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Sample records for junction protein connexin-43

  1. Gap junction protein connexin-43 interacts directly with microtubules

    NARCIS (Netherlands)

    Giepmans, B N; Verlaan, I; Hengeveld, T; Janssen, H; Calafat, J; Falk, M M; Moolenaar, W H

    2001-01-01

    Gap junctions are specialized cell-cell junctions that mediate intercellular communication. They are composed of connexin proteins, which form transmembrane channels for small molecules [1, 2]. The C-terminal tail of connexin-43 (Cx43), the most widely expressed connexin member, has been implicated

  2. Gap junction protein connexin43 exacerbates lung vascular permeability.

    Directory of Open Access Journals (Sweden)

    James J O'Donnell

    Full Text Available Increased vascular permeability causes pulmonary edema that impairs arterial oxygenation and thus contributes to morbidity and mortality associated with Acute Respiratory Distress Syndrome and sepsis. Although components of intercellular adhesive and tight junctions are critical for maintaining the endothelial barrier, there has been limited study of the roles of gap junctions and their component proteins (connexins. Since connexins can modulate inflammatory signaling in other systems, we hypothesized that connexins may also regulate pulmonary endothelial permeability. The relationships between connexins and the permeability response to inflammatory stimuli were studied in cultured human pulmonary endothelial cells. Prolonged treatment with thrombin, lipopolysaccharide, or pathological cyclic stretch increased levels of mRNA and protein for the major connexin, connexin43 (Cx43. Thrombin and lipopolysaccharide both increased intercellular communication assayed by transfer of microinjected Lucifer yellow. Although thrombin decreased transendothelial resistance in these cells, the response was attenuated by pretreatment with the connexin inhibitor carbenoxolone. Additionally, the decreases of transendothelial resistance produced by either thrombin or lipopolysaccharide were attenuated by reducing Cx43 expression by siRNA knockdown. Both carbenoxolone and Cx43 knockdown also abrogated thrombin-induced phosphorylation of myosin light chain. Taken together, these data suggest that increased lung vascular permeability induced by inflammatory conditions may be amplified via increased expression of Cx43 and intercellular communication among pulmonary endothelial cells.

  3. Expression of gap junction protein connexin 43 in bovine urinary bladder tumours.

    Science.gov (United States)

    Corteggio, A; Florio, J; Roperto, F; Borzacchiello, G

    2011-01-01

    The aetiopathogenesis of urinary bladder tumours in cattle involves prolonged ingestion of bracken fern and infection by bovine papillomavirus types 1 or 2 (BPV-1/2). The oncogenic activity of BPV is largely associated with the major oncoprotein E5. Gap junctions are the only communicating junctions found in animal tissues and are composed of proteins known as connexins. Alterations in connexin expression have been associated with oncogenesis. The present study investigated biochemically and immunohistochemically the expression of connexin 43 in samples of normal (n=2), dysplastic (n=3) and neoplastic (n=23) bovine urothelium. The tumours included 10 carcinomas in situ, five papillary urothelial carcinomas and eight invasive urothelial carcinomas. Normal and dysplastic urothelium had membrane expression of connexin 43, but this was reduced in samples of carcinoma in situ. Papillary urothelial carcinomas showed moderate cytoplasmic and membrane labelling, while invasive carcinoma showed loss of connexin 43 expression. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. Role of connexin43-interacting proteins at gap junctions

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2006-01-01

    Gap junctions are arrays of cell-to-cell channels that allow diffusion of small molecules between neighboring cells. The individual channels are formed by the four-transmembrane connexin (Cx) proteins. Recently, multiple proteins have been found to interact at the cytoplasmic site with the most abun

  5. Role of connexin43-interacting proteins at gap junctions

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2006-01-01

    Gap junctions are arrays of cell-to-cell channels that allow diffusion of small molecules between neighboring cells. The individual channels are formed by the four-transmembrane connexin (Cx) proteins. Recently, multiple proteins have been found to interact at the cytoplasmic site with the most

  6. The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein

    NARCIS (Netherlands)

    Giepmans, B N; Moolenaar, W H

    1998-01-01

    Gap junctions mediate cell-cell communication in almost all tissues and are composed of channel-forming integral membrane proteins, termed connexins [1-3]. Connexin43 (Cx43) is the most widely expressed and the most well-studied member of this family. Cx43-based cell-cell communication is regulated

  7. Dynamic changes of connexin-43, gap junctional protein, in outer layers of cumulus cells are regulated by PKC and PI 3-kinase during meiotic resumption in porcine oocytes.

    Science.gov (United States)

    Shimada, M; Maeda, T; Terada, T

    2001-04-01

    Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.

  8. Effects of Angiotensin Ⅱ on Expression of the Gap Junction Channel Protein Connexin 43 in Neonatal Rat Ventricular Myocytes

    Institute of Scientific and Technical Information of China (English)

    Jun Yang; Wei Wu

    2007-01-01

    To study the effects of angiotensin Ⅱ,as a mediator of cardiac hypertrophy,on expression of connexin 43 (Cx43) in cultured neonatal rat ventricular myocytes and correlation of expression of Cx43 and cardiomyocyte hypertrophy.Methods Cardiomyocytes were isolated from newborn SD rats.Angiotensin Ⅱ was added into the media to induce myocyte hypertrophy.Cultures were exposed to 10 ~6 mol/L angiotensin Ⅱ for 72 h,Cx43 expression was characterized by RT-PCR and Immunofluorescence methods.Results Immunofluorescence analysis revealed decreased Cx43 immunoreactivity in cells treated for 72 h with angiotensin Ⅱ.RT-PCR analysis demonstrated there was an obvious decrease of Cx43 mRNA level in cells exposed to angiotensin Ⅱ for 72 h.The changes of expression of connexin 43 were related to its entrance into S phase of the cell cycle.Cultured neonatal rat cardiomyocytes were exposed for 72 h to increase concentrations of angiotensin Ⅱ ( 1.0 × 10-9 ~ 1.0 × 10-6mol/L),resulting in significantly decreased Cx43 expression.Conclusions Angiotensin Ⅱ leads to a concentration-dependent decrease in Cx43 protein in cultured neonatal rat ventricular myocytes by decreasing Cx43 mRNA synthesis.Signal transduction pathways activated by angiotensin Ⅱ under pathophysiologic conditions of cardiac hypertrophy could initiate remodeling of gap junctions.

  9. Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of cell-cell communication

    NARCIS (Netherlands)

    Giepmans, B N; Hengeveld, T; Postma, F R; Moolenaar, W H

    2001-01-01

    Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junct

  10. Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Yi XIA; Kai-zheng GONG; Ming XU; You-yi ZHANG; Ji-hong GUO; Yao SONG; Ping ZHANG

    2009-01-01

    Aim:β-adrenergic receptor (β-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties.Connexin 43 (Cx43),the predominant gap-junction protein in the heart,has an indispensable role in modulating cardiac electric activities by affecting gap-junction function.The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function.Methods:The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay.The GJIC function was evaluated by scrape loading/dye transfer assay.Results:Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level,but not total Cx43.Selective β2-AR inhibitor ICI 118551,but not β-AR inhibitor CGP20712,could fully abolish the effect.Moreover,pretreatment with both protein kinase A inhibitor H89 and G,protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression.Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.Conclusion:Taken together,β2-AR stimulation increases the expression of nonphosphorylated Cx43,thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A-and G1-dependent manner.

  11. Rescue of perfluorooctanesulfonate (PFOS)-mediated Sertoli cell injury by overexpression of gap junction protein connexin 43

    Science.gov (United States)

    Li, Nan; Mruk, Dolores D.; Chen, Haiqi; Wong, Chris K. C.; Lee, Will M.; Cheng, C. Yan

    2016-07-01

    Perfluorooctanesulfonate (PFOS) is an environmental toxicant used in developing countries, including China, as a stain repellent for clothing, carpets and draperies, but it has been banned in the U.S. and Canada since the late 2000s. PFOS perturbed the Sertoli cell tight junction (TJ)-permeability barrier, causing disruption of actin microfilaments in cell cytosol, perturbing the localization of cell junction proteins (e.g., occluden-ZO-1, N-cadherin-ß-catenin). These changes destabilized Sertoli cell blood-testis barrier (BTB) integrity. These findings suggest that human exposure to PFOS might induce BTB dysfunction and infertility. Interestingly, PFOS-induced Sertoli cell injury associated with a down-regulation of the gap junction (GJ) protein connexin43 (Cx43). We next investigated if overexpression of Cx43 in Sertoli cells could rescue the PFOS-induced cell injury. Indeed, overexpression of Cx43 in Sertoli cells with an established TJ-barrier blocked the disruption in PFOS-induced GJ-intercellular communication, resulting in the re-organization of actin microfilaments, which rendered them similar to those in control cells. Furthermore, cell adhesion proteins that utilized F-actin for attachment became properly distributed at the cell-cell interface, resealing the disrupted TJ-barrier. In summary, Cx43 is a good target that might be used to manage PFOS-induced reproductive dysfunction.

  12. Association of connexin43 with a receptor protein tyrosine phosphatase

    NARCIS (Netherlands)

    Giepmans, Ben N G; Feiken, Elles; Gebbink, Martijn F B G; Moolenaar, Wouter H

    2003-01-01

    Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap juncti

  13. Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.

    Science.gov (United States)

    Huang, Yu-Fang; Liao, Chih-Kai; Lin, Jau-Chen; Jow, Guey-Mei; Wang, Hwai-Shi; Wu, Jiahn-Chun

    2013-03-01

    Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS.

  14. Ischaemia-induced autophagy leads to degradation of gap junction protein connexin43 in cardiomyocytes.

    Science.gov (United States)

    Martins-Marques, Tania; Catarino, Steve; Zuzarte, Monica; Marques, Carla; Matafome, Paulo; Pereira, Paulo; Girão, Henrique

    2015-04-15

    GJIC (gap junction intercellular communication) between cardiomyocytes is essential for synchronous heart contraction and relies on Cx (connexin)-containing channels. Increased breakdown of Cx43 has been often associated with various cardiac diseases. However, the mechanisms whereby Cx43 is degraded in ischaemic heart remain unknown. The results obtained in the present study, using both HL-1 cells and organotypic heart cultures, show that simulated ischaemia induces degradation of Cx43 that can be prevented by chemical or genetic inhibitors of autophagy. Additionally, ischaemia-induced degradation of Cx43 results in GJIC impairment in HL-1 cells, which can be restored by autophagy inhibition. In cardiomyocytes, ubiquitin signals Cx43 for autophagic degradation, through the recruitment of the ubiquitin-binding proteins Eps15 (epidermal growth factor receptor substrate 15) and p62, that assist in Cx43 internalization and targeting to autophagic vesicles, via LC3 (light chain 3). Moreover, we establish that degradation of Cx43 in ischaemia or I/R (ischaemia/reperfusion) relies upon different molecular players. Indeed, degradation of Cx43 during early periods of ischaemia depends on AMPK (AMP-activated protein kinase), whereas in late periods of ischaemia and I/R Beclin 1 is required. In the Langendorff-perfused heart, Cx43 is dephosphorylated in ischaemia and degraded during I/R, where Cx43 degradation correlates with autophagy activation. In summary, the results of the present study provide new evidence regarding the molecular mechanisms whereby Cx43 is degraded in ischaemia, which may contribute to the development of new strategies that aim to preserve GJIC and cardiac function in ischaemic heart.

  15. Protein kinase C-dependent regulation of connexin43 gap junctions and hemichannels

    DEFF Research Database (Denmark)

    Alstrøm, Jette Skov; Stroemlund, Line Waring; Nielsen, Morten Schak

    2015-01-01

    and allow transport of molecules such as fluorescent dyes and ATP. A range of phosphorylated amino acids have been detected in the C-terminus of Cx43 and their physiological role has been intensively studied both in the gap junctional form of Cx43 and in its hemichannel configuration. We present the current...

  16. The connexin43 carboxyl terminus and cardiac gap junction organization.

    Science.gov (United States)

    Palatinus, Joseph A; Rhett, J Matthew; Gourdie, Robert G

    2012-08-01

    The precise spatial order of gap junctions at intercalated disks in adult ventricular myocardium is thought vital for maintaining cardiac synchrony. Breakdown or remodeling of this order is a hallmark of arrhythmic disease of the heart. The principal component of gap junction channels between ventricular cardiomyocytes is connexin43 (Cx43). Protein-protein interactions and modifications of the carboxyl-terminus of Cx43 are key determinants of gap junction function, size, distribution and organization during normal development and in disease processes. Here, we review data on the role of proteins interacting with the Cx43 carboxyl-terminus in the regulation of cardiac gap junction organization, with particular emphasis on Zonula Occludens-1. The rapid progress in this area suggests that in coming years we are likely to develop a fuller understanding of the molecular mechanisms causing pathologic remodeling of gap junctions. With these advances come the promise of novel approach to the treatment of arrhythmia and the prevention of sudden cardiac death. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Regulation of connexin43 gap junctional communication by phosphatidylinositol 4,5-bisphosphate

    NARCIS (Netherlands)

    van Zeijl, Leonie; Ponsioen, Bas; Giepmans, Ben N G; Ariaens, Aafke; Postma, Friso R; Várnai, Péter; Balla, Tamas; Divecha, Nullin; Jalink, Kees; Moolenaar, Wouter H

    2007-01-01

    Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosp

  18. Nkx2.5 homeoprotein regulates expression of gap junction protein connexin 43 and sarcomere organization in postnatal cardiomyocytes.

    Science.gov (United States)

    Kasahara, Hideko; Ueyama, Tomomi; Wakimoto, Hiroko; Liu, Margaret K; Maguire, Colin T; Converso, Kimber L; Kang, Peter M; Manning, Warren J; Lawitts, Joel; Paul, David L; Berul, Charles I; Izumo, Seigo

    2003-03-01

    Nkx2.5, an evolutionarily conserved homeodomain containing transcription factor, is one of the earliest cardiogenic markers. Although its expression continues through adulthood, its function in adult cardiomyocytes is not well understood. To examine the effect of Nkx2.5 in terminal differentiated postnatal cardiomyocytes, we generated transgenic mice expressing either wild-type Nkx2.5 (TG-wild), a putative transcriptionally active mutant (carboxyl-terminus deletion mutant: TG-DeltaC) or a DNA non-binding point mutant of Nkx2.5 (TG-I183P) under alpha-myosin heavy chain promoter. Most TG-wild and TG-DeltaC mice died before 4 months of age with heart failure associated with conduction abnormalities. Cardiomyocytes expressing wild-type Nkx2.5 or a putative transcriptionally active mutant (DeltaC) had dramatically reduced expression of connexin 43 and changed sarcomere structure. Wild-type Nkx2.5 adenovirus-infected adult cardiomyocytes demonstrated connexin 43 downregulation as early as 16 h after infection, indicating that connexin 43 downregulation is due to Nkx2.5 overexpression but not due to heart failure phenotype in vivo. These studies indicate that overexpression of Nkx2.5 in terminally differentiated cardiomyocytes dramatically alters cardiac cell structure and function.

  19. Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis.

    Science.gov (United States)

    Khan, Zahidul; Yaiw, Koon-Chu; Wilhelmi, Vanessa; Lam, Hoyin; Rahbar, Afsar; Stragliotto, Giuseppe; Söderberg-Nauclér, Cecilia

    2014-01-01

    A lack of gap junctional intercellular communication (GJIC) is common in cancer. Many oncogenic viruses have been shown to downregulate the junctional protein connexin 43 (Cx43) and reduce GJIC. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific betaherpesvirus that establishes life-long latency after primary infection. It encodes two viral gene products, immediate early (IE) proteins IE1 and IE2, which are crucial in viral replication and pathogenesis of many diseases. Emerging evidence demonstrates that HCMV DNA and proteins are highly prevalent in glioblastoma multiforme (GBM) and in other tumors, but HCMV's role in tumorigenesis remains obscure. In the present study, we examined the effects of HCMV infection on Cx43 expression and GJIC as well as the viral mechanism mediating the effects in human GBM cells and tissue samples. We found that HCMV downregulated Cx43 protein, resulting in disruption of functional GJIC as assayed by fluorescent dye transfer assay. We show that both HCMV-IE72 and IE86 mediate downregulation of Cx43 by silencing RNA targeting either IE72 or IE86 coupled with ganciclovir. This finding was further validated by transfection with expression vectors encoding IE72 or IE86, and we show that viral-mediated Cx43 depletion involved proteasomal degradation. Importantly, we also observed that the Cx43 protein levels and IE staining correlated inversely in 10 human GBM tissue specimens. Thus, HCMV regulates Cx43 expression and GJIC, which may contribute to gliomagenesis.

  20. Connexin43 mutation causes heterogeneous gap junction loss and sudden infant death.

    Science.gov (United States)

    Van Norstrand, David W; Asimaki, Angeliki; Rubinos, Clio; Dolmatova, Elena; Srinivas, Miduturu; Tester, David J; Saffitz, Jeffrey E; Duffy, Heather S; Ackerman, Michael J

    2012-01-24

    An estimated 10% to 15% of sudden infant death syndrome (SIDS) cases may stem from channelopathy-mediated lethal arrhythmias. Loss of the GJA1-encoded gap junction channel protein connexin43 is known to underlie formation of lethal arrhythmias. GJA1 mutations have been associated with cardiac diseases, including atrial fibrillation. Therefore, GJA1 is a plausible candidate gene for premature sudden death. GJA1 open reading frame mutational analysis was performed with polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing on DNA from 292 SIDS cases. Immunofluorescence and dual whole-cell patch-clamp studies were performed to determine the functionality of mutant gap junctions. Immunostaining for gap junction proteins was performed on SIDS-associated paraffin-embedded cardiac tissue. Two rare, novel missense mutations, E42K and S272P, were detected in 2 of 292 SIDS cases, a 2-month-old white boy and a 3-month-old white girl, respectively. Analysis of the E42K victim's parental DNA demonstrated a de novo mutation. Both mutations involved highly conserved residues and were absent in >1000 ethnically matched reference alleles. Immunofluorescence demonstrated no trafficking abnormalities for either mutation, and S272P demonstrated wild-type junctional conductance. However, junctional conductance measurements for the E42K mutation demonstrated a loss of function not rescued by wild type. Moreover, the E42K victim's cardiac tissue demonstrated a mosaic immunostaining pattern for connexin43 protein. This study provides the first molecular and functional evidence implicating a GJA1 mutation as a novel pathogenic substrate for SIDS. E42K-connexin43 demonstrated a trafficking-independent reduction in junctional coupling in vitro and a mosaic pattern of mutational DNA distribution in deceased cardiac tissue, suggesting a novel mechanism of connexin43-associated sudden death.

  1. Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection.

    Science.gov (United States)

    Basu, Rahul; Bose, Abhishek; Thomas, Deepthi; Das Sarma, Jayasri

    2017-09-08

    Gap junctions (GJs) are important for maintenance of CNS homeostasis. GJ proteins, connexin 43 (Cx43) and connexin 47 (Cx47), play a crucial role in production and maintenance of CNS myelin. Cx43 is mainly expressed by astrocytes in the CNS and forms gap junction intercellular communications between astrocytes-astrocytes (Cx43-Cx43) and between astrocytes-oligodendrocytes (Cx43-Cx47). Mutations of these connexin (Cx) proteins cause dysmyelinating diseases in humans. Previously, it has been shown that Cx43 localization and expression is altered due to mouse hepatitis virus (MHV)-A59 infection both in vivo and in vitro; however, its mechanism and association with loss of myelin protein was not elaborated. Thus, we explored potential mechanisms by which MHV-A59 infection alters Cx43 localization and examined the effects of viral infection on Cx47 expression and its association with loss of the myelin marker proteolipid protein. Immunofluorescence and total internal reflection fluorescence microscopy confirmed that MHV-A59 used microtubules (MTs) as a conduit to reach the cell surface and restricted MT-mediated Cx43 delivery to the cell membrane. Co-immunoprecipitation experiments demonstrated that Cx43-β-tubulin molecular interaction was depleted due to protein-protein interaction between viral particles and MTs. During acute MHV-A59 infection, oligodendrocytic Cx47, which is mainly stabilized by Cx43 in vivo, was down-regulated, and its characteristic staining remained disrupted even at chronic phase. The loss of Cx47 was associated with loss of proteolipid protein at the chronic stage of MHV-A59 infection. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Immunohistochemical localization of a gap junction protein (connexin43) in the muscularis externa of murine, canine, and human intestine

    DEFF Research Database (Denmark)

    Mikkelsen, H B; Huizinga, J D; Thuneberg, L

    1993-01-01

    Electron-microscopic studies have revealed a heterogeneous distribution of gap junctions in the muscularis externa of mammalian intestines. This heterogeneity is observed at four different levels: among species; between small and large intestines; between longitudinal and circular muscle layers; ...

  3. Effect of thioridazine on gap junction intercellular communication in connexin 43-expressing cells.

    Science.gov (United States)

    Matesic, D F; Abifadel, D N; Garcia, E L; Jann, M W

    2006-07-01

    Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 micromol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a "hyperphosphorylated" connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in

  4. Gap junction signalling mediated through connexin-43 is required for chick limb development.

    Science.gov (United States)

    Makarenkova, H; Patel, K

    1999-03-15

    During chick limb development the gap junction protein Connexin-43 (Cx43) is expressed in discrete spatially restricted domains in the apical ectodermal ridge (AER) and mesenchyme of the zone of polarising activity. Antisense oligonucleotides (ODNs) were used to investigate the role of Connexin-43 (Cx43) in the development of the chick limb bud. We have used unmodified ODNs in Pluronic F-127 gel, which is liquid at low temperature but sets at room temperature and so remains situated at the point of application. As a mild surfactant, the gel increases antisense ODN penetration and supplies ODNs to the embryo continually for 12-18 h. We have shown a strong decrease in Cx43 protein expression after application of specific antisense oligonucleotides but the abundance of a closely related protein, Connexin-32 (Cx32), was not affected. Application of antisense Cx43 ODNs at stages 8-15 HH before limb outgrowth resulted in dramatic limb phenotypes. About 40% of treated embryos exhibited defects such as truncation of the limb bud, fragmentation into two or more domains, or complete splitting of the limb bud into two or three branches. Molecular analysis of antisense treated embryos failed to detect Shh or Bmp-2 in anterior structures and suggested that extra lobes seen in nicked and split limbs were not a result of establishment of new signalling centres as found after the application of FGF to the flank. However, examination of markers for the AER showed a number of abnormalities. In severely truncated specimens we were unable to detect the expression of either Fgf-4 or Fgf-8. In both nicked and split limbs the expression of these genes was discontinuous. Down-regulation of Cx43 after the antisense application could be comparable to AER removal and results in distal truncation of the limb bud. Taken together these data suggest the existence of a feedback loop between the FGFs and signalling mediated by Cx43.

  5. Classical swine fever virus down-regulates endothelial connexin 43 gap junctions.

    Science.gov (United States)

    Hsiao, Hsiang-Jung; Liu, Pei-An; Yeh, Hung-I; Wang, Chi-Young

    2010-07-01

    Classical swine fever is a contagious disease of pigs characterized by fatal hemorrhagic fever. Classical swine fever virus (CSFV) induces the expression of pro-inflammatory and pro-coagulant factors of vascular endothelial cells and establishes a long-term infection. This study aimed to understand the effect of CSFV on endothelial connexin 43 (Cx43) expression and gap junctional intercellular coupling (GJIC). Porcine aortic endothelial cells were infected with CSFV at different multiplicity of infection for 48 h. Semi-quantitative RT-PCR, immunoconfocal microscopy, and Western blotting showed that the transcription and translation of Cx43 were reduced, and this was associated with an attenuation of GJIC. This decrease occurred in a time-dependent manner. An ERK inhibitor (PD98059), a JNK inhibitor (SP600125), and proteasome/lysosome inhibitors all significantly reversed the reduction in Cx43 protein levels without any influence on the titer of progeny virus. In addition, CSFV activated ERK and JNK in a time-dependent manner and down-regulated Cx43 promoter activity, mainly through decreased AP2 binding. This effect was primarily caused by the replication of CSFV rather than a consequence of cytokines being induced by CSFV infection of endothelial cells.

  6. Interacting Network of the Gap Junction (GJ) Protein Connexin43 (Cx43) is Modulated by Ischemia and Reperfusion in the Heart.

    Science.gov (United States)

    Martins-Marques, Tania; Anjo, Sandra Isabel; Pereira, Paulo; Manadas, Bruno; Girão, Henrique

    2015-11-01

    The coordinated and synchronized cardiac muscle contraction relies on an efficient gap junction-mediated intercellular communication (GJIC) between cardiomyocytes, which involves the rapid anisotropic impulse propagation through connexin (Cx)-containing channels, namely of Cx43, the most abundant Cx in the heart. Expectedly, disturbing mechanisms that affect channel activity, localization and turnover of Cx43 have been implicated in several cardiomyopathies, such as myocardial ischemia. Besides gap junction-mediated intercellular communication, Cx43 has been associated with channel-independent functions, including modulation of cell adhesion, differentiation, proliferation and gene transcription. It has been suggested that the role played by Cx43 is dictated by the nature of the proteins that interact with Cx43. Therefore, the characterization of the Cx43-interacting network and its dynamics is vital to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication, but also to unveil novel and unanticipated biological functions of Cx43. In the present report, we applied a quantitative SWATH-MS approach to characterize the Cx43 interactome in rat hearts subjected to ischemia and ischemia-reperfusion. Our results demonstrate that, in the heart, Cx43 interacts with proteins related with various biological processes such as metabolism, signaling and trafficking. The interaction of Cx43 with proteins involved in gene transcription strengthens the emerging concept that Cx43 has a role in gene expression regulation. Importantly, our data shows that the interactome of Cx43 (Connexome) is differentially modulated in diseased hearts. Overall, the characterization of Cx43-interacting network may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions. Data are available via ProteomeXchange with identifier PXD002331.

  7. Connexin43 in retinal injury and disease.

    Science.gov (United States)

    Danesh-Meyer, Helen V; Zhang, Jie; Acosta, Monica L; Rupenthal, Ilva D; Green, Colin R

    2016-03-01

    Gap junctions are specialized cell-to-cell contacts that allow the direct transfer of small molecules between cells. A single gap junction channel consists of two hemichannels, or connexons, each of which is composed of six connexin protein subunits. Connexin43 is the most ubiquitously expressed isoform of the connexin family and in the retina it is prevalent in astrocytes, Müller cells, microglia, retinal pigment epithelium and endothelial cells. Prior to docking with a neighboring cell, Connexin43 hemichannels have a low open probability as open channels constitute a large, relatively non-specific membrane pore. However, with injury and disease Connexin43 upregulation and hemichannel opening has been implicated in all aspects of secondary damage, especially glial cell activation, edema and loss of vascular integrity, leading to neuronal death. We here review gap junctions and their roles in the retina, and then focus in on Connexin43 gap junction channels in injury and disease. In particular, the effect of pathological opening of gap junction hemichannels is described, and hemichannel mediated loss of vascular integrity explained. This latter phenomenon underlies retinal pigment epithelium loss and is a common feature in several retinal diseases. Finally, Connexin43 channel roles in a number of retinal diseases including macular degeneration, glaucoma and diabetic retinopathy are considered, along with results from related animal models. A final section describes gap junction channel modulation and the ocular delivery of potential therapeutic molecules. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Vascular Pericyte Impairment and Connexin43 Gap Junction Deficit Contribute to Vasomotor Decline in Diabetic Retinopathy.

