrate statistically significant association between the polymorphisms studied and COPD risk. Contrarily, we identified, in our COPD population, a significant association with the CHRM2 (rs1824024) polymorphism and disease severity, with lower lung function test values, frequent exacerbations and poor response to anti‐cholinergic drugs. These results suggest the potential role of receptor polymorphism assessment to discriminate newly COPD phenotypes. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-15T11:13:37.671109-05:
      DOI: 10.1002/jcp.25277
  • Electrically Activated Primary Human Fibroblasts Improve In Vitro and In
           Vivo Skin Regeneration
    • Authors: Mahmoud Rouabhia; Hyun Jin Park, Ze Zhang
      Abstract: Electrical stimulation (ES) changes cellular behaviors and thus constitutes a potential strategy to promote wound healing. However, well‐controlled in vitro findings have yet to be translated to in vivo trials. This study was to demonstrate the feasibility and advantages of transplanting electrically activated cells (E‐Cells) to help wound healing. Primary human skin fibroblasts were activated through well defined ES and cultured with keratinocytes to generate engineered human skin (EHS), which were transplanted to nu/nu mice. The electrically activated EHS grafts were analyzed at 20 and 30 days post‐grafting, showing faster wound closure, thick epidermis, vasculature, and functional basement membrane containing laminin and type IV collagen that were totally produced by the implanted human cells. Since a variety of cells can be electrically activated, E‐Cells may become a new cell source and the transplantation of E‐Cells may represent a new strategy in wound healing and tissue engineering. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-15T11:13:27.590873-05:
      DOI: 10.1002/jcp.25289
  • Effects of a Sublethal and Transient Stress of the Endoplasmic Reticulum
           on the Mitochondrial Population
    • Abstract: Endoplasmic reticulum (ER) and mitochondria are not discrete intracellular organelles but establish close physical and functional interactions involved in several biological processes including mitochondrial bioenergetics, calcium homeostasis, lipid synthesis and the regulation of apoptotic cell death pathways. As many cell types might face a transient and sublethal ER stress during their lifetime, it is thus likely that the adaptive UPR response might affect the mitochondrial population. The aim of this work was to study the putative effects of a non‐lethal and transient endoplasmic reticulum stress on the mitochondrial population in HepG2 cells. The results show that thapsigargin and brefeldin A, used to induce a transient and sublethal ER stress, rapidly lead to the fragmentation of the mitochondrial network associated with a decrease in mitochondrial membrane potential, O2•− production and less efficient respiration. These changes in mitochondrial function are transient and preceeded by the phosphorylation of JNK. Inhibition of JNK activation by SP600125 prevents the decrease in O2•− production and the mitochondrial network fragmentation observed in cells exposed to the ER stress but has no impact on the reduction of the mitochondrial membrane potential. In conclusion, our data shows that a non‐lethal and transient ER stress triggers a rapid activation of JNK without inducing apoptosis, leading to the fragmentation of the mitochondrial network and a reduction of O2•− production. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-15T11:13:00.480515-05:
      DOI: 10.1002/jcp.25292
  • Expression of Muscle‐Specific Ribosomal Protein L3‐Like
           Impairs Myotube Growth
    • Authors: Thomas Chaillou; Xiping Zhang, John J. McCarthy
      Abstract: The ribosome has historically been considered to have no cell‐specific function but rather serve in a “housekeeping” capacity. This view is being challenged by evidence showing that heterogeneity in the protein composition of the ribosome can lead to the functional specialization of the ribosome. Expression profiling of different tissues revealed that ribosomal protein large 3‐like (Rpl3l) is exclusively expressed in striated muscle. In response to a hypertrophic stimulus, Rpl3l expression in skeletal muscle was significantly decreased by 82% whereas expression of the ubiquitous paralog Rpl3 was significantly increased by ∼5‐fold. Based on these findings, we developed the hypothesis that Rpl3l functions as a negative regulator of muscle growth. To test this hypothesis, we used the Tet‐On system to express Rpl3l in myoblasts during myotube formation. In support of our hypothesis, RPL3L expression significantly impaired myotube growth as assessed by myotube diameter (‐23%) and protein content (‐14%). Further analysis showed that the basis of this impairment was caused by a significant decrease in myoblast fusion as the fusion index was significantly lower (‐17%) with RPL3L expression. These findings are the first evidence to support the novel concept of ribosome specialization in skeletal muscle and its role in the regulation of skeletal muscle growth. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-14T05:05:43.80883-05:0
      DOI: 10.1002/jcp.25294
  • A NOXA/MCL‐1 Imbalance Underlies Chemoresistance of Malignant
           Rhabdoid Tumor Cells
    • Authors: Kazutaka Ouchi; Yasumichi Kuwahara, Tomoko Iehara, Mitsuru Miyachi, Yoshiki Katsumi, Kunihiko Tsuchiya, Eiichi Konishi, Akio Yanagisawa, Hajime Hosoi
      Abstract: Malignant rhabdoid tumor (MRT) is a rare aggressive pediatric cancer characterized by inactivation of SNF5, a core subunit of SWI/SNF complexes. Previously, we showed that SNF5 contributes to transcriptional activation of NOXA, a pro‐apoptotic protein that binds and inhibits the anti‐apoptotic protein MCL‐1. In this study, we found that NOXA expression was downregulated in MRT cell lines as well as in clinical MRT samples and that ectopically expressed NOXA bound MCL‐1 and increased the sensitivity of MRT cell lines to doxorubicin (DOX) by promoting apoptosis. Consistent with this finding, knockdown of MCL‐1 in MRT cell lines induced apoptosis and increased DOX sensitivity in MRT cells, and the MCL‐1 inhibitor TW‐37 synergized with DOX to induce MRT cell death. Our results suggest that modulation of the NOXA/MCL‐1 pathway may be a potential strategy for the treatment of patients with MRT. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-14T05:05:40.424859-05:
      DOI: 10.1002/jcp.25293
  • Light/Dark Environmental Cycle Imposes a Daily Profile in the Expression
           of microRNAs in Rat CD133+ Cells
    • Abstract: The phenotype of primary cells in culture varies according to the donor environmental condition. We recently showed that the time of the day imposes a molecular program linked to the inflammatory response that is heritable in culture. Here we investigated whether microRNAs (miRNAs) would show differential expression according to the time when cells were obtained, namely daytime or nighttime. Cells obtained from explants of cremaster muscle and cultivated until confluence (∼20 days) presented high CD133 expression. Global miRNA expression analysis was performed through deep sequencing in order to compare both cultured cells. A total of 504 mature miRNAs were identified, with a specific miRNA signature being associated to the light versus dark phase of a circadian cycle. miR‐1249 and miR‐129‐2‐3p were highly expressed in daytime cells, while miR‐182, miR‐96‐5p, miR‐146a‐3p, miR‐146a‐5p and miR‐223‐3p were highly expressed in nighttime cells. Nighttime cells are regulated for programs involved in cell processes and development, as well as in the inflammation, cell differentiation and maturation; while daytime cells express miRNAs that control stemness and cytoskeleton remodeling. In summary, the time of the day imposes a differential profile regarding to miRNA signature on CD133+ cells in culture. Understanding this daily profile in the phenotype of cultured cells is highly relevant for clinical outputs, including cellular therapy approaches. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-14T04:47:35.517923-05:
      DOI: 10.1002/jcp.25300
  • Cell‐Autonomous Brown‐Like Adipogenesis of Preadipocytes from
           Retinoblastoma Haploinsufficient Mice
    • Authors: Petar D. Petrov; Andreu Palou, M. Luisa Bonet, Joan Ribot
      Abstract: Mechanisms behind the emergence of brown adipocyte‐like (brite or beige) adipocytes within white adipose tissue (WAT) are of interest. Retinoblastoma protein gene (Rb) haploinsufficiency associates in mice with improved metabolic regulation linked to a greater capacity for fatty acid oxidation and thermogenesis in WAT. We aimed to explain a feasible mechanism of WAT‐to‐BAT remodeling in this model. Differentiated primary adipocytes and Sca1‐positive preadipocytes derived from adipose depots of Rb+/− mice and wild‐type siblings were compared. Primary white Rb+/− adipocytes displayed under basal conditions increased glucose uptake and an enhanced expression of brown adipocyte–related genes (Pparg, Ppargc1a, Ppargc1b, Prdm16, Cpt1b), but not of purported beige/brite transcriptional markers (Cd137, Tmem26, Tbx1, Slc27a1, Hoxc9, Shox2). Lack of induction of beige markers phenocopied results in WAT of adult Rb+/− mice. Flow cytometry analysis evidenced an increased number of preadipocytes in WAT depots of Rb+/− mice. Sca1+ preadipocytes from WAT of Rb+/− mice displayed increased gene expression of several transcription factors common to the brown and beige adipogenic programs (Prdm16, Pparg, Ppargc1a) and of receptors of bone morphogenetic proteins (BMPs); however, amongst the recently proposed beige markers, only Tbx1 was upregulated. Adult Rb+/− mice had increased circulating levels of BMP7. These results indicate that preadipose cells resident in WAT depots of Rb+/− mice retain an increased capacity for brown‐like adipogenesis that appears to be different from beige adipogenesis, and suggest that the contribution of these precursors to the Rb+/− adipose phenotype is driven, at least in part, by interaction with BMP7 pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-14T04:47:11.326472-05:
      DOI: 10.1002/jcp.25299
  • IDO‐Expressing Fibroblasts Protect Islet beta Cells from
           Immunological Attack and Reverse Hyperglycemia in Non‐Obese Diabetic
    • Abstract: Indoleamine 2,3‐dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: 1. Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non‐obese diabetic (NOD) mice; 2. Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 106 (10M) or 15 × 106 (15M) IDO‐expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8+ T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL‐ 1β and cleaved caspase‐3 levels were assessed in islets co‐cultured with IDO‐expressing fibroblasts. BGLs in 83% mice treated with 15M IDO‐expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO‐expressing cells were reversed to normoglycemia. 15M IDO‐expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8+ T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co‐cultured with IDO‐ expressing fibroblasts had reduced IL‐1β levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO‐expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8+ T cells and Th17 cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-01-07T08:24:47.026909-05:
      DOI: 10.1002/jcp.25301
  • Table of Contents: Volume 231, Number 5
    • First page: 965
      PubDate: 2016-01-25T11:17:42.404361-05:
      DOI: 10.1002/jcp.25295
  • Editor's Choice
    • First page: 968
      PubDate: 2016-01-25T11:17:46.959262-05:
      DOI: 10.1002/jcp.25296
  • Highlights: Volume 231, Number 5
    • First page: 969
      PubDate: 2016-01-25T11:17:51.748637-05:
      DOI: 10.1002/jcp.25297
  • Molecular Structure and Regulation of P2X Receptors with a Special
           Emphasis on the Role of P2X2 in the Auditory System
    • Authors: Rahul Mittal; Brandon Chan, M'hamed Grati, Jeenu Mittal, Kunal Patel, Luca H. Debs, Amit P. Patel, Denise Yan, Prem Chapagain, Xue Zhong Liu