    Science.gov (United States)

    Ivanova, Elena; Kovacs-Oller, Tamas; Sagdullaev, Botir T

    2017-08-09

    Adequate blood flow is essential to brain function, and its disruption is an early indicator in diseases, such as stroke and diabetes. However, the mechanisms contributing to this impairment remain unclear. To address this gap, in the diabetic and nondiabetic male mouse retina, we combined an unbiased longitudinal assessment of vasomotor activity along a genetically defined vascular network with pharmacological and immunohistochemical analyses of pericytes, the capillary vasomotor elements. In nondiabetic retina, focal stimulation of a pericyte produced a robust vasomotor response, which propagated along the blood vessel with increasing stimulus. In contrast, the magnitude, dynamic range, a measure of fine vascular diameter control, and propagation of vasomotor response were diminished in diabetic retinas from streptozotocin-treated mice. These functional changes were linked to several mechanisms. We found that density of pericytes and their sensitivity to stimulation were reduced in diabetes. The impaired response propagation from the stimulation site was associated with lower expression of connexin43, a major known gap junction unit in vascular cells. Indeed, selective block of gap junctions significantly reduced propagation but not initiation of vasomotor response in the nondiabetic retina. Our data establish the mechanisms for fine local regulation of capillary diameter by pericytes and a role for gap junctions in vascular network interactions. We show how disruption of this balance contributes to impaired vasomotor control in diabetes.SIGNIFICANCE STATEMENT Identification of mechanisms governing capillary blood flow in the CNS and how they are altered in disease provides novel insight into early states of neurological dysfunction. Here, we present physiological and anatomical evidence that both intact pericyte function as well as gap junction-mediated signaling across the vascular network are essential for proper capillary diameter control and vasomotor

  9. Gap junctional communication between vascular cells. Induction of connexin43 messenger RNA in macrophage foam cells of atherosclerotic lesions.

    OpenAIRE

    Polacek, D.; Lal, R; Volin, M. V.; Davies, P F

    1993-01-01

    The structure and function of blood vessels depend on the ability of vascular cells to receive and transduce signals and to communicate with each other. One means by which vascular cells have been shown to communicate is via gap junctions, specifically connexin43. In atherosclerosis, the normal physical patterns of communication are disrupted by the subendothelial infiltration and accumulation of blood monocytes, which in turn can differentiate into resident foam cells. In this paper we repor...

  10. Neuroprotection in the treatment of glaucoma--A focus on connexin43 gap junction channel blockers.

    Science.gov (United States)

    Chen, Ying-Shan; Green, Colin R; Danesh-Meyer, Helen V; Rupenthal, Ilva D

    2015-09-01

    Glaucoma is a form of optic neuropathy and a common cause of blindness, affecting over 60 million people worldwide with an expected rise to 80 million by 2020. Successful treatment is challenging due to the various causes of glaucoma, undetectable symptoms at an early stage and inefficient delivery of drugs to the back of the eye. Conventional glaucoma treatments focus on the reduction of elevated intraocular pressure (IOP) using topical eye drops. However, their efficacy is limited to patients who suffer from high IOP glaucoma and do not address the underlying susceptibility of retinal ganglion cells (RGC) to degeneration. Glaucoma is known as a neurodegenerative disease which starts with RGC death and eventually results in damage of the optic nerve. Neuroprotective strategies therefore offer a novel treatment option for glaucoma by not only preventing neuronal loss but also disease progression. This review firstly gives an overview of the pathophysiology of glaucoma as well as current treatment options including conventional and novel delivery strategies. It then summarizes the rational for neuroprotection as a novel therapy for glaucomatous neuropathies and reviews current potential neuroprotective strategies to preserve RGC, with a focus on connexin43 (Cx43) gap junction channel blockers.

  11. TLR2 mediates gap junctional intercellular communication through connexin-43 in intestinal epithelial barrier injury.

    Science.gov (United States)

    Ey, Birgit; Eyking, Annette; Gerken, Guido; Podolsky, Daniel K; Cario, Elke

    2009-08-14

    Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.

  12. Hyperthermia Differently Affects Connexin43 Expression and Gap Junction Permeability in Skeletal Myoblasts and HeLa Cells

    Directory of Open Access Journals (Sweden)

    Ieva Antanavičiūtė

    2014-01-01

    Full Text Available Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK in hyperthermia-induced changes of connexin43 (Cx43 expression and permeability of Cx43 gap junctions (GJs in the rabbit skeletal myoblasts (SkMs and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC. In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

  13. Hyperthermia differently affects connexin43 expression and gap junction permeability in skeletal myoblasts and HeLa cells.

    Science.gov (United States)

    Antanavičiūtė, Ieva; Mildažienė, Vida; Stankevičius, Edgaras; Herdegen, Thomas; Skeberdis, Vytenis Arvydas

    2014-01-01

    Stress kinases can be activated by hyperthermia and modify the expression level and properties of membranous and intercellular channels. We examined the role of c-Jun NH2-terminal kinase (JNK) in hyperthermia-induced changes of connexin43 (Cx43) expression and permeability of Cx43 gap junctions (GJs) in the rabbit skeletal myoblasts (SkMs) and Cx43-EGFP transfected HeLa cells. Hyperthermia (42°C for 6 h) enhanced the activity of JNK and its target, the transcription factor c-Jun, in both SkMs and HeLa cells. In SkMs, hyperthermia caused a 3.2-fold increase in the total Cx43 protein level and enhanced the efficacy of GJ intercellular communication (GJIC). In striking contrast, hyperthermia reduced the total amount of Cx43 protein, the number of Cx43 channels in GJ plaques, the density of hemichannels in the cell membranes, and the efficiency of GJIC in HeLa cells. Both in SkMs and HeLa cells, these changes could be prevented by XG-102, a JNK inhibitor. In HeLa cells, the changes in Cx43 expression and GJIC under hyperthermic conditions were accompanied by JNK-dependent disorganization of actin cytoskeleton stress fibers while in SkMs, the actin cytoskeleton remained intact. These findings provide an attractive model to identify the regulatory players within signalosomes, which determine the cell-dependent outcomes of hyperthermia.

  14. No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning.

    Science.gov (United States)

    Totzeck, Andreas; Boengler, Kerstin; van de Sand, Anita; Konietzka, Ina; Gres, Petra; Garcia-Dorado, David; Heusch, Gerd; Schulz, Rainer

    2008-11-01

    Cardiac connexin 43 (Cx43) is involved in infarct propagation, and the uncoupling of Cx43-formed channels reduces infarct size. Cx43-formed channels open upon Cx43 dephosphorylation, and ischemic preconditioning (IP) prevents the ischemia-induced Cx43 dephosphorylation. In addition to the sarcolemma, Cx43 is also present in the cardiomyocyte mitochondria. We now examined the interaction of Cx43 with protein phosphatases PP1alpha, PP2Aalpha, and PP2Balpha and the role of such interaction for Cx43 phosphorylation in preconditioned myocardium. Infarct size (in %area at risk) in left ventricular anterior myocardium of Göttinger minipigs subjected to 90 min of low-flow ischemia and 120 min of reperfusion was 23.1 +/- 2.7 [n = 7, nonpreconditioned (NIP) group] and was reduced by IP to 10.0 +/- 3.2 (n = 6, P < 0.05). Mitochondrial and gap junctional Cx43 dephosphorylation increased after 85 min of ischemia in NIP myocardium, whereas Cx43 phosphorylation was preserved with IP. PP2Aalpha and PP1alpha, but not PP2Balpha, were detected by Western blot analysis in the left ventricular myocardium. Cx43 coprecipitated with PP2Aalpha but not with PP1alpha. Although the total PP2Aalpha immunoreactivity (confocal laser scan) was increased to 154 +/- 24% and 194 +/- 13% of baseline (P < 0.05) after 85 min of ischemia in NIP and IP myocardium, respectively, the PP2A activities were similar between the groups. The amount of PP2Aalpha coimmunoprecipitated with Cx43 remained unchanged. Only PP2Aalpha coprecipitates with Cx43 in pig myocardium. This interaction is not affected by IP, suggesting that PP2Aalpha is not involved in the prevention of the ischemia-induced Cx43 dephosphorylation by IP.

  15. Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress.

    Science.gov (United States)

    Le, Hoa T; Sin, Wun Chey; Lozinsky, Shannon; Bechberger, John; Vega, José Luis; Guo, Xu Qiu; Sáez, Juan C; Naus, Christian C

    2014-01-17

    Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H2O2) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H2O2 transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H2O2-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H2O2 insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H2O2 treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.

  16. Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    XING Yifei; XIAO Yajun; ZENG FuQing; ZHAO Jun; XIAO Chuanguo; XIONG Ping; FENG Wei

    2007-01-01

    Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis.mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (χ2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.

  17. Effects of all-trans-retinoic acid on the expression and tyrosine phosphorylation of gap junction connexin 43 in HeLa cell line and its significance

    Institute of Scientific and Technical Information of China (English)

    CHEN Bi-liang; MA Xiang-dong; XIN Xiao-yan; WANG De-tang; WANG Chun-mei

    2001-01-01

    Objective: To investigate the signal transduction mechanism of gap junctional genes connexin43 in human cervical carcinogenesis. Methods: Human cervical carcinoma cell line HeLa was cultured and treated by all-trans-retinoic acid (ATRA). Flow cytometer (FCM) was employed to detect expression of Cx43 protein in HeLa cells. Fluo-3 AM loading and laser scanning confocal microscope (LSCM) were used to measure the concentrations of intracellular calcium ([Ca2+]i) in HeLa cells. Phosphorylation on tyrosine of connexin43 protein was examined by immunoblot. Results: The positive rate of Cx43 protein increased from 1.9% in untreated HeLa cells to 26.3% in RA-treated HeLa cells as shown by FCM. [Ca2+]i was 35.73 nmol/L in untreated HeLa cells which was increased to 58.16 nmol/L in ATRA-treated cells.Immunoblot showed that ATRA-treated HeLa cells had phosphorylation on tyrosine in Cx43 protein whereas untreated cells had not. Conclusions: Carcinogenesis of human cervical carcinoma is related with the abnormal expression of cx43gene and disorder of signal transduction manifested as the decrease of [Ca2+]i and post-translation phosphorylation on tyrosine of Cx43 protein. The anti-tumor effect of ATRA in HeLa cells might be due to the up-regulation of cx43 gene and its signal transduction pathway.

  18. Temporal dynamic changes of connexin 43 expression in C6 cells following lipopolysaccharide stimulation

    Institute of Scientific and Technical Information of China (English)

    Ling Liu; Haiyan Liu; Zhenping Gao; Linbo Zhang; Lue Su; Guojun Dong; Haiyang Yu; Jiayi Tian; Hang Zhao; Yanyan Xu

    2012-01-01

    Connexin 43, a gap junction protein, is expressed mainly in glia in the central nervous system.Neuroinflammation plays an important role in central nervous system injury. Changes to glial connexin 43 levels and neuroinflammation may trigger brain injury and neurodegenerative diseases.To illustrate the relationship between connexin 43 and neuroinflammation, this study investigated how connexin 43 expression levels change in lipopolysaccharide-stimulated rat C6 glioma cells. C6 cells were treated with 0.05, 0.25, 0.5, 1, 2.5 and 5 μg/mL lipopolysaccharide for 24 hours. The nitrite estimation-detected nitric oxide release level was elevated substantially after lipopolysaccharide stimulation. To test the transcriptional level changes of inducible nitric oxide synthase, tumor necrosis factor-α and connexin 43 mRNA, C6 cells were treated with 5 μg/mL lipopolysaccharide for 3-48 hours. Reverse transcription-PCR showed that the expression of inducible nitric oxide synthase and tumor necrosis factor-α mRNA increased over time, but connexin 43 mRNA levels increased in lipopolysaccharide-stimulated C6 cells at 3 and 6 hours, and then decreased from 12 to 48 hours. Connexin 43 protein expression was detected by immunofluorescence staining, and the protein levels matched the mRNA expression levels. These results suggest that connexin 43 expression is biphasic in lipopolysaccharide-inducedneuroinflammation in C6 cells, which may be correlated with the connexin 43 compensatorymechanism.

  19. Hexavalent chromium at low concentration alters Sertoli cell barrier and connexin 43 gap junction but not claudin-11 and N-cadherin in the rat seminiferous tubule culture model

    Energy Technology Data Exchange (ETDEWEB)

    Carette, Diane [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Perrard, Marie-Hélène, E-mail: marie-helene.durand@ens-lyon.fr [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Prisant, Nadia [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Gilleron, Jérome; Pointis, Georges [INSERM U 1065, Team 5 “Physiopathology of Germ Cell Control: Genomic and Non Genomic Mechanisms” C3M, University of Nice Sophia Antipolis, Nice (France); Segretain, Dominique [University of Versailles/St Quentin-en-Yvelines (France); UMR S775, University Paris Descartes, 45 rue des Saints Pères, 75006, Paris (France); Durand, Philippe [Institut de Génomique Fonctionnelle de Lyon, Université de Lyon, Université Lyon I, CNRS, INRA, Ecole Normale Supérieure de Lyon, Lyon (France); Kallistem SAS Ecole Normale Supérieure de Lyon, Lyon (France)

    2013-04-01

    Exposure to toxic metals, specifically those belonging to the nonessential group leads to human health defects and among them reprotoxic effects. The mechanisms by which these metals produce their negative effects on spermatogenesis have not been fully elucidated. By using the Durand's validated seminiferous tubule culture model, which mimics the in vivo situation, we recently reported that concentrations of hexavalent chromium, reported in the literature to be closed to that found in the blood circulation of men, increase the number of germ cell cytogenetic abnormalities. Since this metal is also known to affect cellular junctions, we investigated, in the present study, its potential influence on the Sertoli cell barrier and on junctional proteins present at this level such as connexin 43, claudin-11 and N-cadherin. Cultured seminiferous tubules in bicameral chambers expressed the three junctional proteins and ZO-1 for at least 12 days. Exposure to low concentrations of chromium (10 μg/l) increased the trans-epithelial resistance without major changes of claudin-11 and N-cadherin expressions but strongly delocalized the gap junction protein connexin 43 from the membrane to the cytoplasm of Sertoli cells. The possibility that the hexavalent chromium-induced alteration of connexin 43 indirectly mediates the effect of the toxic metal on the blood–testis barrier dynamic is postulated. - Highlights: ► Influence of Cr(VI) on the Sertoli cell barrier and on junctional proteins ► Use of cultured seminiferous tubules in bicameral chambers ► Low concentrations of Cr(VI) (10 μg/l) altered the trans-epithelial resistance. ► Cr(VI) did not alter claudin-11 and N-cadherin. ► Cr(VI) delocalized connexin 43 from the membrane to the cytoplasm of Sertoli cells.

  20. Close the Gap : a study on the regulation of Connexin43 gap junctional communication

    NARCIS (Netherlands)

    Zeijl, Leonie van

    2009-01-01

    Gap junctions are groups of transmembrane channels that connect the cytoplasms of adjacent cells to mediate the diffusion of small molecules, such as ions, metabolites, second messengers and small peptides. The building blocks of gap junctions are connexin proteins. The most ubiquitous and best stu

  1. Identification of a protein kinase activity that phosphorylates connexin43 in a pH-dependent manner

    Directory of Open Access Journals (Sweden)

    P. Yahuaca

    2000-04-01

    Full Text Available The carboxyl-terminal (CT domain of connexin43 (Cx43 has been implicated in both hormonal and pH-dependent gating of the gap junction channel. An in vitro assay was utilized to determine whether the acidification of cell extracts results in the activation of a protein kinase that can phosphorylate the CT domain. A glutathione S-transferase (GST-fusion protein was bound to Sephadex beads and used as a target for protein kinase phosphorylation. A protein extract produced from sheep heart was allowed to bind to the fusion protein-coated beads. The bound proteins were washed and then incubated with 32P-ATP. Phosphorylation was assessed after the proteins were resolved by SDS-PAGE. Incubation at pH 7.5 resulted in a minimal amount of phosphorylation while incubation at pH 6.5 resulted in significant phosphorylation reaction. Maximal activity was achieved when both the binding and kinase reactions were performed at pH 6.5. The protein kinase activity was stronger when the incubations were performed with manganese rather than magnesium. Mutants of Cx43 which lack the serines between amino acids 364-374 could not be phosphorylated in the in vitro kinase reaction, indicating that this is a likely target of this reaction. These results indicate that there is a protein kinase activity in cells that becomes more active at lower pH and can phosphorylate Cx43.

  2. Connexin 43 ubiquitination determines the fate of gap junctions: restrict to survive.

    Science.gov (United States)

    Ribeiro-Rodrigues, Teresa M; Catarino, Steve; Pinho, Maria J; Pereira, Paulo; Girao, Henrique

    2015-06-01

    Connexins (Cxs) are transmembrane proteins that form channels which allow direct intercellular communication (IC) between neighbouring cells via gap junctions. Mechanisms that modulate the amount of channels at the plasma membrane have emerged as important regulators of IC and their de-regulation has been associated with various diseases. Although Cx-mediated IC can be modulated by different mechanisms, ubiquitination has been described as one of the major post-translational modifications involved in Cx regulation and consequently IC. In this review, we focus on the role of ubiquitin and its effect on gap junction intercellular communication.

  3. Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

    Science.gov (United States)

    Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda S.; Johnson, Keith R.; Mehta, Parmender P.

    2013-01-01

    The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions. PMID:23363606

  4. Short-term pacing in the mouse alters cardiac expression of connexin43

    Directory of Open Access Journals (Sweden)

    Rindler Michael J

    2008-05-01

    Full Text Available Abstract Background Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time. Results The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias. Conclusion Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease.

  5. Role of gap junction and connexin-43 in hypoxic-ischemic brain damage

    Institute of Scientific and Technical Information of China (English)

    Jieying Lin; Niyang Lin

    2006-01-01

    OBJECTEVE:Gap junctin (GJ)is the structural basis for direct intercellular communication of nerve cells . Connexin(Cx) is the protein subunit for constructling GJ channel. Among them, Cx43is closely related with nervous system. Both Cx43 and nervous system play an important role in the pathophysiological development of hypoxic-ischemic injury. We are in attempt to investigate GJ,Cx43 and their correlations with hypoxic-ischemic brain damage by research.DATA SOURCES:Using the terms "brain gap junction"in English and "gap junction"in Chinese, we searched the Medline database and Chinese BioMedical Literature Database as well as China Hospital Knowledge Database to identify the articles published from 1996 to 2006 about GJ and brain hypoxic-ischemic injury.STUDY SELECTION:The articles were selected firstly and abstracts of 250 articles were read thuugh.Articles in which the experimental design met randomized controlled principle were included,and study articles and case reports with repetitve contents were excluded.DATA EXTRACTION:Among 53 included correlative articles, 23 were excluded for repetitive contents and the other 30 were analyzed.DATA SYNTHESIS:GJ,widely esistling in nervous system,plays a key role in maintainling normal differentiation and development as well as physiological function brain tissue.GJ channel is a hydrophilic,low-selectivity and lowohmic channel, which can provide direct channel for intercellular substance transmission and information communication. It plays an important role in the differentiation and development of nerve cells and regulation of physiological function,The funtions of GJ channel are regulated by many factors,which invilved intracellular Ph value, Ca2+concentration, ATP concentration, phosphorylation of Cx, transchannel pressure,some neurohormonal factors,regulatory factors of protein and so on. Cx43 is the main component of GJ channel in the brain tissues. Its expression in the brain tissue of mammal is the strongest

  6. A Cell-Based High-Throughput Assay for Gap Junction Communication Suitable for Assessing Connexin 43-Ezrin Interaction Disruptors Using IncuCyte ZOOM.

    Science.gov (United States)

    Dukic, Aleksandra R; McClymont, David W; Taskén, Kjetil

    2017-01-01

    Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin-protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets. The Cx43-Ezrin interaction is a protein-protein interaction that opens possibilities for targeting with peptides and small molecules. For this reason, we developed a high-throughput cell-based assay in which GJIC can be assessed and new compounds characterized. We used two pools of IAR20 cells, calcein loaded and unloaded, that were mixed and allowed to attach. Next, GJIC was monitored over time using automated imaging via the IncuCyte imager. The assay was validated using known GJ inhibitors and anchoring peptide disruptors, and we further tested new peptides that interfered with the Cx43-Ezrin binding region and reduced GJIC. Although an AlphaScreen assay can be used to screen for Cx43-Ezrin interaction inhibitors, the cell-based assay described is an ideal secondary screen for promising small-molecule hits to help identify the most potent compounds.

  7. 17β estradiol regulation of connexin 43-based gap junction and mechanosensitivity through classical estrogen receptor pathway in osteocyte-like MLO-Y4 cells.

    KAUST Repository

    Ren, Jian

    2013-04-01

    Connexin 43 (Cx43) plays an essential role in osteocyte mechanotransduction. Although estrogen involves in the adaptive responses of bone cells to mechanical loadings, its effects on osteocytic Cx43-based gap junction intercellular communication (GJIC) remain obscure. We found that 17β estradiol (E2) up-regulated Cx43, and enhanced GJIC in osteocyte-like MLO-Y4 cells in fluorescence recovery after photobleaching (FRAP) assay. Combination of E2 pre-treatment and oscillating fluid flow (OFF) further enhanced Cx43 expression and mitogen-activated protein kinase (MAPK) phosphorylation, comparing to E2 or OFF treatment alone. Both blocking of classical estrogen receptors (ERα/β) by fulvestrant and ERα knockdown by small interfering RNA inhibited E2-mediated Cx43 increase, while a GPR30-specific agonist G-1 failed to promote Cx43 expression. Our results suggest that the presence of E2 enhanced Cx43-based GJIC mainly via ERα/β pathway, and sensitized osteocytes to mechanical loading. © 2012 Elsevier Inc. All rights reserved.

  8. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring.

    Science.gov (United States)

    Rossini, Kamila Fernanda; Oliveira, Camila Andrea de; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana

    2017-07-01

    The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. A limitação dietética durante a gravidez influencia o crescimento e desenvolvimento do feto e da prole e sua saúde na vida adulta. Os mecanismos subjacentes dos efeitos adversos da restrição proteica gestacional (RPG) no desenvolvimento dos corações da prole não são bem compreendidos. Avaliar os efeitos da RPG sobre a estrutura cardíaca em filhotes machos de ratas aos 60 dias após o nascimento (d60). Ratos fêmeas Wistar grávidas foram alimentadas com uma dieta de proteína normal (PN, 17% caseína) ou de baixa proteína (BP, caseína 6%). Os valores de pressão arterial (PA) de descendentes do sexo masculino de

  9. Connexin43 hemichannel-mediated regulation of connexin43.

    Directory of Open Access Journals (Sweden)

    Kai Li

    Full Text Available BACKGROUND: Many signaling molecules and pathways that regulate gap junctions (GJs protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43 hemichannels were activated by cadmium (Cd(2+, we tested this hypothesis. PRINCIPAL FINDINGS: Incubation of Cx43-transfected LLC-PK1 cells with Cd(2+ led to an increased expression of Cx43. This effect of Cd(2+ was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd(2+ on JNK activation and Cx43 expression. Indeed, Cd(2+ induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd(2+. CONCLUSIONS: Collectively, our results thus indicate that Cd(2+-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function.

  10. Cardiac Connexin 43 and Ischemic Cardioprotection

    Institute of Scientific and Technical Information of China (English)

    Jin-yi LI; Guo-qiang ZHONG; Yan HE; Yun LING

    2009-01-01

    The connexin 43 (Cx43) proteins, which is the predominant protein that can form gap junctions and non-junctional hemichannels in ventricular myocardium, are central to the cardioprotection afforded by ischemic precondi-tioning (IP) and maybe ischemic postconditioning (PC) too. Recent studies showed that recruitment of Cx43 to the mitochondria in IP might play a role in the production of reactive oxygen species (ROS) that mediates IP. The localiza-tion of Cx43 at mitochondria appears to be important for the achieved cardioprotection and opens a new door for us to reveal the exact mechanisms of ischemia/reperfusion (I/R) injury and cardioprotection, and it might be new targets of pharmacological modulator to achieved cardioprotection.

  11. Ascorbic acid 6-palmitate suppresses gap-junctional intercellular communication through phosphorylation of connexin 43 via activation of the MEK-ERK pathway.

    Science.gov (United States)

    Lee, Kyung Mi; Kwon, Jung Yeon; Lee, Ki Won; Lee, Hyong Joo

    2009-01-15

    Although the health benefits of dietary antioxidants have been extensively studied, their potential negative effects remain unclear. L-Ascorbic acid 6-palmitate (AAP), a synthetic derivative of ascorbic acid (AA), is widely used as an antioxidant and preservative in foods, vitamins, drugs, and cosmetics. Previously, we found that AA exerted an antitumor effect by protecting inhibition of gap-junctional intercellular communication (GJIC), which is closely associated with tumor progression. In this study, we examined whether AAP, an amphipathic derivative of AA, has chemopreventive effects using a GJIC model. AAP and AA exhibited dose-dependent free radical-scavenging activities and inhibited hydrogen peroxide (H(2)O(2))-induced intracellular reactive oxygen species (ROS) production in normal rat liver epithelial cells. Unexpectedly, however, AAP did not protect against the inhibition of GJIC induced by H(2)O(2); instead, it inhibited GJIC synergistically with H(2)O(2). AAP inhibited GJIC in a dose-dependent and reversible manner. This inhibitory effect was not due to the conjugated lipid structure of AAP, as treatment with palmitic acid alone failed to inhibit GJIC under the same conditions. The inhibition of GJIC by AAP was restored in the presence of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126, but not in the presence of other signal inhibitors and antioxidant (PKC inhibitors, EGFR inhibitor, NADPH oxidase inhibitor, catalase, vitamin E, or AA), indicating the critical involvement of MEK signaling in the GJIC inhibitory activity of AAP. Phosphorylation of ERK and connexin 43 (Cx43) was observed following AAP treatment, and this was reversed by U0126. These results suggest that the AAP-induced inhibition of GJIC is mediated by the phosphorylation of Cx43 via activation of the MEK-ERK pathway. Taken together, our results indicate that AAP has a potent carcinogenic effect, and that the influence of dietary

  12. Expression of connexin 43 mRNA and protein in developing follicles of prepubertal porcine ovaries

    Science.gov (United States)

    Melton, C.M.; Zaunbrecher, G.M.; Yoshizaki, G.; Patio, R.; Whisnant, S.; Rendon, A.; Lee, V.H.

    2001-01-01

    A major form of cell-cell communication is mediated by gap junctions, aggregations of intercellular channels composed of connexins (Cxs), which are responsible for exchange of low molecular weight (hybridization revealed that Cx43 mRNA was detectable in granulosa cells of primary follicles but undetectable in dormant primordial follicles. The intensity of the signal increased with follicular growth and was greatest in the large antral follicles. Immunohistochemical evaluation indicated that Cx43 protein expression correlated with the presence of Cx43 mRNA. These results indicate that substantial amounts of Cx43 are first expressed in granulosa cells following activation of follicular development and that this expression increases throughout follicular growth and maturation. These findings suggest an association between the enhancement of intercellular gap-junctional communication and onset of follicular growth. ?? 2001 Elsevier Science Inc. All rights reserved.

  13. Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue

    Directory of Open Access Journals (Sweden)

    Malmström Anders

    2006-05-01

    Full Text Available Abstract Background Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour. Methods Uterine isthmic biopsies were collected from non-pregnant (n = 7, term pregnant women not in labour (n = 14, in normal labour (n = 7 and in prolonged labour (n = 7. mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy. Results In women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p Conclusion The high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility.

  14. Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo.