      Abstract: The P2X purinergic receptors are cation selective channels gated by extracellular adenosine 5'‐triphosphate (ATP). These purinergic receptors are found in virtually all mammalian cell types and facilitate a number of important physiological processes. Within the past few years, the characterization of crystal structures of the zebrafish P2X4 receptor in its closed and open states has provided critical insights into the mechanisms of ligand binding and channel activation. Understanding of this gating mechanism has facilitated to design and interpret new modelling and structure‐function experiments to better elucidate how different agonists and antagonists can affect the receptor with differing levels of potency. This review summarizes the current knowledge on the structure, activation, allosteric modulators, function, and location of the different P2X receptors. Moreover, an emphasis on the P2X2 receptors has been placed in respect to its role in the auditory system. In particular, the discovery of three missense mutations in P2X2 receptors could become important areas of study in the field of gene therapy to treat progressive and noise‐induced hearing loss. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-30T03:25:54.492201-05:
      DOI: 10.1002/jcp.25274
  • All trans‐Retinoic Acid Mediates MED28/HMG Box‐Containing
           Protein 1 (HBP1)/β‐Catenin Signaling in Human Colorectal Cancer
    • Abstract: Vitamin A is required for normal body function, including vision, epithelial integrity, growth, and differentiation. All trans‐retinoic acid (ATRA), a family member of vitamin A, has been explored in treating acute promyelocytic leukemia and other types of cancer. Dysregulated Wnt/β‐catenin signaling and disrupted cadherin‐catenin complex often contribute to colorectal malignancy. MED28, a mammalian Mediator subunit, is found highly expressed in breast and colorectal cancers. Our laboratory has also reported that MED28 regulates cell growth, migration, and invasion in human breast cancer cells. In the current study we investigated the effect of ATRA on MED28 and Wnt/β‐catenin signaling in colorectal cancer. HCT116, HT29, SW480, and SW620, four human colorectal cancer cell lines representing different stages of carcinogenesis and harboring critical genetic changes, were employed. Our data indicated that, regardless of genetic variations among these cells, suppression of MED28 reduced the expression of cyclin D1, c‐Myc, and nuclear β‐catenin, but increased the expression of E‐cadherin and HMG box‐containing protein 1 (HBP1) where HBP1 has been described as a negative regulator of the Wnt/β‐catenin signaling. The reporter activity of an HBP1 promoter increased upon MED28 knockdown, but decreased upon MED28 overexpression. ATRA reduced the expression of MED28 and mimicked the effect of MED28 suppression in down‐regulating Wnt/β‐catenin signaling. Taken together, ATRA can reverse the suppressive effect of MED28 on HBP1 and E‐cadherin and inactivate the Wnt/β‐catenin pathway in colorectal cancer, suggesting a protective effect of ATRA against colorectal cancer. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-30T03:24:43.050634-05:
      DOI: 10.1002/jcp.25285
  • “MicroRNA Expression Signature Is Altered in the Cardiac Remodeling
           Induced by High Fat Diets”
    • Abstract: Recent studies have revealed the involvement of microRNAs (miRNAs) in the control of cardiac hypertrophy and myocardial function. In addition, several reports have demonstrated that high fat (HF) diet induces cardiac hypertrophy and remodeling. In the current study, we investigated the effect of diets containing different percentages of fat on the cardiac miRNA expression signature. To address this question, male C57Bl/6 mice were fed with a low fat (LF) diet or two HF diets, containing 45 kcal% fat (HF45%) and 60 kcal% fat (HF60%) for 10 and 20 weeks. HF60% diet promoted an increase on body weight, fasting glycemia, insulin, leptin, total cholesterol, triglycerides and induced glucose intolerance. HF feeding promoted cardiac remodeling, as evidenced by increased cardiomyocyte transverse diameter and interstitial fibrosis. RNA sequencing analysis demonstrated that HF feeding induced distinct miRNA expression patterns in the heart. HF45% diet for 10 and 20 weeks changed the abundance of 64 and 26 miRNAs in the heart, respectively. On the other hand, HF60% diet for 10 and 20 weeks altered the abundance of 27 and 88 miRNAs in the heart, respectively. Bioinformatics analysis indicated that insulin signaling pathway was overrepresented in response to HF diet. An inverse correlation was observed between cardiac levels of GLUT4 and miRNA‐29c. Similarly, we found an inverse correlation between expression of GSK3β and the expression of miRNA‐21a‐3p, miRNA‐29c‐3p, miRNA‐144‐3p and miRNA‐195a‐3p. In addition, miRNA‐1 overexpression prevented cardiomyocyte hypertrophy. Taken together, our results revealed differentially expressed miRNA signatures in the heart in response to different HF diets. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-30T03:24:20.920844-05:
      DOI: 10.1002/jcp.25280
  • microRNAs and Endothelial (Dys) Function
    • Authors: Gaetano Santulli
      Abstract: Accumulating evidence indicates that microRNAs (miRs) ‐ non‐coding RNAs that can regulate gene expression via translational repression and/or post‐transcriptional degradation ‐ are becoming one of the most fascinating areas of physiology, given their fundamental roles in countless pathophysiological processes. The relative role of different miRs in vascular biology as direct or indirect post‐transcriptional regulators of fundamental genes implied in vascular remodeling designate miRs as potential biomarkers and/or promising drug targets. The mechanistic roles of miRs in modulating endothelial cell (EC) function in physiology and in disease are described here. Drawbacks of currently available therapeutic options are also discussed, pointing at the challenges and clinical opportunities provided by miR‐based treatments. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-30T03:23:57.436402-05:
      DOI: 10.1002/jcp.25276
  • SGLT2 Inhibitors: Glucotoxicity and Tumorigenesis Downstream the Renal
           Proximal Tubule'
    • Abstract: At present, diabetes mellitus is the main cause of end‐stage renal disease. Effective glycaemic management is the most powerful tool to delay the establishment of diabetic complications, such as diabetic kidney disease. Together with reducing blood glucose levels, new anti‐diabetic agents are expected not only to control the progression but also to restore known defects of the diabetic kidney. Sodium‐glucose co‐transporter 2 (SGLT2) inhibitors are promising anti‐diabetic agents that reduce hyperglycaemia by impairing glucose reabsorption in proximal tubule of the kidney and increasing glucosuria. SGLT2 inhibitors have shown to reduce glucotoxicity in isolated proximal tubule cells and also to attenuate expression of markers of overall kidney damage in experimental animal models of diabetes, but the actual renoprotective effect for downstream nephron segments is still unknown and deserves further attention. Here, we briefly discuss possible undesired effects of enhanced glucosuria and albuminuria in nephron segments beyond the proximal tubule after SGLT2 inhibitor treatment, offering new lines of research to further understand the renoprotective action of these anti‐diabetic agents. Strategies blocking glucose reabsorption by renal proximal tubule epithelial cells (RPTEC) may be protective for RPTEC, but downstream nephron segments will still be exposed to high glucose and albumin levels through the luminal face. The actual effect of constant enhanced glucosuria over distal nephron segments remains to be established. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-30T03:07:10.140697-05:
      DOI: 10.1002/jcp.25286
  • Inflammation Controls Sensitivity of Human and Mouse Intestinal Epithelial
           Cells to Galectin‐1
    • Abstract: Galectins play key roles in the inflammatory cascade. In this study we aimed to analyze the effect of galectin‐1 (Gal‐1) on function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro‐inflammatory cytokines, and Gal‐1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal‐1 binding and modulation of IECs. We found an enhanced binding of Gal‐1 to: a) murine IECs exposed to IL‐1β, TNF, and IL‐13, b) IECs from inflamed areas in intestinal tissue from IBD patients, c) small bowel of allergic mice and d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal‐1 favored a tolerogenic microenvironment, while high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase‐9‐ and modulation of Bcl‐2 protein family members. Our results showed that, when added in the presence of diverse pro‐inflammatory cytokines (TNF, IL‐13 and IL‐5), Gal‐1 differentially promoted the secretion of growth factors including TSLP, EGF, IL‐10, IL‐25, and TGF‐β1. In conclusion, we found an augmented binding of Gal‐1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal‐1 concentration. These findings highlight the importance of the inflammatory microenvironment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal‐1 and its role in epithelial cell homeostasis. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:53:53.468735-05:
      DOI: 10.1002/jcp.25249
  • Morphine‐Induced MOR‐1X and ASF/SF2 Expression Is Independent
           of Transcriptional Regulation: Implications for MOR‐1X Signaling
    • Authors: Patrick M. Regan; Ilker K. Sariyer, T. Dianne Langford, Prasun K. Datta, Kamel Khalili
      Abstract: Recently, multiple μ‐opioid receptor (MOR) isoforms have been identified that originate from a single gene, OPRM1; however, both their regulation and their functional significance are poorly characterized. The objectives of this study were to decipher, first, the regulation of alternatively spliced μ‐opioid receptor isoforms and the spliceosome components that determine splicing specificity and, second, the signaling pathways utilized by particular isoforms both constitutively and following agonist binding. Our studies demonstrated that the expression of a particular splice variant, MOR‐1X, was up‐regulated by morphine, and this coincided with an increase in the essential splicing factor ASF/SF2. Structural comparison of this isoform to the prototypical variant MOR‐1 revealed that the unique distal portion of the C‐terminal domain contains additional phosphorylation sites, while functional comparison found distinct signaling differences, particularly in the ERK and p90 RSK pathways. Additionally, MOR‐1X expression significantly reduced Bax expression and mitochondrial dehydrogenase activity, suggesting a unique functional consequence for MOR‐1X specific signaling. Collectively, these findings suggest that alternative splicing of the MOR is altered by exogenous opioids, such as morphine, and that individual isoforms, such as MOR‐1X, mediate unique signal transduction with distinct functional consequence. Furthermore, we have identified for the first time a potential mechanism that involves the essential splicing factor ASF/SF2 through which morphine regulates splicing specificity of the MOR encoding gene, OPRM1. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:53:09.277329-05:
      DOI: 10.1002/jcp.25246
  • Establishment of Immortalized BMP2/4 Double Knock‐Out Osteoblastic
           Cells is Essential for Study of Osteoblast Growth, Differentiation and
    • Abstract: Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock out (ko/ko) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4ko/ko cells were verified by green immunofluorescence and PCR. BMP2/4ko/ko osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone‐relate genes was reduced in the BMP2/4ko/ko cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4ko/ko osteoblasts as reflected by decreased Mmp‐2 and Mmp‐9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:49:20.826641-05:
      DOI: 10.1002/jcp.25266
  • Rab, Arf and Arl‐Regulated Membrane Traffic in Cortical Neuron
    • Authors: Bor Luen Tang
      Abstract: The migration of projection neurons from its birthplace in the subventricular zone to their final destination in the cortical plate is a complex process that requires a series of highly coordinated cellular events. Amongst the key factors involved in the processes would include modulators of cytoskeletal dynamics, as well as cellular membrane traffic. Members of the small GTPases family responsible for the latter process, the Rabs and Arfs, have been recently implicated in cortical neuron migration. Rab5 and Rab11, which are key modulators of endocytosis and endocytic recycling respectively, ensure proper surface expression and distribution of N‐cadherin, a key adhesion protein that tethers migrating neurons to the radial glia fiber tracts during pia‐directed migration. Rab7, which is associated with lysosomal biogenesis and function, is important for the final step of terminal translocation when N‐cadherin is downregulated by lysosomal degradation. Arf6 activity, which is known to be important in neuronal processes outgrowth, may negatively impact the multipolar‐bipolar transition of cortical neurons undergoing radial migration, but the downstream effector of Arf6 in this regard is not yet known. In addition to the above, members of the Arl family which have been recently shown to be important in radial glia scaffold formation, would also be important for cortical neuron migration. In this short review, we discuss recent advances in our understanding in the importance of membrane traffic regulated by the Rab, Arf and Arl family members in cortical neuron migration. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:48:14.648836-05:
      DOI: 10.1002/jcp.25261
  • Valproic Acid Enhances iPSC Induction from Human Bone Marrow‐Derived
           Cells through the Suppression of Reprogramming‐Induced Senescence
    • Abstract: Reprogramming of human somatic cells into pluripotent cells (iPSCs) by defined transcription factors is an extremely inefficient process. Treatment with the histone deacetylase inhibitor valproic acid (VPA) during reprogramming can improve the induction of iPSCs. To examine the specific mechanism underlying the role of VPA in reprogramming, we transfected human bone marrow‐derived cells (HSC‐J2 and HSC‐L1) with lentiviruses carrying defined factors (OCT4, SOX2, KLF4, and c‐MYC, OSKM) in the presence of VPA. We found that OSKM lentiviruses caused significant senescence in transfected cells. Administration of VPA, however, significantly suppressed this reprogramming‐induced stress. Notably, VPA treatment improved cell proliferation in the early stages of reprogramming, and this was related to the down‐regulation of the activated p16/p21 pathway. In addition, VPA also released the G2/M phase blockade in lentivirus‐transfected cells. This study demonstrates a new mechanistic role of the histone deacetylase inhibitor in enhancing the induction of pluripotency. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:45:35.560076-05:
      DOI: 10.1002/jcp.25270
  • Fibronectin Fiber Extension Decreases Cell Spreading and Migration
    • Abstract: The extracellular matrix (ECM) is present in a range of molecular conformations and intermolecular arrangements. Fibronectin (Fn) molecules that constitute fibers within the ECM can exist in a variety of conformations that result from both mechanical stress and chemical factors such as allosteric binding partners. The long‐standing hypothesis that conformational changes regulate the binding of cells to Fn fibers has only been tested for mutated molecules of Fn and has yet to be fully evaluated with Fn fibers. Using time‐lapse microscopy we examined how mechanical extension of single fibers of Fn affects the adhesion and migration of endothelial cells. Using this single fiber adhesion technique, we show that high levels of mechanical strain applied to Fn fibers decreases the rates of both cell spreading and cell migration. These data indicate a fundamental cellular response to mechanical strain in the ECM that might have important implications for understanding how cells are recruited during tissue development and repair. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:45:04.543959-05:
      DOI: 10.1002/jcp.25271
  • Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig
    • Authors: Cristoforo Pomara; Rosario Barone, Antonella Marino Gammazza, Claudia Sangiorgi, Fulvio Barone, Alessandro Pitruzzella, Nicola Locorotondo, Francesca Di Gaudio, Monica Salerno, Francesca Maglietta, Antonio Luciano Sarni, Valentina Di Felice, Francesco Cappello, Emanuela Turillazzi
      Abstract: Anabolic androgenic steroids (AAS) are among the drugs most used by athletes for improving physical performance, as well as for aesthetic purposes. A number of papers have showed the side effects of AAS in different organs and tissues. For example, AAS are known to suppress gonadotropin‐releasing hormone, luteinizing hormone and follicle‐stimulating hormone. This study investigates the effects of nandrolone on testosterone biosynthesis in Leydig cells using various methods, including mass spectrometry, western blotting, confocal microscopy and quantitative real‐time PCR. The results obtained show that testosterone levels increase at a 3.9 µM concentration of nandrolone and return to the basal level a 15.6 µM dose of nandrolone. Nandrolone‐induced testosterone increment was associated with upregulation of the steroidogenic acute regulatory protein (StAR) and downregulation of 17a‐hydroxylase/17, 20 lyase (CYP17A1). Instead, a 15.6 µM dose of nandrolone induced a down‐regulation of CYP17A1. Further in vivo studies based on these data are needed to better understand the relationship between disturbed testosterone homeostasis and reproductive system impairment in male subjects. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:44:39.453482-05:
      DOI: 10.1002/jcp.25272
  • MicroRNAs: Modulators of the Ras Oncogenes in Oral Cancer
    • Authors: Avaniyapuram Kannan Murugan; Arasambattu Kannan Munirajan, Ali S. Alzahrani
      Abstract: Oral squamous cell carcinoma (OSCC) of the head and neck is one of the six most common cancers in the world. OSCC remains the most common cause of cancer deaths in Asian countries. Conventional treatments for OSCC have not improved the overall five year survival and therefore alternative therapeutic targets are often sought. Ras is one of the most frequently deregulated oncogenes in oral cancer. Direct targeting the ras has proven unrealistic and hence, exploring and understanding alternative pathways and/or molecules which regulate ras and its signaling that could pave the way for novel molecular targets and therapy for oral cancer. Recently, microRNAs (miRNAs) have been reported to regulate ras oncogenes in human cancers. In this article, we address the microRNA‐mediated regulation of the ras oncogenes in oral cancer. We describe extensively the tumor suppressive and oncogenic roles of miRNAs in regulation of ras oncogenes in OSCC. We also discuss the role of miRNA‐mediated ras regulation in therapeutic determination in oral cancer. Complete understanding of the miRNA regulation of ras oncogenes in oral cancer may facilitate to plan better strategies for diagnosis, molecular therapeutic targeting and the overall prognosis of this common and deadly cancer. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:44:16.149933-05:
      DOI: 10.1002/jcp.25269
  • E11/Podoplanin Protein Stabilization through Inhibition of the Proteasome
           Promotes Osteocyte Differentiation in Murine In Vitro Models
    • Authors: Katherine A. Staines; Matt Prideaux, Steve Allen, David J. Buttle, Andrew A. Pitsillides, Colin Farquharson
      Abstract: The transmembrane glycoprotein E11 is considered critical in early osteoblast‐osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO‐A5 cell line we reveal increased E11 protein/mRNA expression (P 
      PubDate: 2015-12-28T04:43:33.592579-05:
      DOI: 10.1002/jcp.25282
  • Nuclear Phosphatidylinositol Signaling: Focus on Phosphatidylinositol
           Phosphate Kinases and Phospholipases C
    • Abstract: Phosphatidylinositol (PI) metabolism represents the core of a network of signaling pathways which modulate many cellular functions including cell proliferation, cell differentiation, apoptosis and membrane trafficking. An array of kinases, phosphatases and lipases acts on PI creating an important number of second messengers involved in different cellular processes. Although, commonly, PI signaling was described to take place at the plasma membrane, many evidences indicated the existence of a PI cycle residing in the nuclear compartment of eukaryotic cells. The discovery of this mechanism shed new light on many nuclear functions, such as gene transcription, DNA modifications and RNA expression. As these two PI cycles take place independently of one another, understanding how nuclear lipid signaling functions and modulates nuclear output is fundamental in the study of many cellular processes. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:42:48.594412-05:
      DOI: 10.1002/jcp.25273
  • Cellular Metabolism and Dose Reveal Carnitine‐Dependent and
           –Independent Mechanisms of Butyrate Oxidation in Colorectal Cancer
    • Authors: Anna Han; Natalie Bennett, Amber MacDonald, Megan Johnstone, Jay Whelan, Dallas R. Donohoe
      Abstract: Dietary fiber has been suggested to suppress colorectal cancer development, although the mechanisms contributing to this beneficial effect remain elusive. Butyrate, a fermentation product of fiber, has been shown to have anti‐proliferative and pro‐apoptotic effects on colorectal cancer cells. The metabolic fate of butyrate in the cell is important in determining whether it acts as an HDAC inhibitor or is consumed as a short‐chain fatty acid. Non‐cancerous colonocytes utilize butyrate as the primary energy source whereas cancerous colonocytes increase glucose utilization through the Warburg effect. In this study, we show that butyrate oxidation is decreased in cancerous colonocytes compared to non‐cancerous colonocytes. We demonstrate that colorectal cancer cells utilize both a carnitine‐dependent and carnitine‐independent mechanism that contributes to butyrate oxidation. The carnitine‐dependent mechanism is contingent on butyrate concentration. Knockdown of CPT1A in colorectal cancer cells abolishes butyrate oxidation. In terms of selectivity, the carnitine‐dependent mechanism only regulated butyrate oxidation, as acetate and propionate oxidation were carnitine‐independent. Carnitine decreased the action of butyrate as an HDAC inhibitor and suppressed induction of H3 acetylation by butyrate in colorectal cancer cells. Thus, diminished oxidation of butyrate is associated with decreased HDAC inhibition and histone acetylation. In relation to the mechanism, we find that dichloroacetate, which decreases phosphorylation of pyruvate dehydrogenase, increased butyrate oxidation and that this effect was carnitine‐dependent. In conclusion, these data suggest that colorectal cancer cells decrease butyrate oxidation through inhibition of pyruvate dehydrogenase, which is carnitine‐dependent, and provide insight into why butyrate shows selective effects toward colorectal cancer cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-28T04:41:26.695139-05:
      DOI: 10.1002/jcp.25287
  • Molecular Mechanism of Transcriptional Regulation of Matrix
           Metalloproteinase‐9 in Diabetic Retinopathy
    • Authors: Manish Mishra; Jadwiga Flaga, Renu A. Kowluru
      Abstract: Increase in matrix metalloproteinase‐9 (MMP‐9) is implicated in retinal capillary cell apoptosis, a phenomenon which precedes the development of diabetic retinopathy. MMP‐9 promoter has multiple sites for binding the transcriptional factors, including two for activator protein 1 (AP‐1). The binding of AP‐1, a heterodimer of c‐Jun and c‐Fos, is regulated by posttranslational modifications, and in diabetes, deacetylating enzyme, Sirt1, is inhibited. Our aim is to investigate the molecular mechanism of MMP‐9 transcriptional regulation in diabetes. Binding of AP‐1 (c‐Jun, c‐Fos) at the MMP‐9 promoter, and AP‐1 acetylation were analyzed in retinal endothelial cells incubated in normal or high glucose by chromatin‐immunoprecipitation and co‐immunoprecipitation respectively. Role of AP‐1 in MMP‐9 regulation was confirmed by c‐Jun or c‐Fos siRNAs, and that of its acetylation, by Sirt1 overexpression. In vitro results were validated in the retina from diabetic mice overexpressing Sirt1, and in the retinal microvessels from human donors with diabetic retinopathy. In experimental models, AP‐1 binding was increased at the proximal and distal sites of the MMP‐9 promoter, and similar phenomenon was confirmed in the retinal microvessels from human donors with diabetic retinopathy. Silencing of AP‐1, or overexpression of Sirt1 ameliorated glucose‐induced increase in MMP‐9 expression and cell apoptosis. Thus, in diabetes, due to Sirt1 inhibition, AP‐1 is hyperacetylated, which increases its binding at MMP‐9 promoter, and hence, activation of Sirt1 could inhibit the development of diabetic retinopathy by impeding MMP‐9‐mediated mitochondrial damage. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-22T05:36:54.583902-05:
      DOI: 10.1002/jcp.25268
  • The LPV Motif is Essential for the Efficient Export of Secretory DMP1 from
           the Endoplasmic Reticulum
    • Authors: Tian Liang; Tian Meng, Suzhen Wang, Chunlin Qin, Yongbo Lu
      Abstract: Dentin matrix protein 1 (DMP1) is found abundantly in the extracellular matrices of bone and dentin. Secretory DMP1 begins with a tripeptide of leucine‐proline‐valine (LPV) after the endoplasmic reticulum (ER)‐entry signal peptide is cleaved. The goal of this study was to determine the role of the LPV motif in the secretion of DMP1. A series of DNA constructs was generated to express various forms of DMP1 with or without the LPV motif. These constructs were transfected into a preosteoblast cell line, the MC3T3‐E1 cells, and the subcellular localization and secretion of various forms of DMP1 were examined by immunofluorescent staining and Western‐blotting analyses. Immunofluorescent staining showed that the LPV‐containing DMP1 variants were primarily localized in the Golgi complex, whereas the LPV‐lacking DMP1 variants were found abundantly within the ER. Western‐blotting analyses demonstrated that the LPV‐containing DMP1 variants were rapidly secreted from the transfected cells, as they did not accumulate within the cells, and the amounts increased in the conditioned media over time. In contrast, the LPV‐lacking DMP1 variants were predominantly retained within the cells, and only small amounts were secreted out of the cells over time. These results suggest that the LPV motif is essential for the efficient export of secretory DMP1 from the ER to the Golgi complex. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-14T04:08:58.091611-05:
      DOI: 10.1002/jcp.25265
  • Human Microbiome and Its Association With Health and Diseases
    • Abstract: Human microbiota are distinct communities of microorganisms that resides at different body niches. Exploration of the human microbiome has become a reality due to the availability of powerful metagenomics and metatranscriptomic analysis technologies. Recent advances in sequencing and bioinformatics over the past decade help provide a deep insight into the nature of the host‐microbial interactions and identification of potential deriver genes and pathways associated with human health, well‐being, and predisposition to different diseases. In the present review, we outline recent studies devoted to elucidate the possible link between the microbiota and various type of diseases. The present review also highlights the potential utilization of microbiota as a potential therapeutic option to treat a wide array of human diseases. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-11T04:12:58.062992-05:
      DOI: 10.1002/jcp.25284
  • The Role of Kv1.2 Channel in Electrotaxis Cell Migration
    • Authors: Gaofeng Zhang; Mathew Edmundson, Vsevolod Telezhkin, Yu Gu, Xiaoqing Wei, Paul J. Kemp, Bing Song
      Abstract: Voltage‐gated potassium Kv1.2 channels play pivotal role in maintaining of resting membrane potential and, consequently, regulation of cellular excitability of neurons. Endogenously generated electric field (EF) have been proven as an important regulator for cell migration and tissue repair. The mechanisms of ion channel involvement in EF‐induced cell responses are extensively studied but largely are poorly understood. In this study we generated three COS‐7 clones with different expression levels of Kv1.2 channel, and confirmed their functional variations with patch clamp analysis. Time‐lapse imaging analysis showed that EF‐induced cell migration response was Kv1.2 channel expression level depended. Inhibition of Kv1.2 channels with charybdotoxin (ChTX) constrained the sensitivity of COS‐7 cells to EF stimulation more than their motility. Immunocytochemistry and pull‐down analyses demonstrated association of Kv1.2 channels with actin‐binding protein cortactin and its re‐localization to the cathode‐facing membrane at EF stimulation, which confirms the mechanism of EF‐induced directional migration. This study displays that Kv1.2 channels represent an important physiological link in EF‐induced cell migration. The described mechanism suggests a potential application of EF which may improve therapeutic performance in curing injuries of neuronal and/or cardiac tissue repair, post operational therapy and various degenerative syndromes. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-10T04:11:07.196358-05:
      DOI: 10.1002/jcp.25259
  • Fusion Pore Size Limits 5‐HT Release from Single Enterochromaffin
           Cell Vesicles
    • Authors: Ravinarayan Raghupathi; Claire F. Jessup, Amanda L. Lumsden, Damien J. Keating
      Abstract: Enterochromaffin cells are the major site of serotonin (5‐HT) synthesis and secretion providing ∼95% of the body's total 5‐HT. 5‐HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5‐HT per exocytosis event compared to other endocrine cells. We utilised a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5‐HT packaging and release. Using the fluorescent probe FFN511 we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5‐HT loading into vesicles. Carbon fibre amperometry studies illustrate that the amount of 5‐HT released per exocytosis event from EC cells is dependent on both VMAT and the H+‐ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5‐HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different‐sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5‐HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5‐HT release capacity from individual vesicles. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-10T04:10:17.700098-05:
      DOI: 10.1002/jcp.25256
  • Nuclear Factor κB1/RelA Mediates Inflammation in Human Lung
           Epithelial Cells at Atmospheric Oxygen Levels
    • Authors: Lakshmanan Jagannathan; Cynthia C. Jose, Adriana Arita, Thomas Kluz, Hong Sun, Xiaoru Zhang, Yixin Yao, Andrey V. Kartashov, Artem Barski, Max Costa, Suresh Cuddapah
      Abstract: Oxygen levels range from 2‐9% in vivo. Atmospheric O2 levels (21%) are known to induce cell proliferation defects and cellular senescence in primary cell cultures. However, the mechanistic basis of the deleterious effects of higher O2 levels is not fully understood. On the other hand, immortalized cells including cancer cell lines, which evade cellular senescence are normally cultured at 21% O2 and the effects of higher O2 on these cells are understudied. Here we addressed this problem by culturing immortalized human bronchial epithelial (BEAS‐2B) cells at ambient atmospheric, 21% O2 and lower, 10% O2. Our results show increased inflammatory response at 21% O2 but not at 10% O2. We found higher RelA binding at the NF‐κB1/RelA target gene promoters as well as upregulation of several pro‐inflammatory cytokines in cells cultured at 21% O2. RelA knockdown prevented the upregulation of the pro‐inflammatory cytokines at 21% O2, suggesting NF‐κB1/RelA as a major mediator of inflammatory response in cells cultured at 21% O2. Interestingly, unlike the 21% O2 cultured cells, exposure of 10% O2 cultured cells to H2O2 did not elicit inflammatory response, suggesting increased ability to tolerate oxidative stress in cells cultured at lower O2 levels. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-10T04:09:50.002492-05:
      DOI: 10.1002/jcp.25262
  • Autophagy as a Potential Target for Sarcopenia
    • Authors: Jingjing Fan; Xianjuan Kou, Shaohui Jia, Xiaoqi Yang, Yi Yang, Ning Chen
      Abstract: Sarcopenia is an aging‐related disease with a significant reduction in mass and strength of skeletal muscle due to the imbalance between protein synthesis and protein degradation. The loss of skeletal muscle is an inevitable event during aging process, which can result in the significant impact on the quality of life, and also can increase the risk for other aging‐associated diseases in the elderly. However, the underlying molecular mechanism of aging‐related skeletal muscle loss is still poorly understood. Autophagy is a degradation pathway for the clearance of dysfunctional organelles and damaged macromolecules during aging process. Appropriate induction or accurate regulation of autophagic process and improved quality control of mitochondria through autophagy or other strategies are required for the maintenance of skeletal muscle mass. In this article, we have summarized the current understanding of autophagic pathways in sarcopenia, and discussed the functional status of autophagy and autophagy‐associated quality control of mitochondria in the pathogenesis of sarcopenia. Moreover, this article will provide some theoretical references for the exploration of scientific and optimal intervention strategies such as exercise and caloric restriction for the prevention and treatment of sarcopenia through the regulation of autophagic pathways. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-10T04:09:22.028714-05:
      DOI: 10.1002/jcp.25260
  • Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis and
           Thyroglobulin Internalization in a Thyroid Cell Line
    • Abstract: Rab7 regulates the biogenesis of late endosomes, lysosomes and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over‐expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over‐expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over‐expressing cells, Rab7 and LC3B‐II co‐localized and co‐localization in large circular structures occurred only in Rab7 over‐expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co‐localization of Rab7 with cortactin and f‐actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over‐expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5 or Rab8, was inducible by cAMP and that cAMP‐stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over‐expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-10T04:08:58.636712-05:
      DOI: 10.1002/jcp.25267
  • Stretching Impacts Inflammation Resolution in Connective Tissue
    • Authors: Lisbeth Berrueta; Igla Muskaj, Sara Olenich, Taylor Butler, Gary J. Badger, Romain A. Colas, Matthew Spite, Charles N. Serhan, Helene M. Langevin
      Abstract: Acute inflammation is accompanied from its outset by the release of specialized pro‐resolving mediators (SPMs), including resolvins, that orchestrate the resolution of local inflammation. We showed earlier that, in rats with subcutaneous inflammation of the back induced by carrageenan, stretching for 10 minutes twice daily reduced inflammation and improved pain, two weeks after carrageenan injection. In this study, we hypothesized that stretching of connective tissue activates local pro‐resolving mechanisms within the tissue in the acute phase of inflammation. In rats injected with carrageenan and randomized to stretch vs. no stretch for 48 hours, stretching reduced inflammatory lesion thickness and neutrophil count, and increased resolvin (RvD1) concentrations within lesions. Furthermore, subcutaneous resolvin injection mimicked the effect of stretching. In ex vivo experiments, stretching of connective tissue reduced the migration of neutrophils and increased tissue RvD1 concentration. These results demonstrate a direct mechanical impact of stretching on inflammation‐regulation mechanisms within connective tissue. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-10T04:08:02.893899-05:
      DOI: 10.1002/jcp.25263
  • Thymosin β4 Prevents Angiotensin II‐Induced Cardiomyocyte
           Growth by Regulating Wnt/WISP Signaling
    • Abstract: Thymosin beta‐4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. However, the role of Tβ4 in cardiomyocyte hypertrophy is currently unknown. The purpose of this study was to determine the cardio‐protective effect of Tβ4 in angiotensin II (Ang II)‐induced cardiomyocytes growth. Neonatal rat ventricular cardiomyocytes (NRVM) were pretreated with Tβ4 followed by Ang II stimulation. Cell size, hypertrophy marker gene expression and Wnt signaling components, β‐catenin and Wnt‐induced secreted protein‐1 (WISP‐1) were evaluated by quantitative real‐time PCR, Western blotting and fluorescent microscopy. Pre‐treatment of Tβ4 resulted in reduction of cell size, hypertrophy marker genes and Wnt‐associated gene expression and protein levels; induced by Ang II in cardiomyocytes. WISP‐1 was overexpressed in NRVM and, the effect of Tβ4 in Ang II‐induced cardiomyocytes growth was evaluated. WISP‐1 overexpression promoted cardiomyocytes growth and was reversed by pretreatment with Tβ4. This is the first report which demonstrates that Tβ4 targets Wnt/WISP‐1 to protect Ang II‐induced cardiomyocyte growth. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-02T08:11:29.042492-05:
      DOI: 10.1002/jcp.25275
  • Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was
           Delineated by Nestin Expression
    • Abstract: Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague‐Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I(+)‐fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31(+)‐endothelial cells were detected, exhibited a mesenchymal phenotype and co‐expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co‐expression of E‐cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF‐β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E‐cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein‐7 treatment of alveolar epithelial cells prevented E‐cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I(+)‐fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats. This article is protected by copyright. All rights reserved
      PubDate: 2015-12-02T03:53:17.693136-05:
      DOI: 10.1002/jcp.25257
  • Alternative RUNX1 Promoter Regulation by Wnt/β‐Catenin
           Signaling in Leukemia Cells and Human Hematopoietic Progenitors
    • Abstract: Two distantly located promoter regions regulate the dynamic expression of RUNX genes during development: distal P1 and proximal P2 promoters. We have recently described that β‐catenin increases total Runx1 mRNA levels in human CD34+ hematopoietic progenitors and enhances spatial proximity with its translocation partner ETO. Here we report that induction of Wnt/β‐catenin signaling in HL60 and Jurkat leukemia‐derived cell lines and CD34+ progenitors selectively activate the production of the longer distal P1‐Runx1 mRNA isoform. Gain‐ and loss‐of‐function experiments revealed that the differential increase in P1‐Runx1 expression is accomplished through a minimal β‐catenin responsive region that includes a highly conserved TCF/LEF‐binding element, located ‐20/‐16 bp upstream of the canonical distal P1‐Runx1 transcription start site. We conclude that the distal P1‐Runx1 promoter is a direct transcriptional target of Wnt/β‐catenin signaling that may be important in normal hematopoiesis or its transition into malignant stem cells during the onset or progression of leukemia. This article is protected by copyright. All rights reserved
      PubDate: 2015-11-18T15:47:29.110268-05:
      DOI: 10.1002/jcp.25258
  • High Methionine Diet Poses Cardiac Threat: A Molecular Insight
    • Authors: Pankaj Chaturvedi; Pradip K. Kamat, Anuradha Kalani, Anastasia Familtseva, Suresh C. Tyagi
      Abstract: High methionine diet (HMD) for example red meat which includes lamb, beef, pork can pose cardiac threat and vascular dysfunction but the mechanisms are unclear. We hypothesize that a diet rich in methionine can malfunction the cardiovascular system in three ways: 1) by augmenting oxidative stress; 2) by inflammatory manifestations; and 3) by matrix/vascular remodeling. To test this hypothesis we used four groups of mice 1) WT; 2) WT + methionine; 3) CBS+/−; 4) CBS+/− + methionine. We observed high oxidative stress in mice fed with methionine which was even higher in CBS+/− and CBS+/− + methionine. Higher oxidative stress was indicated by high levels of SOD‐1 in methionine fed mouse hearts while IL‐1, IL‐6, TNFα and TLR4 showed high inflammatory manifestations. The upregulated levels of eNOS/iNOS and upregulated levels of MMP2/MMP9 along with high collagen deposition indicated vascular and matrix remodeling in methionine fed mouse. We evaluated the cardiac function which was dysregulated in the mice fed with HMD. These mice had decreased ejection fraction and left ventricular dysfunction which subsequently leads to adverse cardiac remodeling. In conclusion our study clearly shows that HMD poses a cardiac threat by increasing oxidative stress, inflammatory manifestations, matrix/vascular remodeling and decreased cardiac function. This article is protected by copyright. All rights reserved
      PubDate: 2015-11-13T09:02:23.692201-05:
      DOI: 10.1002/jcp.25247
  • Research Needs for Understanding the Biology of Overdiagnosis in Cancer
    • Authors: Sudhir Srivastava; Brian J. Reid, Sharmistha Ghosh, Barnett S. Kramer
      Abstract: Many cancers offer an extended window of opportunity for early detection and therapeutic intervention that could lead to a reduction in cause‐specific mortality. The pursuit of early detection in screening settings has resulted in decreased incidence and mortality for some cancers (eg, colon and cervical cancers), and increased incidence with only modest or no effect on cause‐specific mortality in others (eg, breast and prostate). Whereas highly sensitive screening technologies are better at detecting a number of suspected “cancers” that are indolent and likely to remain clinically unimportant in the lifetime of a patient, defined as overdiagnosis, they often miss cancers that are aggressive and tend to present clinically between screenings, known as interval cancers. Unrecognized overdiagnosis leads to overtreatment with its attendant (often long‐lasting) side effects, anxiety, and substantial financial harm. Existing methods often cannot differentiate indolent lesions from aggressive ones or understand the dynamics of neoplastic progression. To correctly identify the population that would benefit the most from screening and identify the lesions that would benefit most from treatment, the evolving genomic and molecular profiles of individual cancers during the clinical course of progression or indolence must be investigated, while taking into account an individual's genetic susceptibility, clinical and environmental risk factors, and the tumor microenvironment. Practical challenges lie not only in the lack of access to tissue specimens that are appropriate for the study of natural history, but also in the absence of targeted research strategies. This commentary summarizes the recommendations from a diverse group of scientists with expertise in basic biology, translational research, clinical research, statistics, and epidemiology and public health professionals convened to discuss research directions. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-27T17:47:33.070998-05:
      DOI: 10.1002/jcp.25227
  • Common and Rare Variants Associated with Alzheimer's Disease
    • Authors: Hany Marei; Asmaa Althani, Mohamed El Zowalaty, Mohammad A. Albanna, Carlo Cenciarelli, Tengfei Wang, Thomas Caceci
      Abstract: Alzheimer's disease (AD) is one of the most devastating disorder. Despite the continuing increase of its incidence among aging population, no effective cure has been developed mainly due to difficulties in early diagnosis of the disease before damaging of the brain, and the failure to explore its complex underlying molecular mechanisms. Recent technological advances in genome‐wide association studies (GWAS) and high throughput next generation whole genome, and exome sequencing had deciphered many of AD‐related loci, and discovered single nucleotide polymorphisms (SNPs) that are associated with altered AD molecular pathways. Highlighting altered molecular pathways linked to AD pathogenesis is crucial to identify novel diagnostic and therapeutic AD targets. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-23T17:57:04.715116-05:
      DOI: 10.1002/jcp.25225
  • Effects of Sex Hormones on Ocular Surface Epithelia: Lessons Learned from
           Polycystic Ovary Syndrome
    • Abstract: Polycystic ovary syndrome (PCOS) is the most common endocrine abnormality in women of reproductive age. Although its clinical consequences have been known for a long time to extend beyond the reproductive system, with type‐2 diabetes and obesity being the most common, the involvement of the ocular surface in PCOS has been described only more recently. The ocular surface is a morphofunctional unit comprising eyelid margin, tear film, cornea and conjunctiva. Increasing evidence indicates that these structures are under a sex hormone control and relevant diseases such as ocular allergy and dry eye are often caused by alterations in circulating or local steroid hormones levels. Novel treatments targeting sex hormone receptors on ocular surface epithelial cells are also being developed. In this review we aim to describe the current knowledge on the effects of sex hormones at the ocular surface, with a special focus on the effects of androgen imbalance in PCOS. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-22T17:41:41.611322-05:
      DOI: 10.1002/jcp.25221
  • Non‐hematopoietic β‐arrestin1 confers protection against
           experimental colitis
    • Authors: Taehyung Lee; Eunhee Lee, David Arrollo, Peter C. Lucas, Narayanan Parameswaran
      Abstract: β‐Arrestins are multifunctional scaffolding proteins that modulate G protein‐coupled receptor (GPCR)‐dependent and ‐independent cell signaling pathways in various types of cells. We recently demonstrated that β‐arrestin1 (β‐arr1) deficiency strikingly attenuates dextran sodium sulfate (DSS)‐induced colitis in mice. Since DSS‐induced colitis is in part dependent on gut epithelial injury, we examined the role of β‐arr1 in intestinal epithelial cells (IECs) using a colon epithelial cell line, SW480 cells. Surprisingly, we found that knockdown of β‐arr1 in SW480 cells enhanced epithelial cell death via a caspase‐3‐dependent process. To understand the in vivo relevance and potential cell type‐specific role of β‐arr1 in colitis development, we generated bone marrow chimeras with β‐arr1 deficiency in either the hematopoietic or non‐hematopoietic compartment. Reconstituted chimeric mice were then subjected to DSS‐induced colitis. Similar to our previous findings, β‐arr1 deficiency in the hematopoietic compartment protected mice from DSS‐induced colitis. However, consistent with the role of β‐arr1 in epithelial apoptosis in vitro, non‐hematopoietic β‐arr1 deficiency led to an exacerbated colitis phenotype. To further understand signaling mechanisms, we examined the effect of β‐arr1 on TNF‐α‐mediated NFκB and MAPK pathways. Our results demonstrate that β‐arr1 has a critical role in modulating ERK, JNK and p38 MAPK pathways mediated by TNF‐α in IECs. Together, our results show that β‐arr1‐dependent signaling in hematopoietic and non‐hematopoietic cells differentially regulates colitis pathogenesis and further demonstrates that β‐arr1 in epithelial cells inhibits TNF‐α‐induced cell death pathways. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-19T08:42:37.610904-05:
      DOI: 10.1002/jcp.25216
  • Cathepsin K Deficiency Suppresses Disuse‐Induced Bone Loss
    • Authors: Shuichi Moriya; Yayoi Izu, Smriti Arayal, Makiri Kawasaki, Koki Hata, Chantida Pawaputanon Na Mahasarakhahm, Yuichi Izumi, Paul Saftig, Kazuo Kaneko, Masaki Noda, Yoichi Ezura
      Abstract: Unloading induces bone loss and causes disuse osteoporosis. However, the mechanism underlying disuse osteoporosis is still incompletely understood. Here we examined the effects of cathepsin K (CatK) deficiency on disuse osteoporosis induced by using sciatic neurectomy (Nx) model. After four weeks of surgery, Cat‐K KO and WT mice were sacrificed and subjected to analyses. For cancellous bone rich region, Nx reduced the bone mineral density (BMD) compared to the BMD in the sham operated side in wild type mice. In contrast, CatK deficiency suppressed such Nx‐induced reduction of BMD in cancellous bone. Nx also reduced BMD in the mid shaft cortical bone compared to the BMD in the corresponding region on the sham operated side in wild type mice. In contrast, CatK deficiency suppressed such Nx‐induced reduction of BMD in the mid shaft cortical bone. Bone volume (BV/TV) was reduced by Nx in WT mice. In contrast, Cat‐K deficiency suppressed such reduction in bone volume. Interestingly, CatK deficiency suppressed osteoclast number and osteoclast surface in the Nx side compared to sham side. When bone marrow cells obtained from Nx side femur of CatK‐KO mice were cultured, the levels of the calcified area in culture were increased. Further examination of gene expression indicated that Nx suppressed the expression of genes encoding osteoblast‐phenotype‐related molecules such as Runx2 and alkaline phosphatase in WT mice. In contrast, CatK deficiency suppressed such reduction. These data indicate that CatK is involved in the disuse‐induced bone mass reduction. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-13T11:04:53.470829-05:
      DOI: 10.1002/jcp.25214
  • The IP3R Binding Protein Released with Inositol 1,4,5‐Trisphosphate
           Is Expressed in Rodent Reproductive Tissue and Spermatozoa
    • Authors: Heike Borth; Nele Weber, Dorke Meyer, Andrea Wartenberg, Elisabeth Arlt, Susanna Zierler, Andreas Breit, Gunther Wennemuth, Thomas Gudermann, Ingrid Boekhoff
      Abstract: Besides its capacity to inhibit the 1,4,5‐trisphosphate (IP3) receptor, the regulatory protein IRBIT (IP3 receptor‐binding protein released with IP3) is also able to control the activity of numerous ion channels and electrolyte transporters and thereby creates an optimal electrolyte composition of various biological fluids. Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co‐localize with the proton pumping V‐ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co‐localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturations steps after ejaculation and/or the final fertilization process. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-06T06:35:29.112421-05:
      DOI: 10.1002/jcp.25209
  • Reduced dentin matrix protein expression in Camurati‐Engelmann
           disease transgenic mouse model
    • Authors: Angela Gullard; Christina M. Croney, Xiangwei Wu, Olga Mamaeva, Philip Sohn, Xu Cao, Mary MacDougall
      Abstract: Overexpression of transforming growth factor‐beta 1 (TGF‐β1) has been shown to lead to mineralization defects in both the enamel and dentin layers of teeth. A TGFB1 point mutation (H222D), derived from published cases of Camurati‐Engelmann disease (CED), has been shown to constitutively activate TGF‐β1, leading to excess bone matrix production. Although CED has been well documented in clinical case reports, there are no published studies on the effect of CED on the dentition. The objective of this study was to determine the dental manifestations of hyperactivated TGF‐β1 signaling using an established mouse model of CED‐derived TGF‐β1 mutation. Murine dental tissues were studied via radiography, micro‐CT, immunohistochemistry, and qRT‐PCR. Results showed that initial decreased dental mineralized tissue density is resolved Proliferation assays of incisor pulp and alveolar bone cell cultures revealed that cells from transgenic animals displayed a reduced rate of growth compared to alveolar bone cultures from wild‐type mice. TGF‐β family gene expression analysis indicated significant fold changes in the expression of Alpl, Bmp2‐5, Col‐1, ‐2,‐4 and ‐6, Fgf, Mmp, Runx2, Tgfb3, Tfgbr3, and Vdr genes. Assessment of SIBLINGs reveals downregulation of Ibsp, Dmp1, Dspp, Mepe, and Spp1, as well as reduced staining for BMP‐2 and VDR in mesenchymal‐derived pulp tissue in CED animals. Treatment of dental pulp cells with recombinant human TGF‐β1 resulted in increased SIBLING gene expression. Conclusions: Our results provide in vivo evidence suggesting that TFG‐β1 mediates expression of important dentin extracellular matrix components secreted by dental pulp, and when unbalanced, may contribute to abnormal dentin disorders. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-01T11:13:46.627626-05:
      DOI: 10.1002/jcp.25207
  • Epigenetic Priming Confers Direct Cell Trans‐Differentiation from
           Adipocyte to Osteoblast in a Transgene‐Free State
    • Abstract: The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans‐differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5′‐aza‐dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3‐L1 adipocytes with 5′‐aza‐dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5′‐aza‐dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue‐engineering applications. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-03T17:22:44.577997-05:
      DOI: 10.1002/jcp.25183
  • Cell Surface Human Airway Trypsin‐Like Protease Is Lost During
           Squamous Cell Carcinogenesis
    • Authors: Michael J. Duhaime; Khaliph O. Page, Fausto A. Varela, Andrew S. Murray, Michael E. Silverman, Gina L. Zoratti, Karin List
      Abstract: Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro‐cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin‐like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non‐proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-22T17:23:43.72242-05:0
      DOI: 10.1002/jcp.25173
  • Electrospun Type 1 Collagen Matrices Using a Novel Benign Solvent for
           Cardiac Tissue Engineering
    • Authors: Alan M. Punnoose; Anuradha Elamparithi, Sarah Kuruvilla
      Abstract: Electrospinning is a well‐established technique that uses a high electric field to fabricate ultra fine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers due to its biocompatibility and widespread occurrence in nature. Electrospinning of Collagen alone has been reported with fluoroalcohols such as Hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE), which are toxic to the environment. In this study we describe the use of a novel benign binary solvent to generate nanofibers of Collagen type 1, which is non‐toxic and economical. Transmission electron microscopy (TEM) analysis revealed the characteristic feature of native collagen namely the 67 nm banding pattern, confirming that the triple helical structure was maintained. Scanning Electron Microscopy (SEM) analysis showed the fiber diameters to be in the 200‐800 nm range. Biocompatibility of the three dimensional (3D) scaffolds was established by MTT assays using skeletal myoblasts and Confocal Microscopic analysis of immunofluorescent stained sections for muscle specific markers such as Desmin and Actin. Primary neonatal rat ventricular cardiomyocytes seeded onto the scaffolds were able to maintain their contractile function for a period of 17 days. Our work provides evidence that Collagen 1 can be electrospun without combining with other polymers using a novel benign solvent and we are currently exploring the potential of this approach for cardiac and skeletal muscle tissue engineering. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:48:51.922438-05:
      DOI: 10.1002/jcp.25035
  • Protein Kinase C Is Involved in the Induction of ATP‐Binding
           Cassette Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor
           Agonist in Human Macrophages
    • Authors: Etimad A. Huwait; Nishi N. Singh, Daryn R. Michael, Thomas S. Davies, Joe W. E. Moss, Dipak P. Ramji
      Abstract: The transcription of the ATP‐binding cassette transporter A1 (ABCA1) gene, which plays a key anti‐atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c‐Jun N‐terminal kinase (JNK) and phosphoinositide 3‐kinase (PI3K) in the LXRs‐mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have therefore investigated a potential role for PKC in the action of LXR/RXR agonist 22‐(R)‐hydroxycholesterol (22‐(R)‐HC)/9‐cis‐retinoic acid (9cRA) in THP‐1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein‐1 (AP‐1) DNA binding activity in macrophages treated with 22‐(R)‐HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype‐specific inhibitors. These studies therefore reveal a potentially important role for PKC in the action of 22‐(R)‐HC and 9cRA in human macrophages. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-07T18:01:38.140641-05:
      DOI: 10.1002/jcp.25157
  • Regulation and Functional Implications of Opioid Receptor Splicing in
           Opioid Pharmacology and HIV Pathogenesis
    • Authors: Patrick M. Regan; Dianne Langford, Kamel Khalili
      First page: 976
      Abstract: Despite the identification and characterization of four opioid receptor subtypes and the genes from which they are encoded, pharmacological data does not conform to the predications of a four opioid receptor model. Instead, current studies of opioid pharmacology suggest the existence of additional receptor subtypes; however, no additional opioid receptor subtype has been identified to date. It is now understood that this discrepancy is due to the generation of multiple isoforms of opioid receptor subtypes. While several mechanisms are utilized to generate these isoforms, the primary mechanism involves alternative splicing of the pre‐mRNA transcript. Extensive alternative splicing patterns for opioid receptors have since been identified and discrepancies in opioid pharmacology are now partially attributed to variable expression of these isoforms. Recent studies have been successful in characterizing the localization of these isoforms as well as their specificity in ligand binding; however, the regulation of opioid receptor splicing specificity is poorly characterized. Furthermore, the functional significance of individual receptor isoforms and the extent to which opioid‐ and/or HIV‐mediated changes in the opioid receptor isoform profile contributes to altered opioid pharmacology or the well‐known physiological role of opioids in the exacerbation of HIV neurocognitive dysfunction is unknown. As such, the current review details constitutive splicing mechanisms as well as the specific architecture of opioid receptor genes, transcripts, and receptors in order to highlight the current understanding of opioid receptor isoforms, potential mechanisms of their regulation and signaling, and their functional significance in both opioid pharmacology and HIV‐associated neuropathology. This article is protected by copyright. All rights reserved
      PubDate: 2015-11-24T03:24:07.282774-05:
      DOI: 10.1002/jcp.25237
  • Body Mass Index and Treatment Outcomes in Metastatic Breast Cancer
           Patients Treated with Eribulin
    • First page: 986
      Abstract: Background Eribulin has shown survival advantage and manageable toxicity in heavily pre‐treated metastatic breast cancer (mBC). We assessed whether body mass index (BMI) impacts treatment outcomes in 101 patients treated with eribulin at 6 Italian Oncologic Centers. Methods BMI was addressed as a categorical variable (18.5‐24.9 vs at least 25). Clinical benefit rate (CBR) was assessed overall and in subgroups defined by BMI, line of therapy (LOT) and hormone receptor (HR) status. Analysis of CBR by LOT and HR status were further stratified by BMI. Survival curves were compared using the Kaplan‐Meier method and log‐rank test. Predictors of survival were tested in Cox models. Results Patients treated with eribulin as third line showed greater CBR when their BMI was in the lowest category (77.8 vs 58.1%, p = 0.03). Median progression free survival (PFS) and overall survival (OS) in normal and overweight patients were 4 (95% CI, 3‐5) vs 3 (2.1‐4) months, p = 0.02 and 13 (11‐15) vs 12 (6‐18) months, p = 0.96, respectively. Median PFS and OS in estrogen receptor (ER) positive and negative tumours were 4 (3‐5) vs 3 (2‐4) months, p = 0.005 and 14 (10‐18) vs 7 (4‐10), p = 0.02, respectively. In multivariate analyses, BMI impacted PFS at a nearly significant extent (p = 0.05), while ER expression significantly affected PFS and OS (p = 0.01 and 0.02, respectively). No relevant findings emerged concerning toxicity. Conclusions We found evidence of greater efficacy of eribulin in leaner mBC patients, particularly if given as third line and in ER positive tumours. Further studies are warranted to confirm our findings. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-09T06:41:17.412361-05:
      DOI: 10.1002/jcp.25213
  • Mitotic Inheritance of mRNA Facilitates Translational Activation of the
           Osteogenic‐Lineage Commitment Factor Runx2 in Progeny of
           Osteoblastic Cells
    • Authors: Nelson Varela; Alejandra Aranguiz, Carlos Lizama, Hugo Sepulveda, Marcelo Antonelli, Roman Thaler, Ricardo D. Moreno, Martin Montecino, Gary S. Stein, Andre J. van Wijnen, Mario Galindo
      First page: 1001
      Abstract: Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype‐committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post‐mitotic re‐activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that “bookmark” the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone‐related runt‐related transcription factor Runx2. Here, we characterized a non‐genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self‐renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non‐chromosomal tubulin‐containing compartment. Subsequently, transcription‐independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast‐specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. J. Cell. Physiol. 231: 1001–1014, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-18T04:02:34.052494-05:
      DOI: 10.1002/jcp.25188
  • Glucose and Inflammatory Cells Decrease Adiponectin in Epicardial Adipose
           Tissue Cells: Paracrine Consequences on Vascular Endothelium
    • First page: 1015
      Abstract: Epicardial adipose tissue (EAT) is a source of energy for heart that expresses the insulin‐sensitizer, anti‐inflammatory and anti‐atherogenic protein, adiponectin. But, in coronary artery disease, adiponectin production declines. Our objective was to determine its regulation by glucose and inflammation in stromal cells from EAT and subcutaneous adipose tissue (SAT) and its paracrine effect on endothelial cells. Stromal cells of EAT and SAT were obtained from patients who underwent cardiac surgery. Adipogenesis was induced at 117, 200, or 295 mg/dl glucose, with or without macrophage‐conditioned medium (MCM). Expression of adiponectin, GLUT‐4 and the insulin receptor was analyzed by real‐time PCR. The paracrine effect of stromal cells was determined in co‐cultures with endothelial cells, by exposing them to high glucose and/or MCM, and, additionally, to leukocyte‐conditioned medium from patients with myocardial infarction. The endothelial response was determined by analyzing vascular adhesion molecule expression. Our results showed a U‐shaped dose–response curve of glucose on adiponectin in EAT, but not in SAT stromal cells. Conversely, MCM reduced the adipogenesis‐induced adiponectin expression of EAT stromal cells. The presence of EAT stromal increased the inflammatory molecules of endothelial cells. This deleterious effect was emphasized in the presence of inflammatory cell‐conditioned medium from patients with myocardial infarction. Thus, high glucose and inflammatory cells reduced adipogenesis‐induced adiponectin expression of EAT stromal cells, which induced an inflammatory paracrine process in endothelial cells. This inflammatory effect was lower in presence of mature adipocytes, producers of adiponectin. These results contribute to understanding the role of EAT dysfunction on coronary atherosclerosis progression. J. Cell. Physiol. 231: 1015–1023, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-29T01:50:58.72809-05:0
      DOI: 10.1002/jcp.25189
  • Lipidomics of Mesenchymal Stromal Cells: Understanding the Adaptation of
           Phospholipid Profile in Response to Pro‐Inflammatory Cytokines
    • First page: 1024
      Abstract: Mesenchymal stromal cells (MSCs) present anti‐inflammatory properties and are being used with great success as treatment for inflammatory and autoimmune diseases. In clinical applications MSCs are subjected to a strong pro‐inflammatory environment, essential to their immunosuppressive action. Despite the wide clinical use of these cells, how MSCs exert their effect remains unclear. Several lipids are known to be involved in cell's signaling and modulation of cellular functions. The aim of this paper is to examine the variation in lipid profile of MSCs under pro‐inflammatory environment, induced by the presence of tumor necrosis factor alpha (TNF‐α) and interferon gamma (IFN‐γ), using the most modern lipidomic approach. Major changes in lipid molecular profile of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysoPC (LPC), and sphingomyelin (SM) classes were found. No changes were observed in the phosphatidylinositol (PI) profile. The levels of PC species with shorter fatty acids (FAs), mainly C16:0, decreased under pro‐inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of LPC(18:0), which is known to be an anti‐inflammatory LPC, observed in MSCs subjected to TNF‐α and IFN‐γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF‐α and IFN‐γ also enhanced the levels of PE(40:6) and decreased the levels of PE(O‐38:6). Higher expression of PS(36:1) and SM(34:0) along with a decrease in PS(38:6) levels were observed. These results indicate that lipid metabolism and signaling are modulated during MSCs activation, which suggests that lipids may be involved in MSCs functional and anti‐inflammatory activities. J. Cell. Physiol. 231: 1024–1032, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-10-08T00:32:23.177065-05:
      DOI: 10.1002/jcp.25191
  • HhAntag, a Hedgehog Signaling Antagonist, Suppresses Chondrogenesis and
           Modulates Canonical and Non‐Canonical BMP Signaling
    • Authors: Christina Mundy; Adebayo Bello, Federica Sgariglia, Eiki Koyama, Maurizio Pacifici
      First page: 1033
      Abstract: Chondrogenesis subtends the development of most skeletal elements and involves mesenchymal cell condensations differentiating into growth plate chondrocytes that proliferate, undergo hypertrophy, and are replaced by bone. In the pediatric disorder Hereditary Multiple Exostoses, however, chondrogenesis occurs also at ectopic sites and causes formation of benign cartilaginous tumors—exostoses—near the growth plates. No treatment is currently available to prevent or reverse exostosis formation. Here, we asked whether chondrogenesis could be stopped by targeting the hedgehog pathway, one of its major regulators. Micromass cultures of limb mesenchymal cells were treated with increasing amounts of the hedgehog inhibitor HhAntag or vehicle. The drug effectively blocked chondrogenesis and did so in a dose‐dependent manner as monitored by: alcian blue‐positive cartilage nodule formation; gene expression of cartilage marker genes; and reporter activity in Gli1‐LacZ cell cultures. HhAntag blocked chondrogenesis even when the cultures were co‐treated with bone morphogenetic protein 2 (rhBMP‐2), a strong pro‐chondrogenic factor. Immunoblots showed that HhAntag action included modulation of canonical (pSmad1/5/8) and non‐canonical (pp38) BMP signaling. In cultures co‐treated with HhAntag plus rhBMP‐2, there was a surprising strong up‐regulation of pp38 levels. Implantation of rhBMP‐2‐coated beads near metacarpal elements in cultured forelimb explants induced formation of ectopic cartilage that however, was counteracted by HhAntag co‐treatment. Collectively, our data indicate that HhAntag inhibits not only hedgehog signaling, but also modulates canonical and non‐canonical BMP signaling and blocks basal and rhBMP2‐stimulated chondrogenesis, thus representing a potentially powerful drug‐based strategy to counter ectopic cartilage growth or induce its involution. J. Cell. Physiol. 231: 1033–1044, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-22T03:09:25.944212-05:
      DOI: 10.1002/jcp.25192
  • Downhill Running Excessive Training Inhibits Hypertrophy in Mice Skeletal
           Muscles with Different Fiber Type Composition
    • First page: 1045
      Abstract: The aim of this study was to verify the effects of running overtraining protocols performed in downhill, uphill, and without inclination on the proteins related to hypertrophy signaling pathway in extensor digitorum longus (EDL) and soleus of C57BL/6 mice. We also performed histological and stereological analyses. Rodents were divided into control (CT; sedentary mice), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up), and overtrained by running without inclination (OTR). The incremental load, exhaustive, and grip force tests were used as performance evaluation parameters. 36 h after the grip force test, EDL and soleus were removed and immediately used for immunoblotting analysis or stored at −80°C for histological and stereological analyses. For EDL, OTR/down decreased the protein kinase B (Akt) and tuberous sclerosis protein 2 (TSC2) phosphorylation (p), and increased myostatin, receptor‐activated Smads (pSMAD2‐3), and insulin receptor substrate‐1 (pIRS‐1; Ser307/636). OTR/down also presented low and high relative proportions of cytoplasm and connective tissue, respectively. OTR/up increased the mammalian target of rapamycin (pmTOR), 70‐kDa ribosomal protein S6 kinase 1 (pS6K1) and pSMAD2‐3, and decreased pTSC2. OTR decreased pTSC2 and increased pIRS‐1 (Ser636). For soleus, OTR/down increased S6 ribosomal protein (pS6RP) and pSMAD2‐3, and decreased pIRS‐1 (Ser639). OTR/up decreased pS6K1, pS6RP and pIRS‐1 (Ser639), and increased pTSC2 (Ser939), and pSMAD2‐3. OTR increased pS6RP, 4E‐binding protein‐1 (p4E‐BP1), pTSC2 (Ser939), and pSMAD2‐3, and decreased pIRS‐1 (Ser639). In summary, OTR/down inhibited the skeletal muscle hypertrophy with concomitant signs of atrophy in EDL. The effects of OTR/up and OTR depended on the analyzed skeletal muscle type. J. Cell. Physiol. 231: 1045–1056, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-29T01:53:52.660177-05:
      DOI: 10.1002/jcp.25197
  • Genes in the GABA Pathway Increase in the Lateral Thalamus of
           Sprague–Dawley Rats During the Proestrus/Estrus Phase
    • Authors: Mikhail Umorin; Crystal Stinson, Larry L. Bellinger, Phillip R. Kramer
      First page: 1057
      Abstract: Pain can vary over the estrous cycle as a result of changes in estradiol concentration but the mechanism causing this variation is unclear. Because the thalamus is important in pain control, gene expression in the lateral thalamus (ventral posteromedial, ventral posterolateral, reticular thalamic nuclei) was screened at different phases of the estrous cycle. Gene expression changes in Sprague–Dawley rats were further analyzed by real‐time PCR and ELISA and plasma estradiol levels were measured by RIAs at different phases of the estrous cycle. Our results indicated that both the RNA and protein expression of glutamate decarboxylase 1 and 2 (GAD1, GAD2), GABA(A) receptor‐associated protein like 1 (GABARAPL1), and vesicular GABA transporter (VGAT) significantly increased in the lateral thalamus when plasma estradiol levels were elevated. Estradiol levels were elevated during the proestrus and estrus phases of the estrous cycle. Estrogen receptor α (ERα) was observed to be co‐localized in thalamic cells and thalamic infusion of an ERα antagonist significantly reduced GAD1 and VGAT transcript. GAD1, GAD2, GABARAPL1, and VGAT have been shown to effect neuronal responses suggesting that attenuation of pain during the estrous cycle can be dependent, in part, through estradiol induced changes in thalamic gene expression. J. Cell. Physiol. 231: 1057–1064, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-30T02:18:29.573669-05:
      DOI: 10.1002/jcp.25198
  • Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently
           Generated From Hemogenic Endothelial Cells in Human Pluripotent Stem Cells
    • Authors: Hao Bai; Yanfeng Liu, Yinliang Xie, Dixie L. Hoyle, Robert A. Brodsky, Linzhao Cheng, Tao Cheng, Zack Z. Wang
      First page: 1065
      Abstract: Generation of fully functional hematopoietic multipotent progenitor (MPP) cells from human pluripotent stem cells (hPSCs) has a great therapeutic potential to provide an unlimited cell source for treatment of hematological disorders. We previously demonstrated that CD34+CD31+CD144+ population derived from hPSCs contain hemato‐endothelial progenitors (HEPs) that give rise to hematopoietic and endothelial cells. Here, we report a differentiation system to generate definitive hematopoietic MPP cells from HEPs via endothelial monolayer. In the presence of angiogenic factors, HEPs formed an endothelial monolayer, from which hematopoietic clusters emerged through the process of endothelial‐to‐hematopoietic transition (EHT). EHT was significantly enhanced by hematopoietic growth factors. The definitive MPP cells generated from endothelial monolayer were capable of forming multilineage hematopoietic colonies, giving rise to T lymphoid cells, and differentiating into enucleated erythrocytes. Emergence of hematopoietic cells from endothelial monolayer occurred transiently. Hematopoietic potential was lost during prolonged culture of HEPs in endothelial growth conditions. Our study demonstrated that CD34+CD31+CD144+ HEPs gave rise to hematopoietic MPP cells via hemogenic endothelial cells that exist transiently. The established differentiation system provides a platform for future investigation of regulatory factors involved in de novo generation of hematopoietic MPP cells and their applications in transplantation. J. Cell. Physiol. 231: 1065–1076, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-10-14T03:19:33.314485-05:
      DOI: 10.1002/jcp.25199
  • Evidences of Biological Functions of Biliverdin Reductase A in the Bovine
    • First page: 1077