    Science.gov (United States)

    Basu, Rahul; Banerjee, Kaveri; Bose, Abhishek; Das Sarma, Jayasri

    2015-12-16

    Gap junctions (GJs) form intercellular channels which directly connect the cytoplasm between neighboring cells to facilitate the transfer of ions and small molecules. GJs play a major role in the pathogenesis of infection-associated inflammation. Mutations of gap junction proteins, connexins (Cxs), cause dysmyelination and leukoencephalopathy. In multiple sclerosis (MS) patients and its animal model experimental autoimmune encephalitis (EAE), Cx43 was shown to be modulated in the central nervous system (CNS). The mechanism behind Cx43 alteration and its role in MS remains unexplored. Mouse hepatitis virus (MHV) infection-induced demyelination is one of the best-studied experimental animal models for MS. Our studies demonstrated that MHV infection downregulated Cx43 expression at protein and mRNA levels in vitro in primary astrocytes obtained from neonatal mouse brains. After infection, a significant amount of Cx43 was retained in endoplasmic reticulum/endoplasmic reticulum Golgi intermediate complex (ER/ERGIC) and GJ plaque formation was impaired at the cell surface, as evidenced by a reduction of the Triton X-100 insoluble fraction of Cx43. Altered trafficking and impairment of GJ plaque formation may cause the loss of functional channel formation in MHV-infected primary astrocytes, as demonstrated by a reduced number of dye-coupled cells after a scrape-loading Lucifer yellow dye transfer assay. Upon MHV infection, a significant downregulation of Cx43 was observed in the virus-infected mouse brain. This study demonstrates that astrocytic Cx43 expression and function can be modulated due to virus stress and can be an appropriate model to understand the basis of cellular mechanisms involved in the alteration of gap junction intercellular communication (GJIC) in CNS neuroinflammation. We found that MHV infection leads to the downregulation of Cx43 in vivo in the CNS. In addition, results show that MHV infection impairs Cx43 expression in addition to gap junction

  15. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo.

    Science.gov (United States)

    Wrenzycki, C; Herrmann, D; Carnwath, J W; Niemann, H

    1996-09-01

    In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3'primer. The resultant cDNA was amplified by polymerase chain reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity

  16. Connexin26 Mutations Causing Palmoplantar Keratoderma and Deafness Interact with Connexin43, Modifying Gap Junction and Hemichannel Properties.

    Science.gov (United States)

    Shuja, Zunaira; Li, Leping; Gupta, Shashank; Meşe, Gülistan; White, Thomas W

    2016-01-01

    Mutations in GJB2 (connexin [Cx]26) cause either deafness or deafness associated with skin diseases. That different disorders can be caused by distinct mutations within the same gene suggests that unique channel activities are influenced by each class of mutation. We have examined the functional characteristics of two human mutations, Cx26-H73R and Cx26-S183F, causing palmoplantar keratoderma (PPK) and deafness. Both failed to form gap junction channels or hemichannels when expressed alone. Coexpression of the mutants with wild-type Cx43 showed a transdominant inhibition of Cx43 gap junction channels, without reductions in Cx43 protein synthesis. In addition, the presence of mutant Cx26 shifted Cx43 channel gating and kinetics toward a more Cx26-like behavior. Coimmunoprecipitation showed Cx43 being pulled down more efficiently with mutant Cx26 than wild-type, confirming the enhanced formation of heteromeric connexons. Finally, the formation of heteromeric connexons resulted in significantly increased Cx43 hemichannel activity in the presence of Cx26 mutants. These findings suggest a common mechanism whereby Cx26 mutations causing PPK and deafness transdominantly influence multiple functions of wild-type Cx43. They also implicate a role for aberrant hemichannel activity in the pathogenesis of PPK and further highlight an emerging role for Cx43 in genetic skin diseases.

  17. Involvement of connexin 43 in acupuncture analgesia

    Institute of Scientific and Technical Information of China (English)

    HUANG Guang-ying; ZHENG Cui-hong; YU Wei-chang; TIAN Dai-shi; WANG Wei

    2009-01-01

    Background Connexin 43 (Cx43) is one of the major components of human keratinocyte gap junctions. To study whether gap junctional intercellular communication participates in the transfer of acupoint signals and acupuncture analgesia, the expression of Cx43 was studied in Zusanli (ST36) acupoints compared with control non-acupoint regions in rats after acupuncture. In addition, Cx43 heterozygous gene knockout mice were used to further explore the relationship between Cx43 and acupuncture analgesia. Methods The expression of Cx43 was detected by immunohistochemistry, immunoblotting, and RT-PCR for the Cx43 protein and mRNA. The influence of the Cx43 gene knockout on acupuncture analgesia was measured by a hot plate and observing the writhing response on Cx43 heterozygous gene knockout mice. Results Immunohistochemistry showed abundant Cx43 expression in some cells in the skin and subcutaneous tissue of rat ST36 acupoints. The mRNA and protein levels of Cx43 in acupoints were significantly higher than those in the control points in the non-acupuncture group, and even more so after acupuncture. The hot plate and writhing response experiments showed that partial knockout of the Cx43 gene decreased acupuncture analgesia. Conclusion Cx43 expression and acupuncture analgesia showed a positive correlation.

  18. Connexin43 is dispensable for phagocytosis.

    Science.gov (United States)

    Glass, Aaron M; Wolf, Benjamin J; Schneider, Karin M; Princiotta, Michael F; Taffet, Steven M

    2013-05-01

    Macrophages that lack connexin43 (Cx43), a gap junction protein, have been reported to exhibit dramatic deficiencies in phagocytosis. In this study, we revisit these findings using well-characterized macrophage populations. Cx43 knockout (Cx43(-/-)) mice die soon after birth, making the harvest of macrophages from adult Cx43(-/-) mice problematic. To overcome this obstacle, we used several strategies: mice heterozygous for the deletion of Cx43 were crossed to produce Cx43(+/+) (wild type [WT]) and Cx43(-/-) fetuses. Cells isolated from 12- to 14-d fetal livers were used to reconstitute irradiated recipient animals. After reconstitution, thioglycollate-elicited macrophages were collected by peritoneal lavage and bone marrow was harvested. Bone marrow cells and, alternatively, fetal liver cells were cultured in media containing M-CSF for 7-10 d, resulting in populations of cells that were >95% macrophages based on flow cytometry. Phagocytic uptake was detected using flow cytometric and microscopic techniques. Quantification of phagocytic uptake of IgG-opsonized sheep erythrocytes, zymosan particles, and Listeria monocytogenes failed to show any significant difference between WT and Cx43(-/-) macrophages. Furthermore, the use of particles labeled with pH-sensitive dyes showed equivalent acidification of phagosomes in both WT and Cx43(-/-) macrophages. Our findings suggest that modulation of Cx43 levels in cultured macrophages does not have a significant impact on phagocytosis.

  19. Bone morphogenetic protein-2 (BMP-2 and transforming growth factor-β1 (TGF-β1 alter connexin 43 phosphorylation in MC3T3-E1 Cells

    Directory of Open Access Journals (Sweden)

    Rudkin George H

    2001-07-01

    Full Text Available Abstract Background Bone morphogenetic proteins (BMPs and transforming growth factor-βs (TGF-βs are important regulators of bone repair and regeneration. BMP-2 and TGF-β1 have been shown to inhibit gap junctional intercellular communication (GJIC in MC3T3-E1 cells. Connexin 43 (Cx43 has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-β1 to investigate a possible mechanism for the inhibition of GJIC. Results Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-β1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. Conclusions BMP-2 and TGF-β1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-β1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells.

  20. Premature osteoblast clustering by enamel matrix proteins induces osteoblast differentiation through up-regulation of connexin 43 and N-cadherin.

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    Richard J Miron

    Full Text Available In recent years, enamel matrix derivative (EMD has garnered much interest in the dental field for its apparent bioactivity that stimulates regeneration of periodontal tissues including periodontal ligament, cementum and alveolar bone. Despite its widespread use, the underlying cellular mechanisms remain unclear and an understanding of its biological interactions could identify new strategies for tissue engineering. Previous in vitro research has demonstrated that EMD promotes premature osteoblast clustering at early time points. The aim of the present study was to evaluate the influence of cell clustering on vital osteoblast cell-cell communication and adhesion molecules, connexin 43 (cx43 and N-cadherin (N-cad as assessed by immunofluorescence imaging, real-time PCR and Western blot analysis. In addition, differentiation markers of osteoblasts were quantified using alkaline phosphatase, osteocalcin and von Kossa staining. EMD significantly increased the expression of connexin 43 and N-cadherin at early time points ranging from 2 to 5 days. Protein expression was localized to cell membranes when compared to control groups. Alkaline phosphatase activity was also significantly increased on EMD-coated samples at 3, 5 and 7 days post seeding. Interestingly, higher activity was localized to cell cluster regions. There was a 3 fold increase in osteocalcin and bone sialoprotein mRNA levels for osteoblasts cultured on EMD-coated culture dishes. Moreover, EMD significantly increased extracellular mineral deposition in cell clusters as assessed through von Kossa staining at 5, 7, 10 and 14 days post seeding. We conclude that EMD up-regulates the expression of vital osteoblast cell-cell communication and adhesion molecules, which enhances the differentiation and mineralization activity of osteoblasts. These findings provide further support for the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

  1. Connexin 43 impacts on mitochondrial potassium uptake

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    Kerstin eBoengler

    2013-06-01

    Full Text Available In cardiomyocytes, connexin 43 (Cx43 forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria. Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake.Subsarcolemmal mitochondria were isolated from rat left ventricular (LV myocardium and loaded with the potassium-sensitive dye PBFI. Intramitochondrial potassium was replaced by TEA (tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 µM Gap19, a peptide that specifically inhibits Cx43-dependent hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100±11.6 % in control mitochondria to 65.5±10.7 % (n=6, p<0.05. In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (Cx43Cre-ER(T/fl + 4-OHT. The mitochondria of the Cx43Cre-ER(T/fl + 4-OHT mice contained 3±1% Cx43 (n=6 of that in control mitochondria (100±11%, n=8, p<0.05. The ablation of Cx43 (n=5 reduced the velocity of the potassium influx from 100±11.2 % in control mitochondria (n=9 to 66.6±5.5 % (p<0.05.Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

  2. Role of connexin 43 in cardiovascular diseases.

    Science.gov (United States)

    Michela, Pecoraro; Velia, Verrilli; Aldo, Pinto; Ada, Popolo

    2015-12-05

    Gap junctions (GJs) channels provide the basis for intercellular communication in the cardiovascular system for maintenance of the normal cardiac rhythm, regulation of vascular tone and endothelial function as well as metabolic interchange between the cells. They allow the transfer of small molecules and may enable slow calcium wave spreading, transfer of "death" or of "survival" signals. In the cardiomyocytes the most abundant isoform is Connexin 43 (Cx43). Alterations in Cx43 expression and distribution were observed in myocardium disease; i.e. in hypertrophic cardiomyopathy, heart failure and ischemia. Recent reports suggest the presence of Cx43 in the mitochondria as well, at least in the inner mitochondrial membrane, where it plays a central role in ischemic preconditioning. In this review, the current knowledge on the relationship between the remodeling of cardiac gap junctions and cardiac diseases are summarized. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

    Science.gov (United States)

    Rodriguez-Sinovas, Antonio; Boengler, Kerstin; Cabestrero, Alberto; Gres, Petra; Morente, Miriam; Ruiz-Meana, Marisol; Konietzka, Ina; Miró, Elisabet; Totzeck, Andreas; Heusch, Gerd; Schulz, Rainer; Garcia-Dorado, David

    2006-07-07

    We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

  4. TGF-β1 inhibits connexin-43 expression in cultured smooth muscle cells of human bladder

    Institute of Scientific and Technical Information of China (English)

    Chi Qiang; Zhou Fenghai; Wang Yangmin

    2009-01-01

    Objective: In this research, we studied the TGF-β1 effects on connexin-43 expression in cultured human bladder smooth muscle cells. Methods: Human bladder smooth muscle cells primary cultures, with bladder tissue obtained from patients undergoing cystectomy, were intervened by recombinant human TGF-β1. Connexin-43 expression in human bladder smooth muscle cells was then examined by Western blotting and immunocytochemistry. Results: Stimulation with TGF-β1 led to significant reduction of cormexin-43 immunoreactivity and coupling (P<0.0001). Connexin-43 protein expression was significantly downregnlated (P<0.05). Simultaneously, low phosphorylation species of connexin-43 were particularly affected. Conclusion: Our experiments demonstrated a significant downregulation of connexin-43 by TGF-β1 in cultured human bladder smooth muscle cells. These findings support the view that TGF-β1 is involved in the pathophysiology of urinary bladder dysfunction.

  5. Targeting connexin 43 in diabetic wound healing: Future perspectives

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    Bajpai S

    2009-01-01

    Full Text Available The unknown mechanisms of impaired tissue repair in diabetes mellitus are making this disease a serious clinical problem for the physicians worldwide. The lacuna in the knowledge of the etiology of diabetic wounds necessitates more focused research in order to develop new targeting tools with higher efficacy for their effective management. Gap-junction proteins, connexins, have shown some promising results in the process of diabetic wound healing. Till now the role of connexins has been implicated in peripheral neuropathy, deafness, skin disorders, cataract, germ cell development and treatment of cancer. Recent findings have revealed that gap junctions play a key role in normal as well as diabetic wound healing. The purpose of this review is to provide the information related to etiology, epidemiology, clinical presentation of diabetic wounds and to analyze the role of connexin 43 (Cx43 in the diabetic wound healing process. The current control strategies and the future research challenges have also been discussed briefly in this review.

  6. Sphingosine-1-Phosphate reduces ischemia/reperfusion injury by phosphorylating the gap junction protein Connexin43

    DEFF Research Database (Denmark)

    Morel, Sandrine; Christoffersen, Christina; Axelsen, Lene N;

    2016-01-01

    AIM: Increasing evidence points to lipoprotein composition rather than reverse cholesterol transport in the cardioprotective properties of high-density lipoproteins (HDL). HDL binding to receptors at the surface of cardiomyocytes activates signalling pathways promoting survival, but downstream...... targets are largely unknown. Here, we investigate the pathways by which the Sphingosine-1-Phosphate (S1P) constituent of HDL limits cell death induced by cardiac ischemia/reperfusion (I/R). METHODS AND RESULTS: Apolipoprotein M (ApoM) transgenic (Apom-Tg) mice, in which plasma S1P is increased by 296......%, and wild-type (WT) mice were subjected to in vivo I/R. Infarct size, neutrophil infiltration into the infarcted area and serum Troponin I were less pronounced in Apom-Tg mice. In vitro experiments suggest that this cardioprotection depends on direct effects of S1P on cardiomyocytes, whereas leukocyte...

  7. Mutations of connexin43 in fetuses with congenital heart malformations

    Institute of Scientific and Technical Information of China (English)

    CHEN Ping; XIE Li-jian; HUANG Guo-ying; ZHAO Xiao-qing; CHANG Cai

    2005-01-01

    Background Gap junction channels formed by connexin43 (Cx43) protein are important in cardiac morphogenesis, and Cx43 gene is thought to be associated with congenital heart malformation (CHM). This study was undertaken to detect the mutations of Cx43 in fetuses with CHM.Methods Cx43 extron DNA was amplified by PCR from 16 fetuses with a variety of CHM. The PCR products were analyzed by SSCP and DNA sequencing. Thirty children who had no CHM were selected as controls. Results Eight homozygous mutations of Cx43 were observed in a fetus with double outlet right ventricule (DORV), five of the 8 mutations were missense mutations including Arg239Trp, Ser251Thr, Ala253Pro, Pro283Leu and Thr290Asn, and the remaining 3 were silent polymorphisms including Gly252Gly, Pro256Pro and Thr275Thr. No mutations were found in other fetuses and the control group.Conclusions Mutations of Cx43 may be associated with congenital conotruncal anomalies. PCR-SSCP is an effective method for screening the mutations of Cx43.

  8. Mono-Heteromeric Configurations of Gap Junction Channels Formed by Connexin43 and Connexin45 Reduce Unitary Conductance and Determine both Voltage Gating and Metabolic Flux Asymmetry

    Directory of Open Access Journals (Sweden)

    Guoqiang Zhong

    2017-05-01

    Full Text Available In cardiac tissues, the expression of multiple connexins (Cx40, Cx43, Cx45, and Cx30.2 is a requirement for proper development and function. Gap junctions formed by these connexins have distinct permeability and gating mechanisms. Since a single cell can express more than one connexin isoform, the formation of hetero-multimeric gap junction channels provides a tissue with an enormous repertoire of combinations to modulate intercellular communication. To study further the perm-selectivity and gating properties of channels containing Cx43 and Cx45, we studied two monoheteromeric combinations in which a HeLa cell co-transfected with Cx43 and Cx45 was paired with a cell expressing only one of these connexins. Macroscopic measurements of total conductance between cell pairs indicated a drastic reduction in total conductance for mono-heteromeric channels. In terms of Vj dependent gating, Cx43 homomeric connexons facing heteromeric connexons only responded weakly to voltage negativity. Cx45 homomeric connexons exhibited no change in Vj gating when facing heteromeric connexons. The distributions of unitary conductances (γj for both mono-heteromeric channels were smaller than predicted, and both showed low permeability to the fluorescent dyes Lucifer yellow and Rhodamine123. For both mono-heteromeric channels, we observed flux asymmetry regardless of dye charge: flux was higher in the direction of the heteromeric connexon for MhetCx45 and in the direction of the homomeric Cx43 connexon for MhetCx43. Thus, our data suggest that co-expression of Cx45 and Cx43 induces the formation of heteromeric connexons with greatly reduced permeability and unitary conductance. Furthermore, it increases the asymmetry for voltage gating for opposing connexons, and it favors asymmetric flux of molecules across the junction that depends primarily on the size (not the charge of the crossing molecules.

  9. Connexin 43 is Involved in Aldosterone-Induced Podocyte Injury

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    Min Yang

    2014-11-01

    Full Text Available Background/Aims: Connexin43 (Cx43 belongs to a family of transmembrane proteins that build cell-to-cell channels in gap junctions, which play an essential role in intercellular communication, cell proliferation, differentiation and apoptosis. The aim of this study is to clarify the effect of Cx43 in aldosterone (Aldo-induced podocyte injury and explore the possible molecular mechanism behind this effect. Methods: Uninephrectomized Sprague-Dawley rats were given 1% NaCl (salt in their water and an Aldo infusion (0.75 μg/h for 28 days to induce podocyte injury. Podocytes were incubated in media containing either buffer or increasing concentrations of Aldo (10-9 - 10-7 M for variable time periods. The podocytes were then examined and the mechanism of injury investigated using TUNEL assay, ELISA, immunofluorescence staining, western blot, RNA interference, and DCFDA fluorescence. Results: Here, we report that in vivo administration of Aldo caused greater numbers of TUNEL-positive podocytes, accompanied by increased Cx43 expression, but a reduction in WT1-positive podocytes. In vitro studies indicated that Aldo induces podocyte apoptosis in a dose- and time-dependent manner and is accompanied by increases in Cx43 expression, gap junction intercellular communication (GJIC, ROS production, and the Bax/Bcl-2 ratio. Silencing of Cx43 expression attenuates the increase of ROS production and the subsequent up-regulation of the Bax/Bcl-2 ratio, which partially inhibited the podocytes apoptosis. Conclusions: Our study provides preliminary evidence that upregulation of Cx43 expression is involved in Aldo-induced podocyte injury, and that Cx43 is a potentially relevant therapeutic target for the treatment of chronic kidney diseases (CKD.

  10. 缝隙连接蛋白Cx43在SD大鼠电点燃癫痫模型中的表达%Expression of gap junction connexin43 in the SD rats electrical kindling of epilepsy model

    Institute of Scientific and Technical Information of China (English)

    宋建华; 谢锡驹

    2012-01-01

    Objective To investigate the role of connexin (Cx) in pathogenesis of epilepsy. Methods Thirty - two male SD rats were randomly divided into 4 groups and 4 models were set up. The behaviors of all SD rats were observed meanwhile the EEGs were recorded. Before the end Cx43 in hippocampus was detected with Western blot. The relationship between gap junction connexin and the formation of epilepsy was observed through the intervention of medicine to the kindling - model. Results Rats displayed typical epileptic behaviors,and epileptic discharges were observed in the epilepsy model group. The expression of connexin 43 in the hippocampal tissue was significantly enhanced in the epilepsy model group,but reduced by carbenoxolone and phenytoin sodium. Carbenoxolone and phenytoin sodium reduced frequency and amplitude of epileptic waves. The increasing expression of Cx43 was inhibited. Conclusion Gap junction Cx43 is the material base for the formation of epilepsy. The inhibition of excessive expression of Cx43 can reduce the formation of abnormal gap junction. Consequently the formation of epilepsy is inhibited.%目的 探讨缝隙连接蛋白(Cx)在癫痫形成中的作用.方法 将32只雄性SD大鼠随机分为4组,分别造模.观察SD大鼠行为,记录脑电图.通过免疫蛋白印迹的方法检测Cx43在大鼠海马区的表达变化.通过药物对癫痫电点燃模型成模过程的干预来观察癫痫形成与缝隙连接蛋白间的关系.结果 模型组大鼠出现癫痫发作.蛋白印迹显示Cx43的表达异常增高.药物干预的2组SD大鼠模型痫性发作滞后,发作频率降低,程度减轻,痫性脑电波波幅降低,蛋白印迹显示Cx43的表达增高受到抑制.结论 缝隙连接蛋白是癫痫形成的物质基础.抑制Cx43的过度表达,减少异常缝隙连接的形成,癫痫形成也受到抑制,从而预防癫痫产生.

  11. RXP-E: a connexin43-binding peptide that prevents action potential propagation block

    DEFF Research Database (Denmark)

    Lewandowski, Rebecca; Procida, Kristina; Vaidyanathan, Ravi

    2008-01-01

    Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and ...

  12. 间隙连接蛋白43与妇科疾病%Connexin 43 and Gynecological Diseases

    Institute of Scientific and Technical Information of China (English)

    贾喜菲

    2011-01-01

    The normal and abnormal expression of connexin play a direct role in the function of cell, tissue , and organ, whereas connexin 43 is the most important gap junction protein.The expression of connexin 43 is increased in uterine tissues, as a low-impedance channel for uterine intermuscular cell contraction.Gap junction channels are subject to the penetration of ions, ensuring the metabolism and behavioral coordination of uterine muscular cell populations.The abnormal expression of connexin 43 is closely associated with the occurrence and progress of uterine myoma,endometriosis,cervical cancer,and other gynecological disorders.%间隙连接蛋白的正常和异常表达,可直接影响细胞及组织、器官的功能状态,其中间隙连接蛋白43是最重要的间隙连接蛋白.在子宫组织中,间隙连接蛋白43水平的升高,为子宫肌细胞间收缩控制提供了低电阻通道,使离子很容易通过间隙连接的通道,保证了子宫肌细胞群在代谢及行为上的协调性.其异常表达,与子宫肌瘤、子宫内膜异位症、宫颈癌等多种妇科疾病的发生、发展有着密切的联系.

  13. Viral regulation of aquaporin 4, connexin 43, microcephalin and nucleolin.

    Science.gov (United States)

    Fatemi, S Hossein; Folsom, Timothy D; Reutiman, Teri J; Sidwell, Robert W

    2008-01-01

    The current study investigated whether human influenza viral infection in midpregnancy leads to alterations in proteins involved in brain development. Human influenza viral infection was administered to E9 pregnant Balb/c mice. Brains of control and virally-exposed littermates were subjected to microarray analysis, SDS-PAGE and western blotting at three postnatal stages. Microarray analysis of virally-exposed mouse brains showed significant, two-fold change in expression of multiple genes in both neocortex and cerebellum when compared to sham-infected controls. Levels of mRNA and protein levels of four selected genes were examined in brains of exposed mice. Nucleolin mRNA was significantly decreased in day 0 and day 35 neocortex and significantly increased in day 35 cerebellum. Protein levels were significantly upregulated at days 35 and 56 in neocortex and at day 56 in cerebellum. Connexin 43 protein levels were significantly decreased at day 56 in neocortex. Aquaporin 4 mRNA was significantly decreased in day 0 neocortex. Aquaporin 4 protein levels decreased in neocortex significantly at day 35. Finally, microcephalin mRNA was significantly decreased in day 56 neocortex and protein levels were significantly decreased at 56 cerebellum. These data suggest that influenza viral infection in midpregnancy in mice leads to long-term changes in brain markers for enhanced ribosome genesis (nucleolin), increased production of immature neurons (microcephalin), and abnormal glial-neuronal communication and neuron migration (connexin 43 and aquaporin 4).

  14. Control of Vascular Smooth Muscle Cell Growth by Connexin 43

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    Chintamani eJoshi

    2012-06-01

    Full Text Available Connexin 43 (Cx43, the principal gap junction protein in vascular smooth muscle cells (VSMCs, regulates movement of ions and other signaling molecules through gap junction intercellular communication (GJIC and plays important roles in maintaining normal vessel function; however, many of the signaling mechanisms controlling Cx43 in VSMCs are not clearly described. The goal of this study was to investigate mechanisms of Cx43 regulation with respect to VSMC proliferation. Treatment of rat primary VSMCs with the cAMP analog 8Br-cAMP, the soluble guanylate cyclase (sGC stimulator BAY 41-2272 (BAY, or the Cx inducer diallyl disulfide (DADS significantly reduced proliferation after 72 h compared to vehicle controls. Bromodeoxyuridine uptake revealed reduction (p<.001 in DNA synthesis after 6 h and flow cytometry showed reduced (40% S phase cell numbers after 16 h in DADS-treated cells compared to controls. Cx43 expression significantly increased after 270 min treatment with 8Br-cAMP, 8Br-cGMP, BAY or DADS. Inhibition of PKA, PKG or PKC reversed 8Br-cAMP-stimulated increases in Cx43 expression, whereas only PKG or PKC inhibition reversed 8Br-cGMP- and BAY-stimulated increases in total Cx43. Interestingly, stimulation of Cx43 expression by DADS was not dependent on PKA, PKG or PKC. Using fluorescence recovery after photobleaching, only 8Br-cAMP or DADS increased GJIC with 8Br-cAMP mediated by PKC and DADS mediated by PKG. Further, DADS significantly increased phosphorylation at the MAPK-sensitive serine (Ser255 and Ser279, the cell cycle regulatory kinase-sensitive Ser262 and the PKC-sensitive Ser368 after 30 min while 8Br-cAMP significantly increased phosphorylation only at Ser279 compared to controls. This study demonstrates that 8Br-cAMP- and DADS-enhanced GJIC rather than Cx43 expression and/or phosphorylation plays an important role in regulation of VSMC proliferation and provides new insights into the growth-regulatory capacities of Cx43 in VSMCs.

  15. Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

    Science.gov (United States)

    Chen, Gang; Park, Chul-Kyu; Xie, Rou-Gang; Berta, Temugin; Nedergaard, Maiken

    2014-01-01

    Accumulating evidence suggests that spinal cord astrocytes play an important role in neuropathic pain sensitization by releasing astrocytic mediators (e.g. cytokines, chemokines and growth factors). However, it remains unclear how astrocytes control the release of astrocytic mediators and sustain late-phase neuropathic pain. Astrocytic connexin-43 (now known as GJ1) has been implicated in gap junction and hemichannel communication of cytosolic contents through the glial syncytia and to the extracellular space, respectively. Connexin-43 also plays an essential role in facilitating the development of neuropathic pain, yet the mechanism for this contribution remains unknown. In this study, we investigated whether nerve injury could upregulate connexin-43 to sustain late-phase neuropathic pain by releasing chemokine from spinal astrocytes. Chronic constriction injury elicited a persistent upregulation of connexin-43 in spinal astrocytes for >3 weeks. Spinal (intrathecal) injection of carbenoxolone (a non-selective hemichannel blocker) and selective connexin-43 blockers (connexin-43 mimetic peptides 43Gap26 and 37,43Gap27), as well as astroglial toxin but not microglial inhibitors, given 3 weeks after nerve injury, effectively reduced mechanical allodynia, a cardinal feature of late-phase neuropathic pain. In cultured astrocytes, TNF-α elicited marked release of the chemokine CXCL1, and the release was blocked by carbenoxolone, Gap26/Gap27, and connexin-43 small interfering RNA. TNF-α also increased connexin-43 expression and hemichannel activity, but not gap junction communication in astrocyte cultures prepared from cortices and spinal cords. Spinal injection of TNF-α-activated astrocytes was sufficient to induce persistent mechanical allodynia, and this allodynia was suppressed by CXCL1 neutralization, CXCL1 receptor (CXCR2) antagonist, and pretreatment of astrocytes with connexin-43 small interfering RNA. Furthermore, nerve injury persistently increased excitatory

  16. Immunohistochemistry using an antibody to unphosphorylated connexin 43 to identify human myometrial interstitial cells

    Directory of Open Access Journals (Sweden)

    Van Lommel Alfons

    2008-09-01

    Full Text Available Abstract Background Myometrial smooth myocytes contract as a result of electrical signalling via a process called excitation-contraction coupling. This process is understood in great detail at the cellular level but the generation and coordination of electrical signals throughout the myometrium are incompletely understood. Recent evidence concerning the vital role of interstitial cells of Cajal in tissue-level signalling in gastrointestinal tract, and the presence of similar cells in urinary tract smooth muscle may be relevant for future research into myometrial contractility but there remains a lack of evidence regarding these cells in the myometrium. Methods Single stain immunohistochemical and double stain immunofluorescence techniques visualised antibodies directed against total connexin 43, unphosphorylated connexin 43, KIT, alpha-SMA and prolyl 4-hydroxylase in myometrial biopsies from 26 women representing all stages of reproductive life. Results Myometrial smooth myocytes from term uterine biopsies expressed connexin 43 in a punctate pattern typical of gap junctions. However, on the boundaries of the smooth muscle bundles, cells were present with a more uniform staining pattern. These cells continued to possess the same staining characteristics in non-pregnant biopsies whereas the smooth myocytes no longer expressed connexin 43. Immunohistochemistry using an antibody directed against connexin 43 unphosphorylated at serine 368 showed that it is this isoform that is expressed continually by these cells. Double-stain immunofluorescence for unphosphorylated connexin 43 and KIT, an established marker for interstitial cells, revealed a complete match indicating these cells are myometrial interstitial cells (MICs. MICs had elongated cell processes and were located mainly on the surface of the smooth muscle bundles and within the fibromuscular septum. No particular arrangement of cells as plexuses was observed. Antibody to prolyl 4-hydroxylase

  17. Connexin43 siRNA promotes HUVEC proliferation and inhibits apoptosis induced by ox-LDL: an involvement of ERK signaling pathway.

    Science.gov (United States)

    Yin, Guotian; Yang, Xiuli; Li, Bo; Yang, Meng; Ren, Mingfen

    2014-09-01

    Oxidized low-density lipoprotein (ox-LDL), one of the most important risk factors of atherosclerosis, is a highly antigenic, potent chemoattractant that facilitates the development of atherosclerosis. Gap junctions also play an important in the development of atherosclerosis. In this study, we investigated the effects of ox-LDL on connexin43 and the mechanisms of connexin43 siRNA-inhibited apoptosis induced by ox-LDL in human umbilical vein endothelial cell (HUVEC), to clarify the role of connexin43 in atherosclerosis. Our results showed that ox-LDL significantly inhibited the growth and promoted apoptosis of HUVEC in a dose-dependent manner. Also, ox-LDL upregulated the expression of connexin43. Furthermore, knockdown connexin43 by siRNA promoted proliferation and inhibited apoptosis in ox-LDL-stimulated HUVEC. Moreover, the level of phosphor-ERK1/2 and connexin43 was remarkably attenuated by a ERK pathway inhibitor (PD98059). These results suggest that connexin43 siRNA promotes HUVEC proliferation and inhibits apoptosis induced by ox-LDL, and ERK signaling pathway appears to be involved in these processes.

  18. Adenovirus-mediated delivery of bFGF small interfering RNA increases levels of connexin 43 in the glioma cell line, U251

    Directory of Open Access Journals (Sweden)

    Liu Hongsheng

    2010-01-01

    Full Text Available Abstract Background bFGF is an important growth factor for glioma cell proliferation and invasion, while connexin 43 is implicated in the suppression of glioma growth. Correspondingly, gliomas have been shown to have reduced, or compromised, connexin 43 expression. Methods In this study, a bFGF-targeted siRNA was delivered to the glioma cell line, U251, using adenovirus (Ad-bFGF-siRNA and the expression of connexin 43 and its phosphorylation state were evaluated. U251 cells were infected with Ad-bFGF-siRNA (100, 50, or 25 MOI, and infection with adenovirus expressing green fluorescent protein (Ad-GFP at 100 MOI served as a control. Western blotting and immunofluorescence were used to detect the expression levels, phosphorylation, and localization of connexin 43 in U251 cells infected, and not infected, with Ad-bFGF-siRNA. Results Significantly higher levels of connexin 43 were detected in U251 cells infected with Ad-bFGF-siRNA at 100 and 50 MOI than in cells infected with Ad-GFP, and the same amount of connexin 43 was detected in Ad-GFP-infected and uninfected U251 cells. Connexin 43 phosphorylation did not differ between Ad-bFGF-siRNA-infected and uninfected U251 cells. However, the ratio of phosphorylated to unphosphorylated connexin 43 in Ad-bFGF-siRNA cells was lower, and connexin 43 was predominantly localized to the cytoplasm. Using a scrape loading dye transfer assay, more Lucifer Yellow was transferred to neighboring cells in the Ad-bFGF-siRNA treated group than in the control group. Conclusion To our knowledge, this is the first description of a role for connexin 43 in the inhibition of U251 growth using Ad-bFGF-siRNA.

  19. Adenovirus-mediated delivery of bFGF small interfering RNA increases levels of connexin 43 in the glioma cell line, U251.

    Science.gov (United States)

    Zhang, Biao; Feng, Xuequan; Wang, Jinhuan; Xu, Xinnu; Liu, Hongsheng; Lin, Na

    2010-01-14

    bFGF is an important growth factor for glioma cell proliferation and invasion, while connexin 43 is implicated in the suppression of glioma growth. Correspondingly, gliomas have been shown to have reduced, or compromised, connexin 43 expression. In this study, a bFGF-targeted siRNA was delivered to the glioma cell line, U251, using adenovirus (Ad-bFGF-siRNA) and the expression of connexin 43 and its phosphorylation state were evaluated. U251 cells were infected with Ad-bFGF-siRNA (100, 50, or 25 MOI), and infection with adenovirus expressing green fluorescent protein (Ad-GFP) at 100 MOI served as a control. Western blotting and immunofluorescence were used to detect the expression levels, phosphorylation, and localization of connexin 43 in U251 cells infected, and not infected, with Ad-bFGF-siRNA. Significantly higher levels of connexin 43 were detected in U251 cells infected with Ad-bFGF-siRNA at 100 and 50 MOI than in cells infected with Ad-GFP, and the same amount of connexin 43 was detected in Ad-GFP-infected and uninfected U251 cells. Connexin 43 phosphorylation did not differ between Ad-bFGF-siRNA-infected and uninfected U251 cells. However, the ratio of phosphorylated to unphosphorylated connexin 43 in Ad-bFGF-siRNA cells was lower, and connexin 43 was predominantly localized to the cytoplasm. Using a scrape loading dye transfer assay, more Lucifer Yellow was transferred to neighboring cells in the Ad-bFGF-siRNA treated group than in the control group. To our knowledge, this is the first description of a role for connexin 43 in the inhibition of U251 growth using Ad-bFGF-siRNA.

  20. Common mechanisms linking connexin43 to neural progenitor cell migration and glioma invasion.

    Science.gov (United States)

    Naus, Christian C; Aftab, Qurratulain; Sin, Wun Chey

    2016-02-01

    Cell migration is critical for cell differentiation, tissue formation and organ development. Several mechanisms come to play in the process of cell migration, orchestrating changes in cell polarity, adhesion, process extension and motility. Recent findings have shown that gap junctions, and specifically connexin43 (Cx43), can play a significant role in these processes, impacting adhesion and cytoskeletal rearrangements. Thus Cx43 within a cell regulates its motility and migration via intracellular signaling. Furthermore, Cx43 in the host cells can impact the degree of cellular migration through that tissue. Similarities in these connexin-based processes account for both neural progenitor migration in the developing brain, and for glioma cell invasion in the mature brain. In both cases, Cx43 in the tissue ("soil") in which cells ("seeds") exist facilitates their migration and, for glioma cells, tissue invasion. Cx43 mediates these effects through channel- and non-channel-dependent mechanisms which have similarities in both paradigms of cell migration. This provides insight into developmental processes and pathological situations, as well as possible therapeutic approaches regarding specific functional domains of gap junction proteins.

  1. Two Different Functions of Connexin43 Confer Two Different Bone Phenotypes in Zebrafish.

    Science.gov (United States)

    Misu, Akihiro; Yamanaka, Hiroaki; Aramaki, Toshihiro; Kondo, Shigeru; Skerrett, I Martha; Iovine, M Kathryn; Watanabe, Masakatsu

    2016-06-10

    Fish remain nearly the same shape as they grow, but there are two different modes of bone growth. Bones in the tail fin (fin ray segments) are added distally at the tips of the fins and do not elongate once produced. On the other hand, vertebrae enlarge in proportion to body growth. To elucidate how bone growth is controlled, we investigated a zebrafish mutant, steopsel (stp(tl28d)). Vertebrae of stp(tl28d) (/+) fish look normal in larvae (∼30 days) but are distinctly shorter (59-81%) than vertebrae of wild type fish in adults. In contrast, the lengths of fin rays are only slightly shorter (∼95%) than those of the wild type in both larvae and adults. Positional cloning revealed that stp encodes Connexin43 (Cx43), a connexin that functions as a gap junction and hemichannel. Interestingly, cx43 was also identified as the gene causing the short-of-fin (sof) phenotype, in which the fin ray segments are shorter but the vertebrae are normal. To identify the cause of this difference between the alleles, we expressed Cx43 exogenously in Xenopus oocytes and performed electrophysiological analysis of the mutant proteins. Gap junction coupling induced by Cx43(stp) or Cx43(sof) was reduced compared with Cx43-WT. On the other hand, only Cx43(stp) induced abnormally high (50× wild type) transmembrane currents through hemichannels. Our results suggest that Cx43 plays critical and diverse roles in zebrafish bone growth.

  2. Higher Incidence of Lung Adenocarcinomas Induced by DMBA in Connexin 43 Heterozygous Knockout Mice

    Directory of Open Access Journals (Sweden)

    Krishna Duro de Oliveira

    2013-01-01

    Full Text Available Gap junctions are communicating junctions which are important for tissue homeostasis, and their disruption is involved in carcinogenic processes. This study aimed to verify the influence of deletion of one allele of the Connexin 43 gene on cancer incidence in different organs. The 7, 12-dimethylbenzanthracene (DMBA carcinogenic model, using hebdomadary doses by gavage of 9 mg per animal, was used to induce tumors in Connexin 43 heterozygous or wild-type mice. The experiment began in the eighth week of the mice life, and all of them were euthanized when reaching inadequate physical condition, or at the end of 53 weeks. No statistical differences occurred for weight gain and cancer survival time (P=0.9853 between heterozygous and wild-type mice. Cx43+/− mice presented significantly higher susceptibility to lung cancer (P=0.0200 which was not evidenced for benign neoplasms (P=0.3449. In addition, incidence of ovarian neoplasms was 2.5-fold higher in Cx43+/− mice, although not statistically significant. Other organs showed a very similar cancer occurrence between Cx43 groups. The experiment strengthens the evidence of the relationship between Connexin 43 deficiency and carcinogenesis.

  3. 地西他滨对白血病细胞系中连接蛋白43的作用和机制%Effect and mechanism of decitabine on expression of connexin 43 protein in leukemic cells

    Institute of Scientific and Technical Information of China (English)

    韦炜; 徐娅蓓; 孙云; 莫蓓蕾; 阎骅

    2012-01-01

    目的:探讨地西他滨对白血病细胞中连接蛋门43(Cx43)的作用和机制.方法:蛋白印迹法(Western blot)检测去甲基化药物地西他滨对NB4-MR2、U937细胞中Cx43水平表达的影响以及加入蛋白酶体抑制剂MG132对Cx43表达的影响.实时定量PCR检测地西他滨对NB4-MR2、U937细胞中Cx43 RNA水平的影响.结果:地西他滨明显下调MR2细胞中Cx43表达,对其RNA水平没有明显影响;地西他滨明显缩短Cx43蛋白的半衰期.加入蛋白酶体抑制剂MG132可明显抑制地西他滨引起的Cx43蛋白的减少.结论:地西他滨通过蛋白酶体途径减少Cx43的水平,可能会影响其治疗效果.%Objective To study the effect and mechanism of decitabine on expression of connexin 43(Cx43) protein in leukemic cells. Methods Western blot was used to assess the Cx43 protein level in leukemic NB4-MR2 and U937 ceil lines for determining the effect of decitabine on expression of Cx43 protein and the effect of adding proteasome inhibitor MG132 on the expression. Level of Cx43 RNA expression was determined by real-time PCR. Results Decitabine significantly down-regulated the expression of Cx43 protein in both leukemic cell lines, while no significant change in Cx43 RNA level was found. Moreover, decitabine significantly reduced the half-life of Cx43 protein. The addition of proteasome inhibitor MG132 could block the inhibiting effect of decitabine on Cx43 protein expression. Conclusions Decitabine down-regulated the expression of Cx43 protein in leukemic cells through proteasome pathway.

  4. Expression of Connexin43 in Rat Epithelial Cells and Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To explore the role of connexin43 (Cx43) in gap junctional intercellular communication (GJIC) and propagated sensation along meridians, the expression of Cx43 in the rat epithelial cells and fibroblasts was studied both in vitro and in vivo. With the in vitro study, the rat epithelial cells and fibroblasts were cultured together, and the localization of Cx43 was detected by immunohistochemistry and indirect immunofluorescent cytochemistry and under confocal microscopy . And the expression of Cx43 on the surface of the cells was examined by flow cytometry. With the in vivo examination, 20 SD rats were randomized into control group (n = 10) and electrical acupuncture group (EAgroup, n=10). EA ( 0.5-1.5 V, 4-16 Hz , 30 min) was applied to"Zusanli"acupoint for 30 min at rat's hind paw, the localization of Cx43 was immunohistochemically detected.The immunohistochemical staining and indirect immunfluorescent cytochemistry showed that Cx43was localized on the surface of the cells and in the cytoplasm. The relative expression level of Cx43on the cellular membrane surfaces of the rat epithelial cells and fibroblasts, as determined by FACS, were 13.91 % and 29.53 % respectively. Our studied suggested that Cx43 might be involved in GJIC and propagated sensation along meridians.

  5. Losartan reduced connexin43 expression in left ventricular myocardium of spontaneously hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    Li-li ZHAO; Hong-juan CHEN; Jun-zhu CHEN; Min YU; Yun-lan NI; Wei-fang ZHANG

    2008-01-01

    Objective: To assess the effect of angiotensin Ⅱ type 1 (AT1) receptor antagonist losartan on myocardium connexin43 (Cx43) gap junction (GJ) expression in spontaneously hypertensive rats (SHRs) and investigate possible mechanisms. Methods: Sixteen 9-week-old male SHRs and 8 age-matched male Wistar-Kyoto (WKY) rats were included in this study. SHRs were randomly divided into two groups to receive losartan at 30mg/(kg·d) by oral gavage once daily for 8 weeks (SHR-L) or vehicle (0.9% saline) to act as controls (SHR-V); WKY rats receiving vehicle for 8 weeks served as normotensive controls. At the end of the experiment, rats were sacrificed and the hearts were removed. Expressions of Cx43 and nuclear factor-kappaB p65 (NF-κB p65) proteins in all three groups were observed and further investigations on the effect of angiotensin Ⅱ type 1 receptor antagonist losartan (30mg/(kg·d), 8 weeks) on Cx43 expression were conducted with Western blot and immunohistochemistry. NF-κB p65 protein in nuclear extracts was determined by Western blot. Results: Left ventricular (LV) hypertrophy was prominent in SHRs, Cx43 and NF-κB p65 protein expressions were obviously upregulated and Cx43 distribution was dispersed over the cell surface. Treatment with losarton reduced the over-expressions of Cx43 and NF-κB p65 in LV myocardium. The distribution of Cx43 gap junction also became much regular and confined to intercalated disk after losartan treatment. Conclusion: Cx43 level was upregulated in LV myocardium of SHR during early stage of hypertrophy. Angiotensin Ⅱ type l receptor antagonist losartan prevented Cx43 gap junction remodeling in hypertrophied left ventricles, possibly through the NF-κB pathway.

  6. Effects of ferulic acid on oxidative stress, heat shock protein 70, connexin 43, and monoamines in the hippocampus of pentylenetetrazole-kindled rats.

    Science.gov (United States)

    Hussein, Abdelaziz M; Abbas, Khaled M; Abulseoud, Osama A; El-Hussainy, El-Hussainy M A

    2017-06-01

    The present study investigated the effects of ferulic acid (FA) on pentylenetetrazole (PTZ)-induced seizures, oxidative stress markers (malondialdehyde (MDA), catalase, and reduced glutathione (GSH)), connexin (Cx) 43, heat shock protein 70 (Hsp 70), and monoamines (serotonin (5-HT) and norepinephrine (NE)) levels in a rat model of PTZ-induced kindling. Sixty Sprague Dawley rats were divided into 5 equal groups: (a) normal group; (b) FA group: normal rats received FA at a dose of 40 mg/kg daily; (c) PTZ group: normal rats received PTZ at a dose of 50 mg/kg i.p. on alternate days for 15 days; (d) FA-before group: treatment was the same as for the PTZ group, except rats received FA; and (e) FA-after group: rats received FA from sixth dose of PTZ. PTZ caused a significant increase in MDA, Cx43, and Hsp70 along with a significant decrease in GSH, 5-HT, and NE levels and CAT activity in the hippocampus (p < 0.05). Pre- and post-treatment with FA caused significant improvement in behavioral parameters, MDA, CAT, GSH, 5-HT, NE, Cx43 expression, and Hsp70 expression in the hippocampal region (p < 0.05). We conclude that FA has neuroprotective effects in PTZ-induced epilepsy, which might be due to attenuation of oxidative stress and Cx43 expression and upregulation of neuroprotective Hsp70 and neurotransmitters (5-HT and NE).

  7. Molecular cloning and evolutionary analysis of the GJA1 (connexin43) gene from bats (Chiroptera).

    Science.gov (United States)

    Wang, Li; Li, Gang; Wang, Jinhong; Ye, Shaohui; Jones, Gareth; Zhang, Shuyi

    2009-04-01

    Gap junction protein connexin43 (Cx43), encoded by the GJA1 gene, is the most abundant connexin in the cardiovascular system and was reported as a crucial factor maintaining cardiac electrical conduction, as well as having a very important function in facilitating the recycling of potassium ions from hair cells in the cochlea back into the cochlear endolymph during auditory transduction processes. In mammals, bats are the only taxon possessing powered flight, placing exceptional demand on many organismal processes. To meet the demands of flying, the hearts of bats show many specialties. Moreover, ultrasonic echolocation allows bat species to orientate and often detect and locate food in darkness. In this study, we cloned the full-length coding region of GJA1 gene from 12 different species of bats and obtained orthologous sequences from other mammals. We used the maximum likelihood method to analyse the evolution of GJA1 gene in mammals and the lineage of bats. Our results showed this gene is much conserved in mammals, as well as in bats' lineage. Compared with other mammals, we found one private amino acid substitution shared by bats, which is located on the inner loop domain, as well as some species-specific amino acid substitutions. The evolution rate analyses showed the signature of purifying selection on not only different classification level lineages but also the different domains and amino acid residue sites of this gene. Also, we suggested that GJA1 gene could be used as a good molecular marker to do the phylogenetic reconstruction.

  8. The modulatory effects of connexin 43 on cell death/survival beyond cell coupling.

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    Rodríguez-Sinovas, Antonio; Cabestrero, Alberto; López, Diego; Torre, Iratxe; Morente, Miriam; Abellán, Arancha; Miró, Elisabet; Ruiz-Meana, Marisol; García-Dorado, David

    2007-01-01

    Connexins form a diverse and ubiquitous family of integral membrane proteins. Characteristically, connexins are assembled into intercellular channels that aggregate into discrete cell-cell contact areas termed gap junctions (GJ), allowing intercellular chemical communication, and are essential for propagation of electrical impulses in excitable tissues, including, prominently, myocardium, where connexin 43 (Cx43) is the most important isoform. Previous studies have shown that GJ-mediated communication has an important role in the cellular response to stress or ischemia. However, recent evidence suggests that connexins, and in particular Cx43, may have additional effects that may be important in cell death and survival by mechanisms independent of cell to cell communication. Connexin hemichannels, located at the plasma membrane, may be important in paracrine signaling that could influence intracellular calcium and cell survival by releasing intracellular mediators as ATP, NAD(+), or glutamate. In addition, recent studies have shown the presence of connexins in cell structures other than the plasma membrane, including the cell nucleus, where it has been suggested that Cx43 influences cell growth and differentiation. In addition, translocation of Cx43 to mitochondria appears to be important for certain forms of cardioprotection. These findings open a new field of research of previously unsuspected roles of Cx43 intracellular signaling.

  9. Downregulation of connexin43 by microRNA-130a in cardiomyocytes results in cardiac arrhythmias.

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    Osbourne, Appledene; Calway, Tyler; Broman, Michael; McSharry, Saoirse; Earley, Judy; Kim, Gene H

    2014-09-01

    MicroRNAs (miRNAs) are now recognized as critical regulators of diverse physiological and pathological processes; however, studies of miRNAs and arrhythmogenesis remain sparse. Connexin43 (Cx43), a major cardiac gap junction protein, has elicited great interest in its role in arrhythmias. Additionally, Cx43 was a potential target for miR-130a as predicted by several computational algorithms. This study investigates the effect of miR-130a overexpression in the adult heart and its effect on cardiac rhythm. Using a cardiac-specific inducible system, transgenic mice demonstrated both atrial and ventricular arrhythmias. We performed ventricular-programmed electrical stimulation and found that the αMHC-miR130a mice developed sustained ventricular tachycardia beginning 6weeks after overexpression. Western blot analysis demonstrated a steady decline in Cx43 after 2weeks of overexpression with over a 90% reduction in Cx43 levels by 10weeks. Immunofluorescent staining confirmed a near complete loss of Cx43 throughout the heart. To validate Cx43 as a direct target of miR-130a, we performed in vitro target assays in 3T3 fibroblasts and HL-1 cardiomyocytes, both known to endogenously express miR-130a. Using a luciferase reporter fused to the 3'UTR of Cx43, we found a 52.9% reduction in luciferase activity in 3T3 cells (parrhythmias.

  10. Connexin 43 expression in human and mouse testes with impaired spermatogenesis

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    M Kotula-Balak

    2009-08-01

    Full Text Available Connexin 43 (Cx43 belongs to a family of proteins that form gap junction channels. The aim of this study was to examine the expression of Cx43 in the testis of a patient with Klinefelter’s syndrome and of mice with the mosaic mutation and a partial deletion in the long arm of the Y chromosome. These genetic disorders are characterized by the presence of numerous degenerated seminiferous tubules and impaired spermatogenesis. In mouse testes, the expression and presence of Cx43 were detected by means of immunohistochemistry and Western blot analysis, respectively. In testes of Klinefelter’s patient only immunoexpression of Cx43 was detected. Regardless of the species Cx43 protein was ubiquitously distributed in testes of reproductively normal males, whereas in those with testicular disorders either a weak intensity of staining or no staining within the seminiferous tubules was observed. Moderate to strong or very strong staining was confined to the interstitial tissue. In an immunoblot analysis of testicular homogenates Cx43 appeared as one major band of approximately 43 kDa. Our study adds three more examples of pathological gonads in which the absence or apparent decrease of Cx43 expression within the seminiferous tubules was found. A positive correlation between severe spermatogenic impairment and loss of Cx43 immunoreactivity observed in this study supports previous data that gap junctions play a crucial role in spermatogenesis. Strong Cx43 expression detected mostly in the interstitial tissue of the Klinefelter’s patient may presumably be of importance in sustaining Leydig cell metabolic activity. However, the role of gap junction communication in the control of Leydig cell function seems to be more complex than originally thought.

  11. Atorvastatin prevents connexin43 remodeling in hypertrophied left ventricular myocardium of spontaneously hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-juan; YAO Lei; CHEN Tu-gang; YU Min; WANG Li-hong; CHEN Jun-zhu

    2007-01-01

    Background Connexin43 (Cx43) is the predominant gap junction protein in heart and is involved in the control of cell-to-cell communication to modulate the contractility and the electrical coupling of cardiac myocytes. Left ventricular(LV) hypertrophy is accompanied by changes of Cx43 expression. Recent studies have demonstrated that statins reduced cardiac hypertrophy. However, it is unknown whether statins can affect Cx43 expression in hypertrophied left ventricular myocardium. This study was designed to assess the effects of atorvastatin on LV hypertrophy and Cx43 expression in spontaneously hypertensive rats (SHR).Methods Nine-week old SHRs were randomly divided into two groups. Some received atorvastatin at 30 mg/kg by oral gavage once daily for 8 weeks (SHR-A); others received vehicle. Age-matched Wistar-Kyoto rats (WKY) received atorvastatin or vehicle for 8 weeks were used as controls. At the end of the experiment, we investigated LV hypertrophy and the expression of Cx43 in LV myocardium in four groups. Cx43 expression was investigated by the methods of Western blotting, immunohistochemistry, and transmission electron microscope. LV hypertrophy was accessed by pathological analysis and plasma brain natriuretic peptide (BNP) level.Results LV hypertrophy was prominent in untreated SHR. In SHR, LV myocardium Cx43 level was upregulated, and the distribution of Cx43 was displaced from their usual locations to other sites at various distances away from the intercalated disks. After atorvastatin treatment, myocardium Cx43 level was reduced in SHR-A, and the distribution of Cx43 gap junction became much regular and confined to intercalated disk. Statins also prevented LV hypertrophy in SHR.Conclusions These results provide novel in vivo evidence for the key role of Cx43 gap junctions in LV hypertrophy and the possible mechanism in anti-hypertrophic effect of statins. Atorvastatin treatment may have beneficial effects on LV hypertrophy in spontaneously hypertensive

  12. Connexin43与心血管疾病关系的研究进展%Progress on correlation between connexin 43 and cardiovascular disease

    Institute of Scientific and Technical Information of China (English)

    吴瑶

    2011-01-01

    Connexin( Cx43)作为最重要的间隙连接蛋白,是哺乳动物心脏中最主要的连接蛋白。Cx43对心脏发育、心脏电生理活动、心肌的缺血后修复起重要作用,Cx43异常是很多心血管疾病包括先天性心脏病、缺血性心脏病、心律失常、高血压的重要病因。Cx43表达受多种因素影响,包括甲基化修饰、组蛋白修饰和非编码RNA调控等,这些调控机制的异常可能是其影响疾病发生、发展的重要因素。%As the most important gap junction protein, connexin 43 ( Cx43 ) is the most important connexin in the mammalian heart. Recent studies suggest that it plays an important role in heart development, cardiac electrophysiological activity and cell repair after myocardial ischemia. Cx43 abnormity is an important cause of many cardiovascular diseases including congenital heart disease, ischemic heart disease, arrhythmias and hypertension. The expression of Cx43 is regulated by various factors, including methylation, histone modification and non-coding RNA regulation, etc. These abnormal regulations may be important for the development of diseases.