      Abstract: Epididymal sperm binding protein 1 (ELSPBP1) is secreted by the epididymal epithelium via epididymosomes and is specifically transferred to dead spermatozoa during epididymal transit. We identified biliverdin reductase A (BLVRA) as a partner of ELSPBP1 by immunoprecipitation followed by tandem mass spectrometry. Pull down assays showed that these two proteins interact in the presence of zinc ions. The BLVRA enzyme is known to convert biliverdin to bilirubin, both of which possess antioxidant activity. Assessment by real‐time RT‐PCR showed that BLVRA is highly expressed in the caput and the corpus epididymis, but is expressed at lower levels in the testis and the cauda epididymis. It is primarily found in the soluble fraction of the caput epididymal fluid, is barely detectable in the cauda fluid, and is detectable to a lesser extent in the epididymosome fraction of both caput and cauda fluids. Immunocytometry on epididymal sperm showed that BLVRA is found on all sperm recovered from the caput region, whereas it is undetectable on cauda sperm. Biliverdin and bilirubin are found in higher concentrations in the caput epididymal fluid, as measured by mass spectrometry. Lipid peroxidation was limited by 1 μM of biliverdin, but not bilirubin when caput spermatozoa were challenged with 500 μM H2O2. Since immature spermatozoa are a source of reactive oxygen species, BLVRA may be involved in the protection of maturing spermatozoa. It is also plausible that BLVRA is implicated in haemic protein catabolism in the epididymal luminal environment. J. Cell. Physiol. 231: 1077–1089, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-09-30T02:18:00.222198-05:
      DOI: 10.1002/jcp.25200
  • Epigenetic Modulation in Periodontitis: Interaction of Adiponectin and
           JMJD3‐IRF4 Axis in Macrophages
    • Authors: Dongying Xuan; Qianqian Han, Qisheng Tu, Lan Zhang, Liming Yu, Dana Murry, Tianchi Tu, Yin Tang, Jane B. Lian, Gary S. Stein, Paloma Valverde, Jincai Zhang, Jake Chen
      First page: 1090
      Abstract: Emerging evidence suggests an important role for epigenetic mechanisms in modulating signals during macrophage polarization and inflammation. JMJD3, a JmjC family histone demethylase necessary for M2 polarization is also required for effective induction of multiple M1 genes by lipopolysaccharide (LPS). However, the effects of JMJD3 to inflammation in the context of obesity remains unknown. To address this deficiency, we firstly examined the expression of JMJD3 in macrophage isolated from bone marrow and adipose tissue of diet induced obesity (DIO) mice. The results indicated that JMJD3 was down‐regulated in obesity. Adiponectin (APN), a factor secreted by adipose tissue which is down‐regulated in obesity, functions to switch macrophage polarization from M1 to M2, thereby attenuating chronic inflammation. Intriguingly, our results indicated that APN contributed to JMJD3 up‐regulation, reduced macrophage infiltration in obese adipose tissue, and abolished the up‐regulation of JMJD3 in peritoneal macrophages isolated from DIO mice when challenged with Porphyromonas gingivalis LPS (pg.lps). To elucidate the interaction of APN and JMJD3 involved in macrophage transformation in the context of inflammation, we designed the loss and gain‐function experiments of APN in vivo with APN−/− mice with experimental periodontitis and in vitro with macrophage isolated from APN−/− mice. For the first time, we found that APN can help to reduce periodontitis‐related bone loss, modulate JMJD3 and IRF4 expression, and macrophage infiltration. Therefore, it can be inferred that APN may contribute to anti‐inflammation macrophage polarization by regulating JMJD3 expression, which provides a basis for macrophage‐centered epigenetic therapeutic strategies. J. Cell. Physiol. 231: 1090–1096, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-10-06T05:17:20.57319-05:0
      DOI: 10.1002/jcp.25201
  • Involvement of Notch‐1 in Resistance to Regorafenib in Colon Cancer
    • Authors: Giovanna Mirone; Stefania Perna, Arvind Shukla, Gabriella Marfe
      First page: 1097
      Abstract: Regorafenib, an oral small‐molecule multi kinase inhibitor, is able to block Vascular Endothelial Growth Factor Receptors (VEGFR‐1, 2, and 3), Platelet‐Derived Growth Factor Receptors (PDGF), Fibroblast Growth Factor (FGF) receptor 1, Raf, TIE‐2, and the kinases KIT, RET, and BRAF. Different studies have displayed its antitumor activity in several cancer models (both in vitro and in vivo), particularly in colorectal and gastrointestinal stromal cancers. The mechanism of resistance to regorafenib is largely unknown. In our investigation, we have generated regorafenib‐resistant SW480 cells (Reg‐R‐SW480 cells) by culturing such cells with increasing concentration of regorafenib. Examination of intracellular signaling found that Akt signaling was activated in Reg‐R‐SW480 cells but not in wild–type SW480 cells, after regorafenib treatment as measured by Western Blot. The Notch pathway is a fundamental signaling system in the development and homeostasis of tissues since it regulates different cellular process such as proliferation, differentiation, and apoptosis and it can be a potential driver of resistance to a wide array of targeted therapies. In this study, we found that Notch‐1 was significantly up‐regulated in resistant tumor cells as well as HES1 and HEY. Additionally, inhibition of Notch‐1 in resistant cells partially restored sensitivity to regorafenib treatment in vitro. Collectively, these data suggest a key role of Notch‐1 in mediating the resistant effects of regorafenib in colorectal cancer cells, and also provide a rationale to improve the therapeutic efficacy of regorafenib. J. Cell. Physiol. 231: 1097–1105, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-10-08T00:34:46.996293-05:
      DOI: 10.1002/jcp.25206
  • The Nox1/4 Dual Inhibitor GKT137831 or Nox4 Knockdown Inhibits
           Angiotensin‐II‐Induced Adult Mouse Cardiac Fibroblast
           Proliferation and Migration. AT1 Physically Associates With Nox4
    • Authors: Naveen K. Somanna; Anthony J. Valente, Maike Krenz, William P. Fay, Patrice Delafontaine, Bysani Chandrasekar
      First page: 1130
      Abstract: Both oxidative stress and inflammation contribute to chronic hypertension‐induced myocardial fibrosis and adverse cardiac remodeling. Here we investigated whether angiotensin (Ang)‐II‐induced fibroblast proliferation and migration are NADPH oxidase (Nox) 4/ROS and IL‐18 dependent. Our results show that the potent induction of mouse cardiac fibroblast (CF) proliferation and migration by Ang‐II is markedly attenuated by Nox4 knockdown and the Nox inhibitor DPI. Further, Nox4 knockdown and DPI pre‐treatment attenuated Ang‐II‐induced IL‐18, IL‐18Rα and collagen expression, and MMP9 and LOX activation. While neutralization of IL‐18 blunted Ang‐II‐induced CF proliferation and migration, knockdown of MMP9 attenuated CF migration. The antioxidant NAC and the cell‐permeable SOD mimetics Tempol, MnTBAP, and MnTMPyP attenuated oxidative stress and inhibited CF proliferation and migration. The Nox1/Nox4 dual inhibitor GKT137831 also blunted Ang‐II‐induced H2O2 production and CF proliferation and migration. Further, AT1 bound Nox4, and Ang‐II enhanced their physical association. Notably, GKT137831 attnuated the AT1/Nox4 interaction. These results indicate that Ang‐II induces CF proliferation and migration in part via Nox4/ROS‐dependent IL‐18 induction and MMP9 activation, and may involve AT1/Nox4 physical association. Thus, either (i) neutralizing IL‐18, (ii) blocking AT1/Nox4 interaction or (iii) use of the Nox1/Nox4 inhibitor GKT137831 may have therapeutic potential in chronic hypertension‐induced adverse cardiac remodeling. J. Cell. Physiol. 231: 1130–1141, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-10-19T04:07:39.502506-05:
      DOI: 10.1002/jcp.25210
  • Appl1 and Appl2 are Expendable for Mouse Development But Are Essential for
           HGF‐Induced Akt Activation and Migration in Mouse Embryonic
    • Authors: Yinfei Tan; Xiaoban Xin, Francis J. Coffey, David L. Wiest, Lily Q. Dong, Joseph R. Testa
      First page: 1142
      Abstract: Although Appl1 and Appl2 have been implicated in multiple cellular activities, we and others have found that Appl1 is dispensable for mouse embryonic development, suggesting that Appl2 can substitute for Appl1 during development. To address this possibility, we generated conditionally targeted Appl2 mice. We found that ubiquitous Appl2 knockout (Appl2−/−) mice, much like Appl1−/− mice, are viable and grow normally to adulthood. Intriguingly, when Appl1−/− mice were crossed with Appl2−/− mice, we found that homozygous Appl1;Appl2 double knockout (DKO) animals are also viable and grossly normal with regard to reproductive potential and postnatal growth. Appl2‐null and DKO mice were found to exhibit altered red blood cell physiology, with erythrocytes from these mice generally being larger and having a more irregular shape than erythrocytes from wild type mice. Although Appl1/2 proteins have been previously shown to have a very strong interaction with phosphatidylinositol‐3 kinase (Pi3k) in thymic T cells, Pi3k‐Akt signaling and cellular differentiation was unaltered in thymocytes from Appl1;Appl2 (DKO) mice. However, Appl1/2‐null mouse embryonic fibroblasts exhibited defects in HGF‐induced Akt activation, migration, and invasion. Taken together, these data suggest that Appl1 and Appl2 are required for robust HGF cell signaling but are dispensable for embryonic development and reproduction. J. Cell. Physiol. 231: 1142–1150, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-10-21T04:01:52.596544-05:
      DOI: 10.1002/jcp.25211
  • Calcineurin‐NFAT Signaling Controls Somatic Cell Reprogramming in a
           Stage‐Dependent Manner
    • Authors: Ming Sun; Bing Liao, Yu Tao, Hao Chen, Feng Xiao, Junjie Gu, Shaorong Gao, Ying Jin
      First page: 1151
      Abstract: Calcineurin‐NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin‐NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin‐NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin‐NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin‐NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1+ intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin‐NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming. J. Cell. Physiol. 231: 1151–1162, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-10-20T04:39:46.312467-05:
      DOI: 10.1002/jcp.25212
  • Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing
           Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells
    • First page: 1171
      Abstract: Acetylcholine (ACh) protected against cardiac injury via promoting autophagy and mitochondrial biogenesis, however, the involvement of mitophagy in ACh‐elicited cardioprotection remains unknown. In the present study, H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) and ACh treatment during reoxygenation. Mitophagy markers PTEN‐induced kinase 1 (PINK1) and Parkin translocation were examined using western blot and confocal fluorescence microscopy. Mitochondrial membrane potential and reactive oxygen species (ROS) were detected with fluorescence staining. We found that H/R‐treated cells exhibited reduced levels of PINK1 and Parkin in mitochondria, accompanied with decreased autophagy flux (reduced LC3‐II/LC3‐I and increased p62). Conversely, ACh increased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. Confocal imaging of Parkin and MitoTracker Green‐labeled mitochondria further confirmed ACh‐induced mitochondrial translocation of Parkin, which was reversed by M2 receptor antagonist methoctramine and M2 receptor siRNA, suggesting ACh could induce mitophagy by M2 receptor after H/R. Mitophagy inhibitor 3‐methaladenine abolished ACh‐induced mitoprotection, manifesting as aggravated mitochondrial morphology disruption, ATP and membrane potential depletion, increased ROS overproduction, and apoptosis. Furthermore, PINK1/Parkin siRNA attenuated the protective effects of ACh against ATP loss and oxidative stress due to mitochondrial‐dependent injury. Taken together, ACh promoted mitochondrial translocation of PINK1/Parkin to stimulate cytoprotective mitophagy via M2 receptor, which may provide beneficial targets in the preservation of cardiac homeostasis against H/R injury. J. Cell. Physiol. 231: 1171–1181, 2016. © 2015 Wiley Periodicals, Inc.
      PubDate: 2015-10-23T03:05:41.843665-05:
      DOI: 10.1002/jcp.25215
School of Mathematical and Computer Sciences
Heriot-Watt University
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