  13. Sertoli-cell-specific knockout of connexin 43 leads to multiple alterations in testicular gene expression in prepubertal mice

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    Sarah Giese

    2012-11-01

    A significant decline in human male reproductive function has been reported for the past 20 years but the molecular mechanisms remain poorly understood. However, recent studies showed that the gap junction protein connexin-43 (CX43; also known as GJA1 might be involved. CX43 is the predominant testicular connexin (CX in most species, including in humans. Alterations of its expression are associated with different forms of spermatogenic disorders and infertility. Men with impaired spermatogenesis often exhibit a reduction or loss of CX43 expression in germ cells (GCs and Sertoli cells (SCs. Adult male transgenic mice with a conditional knockout (KO of the Gja1 gene [referred to here as connexin-43 (Cx43] in SCs (SCCx43KO show a comparable testicular phenotype to humans and are infertile. To detect possible signaling pathways and molecular mechanisms leading to the testicular phenotype in adult SCCx43KO mice and to their failure to initiate spermatogenesis, the testicular gene expression of 8-day-old SCCx43KO and wild-type (WT mice was compared. Microarray analysis revealed that 658 genes were significantly regulated in testes of SCCx43KO mice. Of these genes, 135 were upregulated, whereas 523 genes were downregulated. For selected genes the results of the microarray analysis were confirmed using quantitative real-time PCR and immunostaining. The majority of the downregulated genes are GC-specific and are essential for mitotic and meiotic progression of spermatogenesis, including Stra8, Dazl and members of the DM (dsx and map-3 gene family. Other altered genes can be associated with transcription, metabolism, cell migration and cytoskeleton organization. Our data show that deletion of Cx43 in SCs leads to multiple alterations of gene expression in prepubertal mice and primarily affects GCs. The candidate genes could represent helpful markers for investigators exploring human testicular biopsies from patients showing corresponding spermatogenic deficiencies and for

  14. Ischemic preconditioning preserves connexin 43 phosphorylation during sustained ischemia in pig hearts in vivo.

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    Schulz, Rainer; Gres, Petra; Skyschally, Andreas; Duschin, Alexej; Belosjorow, Sergej; Konietzka, Ina; Heusch, Gerd

    2003-07-01

    During myocardial ischemia, connexin 43 (Cx43) is dephosphorylated in vitro, and the subsequent opening of gap junctions formed by two opposing Cx43 hexamers was suggested to propagate ischemia/reperfusion injury. Reduction of infarct size (IS) by ischemic preconditioning (IP) involves activation of protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK), both of which can phosphorylate Cx43. We now studied in anesthetized pigs whether IP impacts on Cx43 phosphorylation by measuring the density of non-phosphorylated and total Cx43 (confocal laser) during normoperfusion and 90-min ischemia in non-preconditioned and preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 and the activity of p38MAPK were assessed. IP by 10 min ischemia and 15 min reperfusion reduced IS. Non-phosphorylated Cx43 remained unchanged during ischemia in preconditioned hearts, while it increased from 35+/-3 to 75+/-8 AU (P<0.05) in non-preconditioned hearts. Co-localization of PKCalpha, p38MAPKalpha, and p38MAPKbeta with Cx43 during ischemia increased only in preconditioned hearts. While the ischemia-induced increase in p38MAPKalpha activity was comparable in preconditioned and non-preconditioned hearts, p38MAPKbeta activity was increased only in preconditioned hearts. Blockade of p38MAPK by SB203580 attenuated the IS-reduction and the increased p38MAPK-Cx43 co-localization by IP. We conclude that IP increases co-localization of protein kinases with Cx43 and preserves phosphorylation of Cx43 during ischemia.

  15. Connexin-43 channels are a pathway for discharging lactate from glycolytic pancreatic ductal adenocarcinoma cells.

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    Dovmark, T H; Saccomano, M; Hulikova, A; Alves, F; Swietach, P

    2017-08-10

    Glycolytic cancer cells produce large quantities of lactate that must be removed to sustain metabolism in the absence of oxidative phosphorylation. The only venting mechanism described to do this at an adequate rate is H(+)-coupled lactate efflux on monocarboxylate transporters (MCTs). Outward MCT activity is, however, thermodynamically inhibited by extracellular acidity, a hallmark of solid tumours. This inhibition would feedback unfavourably on metabolism and growth, raising the possibility that other venting mechanisms become important in under-perfused tumours. We investigated connexin-assembled gap junctions as an alternative route for discharging lactate from pancreatic ductal adenocarcinoma (PDAC) cells. Diffusive coupling (calcein transmission) in vitro was strong between Colo357 cells, weaker yet hypoxia-inducible between BxPC3 cells, and very low between MiaPaCa2 cells. Coupling correlated with levels of connexin-43 (Cx43), a protein previously linked to late-stage disease. Evoked lactate dynamics, imaged in Colo357 spheroids using cytoplasmic pH as a read-out, indicated that lactate anions permeate gap junctions faster than highly-buffered H(+) ions. At steady-state, junctional transmission of lactate (a chemical base) from the spheroid core had an alkalinizing effect on the rim, producing therein a milieu conducive for growth. Metabolite assays demonstrated that Cx43 knockdown increased cytoplasmic lactate retention in Colo357 spheroids (diameter ~150 μm). MiaPaCa2 cells, which are Cx43 negative in monolayer culture, showed markedly increased Cx43 immunoreactivity at areas of invasion in orthotopic xenograft mouse models. These tissue areas were associated with chronic extracellular acidosis (as indicated by the marker LAMP2 near/at the plasmalemma), which can explain the advantage of junctional transmission over MCT in vivo. We propose that Cx43 channels are important conduits for dissipating lactate anions from glycolytic PDAC cells. Furthermore

  16. c-Src Kinase Inhibition Reduces Arrhythmia Inducibility and Connexin43 Dysregulation after Myocardial Infarction

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    Rutledge, Cody A.; Ng, Fu Siong; Sulkin, Matthew S.; Greener, Ian D.; Sergeyenko, Artem M.; Liu, Hong; Gemel, Joanna; Beyer, Eric C.; Sovari, Ali A.; Efimov, Igor R.; Dudley, Samuel C.

    2014-01-01

    Objectives The aim of this study was to evaluate the role of c-Src inhibition on connexin43 (Cx43) regulation in a mouse model of myocardial infarction (MI). Background MI is associated with decreased expression of Cx43, the principal gap junction protein responsible for propagating current in ventricles. Activated c-Src has been linked to Cx43 dysregulation. Methods MI was induced in 12-week-old mice by coronary artery occlusion. MI mice were treated with c-Src inhibitors (PP1 or AZD0530), PP3 (an inactive analogue of PP1), or saline. Treated hearts were compared to sham mice by echocardiography, optical mapping, telemetry ECG monitoring, and inducibility studies. Tissues were collected for immunoblotting, quantitative PCR, and immunohistochemistry. Results Active c-Src was elevated in PP3-treated MI mice compared to sham at the scar border (280%, p=0.003) and distal ventricle (346%, p=0.013). PP1 treatment restored active c-Src to sham levels at the scar border (86%, p=0.95) and distal ventricle (94%, p=1.0). PP1 raised Cx43 expression by 69% in the scar border (p=0.048) and by 73% in distal ventricle (p=0.043) compared to PP3 mice. PP1-treated mice had restored conduction velocity at the scar border (PP3: 32 cm/s, PP1: 41 cm/s, p < 0.05) and lower arrhythmic inducibility (PP3: 71%, PP1: 35%, p < 0.05) than PP3 mice. PP1 did not change infarct size, ECG pattern, or cardiac function. AZD0530 treatment demonstrated restoration of Cx43 comparable to PP1. Conclusions c-Src inhibition improved Cx43 levels and conduction velocity and lowered arrhythmia inducibility after MI, suggesting a new approach for arrhythmia reduction following MI. PMID:24361364

  17. Estradiol Receptors Regulate Differential Connexin 43 Expression in F98 and C6 Glioma Cell Lines.

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    Zahra Moinfar

    Full Text Available Glioma is the most common malignant primary brain tumour with male preponderance and poor prognosis. Glioma cells express variable amounts of connexin 43 (Cx43 and estrogen receptors (ERs. Both, Cx43 and ERs, play important roles in cell proliferation and migration. Therefore, we investigated the effects of 17-ß estradiol (E2 on Cx43 expression in two glioma cell lines with variable native expression of Cx43.F98 and C6 rat glioma cells were cultured for 24 h in the presence of 10 nM or 100 nM E2, and the E2-antagonist, Fulvestrant. An MTT assay was performed to evaluate cell viability. ERα, ERβ and Cx43 protein expressions were analysed by western blotting and Cx43 mRNA expression was analysed by real-time polymerase chain reaction. To quantify cell migration, an exclusive zone migration assay was used. Functional coupling of cells via gap junctions was examined using whole-cell patch-clamp technique.E2 reduced Cx43 expression in C6 cells, but increased Cx43 expression in F98 cultures. These effects were mediated via ERs. Moreover, E2 promoted C6 cell migration, but it did not affect F98 cell migration. The expression level of ERα was found to be high in C6, but low in F98 cells. ERβ was exclusively expressed in C6 cells. In addition, E2 treatment induced a significant decrease of ERβ in C6 cultures, while it decreased ERα expression in F98 glioma cells.These findings show that E2 differentially modulates Cx43 expression in F98 and C6 glioma cells, likely due to the differential expression of ERs in each of these cell lines. Our findings point to the molecular mechanisms that might contribute to the gender-specific differences in the malignancy of glioma and could have implications for therapeutic strategies against glioma.

  18. Bisphosphonates improve trabecular bone mass and normalize cortical thickness in ovariectomized, osteoblast connexin43 deficient mice.

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    Watkins, Marcus P; Norris, Jin Yi; Grimston, Susan K; Zhang, Xiaowen; Phipps, Roger J; Ebetino, Frank H; Civitelli, Roberto

    2012-10-01

    The gap junction protein, connexin43 (Cx43) controls both bone formation and osteoclastogenesis via osteoblasts and/or osteocytes. Cx43 has also been proposed to mediate an anti-apoptotic effect of bisphosphonates, potent inhibitors of bone resorption. We studied whether bisphosphonates are effective in protecting mice with a conditional Cx43 gene deletion in osteoblasts and osteocytes (cKO) from the consequences of ovariectomy on bone mass and strength. Ovariectomy resulted in rapid loss of trabecular bone followed by a slight recovery in wild type (WT) mice, and a similar degree of trabecular bone loss, albeit slightly delayed, occurred in cKO mice. Treatment with either risedronate (20 μg/kg) or alendronate (40 μg/kg) prevented ovariectomy-induced bone loss in both genotypes. In basal conditions, bones of cKO mice have larger marrow area, higher endocortical osteoclast number, and lower cortical thickness and strength relative to WT. Ovariectomy increased endocortical osteoclast number in WT but not in cKO mice. Both bisphosphonates prevented these increases in WT mice, and normalized endocortical osteoclast number, cortical thickness and bone strength in cKO mice. Thus, lack of osteoblast/osteocyte Cx43 does not alter bisphosphonate action on bone mass and strength in estrogen deficiency. These results support the notion that one of the main functions of Cx43 in cortical bone is to restrain osteoblast and/or osteocytes from inducing osteoclastogenesis at the endocortical surface. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. A role for connexin43 in macrophage phagocytosis and host survival after bacterial peritoneal infection.

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    Anand, Rahul J; Dai, Shipan; Gribar, Steven C; Richardson, Ward; Kohler, Jeff W; Hoffman, Rosemary A; Branca, Maria F; Li, Jun; Shi, Xiao-Hua; Sodhi, Chhinder P; Hackam, David J

    2008-12-15

    The pathways that lead to the internalization of pathogens via phagocytosis remain incompletely understood. We now demonstrate a previously unrecognized role for the gap junction protein connexin43 (Cx43) in the regulation of phagocytosis by macrophages and in the host response to bacterial infection of the peritoneal cavity. Primary and cultured macrophages were found to express Cx43, which localized to the phagosome upon the internalization of IgG-opsonized particles. The inhibition of Cx43 using small interfering RNA or by obtaining macrophages from Cx43 heterozygous or knockout mice resulted in significantly impaired phagocytosis, while transfection of Cx43 into Fc-receptor expressing HeLa cells, which do not express endogenous Cx43, conferred the ability of these cells to undergo phagocytosis. Infection of macrophages with adenoviruses expressing wild-type Cx43 restored phagocytic ability in macrophages from Cx43 heterozygous or deficient mice, while infection with viruses that expressed mutant Cx43 had no effect. In understanding the mechanisms involved, Cx43 was required for RhoA-dependent actin cup formation under adherent particles, and transfection with constitutively active RhoA restored a phagocytic phenotype after Cx43 inactivation. Remarkably, mortality was significantly increased in a mouse model of bacterial peritonitis after Cx43 inhibition and in Cx43 heterozygous mice compared with untreated and wild-type counterparts. These findings reveal a novel role for Cx43 in the regulation of phagocytosis and rearrangement of the F-actin cytoskeleton, and they implicate Cx43 in the regulation of the host response to microbial infection.

  20. Hindlimb unloading results in increased predisposition to cardiac arrhythmias and alters left ventricular connexin 43 expression.

    Science.gov (United States)

    Moffitt, Julia A; Henry, Matthew K; Welliver, Kathryn C; Jepson, Amanda J; Garnett, Emily R

    2013-03-01

    Hindlimb unloading (HU) is a well-established animal model of cardiovascular deconditioning. Previous data indicate that HU results in cardiac sympathovagal imbalance. It is well established that cardiac sympathovagal imbalance increases the risk for developing cardiac arrhythmias. The cardiac gap junction protein connexin 43 (Cx43) is predominately expressed in the left ventricle (LV) and ensures efficient cell-to-cell electrical coupling. In the current study we wanted to test the hypothesis that HU would result in increased predisposition to cardiac arrhythmias and alter the expression and/or phosphorylation of LV-Cx43. Electrocardiographic data using implantable telemetry were obtained over a 10- to 14-day HU or casted control (CC) condition and in response to a sympathetic stressor using isoproterenol administration and brief restraint. The arrhythmic burden was calculated using a modified scoring system to quantify spontaneous and provoked arrhythmias. In addition, Western blot analysis was used to measure LV-Cx43 expression in lysates probed with antibodies directed against the total and an unphosphorylated form of Cx43 in CC and HU rats. HU resulted in a significantly greater total arrhythmic burden during the sympathetic stressor with significantly more ventricular arrhythmias occurring. In addition, there was increased expression of total LV-Cx43 observed with no difference in the expression of unphosphorylated LV-Cx43. Specifically, the increased expression of LV-Cx43 was consistent with the phosphorylated form. These data taken together indicate that cardiovascular deconditioning produced through HU results in increased predisposition to cardiac arrhythmias and increased expression of phosphorylated LV-Cx43.

  1. Silencing of desmoplakin decreases connexin43/Nav1.5 expression and sodium current in HL‑1 cardiomyocytes.

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    Zhang, Qianhuan; Deng, Chunyu; Rao, Fang; Modi, Rohan M; Zhu, Jiening; Liu, Xiaoying; Mai, Liping; Tan, Honghong; Yu, Xiyong; Lin, Qiuxiong; Xiao, Dingzhang; Kuang, Sujuan; Wu, Shulin

    2013-09-01

    Desmosomes and gap junctions are situated in the intercalated disks of cardiac muscle and maintain the integrity of mechanical coupling and electrical impulse conduction between cells. The desmosomal plakin protein, desmoplakin (DSP), also plays a crucial role in the stability of these interconnected components as well as gap junction connexin proteins. In addition to cell‑to‑cell junctions, other molecules, including voltage‑gated sodium channels (Nav1.5) are present in the intercalated disk and support the contraction of cardiac muscle. Mutations in genes encoding desmosome proteins may result in fatal arrhythmias, including arrhythmogenic right ventricular cardiomyopathy (ARVC). Therefore, the aim of the present study was to determine whether the presence of DSP is necessary for the normal function and localization of gap junction protein connexin43 (Cx43) and Nav1.5. To examine this hypothesis, RNA interference was utilized to knock down the expression of DSP in HL‑1 cells and the content, distribution and function of Cx43 and Nav1.5 was assessed. Western blotting and flow cytometry experiments revealed that Cx43 and Nav1.5 expression decreased following DSP silencing. In addition, immunofluorescence studies demonstrated that a loss of DSP expression led to an abnormal distribution of Cx43 and Nav1.5, while scrape‑loading dye/transfer revealed a decrease in dye transfer in DSP siRNA‑treated cells. The sodium current was also recorded by the whole‑cell patch clamp technique. The results indicated that DSP suppression decreased sodium current and slowed conduction velocity in cultured cells. The present study indicates that impaired mechanical coupling largely affects electrical synchrony, further uncovering the pathogenesis of ARVC.

  2. Contribution of intracellular calcium and pH in ischemic uncoupling of cardiac gap junction channels formed of connexins 43, 40, and 45: a critical function of C-terminal domain.

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    Giriraj Sahu

    Full Text Available Ischemia is known to inhibit gap junction (GJ mediated intercellular communication. However the detail mechanisms of this inhibition are largely unknown. In the present study, we determined the vulnerability of different cardiac GJ channels formed of connexins (Cxs 43, 40, and 45 to simulated ischemia, by creating oxygen glucose deprived (OGD condition. 5 minutes of OGD decreased the junctional conductance (Gj of Cx43, Cx40 and Cx45 by 53±3%, 64±1% and 85±2% respectively. Reduction of Gj was prevented completely by restricting the change of both intracellular calcium ([Ca(2+]i and pH (pHi with potassium phosphate buffer. Clamping of either [Ca(2+]i or pHi, through BAPTA (2 mM or HEPES (80 mM respectively, offered partial resistance to ischemic uncoupling. Anti-calmodulin antibody attenuated the uncoupling of Cx43 and Cx45 significantly but not of Cx40. Furthermore, OGD could reduce only 26±2% of Gj in C-terminus (CT truncated Cx43 (Cx43-Δ257. Tethering CT of Cx43 to the CT-truncated Cx40 (Cx40-Δ249, and Cx45 (Cx45-Δ272 helped to resist OGD mediated uncoupling. Moreover, CT domain played a significant role in determining the junction current density and plaque diameter. Our results suggest; OGD mediated uncoupling of GJ channels is primarily due to elevated [Ca(2+]i and acidic pHi, though the latter contributes more. Among Cx43, Cx40 and Cx45, Cx43 is the most resistant to OGD while Cx45 is the most sensitive one. CT of Cx43 has major necessary elements for OGD induced uncoupling and it can complement CT of Cx40 and Cx45.

  3. Effect of calcium channel blocker on gap junctional connexin 43 in infarcted myocardium in rats%钙通道阻滞剂对梗死心肌中不同部位心肌连接蛋白43表达的影响

    Institute of Scientific and Technical Information of China (English)

    杨永健; 速晓华; 唐兵; 李德; 杨大春

    2011-01-01

    目的:研究L和L/T型钙通道阻制剂对梗死心脏不同部位(坏死区域、肥厚区域等)心肌组织中连接蛋白43(Cx43)的影响.方法:随机将大鼠48只分为假手术组、心肌梗死(MI)组、阿莫地平(L型钙通道阻滞剂)组和米贝拉地尔(L/T型钙通道阻滞剂)组(每组n=12只).通过结扎大鼠左冠状动脉建立MI模型,术前7 d,上述4个组分别用安慰剂、L型钙通道阻滞剂阿莫地平4 mg/(kg·d)和L/T型钙通道阻滞剂米贝拉地尔10 mg/(kg·d).术后1、3、7 d,分别检测左心室游离壁(LVFW,梗死区)、心室间隔(IS,肥厚区)和右心室壁(Rv),正常心肌组织中Cx43蛋白的表达.术后7 d显微直视下测LVFW处MI病灶的大小、IS的厚度及左心室的大小.结果:IS中Cx43蛋白表达于术后1、3、7 d呈逐渐增加的趋势;LVFW中Cx43蛋白的表达于术后1、3、7 d时均处于低水平,与对照组相比差异显著(P<0.05).RV中Cx43蛋白的表达于术后1、3、7 d无显著差异,与对照组相比也无显著性差异.米贝拉地尔能明显地抑制LVFW心肌组织中Cx43表达的下调,缩小MI病灶;阿莫地平则抑制肥厚心肌中Cx43蛋白的表达,明显抑制IS的肥厚.结论:MI病理过程中,梗死病灶内Cx43的表达下调,肥厚组织中Cx43的表达上调.L和L/T型钙通道阻滞剂均能减轻心肌重构与选择性地调节心肌组织中Cx43的表达有关.%AIM: To investigate the effect of cardiac L- and L/T-type Ca2+ channels on gap junctional connexin 43 ( Cx43 ) in myocardium infarcted heart remodeling of rats. METHODS: Rat myocardium infarction model was established by permanent ligation of the left coronary artery. Infarcted rats were treated with oral placebo, amlodipine [ L-channel blockade, 4 mg/( kg · day) ] or mibefradil [ L/T-channel blockade, 10 mg/(kg·day] beginning 7 days before induction of myocardial infarction (MI). Protein levels of Cx43 were measured 1, 3 and 7 days postcoronary occlusion in the noninfarcted and

  4. The long-term effects of FSH and triiodothyronine administration during the pubertal period on Connexin 43 expression and spermatogenesis efficiency in adult rats.

    Science.gov (United States)

    Marchlewska, Katarzyna; Slowikowska-Hilczer, Jolanta; Walczak-Jedrzejowska, Renata; Oszukowska, Elzbieta; Filipiak, Eliza; Kula, Krzysztof

    2015-04-01

    Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Hyperstimulation of both hormones evokes regressional changes in connexin 43 expression and the seminiferous epithelium in young rats during testicular maturation. However, separate treatments with T3 reduce Sertoli cell number, which seems to be closely connected with the maturation of connexin 43 gap junctions. FSH elevates Sertoli cell number and function, but this effect may take place regardless of the presence of connexin 43-dependent intercellular communication. The aim of the study was to evaluate the later effects of such treatments. Newborn, male Wistar rats were divided randomly into experimental groups receiving daily subcutaneous injections of either 7.5 IU/animal FSH, or 100 mg/kg b.w. T3, or both substances or the same volume of vehicle (control group) until day 15 of life. The animals were sacrificed on day 50. Morphometric analysis and immunohistochemical reactions were performed using antibodies against Vimentin, Proliferating Cell Nuclear Antigen and Connexin 43 in the testis. Sertoli cell count, efficiency of spermatogenesis, and hormonal pattern were examined. Disturbances in the connexin 43 expression reduced the number of Sertoli cells, the efficiency of spermatogenesis and impaired endocrine function of testes in adult rats treated with FSH and T3 during puberty. Stimulation with FSH alone increased Sertoli cell number, but was associated with a negative effect on cell-to-cell connexin 43-dependent communication, with a consequential reduction of spermatogenesis efficiency. J. Exp. Zool. 323A: 256-265, 2015. © 2015 Wiley Periodicals, Inc.

  5. Effect of Beta-Carotene on Oxidative Stress and Expression of Cardiac Connexin 43

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    Novo, Rosangela; Azevedo, Paula S.; Minicucci, Marcos F.; Zornoff, Leonardo A. M., E-mail: lzornoff@fmb.unesp.br; Paiva, Sergio A. R. [Faculdade de Medicina de Botucatu - Universidade Estadual Paulista ' Júlio de Mesquita Filho' , Botucatu, SP (Brazil)

    2013-09-15

    Intervention studies have shown an increased mortality in patients who received beta-carotene. However, the mechanisms involved in this phenomenon are still unknown. Evaluate the influence of beta-carotene on oxidative stress and the expression of connexin 43 in rat hearts. Wistar rats, weighing approximately 100 g, were allocated in two groups: Control Group (n = 30), that received the diet routinely used in our laboratory, and Beta-Carotene Group (n = 28), which received beta-carotene (in crystal form, added and mixed to the diet) at a dose of 500 mg of beta carotene/kg of diet. The animals received the treatment until they reached 200-250g, when they were sacrificed. Samples of blood, liver and heart were collected to perform Western blotting and immunohistochemistry for connexin 43; morphometric studies, dosages of beta carotene by high performance liquid chromatography as well as reduced glutathione, oxidized glutathione and lipids hydroperoxides were performed by biochemical analysis. Beta-carotene was detected only in the liver of Beta-Carotene Group animals (288 ± 94.7 μg/kg). Levels of reduced/ oxidized glutathione were higher in the liver and heart of Beta-Carotene Group animals (liver - Control Group: 42.60 ± 1.62; liver - Beta-Carotene Group: 57.40 ± 5.90; p = 0.04; heart: - Control Group: 117.40 ± 1.01; heart - Beta-Carotene Group: 121.81 ± 1.32 nmol/mg protein; p = 0.03). The content of total connexin 43 was larger in Beta-Carotene Group. Beta-carotene demonstrated a positive effect, characterized by the increase of intercellular communication and improvement of anti-oxidizing defense system. In this model, mechanism does not explain the increased mortality rate observed with the beta-carotene supplementation in clinical studies.

  6. Connexin43 recruits PTEN and Csk to inhibit c-Src activity in glioma cells and astrocytes

    Science.gov (United States)

    González-Sánchez, Ana; Jaraíz-Rodríguez, Myriam; Domínguez-Prieto, Marta; Herrero-González, Sandra; Medina, José M.; Tabernero, Arantxa

    2016-01-01

    Connexin43 (Cx43), the major protein forming gap junctions in astrocytes, is reduced in high-grade gliomas, where its ectopic expression exerts important effects, including the inhibition of the proto-oncogene tyrosine-protein kinase Src (c-Src). In this work we aimed to investigate the mechanism responsible for this effect. The inhibition of c-Src requires phosphorylation at tyrosine 527 mediated by C-terminal Src kinase (Csk) and dephosphorylation at tyrosine 416 mediated by phosphatases, such as phosphatase and tensin homolog (PTEN). Our results showed that the antiproliferative effect of Cx43 is reduced when Csk and PTEN are silenced in glioma cells, suggesting the involvement of both enzymes. Confocal microscopy and immunoprecipitation assays confirmed that Cx43, in addition to c-Src, binds to PTEN and Csk in glioma cells transfected with Cx43 and in astrocytes. Pull-down assays showed that region 266–283 in Cx43 is sufficient to recruit c-Src, PTEN and Csk and to inhibit the oncogenic activity of c-Src. As a result of c-Src inhibition, PTEN was increased with subsequent inactivation of Akt and reduction of proliferation of human glioblastoma stem cells. We conclude that the recruitment of Csk and PTEN to the region between residues 266 and 283 within the C-terminus of Cx43 leads to c-Src inhibition. PMID:27391443

  7. The protective effect of functional connexin43 channels on a human epithelial cell line exposed to oxidative stress.

    Science.gov (United States)

    Hutnik, Cindy M L; Pocrnich, Cady E; Liu, Hong; Laird, Dale W; Shao, Qing

    2008-02-01

    To determine the role of connexin43 (Cx43) and gap junctional intercellular communication (GJIC) in the response of the human retinal pigment epithelial cell line ARPE-19 to oxidative stress. ARPE-19 cells were treated with the chemical oxidant tert-butyl hydroperoxide (t-BOOH), and cell viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. GJIC was evaluated by scrape loading/dye transfer and microinjection assays, and Cx43 expression was detected by Western blot and immunofluorescent staining combined with confocal microscopy analysis. Retroviral infection of ARPE-19 cells with shRNA vectors targeting Cx43 or vectors encoding Cx43, Cx26, and a disease-linked dominant negative Cx43 mutant (G21R) were used, and the effect on cell viability was assessed. t-BOOH-induced ARPE-19 cell death was correlated with reductions in GJIC and in the total level of Cx43 protein expression. Overexpression of Cx26 and Cx43 increased the viability of oxidant-treated ARPE-19 cells. Conversely, shRNA knockdown of Cx43, expression of a disease-linked dominant negative Cx43 mutant, and blocking GJIC with 18beta-glycyrrhetinic acid and flufenamic acid all increased t-BOOH-induced ARPE-19 cell death. Cx43-mediated protection of ARPE-19 cells from oxidative stress-induced death is dependent on functional Cx43 channels.

  8. Omega-3 fatty acids and atorvastatin affect connexin 43 expression in the aorta of hereditary hypertriglyceridemic rats.

    Science.gov (United States)

    Dlugosová, Katarína; Weismann, Peter; Bernátová, Iveta; Sotníková, Ruzena; Slezák, Ján; Okruhlicová, Ludmila

    2009-12-01

    Statins and omega-3 polyunsaturated fatty acids (n-3 PUFA) reduce cardiovascular disease incidence during hypertriglyceridemia (HTG). To elucidate possible cardioprotective mechanisms, we focused on gap junction protein connexin 43 (Cx43). Its expression is disturbed during atherogenesis, but little information is available on its expression during HTG. Experiments were performed on adult male hereditary HTG (hHTG) rats treated with n-3 PUFA (30 mg/day) and atorvastatin (0.5 mg/100 g body weight per day) for 2 months. Cx43 expression and distribution in the aorta were investigated by using Western blotting and immunolabeling, followed by quantitative analysis. Transmission electronmicroscopy was used to study ultrastructure of endothelial contact sites. In contrast to age-matched Wistar, Cx43 expression in aorta of hHTG rats was significantly higher (p < 0.05), and prominent Cx43 immunospots were seen in tunica media and less in endothelium of hHTG rats. Changes in Cx43 expression were accompanied by local qualitative subcellular alterations of interendothelial connections. Treatment of hHTG rats with n-3 PUFA and atorvastatin markedly lowered Cx43 expression in aorta and modified connexin distribution in endothelium and media (p < 0.05 vs. untreated hHTG). The protective effect of treatment of HTG was observed on the structural integrity of the endothelium and was readily visible at the molecular level. Results indicate the involvement of altered Cx43 expression in vascular pathophysiology during HTG and during HTG treatment.

  9. [Impacts of early metoprolol intervention on connexin 43 and phosphorylated connexin 43 expression in rabbits with experimental myocardial infarction].

    Science.gov (United States)

    Zhou, M; Lu, Q; Jiang, J Q; Chen, Z N; Gong, Z G; Li, Z G; Fu, W W; Ding, S F

    2017-04-24

    Objective: To investigate the early intervention effects of metoprolol on connexin 43(Cx43) and phosphorylated Cx43 (p-Cx43) expression in rabbits with post myocardial infarction. Methods: A total of 24 adult male New Zealand white rabbits were divided into sham group (n=6), early treatment group(n=6), routine treatment group(n=6), and myocardial infarction group(n=6) with a randomized block design blocked by weight. Myocardial infarction was induced by left anterior descending coronary artery (LAD) ligation. Rabbits in sham group received similar surgical procedure without LAD ligation. Metoprolol (12.5 mg/kg dissolved in 2 ml distilled water) was applied to rabbits in early treatment group and routine treatment group per gavage immediately after recovery from anesthesia and at 24 hours after myocardial infarction, respectively, then treated daily for 40 days. Rabbits in sham group and myocardial infarction group received 2 ml distilled water per gavage daily for 40 days. Plasma lactate dehydrogenase (LDH) and creatine kinase (CK) level were detected by automatic biochemistry analyzer after 6 hours in all rabbits. Ventricular fibrillation threshold (VFT) was measured in vivo by bipolar pacing electrodes at 40 days. Cx43 and p-Cx43 distribution in ventricular tissue was detected by immunofluorescence analyses. Cx43 and p-Cx43 protein level in ventricular tissue was determined by Western blot. Results: (1) Plasma LDH ((851.7±85.9)U/L vs. (332.3±39.6)U/L, Pmyocardial infarction group than in sham group (both Pmyocardial infarction group than that in sham group ((470.0±91.0) beats per minute vs. (683.3±60.9) beats per minute, Pmyocardial infarction group (both Pmyocardial infarction group. The expression of p-Cx43 in myocardial infarction group was less than in sham group, which was significantly upregulated in in early treatment group and routine treatment group when compared with myocardial infarction group, and expression of p-Cx43 was significantly higher in

  10. Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content.

    Science.gov (United States)

    Oloris, Silvia Catarina Salgado; Mesnil, Marc; Reis, Viviane Neri de Souza; Sakai, Mônica; Matsuzaki, Patrícia; Fonseca, Evelise de Souza Monteiro; da Silva, Tereza Cristina; Avanzo, José Luís; Sinhorini, Idércio Luiz; Guerra, José Luiz; Costa-Pinto, Frederico Azevedo; Maiorka, Paulo Cesar; Dagli, Maria Lúcia Zaidan

    2007-03-06

    Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.

  11. [The effect of Connexin43 downregulation on biological functions of HUVEC].

    Science.gov (United States)

    Zhang, Cai-zhen; Mu, Xiao-feng; Xu, Xian-xiang; Qiu, Fei; Lin, Jun-sheng; Diao, Yong

    2015-03-01

    Connexin43 has been shown to play a pivotal role in wound healing process. Wound repair is enhanced by acute downregulation of connexin43, by increasing proliferation and migration of keratinocyte and fibroblast. Angiogenesis is also a central feature of wound repair, but little is known about the effects of connexin43 modulation on functions of endothelial cells. We used connexin43 specific small interference RNA (siRNA) to reduce the expression of connexin43 in human umbilical vein endothelial cell (HUVEC), and investigated the effects of connexin43 downregulation on intercellular communication, viability, proliferation, migration and angiogenic activity of HUVEC. Treatment of siRNA markedly reduced the expression of connexin43 by -80% in HUVEC (P HUVEC decreased significantly (P < 0.05), compared with that of the normal cells. The results suggest that temporally downregulation of connexin43 expression at early stage of wound to inhibit the abnormal angiogenesis characterized with leaky and inflamed blood vessels, maybe a prerequisite for coordinated normal healing process.

  12. Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts.

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    Shuo Liu

    Full Text Available Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases

  13. 3,5,4′-tri-O-acetylresveratrol Ameliorates Seawater Exposure-Induced Lung Injury by Upregulating Connexin 43 Expression in Lung

    Directory of Open Access Journals (Sweden)

    Lijie Ma

    2013-01-01

    Full Text Available The aim of the present study was to examine the effects of 3,5,4′-tri-O-acetylresveratrol on connexin 43 (Cx43 in acute lung injury (ALI in rats induced by tracheal instillation of artificial seawater. Different doses (50, 150, and 450 mg/kg of 3,5,4′-tri-O-acetylresveratrol were administered orally for 7 days before modeling. Four hours after seawater inhalation, histological changes, contents of TNF-α, IL-1β and IL-10, and the expression of Cx43 in lungs were detected. Besides, the gap junction communication in A549 cells and human umbilical vein endothelial cells (HUVECs challenged by seawater was also evaluated. Histological changes, increased contents of inflammatory factors, upregulation in gene level, and deregulation in protein level of Cx43 in lungs stimulated by seawater were observed. On the other hand, pretreatment with 3,5,4′-tri-O-acetylresveratrol significantly inhibited infiltration of inflammation, development of pulmonary edema, and contents of inflammatory mediators in lungs. Above all, 3,5,4′-tri-O-acetylresveratrol upregulated the expression of Cx43 in both gene and protein levels, and its intermediate metabolite, resveratrol, also enhanced the gap junction communication in the two cell lines. The results of the present study suggested that administration of 3,5,4′-tri-O-acetylresveratrol may be beneficial for treatment of inflammatorycellsin lung.

  14. Beta Lactams Antibiotic Ceftriaxone Modulates Seizures, Oxidative Stress and Connexin 43 Expression in Hippocampus of Pentylenetetrazole Kindled Rats

    Science.gov (United States)

    Hussein, Abdelaziz M.; Ghalwash, Mohammed; Magdy, Khaled; Abulseoud, Osama A.

    2016-01-01

    Background and Purpose: This study aimed to investigate the effect of ceftriaxone on oxidative stress and gap junction protein (connexin 43, Cx-43) expression in pentylenetetrazole (PTZ) induced kindling model. Methods: Twenty four Sprague dawely rats were divided into 3 equal groups (a) normal group: normal rats. (b) PTZ kindled group: received PTZ at the dose of 50 mg/kg via intraperitoneal injection (i.p.) every other day for 2 weeks (c) ceftriaxone treated group: received ceftriaxone at the dose 200 mg\\kg/12 hrs via i.p. injection daily from the 6th dose of PTZ for 3 days. Racine score, latency before beginning the first myoclonic jerk and duration of the jerks used as parameters of behavioral assessment. Immunohistopathological study for Cx-43 expression in hippocampus and measurement of markers of oxidative stress (malondialdehyde [MDA], low reduced glutathione [GSH] and catalase [CAT]) in hippocampal neurons were done. Results: PTZ kindling was associated with behavioral changes (in the form high stage of Racine score, long seizure duration and short latency for the first jerk), enhanced oxidative stress state (as demonstrated by high MDA, low GSH and CAT) and up regulation of Cx43 in hippocampal regions. While, ceftriaxone treatment ameliorated, significantly, PTZ-induced convulsions and caused significant improvement in oxidative stress markers and Cx-43 expression in hippocamal regions (p < 0.05). Conclusions: These findings support the anticonvulsive effects of some beta-lactams antibiotics which could offer a possible contributor in the basic treatment of temporal lobe epilepsy. This effect might be due to reduction of oxidative stress and Cx43 expression. PMID:27390674

  15. High glucose induces dysfunction of airway epithelial barrier through down-regulation of connexin 43.

    Science.gov (United States)

    Yu, Hongmei; Yang, Juan; Zhou, Xiangdong; Xiao, Qian; Lü, Yang; Xia, Li

    2016-03-01

    The airway epithelium is a barrier to the inhaled antigens and pathogens. Connexin 43 (Cx43) has been found to play critical role in maintaining the function of airway epithelial barrier and be involved in the pathogenesis of the diabetic retinal vasculature, diabetes nephropathy and diabetes skin. Hyperglycemia has been shown to be an independent risk factor for respiratory infections. We hypothesize that the down-regulation of Cx43 induced by HG alters the expression of tight junctions (zonula occludens-1 (ZO-1) and occludin) and contributes to dysfunction of airway epithelial barrier, and Cx43 plays a critical role in the process in human airway epithelial cells (16 HBE). We show that high glucose (HG) decreased the expression of ZO-1 and occludin, disassociated interaction between Cx43 and tight junctions, and then increased airway epithelial transepithelial electrical resistance (TER) and permeability by down-regulation of Cx43 in human airway epithelial cells. These observations demonstrate an important role for Cx43 in regulating HG-induced dysfunction of airway epithelial barrier. These findings may bring new insights into the molecular pathogenesis of pulmonary infection related to diabetes mellitus and lead to novel therapeutic intervention for the dysfunction of airway epithelial barrier in chronic inflammatory airway diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Transition from Preinvasive Carcinoma In Situ to Seminoma Is Accompanied by a Reduction of Connexin 43 Expression in Sertoli Cells and Germ Cells1

    Science.gov (United States)

    Brehm, Ralph; Rüttinger, Christina; Fischer, Petra; Gashaw, Isabella; Winterhager, Elke; Kliesch, Sabine; Bohle, Rainer M; Steger, Klaus; Bergmann, Martin

    2006-01-01

    Abstract Carcinoma in situ (CIS) represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap junctional proteins, connexin 43 (Cx43) represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development. PMID:16820096

  17. HIV-Associated Cardiovascular Disease: Role of Connexin 43.

    Science.gov (United States)

    Prevedel, Lisa; Morocho, Camilla; Bennett, Michael V L; Eugenin, Eliseo A

    2017-09-01

    Chronic HIV infection due to effective antiretroviral treatment has resulted in a broad range of clinical complications, including accelerated heart disease. Individuals with HIV infection have a 1.5 to 2 times higher incidence of cardiovascular diseases than their uninfected counterparts; however, the underlying mechanisms are poorly understood. To explore the link between HIV infection and cardiovascular diseases, we used postmortem human heart tissues obtained from HIV-infected and control uninfected individuals to examine connexin 43 (Cx43) expression and distribution and HIV-associated inflammation. Here, we demonstrate that Cx43 is dysregulated in the hearts of HIV-infected individuals. In all HIV heart samples analyzed, there were areas where Cx43 was overexpressed and found along the lateral membrane of the cardiomyocyte and in the intercalated disks. Areas of HIV tissue with anomalous Cx43 expression and localization also showed calcium overload, sarcofilamental atrophy, and accumulation of collagen. All these changes were independent of viral replication, CD4 counts, inflammation, and type of antiretroviral treatment. Overall, we propose that HIV infection increases Cx43 expression in heart, resulting in tissue damage that likely contributes to the high rates of cardiovascular disease in HIV-infected individuals. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  18. Connexin43 expression levels influence intercellular coupling and cell proliferation of native murine cardiac fibroblasts.

    Science.gov (United States)

    Zhang, Yan; Kanter, Evelyn M; Laing, James G; Aprhys, Colette; Johns, David C; Kardami, Elissavet; Yamada, Kathryn A

    2008-09-01

    Little is known about connexin expression and function in murine cardiac fibroblasts. The authors isolated native ventricular fibroblasts from adult mice and determined that although they expressed both connexin43 (Cx43) and connexin45 (Cx45), the relative abundance of Cx45 was greater than that of Cx43 in fibroblasts compared to myocytes, and the electrophoretic mobility of both Cx43 and Cx45 differed in fibroblasts and in myocytes. Increasing Cx43 expression by adenoviral infection increased intercellular coupling, whereas decreasing Cx43 expression by genetic ablation decreased coupling. Interestingly, increasing Cx43 expression reduced fibroblast proliferation, whereas decreasing Cx43 expression increased proliferation. These data demonstrate that native fibroblasts isolated from the mouse heart exhibit intercellular coupling via gap junctions containing both Cx43 and Cx45. Fibroblast proliferation is inversely related to the expression level of Cx43. Thus, connexin expression and remodeling is likely to alter fibroblast function, maintenance of the extracellular matrix, and ventricular remodeling in both normal and diseased hearts.

  19. Inhibition of Connexin43 Hemichannels Impairs Spatial Short-Term Memory without Affecting Spatial Working Memory.

    Science.gov (United States)

    Walrave, Laura; Vinken, Mathieu; Albertini, Giulia; De Bundel, Dimitri; Leybaert, Luc; Smolders, Ilse J

    2016-01-01

    Astrocytes are active players in higher brain function as they can release gliotransmitters, which are essential for synaptic plasticity. Various mechanisms have been proposed for gliotransmission, including vesicular mechanisms as well as non-vesicular ones, for example by passive diffusion via connexin hemichannels (HCs). We here investigated whether interfering with connexin43 (Cx43) HCs influenced hippocampal spatial memory. We made use of the peptide Gap19 that blocks HCs but not gap junction channels and is specific for Cx43. To this end, we microinfused transactivator of transcription linked Gap19 (TAT-Gap19) into the brain ventricle of male NMRI mice and assessed spatial memory in a Y maze. We found that the in vivo blockade of Cx43 HCs did not affect the locomotor activity or spatial working memory in a spontaneous alternation Y maze task. Cx43 blockade did however significantly impair the spatial short-term memory in a delayed spontaneous alternation Y maze task. These results indicate that Cx43 HCs play a role in spatial short-term memory.

  20. Inhibition of Connexin43 Hemichannels Impairs Spatial Short-Term Memory without Affecting Spatial Working Memory

    Science.gov (United States)

    Walrave, Laura; Vinken, Mathieu; Albertini, Giulia; De Bundel, Dimitri; Leybaert, Luc; Smolders, Ilse J.

    2016-01-01

    Astrocytes are active players in higher brain function as they can release gliotransmitters, which are essential for synaptic plasticity. Various mechanisms have been proposed for gliotransmission, including vesicular mechanisms as well as non-vesicular ones, for example by passive diffusion via connexin hemichannels (HCs). We here investigated whether interfering with connexin43 (Cx43) HCs influenced hippocampal spatial memory. We made use of the peptide Gap19 that blocks HCs but not gap junction channels and is specific for Cx43. To this end, we microinfused transactivator of transcription linked Gap19 (TAT-Gap19) into the brain ventricle of male NMRI mice and assessed spatial memory in a Y maze. We found that the in vivo blockade of Cx43 HCs did not affect the locomotor activity or spatial working memory in a spontaneous alternation Y maze task. Cx43 blockade did however significantly impair the spatial short-term memory in a delayed spontaneous alternation Y maze task. These results indicate that Cx43 HCs play a role in spatial short-term memory. PMID:28066184

  1. Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex.

    Science.gov (United States)

    Liu, Xiuxin; Sun, Lin; Torii, Masaaki; Rakic, Pasko

    2012-05-22

    The prospective pyramidal neurons, migrating from the proliferative ventricular zone to the overlaying cortical plate, assume multipolar morphology while passing through the transient subventricular zone. Here, we show that this morphogenetic transformation, from the bipolar to the mutipolar and then back to bipolar again, is associated with expression of connexin 43 (Cx43) and, that knockdown of Cx43 retards, whereas its overexpression enhances, this morphogenetic process. In addition, we have observed that knockdown of Cx43 reduces expression of p27, whereas overexpression of p27 rescues the effect of Cx43 knockdown in the multipolar neurons. Furthermore, functional gap junction/hemichannel domain, and the C-terminal domain of Cx43, independently enhance the expression of p27 and promote the morphological transformation and migration of the multipolar neurons in the SVZ/IZ. Collectively, these results indicate that Cx43 regulates the passage of migrating neurons through their multipolar stage via p27 signaling and that interference with this process, by either genetic and/or environmental factors, may cause cortical malformations.

  2. Gap junctions - guards of excitability.

    Science.gov (United States)

    Stroemlund, Line Waring; Jensen, Christa Funch; Qvortrup, Klaus; Delmar, Mario; Nielsen, Morten Schak

    2015-06-01

    Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV1.5 to the membrane.

  3. Downregulation of Rho associated coiled-coil forming protein kinase 1 in the process of delayed myocardialization of cardiac proximal outflow tract septum in connexin 43 knockout mice embryo

    Institute of Scientific and Technical Information of China (English)

    QI Chun-hua; ZHAO Xiao-qing; MA Duan; MA Xiao-jing; ZHOU Guo-min; HUANG Guo-ying

    2011-01-01

    Background The connexln43 knockout (Cx43 KO) mouse dies at birth with an enlarged conotruncal region, which leads to the obstruction of the right outflow tract (OFT). Since myocardialization of the proximal OFT septum is one of the key events during heart development, we investigated the process in the Cx43 KO embryo hearts. Rho associated coiled-coil forming protein kinase 1 (ROCK1), is a recently found key molecule to regulate the myocardialization of OFT, but its spatiotemporal expression pattern during myocardialization remains unknown. The objective of this study was to investigate the differentially expressed pattern of ROCK1 between Cx43 KO and wild type embryo hearts, and its relationship with the delayed myocardialization in Cx43 KO embryo hearts.Methods Using immunohistochemistry, the processes of myocardiolization were investigated both in Cx43 KO and wild type embryo hearts. The differentially expressed pattern of ROCK1 between Cx43 KO and wildtype embryo hearts was evaluated both at the mRNA and protein level by real-time RT-PCR and immunohistochemistry.Results The expression of α-sarcomeric actin (α-SCA) in the proximal OFT septum of Cx43 KO embryos was delayed. Meanwhile, it was shown that the downregulation of ROCK1 coincided with delayed myocardialization. The expression of ROCK1 protein was mainly limited to the proximal outflow tract septum from embryo day (E) E11.5 to E15.5. Its expression pattern was similar with that of α-SCA. Real-time RT-PCR found that the expression level of Rock-1 mRNA began at a low level on E11.5 and reached peak at E13.5 and E14.5.Conclusions ROCK1 may have an important role in the process of myocardialization of the proximal OFT septum. Downregulation of ROCK1 is likely to contribute to the aberrant myocardialization in Cx43 KO embryo hearts.

  4. Suppression of cell membrane permeability by suramin: involvement of its inhibitory actions on connexin 43 hemichannels.

    Science.gov (United States)

    Chi, Yuan; Gao, Kun; Zhang, Hui; Takeda, Masayuki; Yao, Jian

    2014-07-01

    Suramin is a clinically prescribed drug for treatment of human African trypanosomiasis, cancer and infection. It is also a well-known pharmacological antagonist of P2 purinoceptors. Despite its clinical use and use in research, the biological actions of this molecule are still incompletely understood. Here, we investigated the effects of suramin on membrane channels, as exemplified by its actions on non-junctional connexin43 (Cx43) hemichannels, pore-forming α-haemolysin and channels involved in ATP release under hypotonic conditions. Hemichannels were activated by removing extracellular Ca(2+) . The influences of suramin on hemichannel activities were evaluated by its effects on influx of fluorescent dyes and efflux of ATP. The membrane permeability and integrity were assessed through cellular retention of preloaded calcein and LDH release. Suramin blocked Cx43 hemichannel permeability induced by removal of extracellular Ca(2+) without much effect on Cx43 expression and gap junctional intercellular communication. This action of suramin was mimicked by its analogue NF023 and NF449 but not by another P2 purinoceptor antagonist PPADS. Besides hemichannels, suramin also significantly blocked intracellular and extracellular exchanges of small molecules caused by α-haemolysin from Staphylococcus aureus and by exposure of cells to hypotonic solution. Furthermore, it prevented α-haemolysin- and hypotonic stress-elicited cell injury. Suramin blocked membrane channels and protected cells against toxin- and hypotonic stress-elicited injury. Our finding provides novel mechanistic insights into the pharmacological actions of suramin. Suramin might be therapeutically exploited to protect membrane integrity under certain pathological situations. © 2014 The British Pharmacological Society.

  5. Involvement of connexin43 hemichannel in ATP release after γ-irradiation

    Science.gov (United States)

    Ohshima, Yasuhiro; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Kojima, Shuji

    2012-01-01

    Ionizing radiation induces biological effects not only in irradiated cells but also in non-irradiated cells, which is called the bystander effect. Recently, in vivo and in vitro experiments have suggested that both gap junction hemichannel connexin43 (Cx43) and extracellular adenosine triphosphate (ATP) released from cells play a role in the bystander effect. We have reported that γ-irradiation induces ATP release from B16 melanoma cells, which is dependent on the P2X7 receptor. However, the mechanism of ATP release caused by irradiation remains unclear. We here show the involvement of Cx43 in P2X7 receptor-dependent ATP release after 0.5 Gy γ-irradiation. Inhibitors of gap junction hemichannels and an inhibitory peptide for Cx43 (gap26), but not an inhibitory peptide for pannexin1 (Panx1), significantly blocked γ-irradiation-induced ATP release from B16 melanoma cells. We confirmed high expression of Cx43 mRNA in B16 melanoma cells. These results suggest involvement of Cx43 in radiation-induced ATP release. We found that after 0.5 Gy γ-irradiation tyrosine phosphorylation was significantly blocked by P2X7 receptor antagonist, but not gap26, suggesting that tyrosine phosphorylation is a downstream event from the P2X7 receptor. Since tyrosine kinase inhibitor significantly suppressed radiation-induced ATP release, tyrosine phosphorylation appears to play an important role in the Cx43-mediated ATP release downstream of the P2X7 receptor. In conclusion, the Cx43 hemichannel, which lies downstream of the P2X7 receptor, is involved in ATP release in response to radiation. Our results suggest a novel mechanism for radiation-induced biological effects mediated by both ATP and Cx43. PMID:22843620

  6. Connexin43 contributes to electrotonic conduction across scar tissue in the intact heart

    Science.gov (United States)

    Mahoney, Vanessa M.; Mezzano, Valeria; Mirams, Gary R.; Maass, Karen; Li, Zhen; Cerrone, Marina; Vasquez, Carolina; Bapat, Aneesh; Delmar, Mario; Morley, Gregory E.

    2016-05-01

    Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression.

  7. HIF-1 and c-Src mediate increased glucose uptake induced by endothelin-1 and connexin43 in astrocytes.

    Directory of Open Access Journals (Sweden)

    José Carlos Valle-Casuso

    Full Text Available In previous work we showed that endothelin-1 (ET-1 increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43, the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism.

  8. Effect of doxycycline on connexin 43 protein expression of cardiomyoblast cells in anoxic rat%多西环素对缺氧大鼠心肌细胞缝隙连接蛋白43表达的影响

    Institute of Scientific and Technical Information of China (English)

    罗刚; 巫相宏; 安迪; 黄文

    2013-01-01

    目的 探讨多西环素对缺氧大鼠H9c2心肌细胞缝隙连接蛋白43 (connexin 43,Cx 43)表达的影响及其可能的机制.方法 培养大鼠H9c2心肌细胞,用Western Blotting法检测缺氧6h、12h、24h后Cx43总蛋白的表达量及基质金属酶抑制剂(多西环素)、PI3K抑制剂(LY294002)、ERK1/2抑制剂(U0126)干预缺氧H9c2心肌细胞后Cx43总蛋白的表达量改变.结果 缺氧组较空白对照组Cx43总蛋白表达量明显降低,差异有统计学意义(P<0.01).缺氧6h时,多西环素和U0126干预组较缺氧组Cx43总蛋白表达量明显增高,差异有统计学意义(P<0.05);缺氧12h时,U0126干预组Cx43总蛋白表达量仍较缺氧组增高,差异有统计学意义(P<0.05);缺氧24 h时,多西环素和U0126干预后Cx43总蛋白表达量与缺氧组比较,差异无统计学意义(P>0.0.05).LY294002干预组Cx43总蛋白表达量在观察时间范围内均较缺氧组显著降低,差异有统计学意义(P<0.01).结论 在培养的大鼠H9c2心肌细胞中,多西环素通过抑制基质金属酶增加缺氧心肌细胞Cx43总蛋白表达量,该作用至少部分抑制由ERK1/2信号介导的传导通路.%Objectives To investigate the effects and the possible mechanisms of doxycycline on the expression of connexin 43 (Cx43) in rat H9c2 cardiomyoblast cells under hypoxic conditions.Methods H9c2 cardiomyoblast cells were cultured with hypoxia treatment for 6 h,12 h and 24 h.Expressions of Cx43 in cardiomyocytes were quantified by Western Blotting,as well as the changes intervened by matrix metalloproteinases (MMPs) inhibitor (doxycycline),PI3K inhibitor (LY294002),ERK1/2 inhibitor (U0126) under hypoxic conditions.Results Expression of Cx43 in hypoxia group was obviously lower than that in normal control group (P<0.01).Expressions of Cx43 in doxycycline group and U0126 group were obviously higher than that in hypoxia group after 6 h of hypoxia (P<0.05).Expression of Cx43 in U0126 group was still obviously

  9. Altered expression of connexin43 and its possible role in endothelin - 1 - induced contraction in rabbit basilar artery%缝隙连接蛋白43在内皮素诱导的脑基底动脉收缩中的表达变化

    Institute of Scientific and Technical Information of China (English)

    赵万巨; 王海涛; 洪涛

    2011-01-01

    目的 探讨Cx43在内皮素诱导的脑基底动脉收缩中的表达变化及其可能的作用.方法 血管环张力实验检测内皮素诱导的脑基底动脉的收缩变化并应用Western blot检测基底动脉Cx43蛋白的表达变化,染料传输实验用来检测脑基底动脉收缩过程中平滑肌细胞间缝隙连接的功能变化.结果 浓度递增的内皮素导致脑基底动脉呈显著浓度依赖性的收缩,一定浓度缝隙连接阻断剂苷珀酸显著缓解该收缩;收缩过程中,Cx43的蛋白表达呈显著时间依赖性的升高,苷珀酸减弱该表达的升高;内皮素刺激下,血管平滑肌细胞间的染料传输呈时间依赖性的升高,苷珀酸显著减少染料在细胞间的传输.结论 脑血管痉挛过程中,通过增加Cx43的表达,血管细胞间缝隙连接的功能被内皮素激活并在血管痉挛病理过程中发挥重要作用;抑制缝隙连接的功能是有效缓解蛛网膜下腔出血后脑血管痉挛的新途径.%Objective To investigate the role of connexin43 in ET- 1 -induced contraction in rabbit basilar artery.Methods The ET - 1 - induced contraction without or with carbenoxolone was studied with an isometric tension system.The expression of connexin43 protein in ET - t stimulated basilar arteries was studied with Western blot.Scrape/scratch method was used to analyze the function of gap junction in cultured rabbit cerebrovascular smooth muscle ceils.Results ET - 1 produced a concentration - dependent contraction.Carbenoxolone inhibited ET- 1 induced contraction.The connexin43 protein level was increased in ET- 1stimulated basilar arteries.Carbenoxolone decreased the connexin43 protein level increased by ET - 1.Cells treated with ET - 1 appeared positive communication and the dye transfer was increased in a time - dependent fashion.Carbenoxolone suppressed the ET - 1 - induced increasement of dye transfer.Conclusions The enhancement of gap junction intercellular communication is

  10. Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria.

    Science.gov (United States)

    Boengler, Kerstin; Stahlhofen, Sabine; van de Sand, Anita; Gres, Petra; Ruiz-Meana, Marisol; Garcia-Dorado, David; Heusch, Gerd; Schulz, Rainer

    2009-03-01

    Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase alpha (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 microg/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 microg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 +/- 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 +/- 4%, n = 7), whereas the immunoreactivities of ATP synthase alpha (49.1 +/- 6.4%, n = 7) and MnSOD (79.9 +/- 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP's cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of

  11. Knockdown of connexin 43 attenuates balloon injury-induced vascular restenosis through the inhibition of the proliferation and migration of vascular smooth muscle cells.

    Science.gov (United States)

    Han, Xiao-Jian; He, Dan; Xu, Liang-Jing; Chen, Min; Wang, Yi-Qi; Feng, Jiu-Geng; Wei, Min-Jun; Hong, Tao; Jiang, Li-Ping

    2015-11-01

    Coronary artery disease (CAD) or atherosclerotic heart disease is one of the most common types of cardiovascular disease. Although percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] is a mature, well-established technique used to treat atherosclerotic heart disease, its long‑term therapeutic effects are compromised by a high incidence of vascular restenosis (RS) following angioplasty. In our previous study, we found that the principal gap junction protein, connexin 43 (Cx43), in vascular smooth muscle cells (VSMCs) was involved in the development of vascular RS following angioplasty-induced balloon injury. However, the exact role action of Cx43 in vascular RS remains unclear. In the present study, we aimed to further examine whether the knockdown of Cx43 attenuates the development of vascular RS through the inhibition of the proliferation and migration of VSMCs. We found that the use of a lentiviral vector expressing shRNA targeting Cx43 (Cx43‑RNAi-LV) efficiently silenced the mRNA and protein expression of Cx43 in cultured VSMCs. In addition, MTT and Transwell assays were used to examined the proliferation and migration of the VSMCs, respectively. The results revealed that the knockdown of Cx43 by Cx43-RNAi-LV at a multiplicity of infection (MOI) of 100 significantly inhibited the proliferation and migration of the VSMCs in vitro. Notably, the knockdown of Cx43 also effectively attenuated the development of vascular RS and intimal hyperplasia following balloon injury in vivo. Taken together, our data suggest that Cx43 is involved in the development of vascular RS and intimal hyperplasia through the regulation of the proliferation and migration of VSMCs. Thus, the present study provides new insight into the pathogenesis of vascular RS, and suggests that further comfirms that Cx43 may well be a novel potential pharmacological target for preventing vascular RS following PCI.

  12. Depression of Intraocular Pressure Following Inactivation of Connexin43 in the Nonpigmented Epithelium of the Ciliary Body

    Science.gov (United States)

    Calera, Mónica R.; Wang, Zhao; Sanchez-Olea, Roberto; Paul, David L.; Civan, Mortimer M.; Goodenough, Daniel A.

    2010-01-01

    Purpose Conditional inactivation of connexin43 (Cx43) in the pigmented epithelium of the mouse eye results in a reduction in aqueous humor production and complete loss of the vitreous chamber. It was proposed that gap junctions between pigmented and nonpigmented epithelia of the ciliary body are critical for the production of the aqueous humor. To form such junctions, Cx43 in the pigmented epithelium must interact with connexin(s) present in the adjacent cells of the nonpigmented epithelium. The importance of Cx43 expression in the nonpigmented epithelium for the establishment of gap junctions and the regulation of intraocular pressure was tested. Methods To inactivate Cx43 in the nonpigmented epithelium of the mouse eye, a mouse line was crossed with a floxed Cx43 locus (Cx43flox/flox) and a transgenic mouse line expressing cre recombinase under the control of the Pax6α promoter. General eye structure was evaluated by light microscopy, gap junctions were analyzed by electron microscopy, and intraocular pressure was directly assessed with micropipettes. Results In Pax6α-cre/Cx43flox/flox mice, Cx43 was partially inactivated in the nonpigmented epithelium of the ciliary body and iris. Animals developed dilatations between the pigmented and nonpigmented epithelia and displayed a significant reduction in intraocular pressure. However, gap junctions between the ciliary epithelial layers were decreased but not eliminated. Conclusions Cx43 expression in the nonpigmented epithelium of the ciliary body contributes to the formation of gap junctions with the cells of the pigmented epithelium. These gap junctions play a critical role in maintaining the physical integrity of the ciliary body epithelium. Although the partial loss of Cx43 from the nonpigmented epithelium was correlated with a measurable drop in intraocular pressure, possible changes in Cx43 in the aqueous outflow pathway may provide an additional contribution to the observed phenotype. PMID:19168903

  13. Hypoxia in high glucose followed by reoxygenation in normal glucose reduces the viability of cortical astrocytes through increased permeability of connexin 43 hemichannels

    Science.gov (United States)

    Orellana, Juan A.; Hernández, Diego E.; Ezan, Pascal; Velarde, Victoria; Bennett, Michael V. L.; Giaume, Christian; Sáez, Juan C.

    2009-01-01

    Brain ischemia causes more extensive injury in hyperglycemic than normoglycemic subjects, and the increased damage is to astroglia as well as neurons. In the present work, we found that in cortical astrocytes from rat or mouse, reoxygenation after hypoxia in a medium mimicking interstitial fluid during ischemia increases hemichannel activity and decreases cell-cell communication via gap junctions as indicated by dye uptake and dye coupling, respectively. These effects were potentiated by high glucose during the hypoxia in a concentration-dependent manner (and by zero glucose) and were not observed in connexin 43−/− astrocytes. The responses were transient or persistent after short and long periods of hypoxia, respectively. The persistent responses were associated with a progressive reduction in cell viability that was prevented by La3+ or peptides that block connexin 43 (Cx43) hemichannels or by inhibition of p38 MAP kinase prior to hypoxia-reoxygenation but not by treatments that block pannexin hemichannels. Block of Cx43 hemichannels did not affect the reduction in gap junction mediated dye coupling observed during reoxygenation. Cx43 hemichannels may be a novel therapeutic target to reduce cell death following stroke, particularly in hyperglycemic conditions. PMID:19705457

  14. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.

    Science.gov (United States)

    Boengler, Kerstin; Dodoni, Giuliano; Rodriguez-Sinovas, Antonio; Cabestrero, Alberto; Ruiz-Meana, Marisol; Gres, Petra; Konietzka, Ina; Lopez-Iglesias, Carmen; Garcia-Dorado, David; Di Lisa, Fabio; Heusch, Gerd; Schulz, Rainer

    2005-08-01

    Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP. Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without (n=13) or with (n=13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4+/-0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1+/-0.2, p<0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262+/-63% of baseline. These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.

  15. Connexin 43 reboots meiosis and reseals blood-testis barrier following toxicant-mediated aspermatogenesis and barrier disruption.

    Science.gov (United States)

    Li, Nan; Mruk, Dolores D; Mok, Ka-Wai; Li, Michelle W M; Wong, Chris K C; Lee, Will M; Han, Daishu; Silvestrini, Bruno; Cheng, C Yan

    2016-04-01

    Earlier studies have shown that rats treated with an acute dose of 1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide (adjudin, a male contraceptive under development) causes permanent infertility due to irreversible blood-testis barrier (BTB) disruption even though the population of undifferentiated spermatogonia remains similar to normal rat testes, because spermatogonia fail to differentiate into spermatocytes to enter meiosis. Since other studies have illustrated the significance of connexin 43 (Cx43)-based gap junction in maintaining the homeostasis of BTB in the rat testis and the phenotypes of Sertoli cell-conditional Cx43 knockout mice share many of the similarities of the adjudin-treated rats, we sought to examine if overexpression of Cx43 in these adjudin-treated rats would reseal the disrupted BTB and reinitiate spermatogenesis. A full-length Cx43 cloned into mammalian expression vector pCI-neo was used to transfect testes of adjudin-treated ratsversusempty vector. It was found that overexpression of Cx43 indeed resealed the Sertoli cell tight junction-permeability barrier based on a functionalin vivoassay in tubules displaying signs of meiosis as noted by the presence of round spermatids. Thus, these findings suggest that overexpression of Cx43 reinitiated spermatogenesis at least through the steps of meiosis to generate round spermatids in testes of rats treated with an acute dose of adjudin that led to aspermatogenesis. It was also noted that the round spermatids underwent eventual degeneration with the formation of multinucleated cells following Cx43 overexpression due to the failure of spermiogenesis because no elongating/elongated spermatids were detected in any of the tubules examined. The mechanism by which overexpression of Cx43 reboots meiosis and rescues BTB function was also examined. In summary, overexpression of Cx43 in the testis with aspermatogenesis reboots meiosis and reseals toxicant-induced BTB disruption, even though it fails to

  16. CONNEXIN-47 AND CONNEXIN-32 IN GAP JUNCTIONS OF OLIGODENDROCYTE SOMATA, MYELIN SHEATHS, PARANODAL LOOPS AND SCHMIDT-LANTERMAN INCISURES: IMPLICATIONS FOR IONIC HOMEOSTASIS AND POTASSIUM SIPHONING

    OpenAIRE

    KAMASAWA, N.; SIK, A.; Morita, M; Yasumura, T; Davidson, K. G. V.; Nagy, J.I.; RASH, J.E.

    2005-01-01

    The subcellular distributions and co-associations of the gap junction-forming proteins connexin47 and connexin32 were investigated in oligodendrocytes of adult mouse and rat CNS. By confocal immunofluorescence light microscopy, abundant connexin47 was co-localized with astrocytic connexin43 on oligodendrocyte somata, and along myelinated fibers, whereas connexin32 without connexin47 was co-localized with contactin-associated protein (caspr) in paranodes. By thin-section transmission electron ...

  17. Photoperiod-Dependent Effects of 4-tert-Octylphenol on Adherens and Gap Junction Proteins in Bank Vole Seminiferous Tubules

    Directory of Open Access Journals (Sweden)

    Anna Hejmej

    2013-01-01

    Full Text Available In the present study we evaluated in vivo and in vitro effects of 4-tert-octylphenol (OP on the expression and distribution of adherens and gap junction proteins, N-cadherin, β-catenin, and connexin 43 (Cx43, in testes of seasonally breeding rodents, bank voles. We found that in bank vole testes expression and distribution of N-cadherin, β-catenin, and Cx43 were photoperiod dependent. Long-term treatment with OP (200 mg/kg b.w. resulted in the reduction of junction proteins expressions (P<0.05, P<0.01 and their delocalization in the testes of males kept in long photoperiod, whereas in short-day animals slight increase of Cx43 (P<0.05, N-cadherin, and β-catenin (statistically nonsignificant levels was observed. Effects of OP appeared to be independent of FSH and were maintained during in vitro organ culture, indicating that OP acts directly on adherens and gap junction proteins in the testes. An experiment performed using an antiestrogen ICI 182,780 demonstrated that the biological effects of OP on β-catenin and Cx43 involve an estrogen receptor-mediated response. Taken together, in bank vole organization of adherens and gap junctions and their susceptibility to OP are related to the length of photoperiod. Alterations in cadherin/catenin and Cx43-based junction may partially result from activation of estrogen receptor α and/or β signaling pathway.

  18. Effects of extremely low frequency magnetic fields on gap junctional intercellular communication and its mechanism

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The study on biological effect of electromagnetic fields has been paid close attention in recent years. Gap junctional intercellular communication (GJIC) plays an important role in the maintenance of cell proliferation and differentiation, and in the multistage process of carcinogenesis. A series of researches showed that xtremely low frequency (ELF) magnetic fields not only enhance the inhibition of GJIC induced by 12-O-tetradecanoylphorbol-13-acetate, but also inhibit GJIC directly when the intensity is equal to or more than 0.4  mT, and that the mechanisms of GJIC inhibition by ELF magnetic fields are due to hyperphosphorylation of connexin 43, which is mediated by protein kinase C-activated signal transduction, and the internalization of connexin 43 from plasma membrane to cytoplasm.

  19. Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

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    Mehta Bhavya C

    2005-10-01

    Full Text Available Abstract Background The arachnoid granulations (AGs are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. Methods Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7–10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7–10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144 and their expression was quantified using flow cytometry analysis. Results Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. Conclusion To our knowledge, this is the first report of

  20. Occludin and connexin 43 expression contribute to the pathogenesis of traumatic brain edema*

    Institute of Scientific and Technical Information of China (English)

    Wanyin Ren; Guojie Jing; Qin Shen; Xiaoteng Yao; Yingchao Jing; Feng Lin; Weidong Pan

    2013-01-01

    The experimental model of traumatic brain injury was established in Sprague-Dawley rats according to Feeney’s free fal ing method. The brains were harvested at 2, 6 and 24 hours, and at 3 and 5 days after injury. Changes in brain water content were determined using the wet and dry weights. Our results showed that water content of tissue significantly increased after traumatic brain injury, and reached minimum at 24 hours. Hematoxylin-eosin staining revealed pathological impairment of brain tissue at each time point after injury, particularly at 3 days, with nerve celledema, degenera-tion, and necrosis observed, and the apoptotic rate significantly increased. Immunohistochemistry and western blot analysis revealed that the expression of occludin at the injured site gradual y de-creased as injury time advanced and reached a minimum at 3 days after injury; the expression of connexin 43 gradual y increased as injury time advanced and reached a peak at 24 hours after in-jury. The experimental findings indicate that changes in occludin and connexin 43 expression were consistent with the development of brain edema, and may reflect the pathogenesis of brain injury.

  1. Mechanism of Mitochondrial Connexin43′s Protection of the Neurovascular Unit under Acute Cerebral Ischemia-Reperfusion Injury

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    Shuai Hou

    2016-05-01

    Full Text Available We observed mitochondrial connexin43 (mtCx43 expression under cerebral ischemia-reperfusion (I/R injury, analyzed its regulation, and explored its protective mechanisms. Wistar rats were divided into groups based on injections received before middle cerebral artery occlusion (MCAO. Cerebral infarction volume was detected by 2,3,5-triphenyltetrazolim chloride staining, and cell apoptosis was observed by transferase dUTP nick end labeling. We used transmission electron microscopy to observe mitochondrial morphology and determined superoxide dismutase (SOD activity and malondialdehyde (MDA content. MtCx43, p-mtCx43, protein kinase C (PKC, and p-PKC expression were detected by Western blot. Compared with those in the IR group, cerebral infarction volumes in the carbenoxolone (CBX and diazoxide (DZX groups were obviously smaller, and the apoptosis indices were down-regulated. Mitochondrial morphology was damaged after I/R, especially in the IR and 5-hydroxydecanoic acid (5-HD groups. Similarly, decreased SOD activity and increased MDA were observed after MCAO; CBX, DZX, and phorbol-12-myristate-13-acetate (PMA reduced mitochondrial functional injury. Expression of mtCx43 and p-mtCx43 and the p-Cx43/Cx43 ratio were significantly lower in the IR group than in the sham group. These abnormalities were ameliorated by CBX, DZX, and PMA. MtCx43 may protect the neurovascular unit from acute cerebral IR injury via PKC activation induced by mitoKATP channel agonists.

  2. Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice.

    Science.gov (United States)

    Ganesan, Shanthi; Nteeba, Jackson; Keating, Aileen F

    2015-01-01

    The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) were dosed with sesame oil or DMBA (1mg/kg; ip) for 14days and ovaries collected 3days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (Pobese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (Pobesity while total CX37 protein was reduced (Pobese ovaries. Cx43 mRNA and total protein levels were decreased (Pobese ovaries while basal protein staining intensity was reduced (Pobese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (Pobesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Morphology and the expression of connexin 43 and 40 in bundles of transition cells in the adult atrioventricular junction%成人房室交界区过渡细胞束形态学及连接蛋白43和40的表达

    Institute of Scientific and Technical Information of China (English)

    宋慧芳; 吕华; 范益民; 夏仲年; 阎八一; 高雨仁; 杨桂姣

    2011-01-01

    目的 观察临床上心律失常常见发生部位及射频消融治疗靶点部位--房室交界区和邻近区域的形态学特点及连接蛋白(Cx)43和40的表达,为心律失常发生机制及可能的有效治疗部位提供形态学依据.方法 10例正常成人心脏,选取房室交界区及其邻近部位,常规石蜡包埋,HE、Masson染色,选定目标部位行Cx43、Cx40免疫组织化学染色,并进行图像及统计学分析.结果 峡部、冠状窦口周围和远端、二尖瓣纤维环处及与右房瓣上肌环邻近的中心纤维体等处可见较多过渡细胞束走行,并从不同方向与房室结及结后延伸不同部位相连.过渡细胞组成、排布、心肌纤维走行和相互间的联系有别于普通工作心肌.Cx43、Cx40在过渡细胞束1~4及右房瓣上肌环中分布范围小但集中,而在左房近膜部处的过渡细胞中表达较多;峡部Cx43、Cx40表达较集中.结论 房室交界区及其邻近区域存在双(多)径传导的形态学基础,且与目前常用的射频消融治疗心律失常靶点部位具有对应关系.%Objective To observe the morphological feature and the expression of connexin( Cx )43 and Cx40 in the common arrhythmogenesis and radiofrequenc:y ahlation( RFA ) region, the atrioventricular junction and its surrounding area, in order to provide the morphological basis for understanding the mechanism of arrhythmogenesis and possible sites for therapy.Methods Ten fresh normal adult hearts were used in this study.The specimens containing the atrioventricular node( AVN ) was harvested, embedded in paraffin and sectioned.The sections were stained with HE.Masson staining.Expression of Cx43 and Cx40 was examined by immunohistochemistry and image analysis.Results There were the bundles of transition cells at cavo-tricuspid isthmus, around the opening and distal end of coronary sinus, at the annulus fibrosus of bicuspid valve, and at the central fibrous body( CFB ) near the annulus of right

  4. Impact of obesity on 7,12-dimethylbenz[a]anthracene-induced altered ovarian connexin gap junction proteins in female mice

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-01-01

    The ovarian gap junction proteins alpha 4 (GJA4 or connexin 37; CX37), alpha 1 (GJA1 or connexin 43; CX43) and gamma 1 (GJC1 or connexin 45; CX45) are involved in cell communication and folliculogenesis. 7,12-dimethylbenz[a]anthracene (DMBA) alters Cx37 and Cx43 expression in cultured neonatal rat ovaries. Additionally, obesity has an additive effect on DMBA-induced ovarian cell death and follicle depletion, thus, we investigated in vivo impacts of obesity and DMBA on CX protein levels. Ovaries were collected from lean and obese mice aged 6, 12, 18, or 24 wks. A subset of 18 wk old mice (lean and obese) were dosed with sesame oil or DMBA (1 mg/kg; ip) for 14 days and ovaries collected 3 days thereafter. Cx43 and Cx45 mRNA and protein levels decreased (P < 0.05) after 18 wks while Cx37 mRNA and protein levels decreased (P < 0.05) after 24 wks in obese ovaries. Cx37 mRNA and antral follicle protein staining intensity were reduced (P < 0.05) by obesity while total CX37 protein was reduced (P < 0.05) in DMBA exposed obese ovaries. Cx43 mRNA and total protein levels were decreased (P < 0.05) by DMBA in both lean and obese ovaries while basal protein staining intensity was reduced (P < 0.05) in obese controls. Cx45 mRNA, total protein and protein staining intensity level were decreased (P < 0.05) by obesity. These data support that obesity temporally alters gap junction protein expression and that DMBA-induced ovotoxicity may involve reduced gap junction protein function. - Highlights: • Ovarian gap junction proteins are affected by ovarian aging and obesity. • DMBA exposure negatively impacts gap junction proteins. • Altered gap junction proteins may contribute to infertility.

  5. Elevated connexin 43 expression in arsenite-and cadmium-transformed human bladder cancer cells, tumor transplants and selected high grade human bladder cancers.

    Science.gov (United States)

    Zhang, Ruowen; Wang, Liping; Garrett, Scott H; Sens, Donald A; Dunlevy, Jane R; Zhou, Xu Dong; Somji, Seema

    2016-10-01

    Connexin 43 has been shown to play a role in cell migration and invasion; however, its role in bladder cancer is not well defined. Previous studies from our laboratory have shown that the environmental pollutants arsenite and cadmium can cause malignant transformation of the immortalized urothelial cell line UROtsa. These transformed cells can form tumors in immune-compromised mice. The goal of the present study was to determine if connexin 43 is expressed in the normal human bladder, the arsenite and cadmiun-transformed UROtsa cells as well as human urothelial cancer. The results obtained showed that connexin 43 is not expressed in the epithelial cells of the human bladder but is expressed in immortalized cultures of human urothelial cells and the expression is variable in the arsenite and cadmium- transformed urothelial cell lines derived from these immortalized cells. Tumor heterotransplants generated from the transformed cells expressed connexin 43 and the expression was localized to areas of squamous differentiation. Immuno-histochemical analysis of human bladder cancers also showed that the expression of connexin 43 was localized to areas of the tumor that showed early features of squamous differentiation. Treatment of UROtsa cells with various concentrations of arsenite or cadmium did not significantly alter the expression level of connexin 43. In conclusion, our results show that the expression of connexin 43 is localized to the areas of the tumor that show squamous differentiation, which may be an indicator of poor prognosis. This suggests that connexin 43 has the potential to be developed as a biomarker for bladder cancer that may have the ability to invade and metastasize.

  6. Connexin 43 Gene Therapy Delivered by Polymer-Modified Salmonella in Murine Tumor Models

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    Wei-Kuang Wang

    2014-04-01

    Full Text Available The use of preferentially tumor-targeting bacteria as vectors is one of the most innovative approaches for the treatment of cancer. This method is based on the observation that some obligate or facultative anaerobic bacteria are capable of selectively multiplying in tumors and inhibiting their growth. Previously, we found that the tumor-targeting efficiency of Salmonella could be modulated by modifying the immune response to these bacteria by coating them with poly(allylamine hydrochloride (PAH, and these organisms are designated PAH-S.C. (S. choleraesuis. PAH can provide a useful platform for the chemical modification of Salmonella, perhaps by allowing a therapeutic gene to bind to tumor-targeting Salmonella. This study aimed to investigate the benefits of the use of PAH-S.C. for gene delivery. To evaluate this modulation, the invasion activity and gene transfer of DNA-PAH-S.C. were measured in vitro and in vivo. Treatment with PAH-S.C. carrying a tumor suppressor gene (connexin 43 resulted in inhibition of tumor growth, which suggested that tumor-targeted gene therapy using PAH-S.C. carrying a therapeutic gene could exert antitumor activities. This technique represents a promising strategy for the treatment of tumors.

  7. Osteoblast connexin43 modulates skeletal architecture by regulating both arms of bone remodeling.

    Science.gov (United States)

    Watkins, Marcus; Grimston, Susan K; Norris, Jin Yi; Guillotin, Bertrand; Shaw, Angela; Beniash, Elia; Civitelli, Roberto

    2011-04-15

    Connexin43 (Cx43) has an important role in skeletal homeostasis, and Cx43 gene (Gja1) mutations have been linked to oculodentodigital dysplasia (ODDD), a human disorder characterized by prominent skeletal abnormalities. To determine the function of Cx43 at early steps of osteogenesis and its role in the ODDD skeletal phenotype, we have used the Dermo1 promoter to drive Gja1 ablation or induce an ODDD mutation in the chondro-osteogenic linage. Both Gja1 null and ODDD mutant mice develop age-related osteopenia, primarily due to a progressive enlargement of the medullary cavity and cortical thinning. This phenotype is the consequence of a high bone turnover state, with increased endocortical osteoclast-mediated bone resorption and increased periosteal bone apposition. Increased bone resorption is a noncell autonomous defect, caused by exuberant stimulation of osteoclastogenesis by Cx43-deficient bone marrow stromal cells, via decreased Opg production. The latter is part of a broad defect in osteoblast differentiation and function, which also results in abnormal structural and material properties of bone leading to decreased resistance to mechanical load. Thus Cx43 in osteogenic cells is a critical regulator of both arms of the bone remodeling cycle, its absence causing structural changes remindful of aged or disused bone.

  8. Targeting connexin 43 protects against the progression of experimental chronic kidney disease in mice.

    Science.gov (United States)

    Abed, Ahmed; Toubas, Julie; Kavvadas, Panagiotis; Authier, Florence; Cathelin, Dominique; Alfieri, Carlo; Boffa, Jean-Jacques; Dussaule, Jean-Claude; Chatziantoniou, Christos; Chadjichristos, Christos E

    2014-10-01

    Excessive recruitment of monocytes and progression of fibrosis are hallmarks of chronic kidney disease (CKD). Recently we reported that the expression of connexin 43 (Cx43) was upregulated in the kidney during experimental nephropathy. To investigate the role of Cx43 in the progression of CKD, we interbred RenTg mice, a genetic model of hypertension-induced CKD, with Cx43+/- mice. The renal cortex of 5-month-old RenTgCx43+/- mice showed a marked decrease of cell adhesion markers leading to reduced monocyte infiltration and interstitial renal fibrosis compared with their littermates. In addition, functional and histological parameters such as albuminuria and glomerulosclerosis were ameliorated in RenTgCx43+/- mice. Interestingly, treatment with Cx43 antisense produced remarkable improvement of renal function and structure in 1-year-old RenTg mice. Similar results were found in Cx43+/- or wild-type mice treated with Cx43 antisense after obstructive nephropathy. Furthermore, in these mice, Cx43 antisense attenuated E-cadherin downregulation and phosphorylation of the transcription factor Sp1 by the ERK pathway resulting in decreased transcription of type I collagen gene. Interestingly, Cx43-specific blocking peptide inhibited monocyte adhesion in activated endothelium and profibrotic pathways in tubular cells. Cx43 was highly increased in biopsies of patients with CKD. Thus, Cx43 may represent a new therapeutic target against the progression of CKD.

  9. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the best-studied

  10. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the

  11. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    Giepmans, Ben N G

    2004-01-01

    Gap junctions form channels between adjacent cells. The core proteins of these channels are the connexins. Regulation of gap junction communication (GJC) can be modulated by connexin-associating proteins, such as regulatory protein phosphatases and protein kinases, of which c-Src is the best-studied

  12. Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: Role of β-catenin/connexin43 association

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    Talhouk, Rabih S., E-mail: rtalhouk@aub.edu.lb [Department of Biology, Faculty of Arts and Sciences, American University of Beirut, P.O. Box 11-0236, Beirut (Lebanon); Fares, Mohamed-Bilal; Rahme, Gilbert J.; Hariri, Hanaa H.; Rayess, Tina; Dbouk, Hashem A.; Bazzoun, Dana; Al-Labban, Dania [Department of Biology, Faculty of Arts and Sciences, American University of Beirut, P.O. Box 11-0236, Beirut (Lebanon); El-Sabban, Marwan E., E-mail: me00@aub.edu.lb [Department of Anatomy, Cell Biology and Physiology, Faculty of Medicine, American University of Beirut, P.O. Box 11-0236, Beirut (Lebanon)

    2013-12-10

    Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of β-catenin, α-catenin and ZO-2, it decreased nuclear levels of β-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, β-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, β-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering β-catenin away from nucleus. - Highlights: • Cx43 over-expressing MCF-7 and MDA-MB-231 were grown in 2D and 3D cultures. • Proliferation and growth morphology were affected in a context dependent manner. • Extravasation ability of both MCF

  13. Effects of rotigaptide (ZP123) on connexin43 remodeling in canine ventricular fibrillation.

    Science.gov (United States)

    Su, Guo-Ying; Wang, Jing; Xu, Zhen-Xing; Qiao, Xiao-Jun; Zhong, Jing-Quan; Zhang, Yun

    2015-10-01

    The present study investigated the effects of rotigaptide (ZP123) on the expression, distribution and phosphorylation of connexin43 (Cx43) in myocardial cell membranes in cardioversion of ventricular fibrillation (VF). A model of prolonged VF (8, 12 and 30 min) was established in mongrel dogs (n=8/group), following treatment with ZP123 or normal saline (NS control). A sham control was included. Cardiopulmonary resuscitation was begun at the start of VF followed by defibrillation. Animals received a maximum of three defibrillations of increasing energy (70, 100 and 150 J biphasic shock) as required. The average defibrillation energy, defibrillation success rate, return of spontaneous circulation and survival rate were recorded. Cx43 and phosphorylated (p-)Cx43 expression in cardiomyocyte membranes was detected by western blot and immunofluorescence analyses. Compared with the NS-treated control groups, the success defibrillation rate in the 8-min and 12-min ZP123 groups was significantly higher (P<0.05), while the average defibrillation energy was significantly lower (P<0.05). Cx43 expression in the VF groups was significantly lower than that in the sham control group (P<0.05). Cx43 expression was higher in the 12-min and 30-min ZP123 groups than that in the NS control group (P<0.05), while p-Cx43 expression decreased, although the levels were significantly higher than those in the control groups (P<0.05). Cx43 expression was positively correlated with the defibrillation success rate (r=0.91; P<0.01) and negatively with the mean defibrillation energy (r=-0.854; P<0.01), while p-Cx43 expression was positively correlated with the success rate of the previous three defibrillations (r=0.926; P<0.01).In conclusion, ZP123 reduced Cx43 remodeling through regulating the expression, distribution and phosphorylation of Cx43, thereby reducing the defibrillation energy required for successful cardioversion.

  14. Maturational changes in connexin 43 expression in the seminiferous tubules may depend on thyroid hormone action

    Science.gov (United States)

    Marchlewska, Katarzyna; Kula, Krzysztof; Walczak-Jedrzejowska, Renata; Kula, Wojciech; Oszukowska, Elzbieta; Filipiak, Eliza; Moszura, Tomasz

    2013-01-01

    Introduction Connexin 43 (Cx43) mediates the effect of thyroid hormone on Sertoli cell maturation in vitro. We investigated the influence of triiodothyronine (T3) administration on Cx43 expression in relation to the progress in seminiferous tubule maturation. Material and methods Male rats were daily injected with 100 µg T3/kg body weight from birth until postnatal day (pnd) 5 (transient treatment – tT3) or until pnd 15 (continuous treatment – cT3) or solvent – control (C). On pnd 16 serum hormone levels, body and testes weight, seminiferous tubule morphometry, Cx43 immunostaining and germ cell degeneration were investigated. Cx43 expression was also assessed in six 50-day-old adult untreated rats. Result tT3 increased 2.6-fold serum level of T3, testes weight, and seminiferous tubule diameter, and induced maturation-like dislocation of Cx43 expression from the apical to the peripheral region of Sertoli cell cytoplasm. In addition, incidence of Cx43-positive tubules declined from 86% in C to 46% after tT3, being similar to the adult value (30% of tubules Cx43-positive). In turn, cT3 increased serum T3 level 12-fold, and decreased body weight. Seminiferous tubules became shortened and distended, Sertoli cell cytoplasm vacuolated, Cx43 expression had minimal intensity and germ cell degeneration increased. Conclusions Cx43 might intermediate a short and transient stimulatory effect of T3 on seminiferous tubule maturation that disappeared together with exposure to the toxic effect of a continuously high level of the hormone. PMID:23515877

  15. Loss of cadherin-binding proteins β-catenin and plakoglobin in the heart leads to gap junction remodeling and arrhythmogenesis.

    Science.gov (United States)

    Swope, David; Cheng, Lan; Gao, Erhe; Li, Jifen; Radice, Glenn L

    2012-03-01

    Arrhythmic right ventricular cardiomyopathy (ARVC) is a hereditary heart muscle disease that causes sudden cardiac death (SCD) in young people. Almost half of ARVC patients have a mutation in genes encoding cell adhesion proteins of the desmosome, including plakoglobin (JUP). We previously reported that cardiac tissue-specific plakoglobin (PG) knockout (PG CKO) mice have no apparent conduction abnormality and survive longer than expected. Importantly, the PG homolog, β-catenin (CTNNB1), showed increased association with the gap junction protein connexin43 (Cx43) in PG CKO hearts. To determine whether β-catenin is required to maintain cardiac conduction in the absence of PG, we generated mice lacking both PG and β-catenin specifically in the heart (i.e., double knockout [DKO]). The DKO mice exhibited cardiomyopathy, fibrous tissue replacement, and conduction abnormalities resulting in SCD. Loss of the cadherin linker proteins resulted in dissolution of the intercalated disc (ICD) structure. Moreover, Cx43-containing gap junction plaques were reduced at the ICD, consistent with the arrhythmogenicity of the DKO hearts. Finally, ambulatory electrocardiogram monitoring captured the abrupt onset of spontaneous lethal ventricular arrhythmia in the DKO mice. In conclusion, these studies demonstrate that the N-cadherin-binding partners, PG and β-catenin, are indispensable for maintaining mechanoelectrical coupling in the heart.

  16. Late cardioprotection of exercise preconditioning against exhaustive exercise-induced myocardial injury by up-regulatation of connexin 43 expression in rat hearts

    Institute of Scientific and Technical Information of China (English)

    Kai Wang; Bai-Chao Xu; Hai-Yun Duan; Hua Zhang; Fu-Song Hu

    2015-01-01

    Objective:To investigate the expression of myocardium connexin 43 (Cx43) in late exercise preconditioning (LEP) cardioprotection.Methods: Eight-week-old adult male Sprague Dawley rats were randomly assigned into four groups (n=8). Myocardial injury was judged in accordance with serum levels of cTnⅠ and NT-proBNP as well as hematoxylin basicfuchsin picric acid staining of myocardium.Cx43mRNA was detected byin situhybridization and qualified by real-time fluorescence quantitative PCR. Cx43 protein was localized by immunohistochemistry and its expression level was determined by western blotting.Results:The LEP obviously attenuated the myocardial ischemia/hypoxia injury caused by exhaustive exercise. There was no significant difference of Cx43mRNA level between the four groups. Cx43 protein level was decreased significantly in group EE (P<0.05). However, LEP produced a significant increase in Cx43 protein level (P<0.05), and the decreased Cx43 protein level in exhaustive exercise was significantly up-regulated by LEP (P<0.05).Conclusions:LEP protects rat heart against exhaustive exercise-induced myocardial injury by up-regulating the expression of myocardial Cx43.

  17. Transition from Preinvasive Carcinoma In Situ to Seminoma Is Accompanied by a Reduction of Connexin 43 Expression in Sertoli Cells and Germ Cells

    Directory of Open Access Journals (Sweden)

    Ralph Brehm

    2006-06-01

    Full Text Available Carcinoma in situ (CIS represents the preinvasive stage of human germ cell tumors, but the mechanism leading to pubertal proliferation and invasive malignancy remains unknown. Among testicular gap functional proteins, connexin 43 (Cx43 represents the predominant Cx, and, previously, an inverse correlation between synthesis of Cx43 protein and progression of tumor development was detected. In the present study, using cDNA microarray analysis, in situ hybridization, semi quantitative reverse transcription-polymerase chain reaction (RT-PCR from tissue homogenates, RT-PCR from microdissected tubules with normal spermatogenesis and CIS, and seminoma cells from invasive seminoma, we asked whether reduction of Cx43 protein is accompanied by a change of Cx43 transcripts. We detected a significant downregulation of Cx43 at mRNA level in Sertoli and germ cells starting in seminiferous tubules infiltrated with CIS and resulting in a complete loss in seminoma cells. It was demonstrated that downregulation of Cx43 expression in neoplastic human testis takes place at the transcriptional level and starts in CIS. This reduction of Cx43 expression further suggests that early intratubular derangement in Cx43 gene expression and disruption of intercellular communication between Sertoli cells and/or Sertoli and preinvasive tumor cells may play a role in the progression phase of human seminoma development.

  18. Connexin 43 enhances Bax activation via JNK activation in sunitinib-induced apoptosis in mesothelioma cells.

    Science.gov (United States)

    Uzu, Miaki; Sato, Hiromi; Shimizu, Ayaka; Shibata, Yukihiro; Ueno, Koichi; Hisaka, Akihiro

    2017-06-01

    The constituent protein of gap junctions, connexin (Cx), interacts with various proteins via its C-terminus region, including kinases, cell-adhesion proteins, and a pro-apoptotic protein, Bax. This molecular interaction may affect expression and functioning of the interacting proteins and modulate the cellular physiology. In our previous work, Cx43 was found to interact directly with Bax and in the presence of sunitinib, lead to the Bax-mediated apoptosis in mesothelioma cells. In this study, we investigated the mechanism of how Cx43 promotes Bax-mediated apoptosis using the same cell line. Treatment with sunitinib increased the expression of the active conformation of the Bax protein, which was predominantly localized at the mitochondria, only in Cx43-transfected cells. Bax oligomerization and decrease in the mitochondrial membrane potential were also observed. The involvement of c-Jun N-terminal kinase (JNK) in the interaction of Cx43 and Bax was further examined. Treatment with sunitinib increased the expression of phosphorylated (active) form of JNK only in the Cx43-transfected cells. Phosphorylated JNK and active Bax were co-localized, and the co-localization was suppressed by the knockdown of Cx43. Moreover, JNK inhibition clearly suppressed Bax activation. In conclusion, we identified a novel Cx43-JNK-Bax axis regulating the process of apoptosis for the first time. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  19. Mechanism of Regulatory Effect of MicroRNA-206 on Connexin 43 in Distant Metastasis of Breast Cancer

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    Zi-Jing Lin; Jia Ming; Lu Yang; Jun-Ze Du; Ning Wang; Hao-Jun Luo

    2016-01-01

    Background: MicroRNA-206 (miR-206) and connexin 43 (Cx43) are related with the distant metastasis of breast cancer.It remains unclear whether the regulatory effect of miR-206 on Cx43 is involved in metastasis of breast cancer.Methods: Using quantitative real-time polymerase chain reaction and Western blot, the expressions of miR-206 and Cx43 were determined in breast cancer tissues, hepatic and pulmonary metastasis (PM), and cell lines (MCF-10A, MCF-7, and MDA-MB-231).MCF-7/MDA-M-231 cells were transfected with lentivirus-shRNA vectors to enhance/inhibit miR-206, and then Cx43 expression was observed.Cell counting kit-8 assay and Transwell method were used to detect their changes in proliferation, migration, and invasion activity.The mutant plasmids of Cx43-3' untranslated region (3'UTR) at position 478-484 and position 1609-1615 were constructed.Luciferase reporter assay was performed to observe the effects of miR-206 on luciferase expression of different mutant plasmids and to confirm the potential binding sites of Cx43.Results: Cx43 protein expression in hepatic and PM was significantly higher than that in the primary tumor, while no significant difference was showed in messenger RNA (mRNA) expression.MiR-206 mRNA expression in hepatic and PM was significantly lower than that in the primary tumor.Cx43 mRNA and protein levels, as well as cell proliferation, migration, and invasion capabilities, were all significantly improved in MDA-MB-231 cells after reducing miR-206 expression but decreased in MCF-7 cells after elevating miR-206 expression, which demonstrated a significantly negative correlation between miR-206 and Cx43 expression (P =0.03).MiR-206 can drastically decrease Cx43 expression of MCF-7 cells but exerts no effects on Cx43 expression in 293 cells transfected with the Cx43 coding region but the lack of Cx43-3'UTR, suggesting that Cx43-3'UTR may be the key in Cx43 regulated by miR-206.Luciferase expression showed that the inhibition efficiency was

  20. 产妇子宫平滑肌组织中MAPK信号转导通路及CX43表达与产后出血关系的研究%Expression and Significance of Mitogen-Activated Protein Kinase Pathway and Connexin43 in Myometrium of Parturient Women with Postpartum Hemorrhage

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    颜建英; 黄科华; 刘青闽; 黄晓燕; 徐榕莉; 余爱丽; 邓婷

    2012-01-01

    切相关,提示MAPK信号通路活化受阻和CX43表达降低可能是宫缩乏力性产后出血发病的重要因素之一,两者相互调控共同参与该病的发生发展.%Objective:To investigate the contractility and contract potentiality of myometrium from parturient women with postpartum hemorrhage by uterine atony,and to determine the expression and significance of mitogen-activated protein kinase (MAPK) pathway and CX43 in the pathogenesis of postpartum hemorrhage ( PPH). Methods:30 parturient with PPH, who presented to Fujian Maternity and Child Health Hospital for regular prenatal care and parturition from November 2009 to December 2010, were selected as the study group. 30 health parturient at the same period were chosen as control. Isometric tension recording was used to detect the contractility and its potentiality induced by oxytocin. The levels of CX43, phosphorylation of ERKi/2,JNK1/2 and p38MAPK in myometrium of study group and control were determined by western blotting: The mRNA of CX43 was detected by Real-time fluorescent quantitative reverse transcription PCR. Results : (1)The spontaneous contractility and frequency of myometriums in the study group were significanctly lower than those of control(P<0.05). The frequency,range ,contractility and potentiality of the constriction induced by oxytocin in study group were lower than those of control( P<0.05, P<0.01). (2) When contractility of 72.66 g · times per hour was chosen as diagnostic point of postpartum hemorrhage by uterine atony , the area under curve of ROC was 0.802. (3)In the study group,the expressions of p-ERK1/2,p-JNK1/2, p-P38,CX43mRNA and their protein levels in myometrium were significantly lower than those of control (P< 0.05). (4)There were positive correlations between the expressions of p-ERK1/2, p-JNK1/2, p-P38, CX43mRNA and the protein levels in myometrium and the contraction activity in both groups(P<0.05). (5) The CX43mRNA levels and its protein quantum were

  1. Connexin 43 Upregulation in Mouse Lungs during Ovalbumin-Induced Asthma.

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    Yin Yao

    Full Text Available Connexin (Cx-based gap junction channels play important roles in the inflammatory response. Cx43 is involved in the pathogenesis of some lung diseases such as acute lung injury. However, the Cx43 expression in asthma is unclear. In the present study, we used a murine model of ovalbumin (OVA-induced allergic airway disease to examine the levels of Cx43 and analyze the relationship between Cx43 and airway inflammation in allergic airway disease.Asthma was induced in mice via sensitization and challenge with OVA. Cx43 mRNA and protein expression levels were investigated via QT-PCR, western blot, and immunohistochemistry 0 h, 8 h, 1 d, 2 d and 4 d after the first challenge. The relationship between Cx43 protein levels and inflammatory cell infiltration, cytokine levels was analyzed.The OVA-induced mice exhibited typical pathological features of asthma, including airway hyper-responsiveness; strong inflammatory cell infiltration surrounding the bronchia and vessels; many inflammatory cells in the bronchoalveolar lavage fluid (BALF; higher IL-4, IL-5 and IL-13 levels; and high OVA specific IgE levels. Low Cx43 expression was detected in the lungs of control (PBS mice. A dramatic increase in the Cx43 mRNA and protein levels was found in the asthmatic mice. Cx43 mRNA and protein expression levels increased in a time-dependent manner in asthma mice, and Cx43 was mostly localized in the alveolar and bronchial epithelial layers. Moreover, lung Cx43 protein levels showed a significant positive correlation with inflammatory cell infiltration in the airway and IL-4 and IL-5 levels in the BALF at different time points after challenge. Interestingly, the increase in Cx43 mRNA and protein levels occurred prior to the appearance of the inflammatory cell infiltration.Our data suggest that there is a strong upregulation of Cx43 mRNA and protein levels in the lungs in asthma. Cx43 levels also exhibited a positive correlation with allergic airway inflammation. Cx43

  2. Gap junction intercellular communication and benzene toxicity.

    Science.gov (United States)

    Rivedal, Edgar; Witz, Gisela; Leithe, Edward

    2010-03-19

    Aberrant regulation of gap junction intercellular communication (GJIC) has been linked to several human diseases, including cancer and abnormal hematopoietic development. Benzene exposure has been shown to cause hematotoxicity and leukemia, but the underlying mechanisms involved remain unclear. We have observed that several metabolites of benzene have the ability to block gap junction intercellular communication. The ring-opened trans,trans-muconaldehyde (MUC) was found to be the most potent inhibitor of gap junction channels. MUC was found to induce cross-linking of the gap junction protein connexin43, which seemed to be responsible for the induced inhibition of GJIC. Glutaraldehyde, which has a similar molecular structure as MUC, was found to possess similar effects on gap junctions as MUC, while the mono-aldehyde formaldehyde shows lower potency, both as a connexin cross-linker, and as an inhibitor of GJIC. Both glutaraldehyde and formaldehyde have previously been associated with induction of leukemia and disturbance of hematopoiesis. Taken together, the data support a possible link between the effect of MUC on gap junctions, and the toxic effects of benzene. Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.

  3. Chronic exposure to hexachlorobenzene results in down-regulation of connexin43 in the breast.

    Science.gov (United States)

    Delisle, Ariane; Ferraris, Emanuelle; Plante, Isabelle

    2015-11-01

    Decreased expression of connexins has been associated with cancer, but the underlying mechanisms are poorly understood. We have previously shown that a 5 day exposure to hexachlorobenzene (HCB) resulted in decreased connexins expression in hepatocytes 45 days later, and that this down-regulation was linked to activation of Akt through the ILK pathway. Because HCB promotes cancer in both the liver and breast, the present study aimed to determine if the mechanisms are similar in both tissues. MCF-12A breast cells were thus transfected with vectors coding for either Akt or a constitutively active form of Akt. In those cells, activation of Akt was correlated with decreased Cx43 levels. Female rats were then exposed to HCB by gavage either following the same protocol used previously for the liver or through a chronic exposure. While no changes were observed after the 5 days exposure protocol, chronic exposure to HCB resulted in increased Akt levels and decreased Cx43 levels in breast cells. In vitro, Akt was activated in MCF-12A cells exposed to HCB either for 7 days or chronically, but no changes were observed in junctional proteins. Together, these results suggested that, while activation of Akt can decrease Cx43 expression in breast cells in vitro, other mechanisms are involved during HCB exposure, leading to a decrease in Cx43 levels in a model- and duration-dependent manner. Finally, we showed that HCB effects are tissue specific, as we did not observe the same results in breast and liver tissues.

  4. Connexin 43 Is a Potential Prognostic Biomarker for Ewing Sarcoma/Primitive Neuroectodermal Tumor

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    Marilyn M. Bui

    2011-01-01

    Full Text Available Connexins (Cxs are building unit proteins of gap junctions (GJs that are prognostic markers in carcinomas. To investigate the role of Cx in Ewing sarcoma (EWS/primitive neuroectodermal tumor (PNET, we examined the expression of Cx43 and Cx26 in 36 EWS/PNETs and found (1 cytoplasmic Cx43 reactivity in 28/36 (78% cases. (2 Cx43 score was significantly correlated with overall survival (P=.025. The average scores for patients alive and dead at 3 years are 46.08 and 96.98 (P=.004 at 5 years are 46.06 and 96.42 (P=.002. (3 Metastasis had a significant effect on the overall survival (P=.003. (4 Cytoplasmic Cx26 reactivity was detected in 2 of 36 (6% patients who died with metastasis. Our results suggest a possible oncogenic and prognostic role for Cx43 and Cx26 in EWS/PNET. The lack of membranous immunoreactivity suggests that the effect of Cx in EWS/PNET is via a GJ function-independent mechanism.

  5. On the role of the gap junction protein Cx43 (GJA1 in human cardiac malformations with Fallot-pathology. a study on paediatric cardiac specimen.

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    Aida Salameh

    Full Text Available INTRODUCTION: Gap junction channels are involved in growth and differentiation. Therefore, we wanted to elucidate if the main cardiac gap junction protein connexin43 (GJA1 is altered in patients with Tetralogy of Fallot or double-outlet right ventricle of Fallot-type (62 patients referred to as Fallot compared to other cardiac anomalies (21 patients referred to as non-Fallot. Patients were divided into three age groups: 0-2years, 2-12years and >12years. Myocardial tissue samples were collected during corrective surgery and analysis of cell morphology, GJA1- and N-cadherin (CDH2-distribution, as well as GJA1 protein- and mRNA-expression was carried out. Moreover, GJA1-gene analysis of 16 patients and 20 healthy subjects was performed. RESULTS: Myocardial cell length and width were significantly increased in the oldest age group compared to the younger ones. GJA1 distribution changed significantly during maturation with the ratio of polar/lateral GJA1 increasing from 2.93±0.68 to 8.52±1.41. While in 0-2years old patients ∼6% of the lateral GJA1 was co-localised with CDH2 this decreased with age. Furthermore, the changes in cell morphology and GJA1-distribution were not due to the heart defect itself but were significantly dependent on age. Total GJA1 protein expression decreased during growing-up, whereas GJA1-mRNA remained unchanged. Sequencing of the GJA1-gene revealed only few heterozygous single nucleotide polymorphisms within the Fallot and the healthy control group. CONCLUSION: During maturation significant changes in gap junction remodelling occur which might be necessary for the growing and developing heart. In our study point mutations within the Cx43-gene could not be identified as a cause of the development of TOF.

  6. Construction of Mouse Melanoma Cell Models of Wild-type Overexpression Connexin 43 and Site-specific Mutant Connexin 43%过表达及定点突变缝隙连接蛋白Cx43小鼠黑色素瘤细胞模型的构建

    Institute of Scientific and Technical Information of China (English)

    李玢; 张广献; 刘娟; 赵青; 谭宇蕙; 吴映雅; 易华; 杜标炎

    2014-01-01

    目的:构建小鼠黑色素瘤细胞(B16)过表达野生型及点突变型缝隙连接蛋白43(Connexin43, Cx43)细胞模型,为以缝隙连接(Gap Junction, GJ)为靶点的中药复方、中药单药及药物单体的研究提供可靠阳性对照和阴性对照。方法构建野生型Cx43、突变型Cx43G21R、突变型Cx43G138R重组荧光蛋白融合慢病毒表达质粒,用定点突变技术获得Cx43G21R和Cx43G138R突变体,对上述3种质粒进行双酶切和测序鉴定,并分别包装病毒感染B16细胞,使B16细胞过表达野生型Cx43、突变型Cx43G21R、突变型Cx43G138R。 Western blot检测Cx43蛋白表达水平变化,荧光示踪法观察缝隙连接通讯(Gap Junction Intercellular Communication, GJIC)功能变化。结果①酶切及测序证明,成功构建 pLVCx43-mCherry、 pLVCx43-mCherryG21R、 pLVCx43-mCherryG138R重组荧光蛋白融合慢病毒表达质粒。②成功感染B16细胞并筛选稳定过表达Cx43、 Cx43G21R、Cx43G138R细胞株, Western blot检测显示上述细胞株Cx43蛋白表达均高于对照组。③过表达野生型Cx43后B16细胞GJIC功能较对照组增强;过表达突变型Cx43后B16细胞GJIC功能较对照组减弱。结论过表达野生型Cx43可增强B16细胞GJIC功能,过表达突变型Cx43可抑制B16细胞GJIC功能。%Objective To construct mouse B16 melanoma cell models of wild-type overexpression Cx43 and site-specific mutant Cx43, thus to provide reliable positive and negative control for the study of gap junction targeted Chinese herbal formula, Chinese medicine ingredient, and drug monomer. Methods We constructed the recombined fluorescent protein(mCherry) infused with Lentivirus expression plasmid of wild-type Cx43(pLVCx43-mCherry), mutant R (pLVCx43G21R-mCherry) and mutant Cx43G138R(pLVCx43 G138R-mCherry). The Cx43G21R and Cx43G138R mutants were obtained by site-specific mutagenesis. The three kinds of plasmid were performed double restriction

  7. Connexin 43 is not essential for the control of renin synthesis and secretion.

    Science.gov (United States)

    Gerl, Melanie; Kurt, Birgül; Kurtz, Armin; Wagner, Charlotte

    2014-05-01

    The juxtaglomerular areas of mammalian kidneys express the gap junction proteins connexin 37, 40, 43, and 45. Among these, Cx40 plays a major role for the function of juxtaglomerular renin-expressing cells, while Cx37 and Cx45 appear to be less relevant in this context. Since the role of the remaining Cx43 for the function of renin expression is not well understood, this study aimed to systematically characterize the direct role of Cx43 for renin expression and secretion. For this aim, we generated mice with endothelium and with renin cell-specific deletions of Cx43, and we characterized the regulation of renin expression and renin secretion in the kidneys of these mice on normal salt diet and during chronic challenge of the renin system by pretreatment of mice with a low-salt diet in combination with an angiotensin I-converting enzyme inhibitor. We found that renal renin mRNA abundance, plasma renin concentration, and systolic blood pressure did not differ between wild-type, Cx43(fl/fl) Ren1d(+/Cre) mice as well as Cx43(fl/fl) Tie-2(+/Cre) mice under basal conditions nor under chronic stimulation by salt depletion. The localization of renin-expressing cells was also regular in kidneys of all genotypes, and moreover, regulation of renin secretion by beta-adrenergic stimulation and renal perfusion pressure measured in isolated perfused kidneys of Cx43(fl/fl) Ren1d(+/Cre) and Cx43(fl/fl) Tie-2(+/Cre) mice was not different from control. We infer from these results that Cx43 plays if at all only a minor role for the functional control of renin-producing cells in the kidney.

  8. Expression and function of connexin 43 in human gingival wound healing and fibroblasts.

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    Rana Tarzemany

    Full Text Available Connexins (C×s are a family of transmembrane proteins that form hemichannels and gap junctions (GJs on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of

  9. Connexin 43 astrocytopathy linked to rapidly progressive multiple sclerosis and neuromyelitis optica.

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    Katsuhisa Masaki

    Full Text Available BACKGROUND: Multiple sclerosis (MS and neuromyelitis optica (NMO occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx pathology and disease aggressiveness in Asian patients with MS and NMO. METHODS/PRINCIPAL FINDINGS: Samples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD, six with MS, and 20 with other neurological diseases (OND. Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59, chronic active (n=58 and chronic inactive (n=23. Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, P=0.0090. Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, P=0.0296. Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients. CONCLUSIONS

  10. RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

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    Xie, Xi [Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Department of Pharmaceutical Engineering, Ocean College, Hainan University, Haikou 570228 (China); Chen, Cheng; Huang, Kaipeng; Wang, Shaogui; Hao, Jie; Huang, Junying [Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Huang, Heqing, E-mail: huangheq@mail.sysu.edu.cn [Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China)

    2014-10-01

    RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs. - Highlights: • RhoA/ROCK signaling induces Cx43 degradation in GMCs. • F-actin and ZO-1 have functions in the regulation of Cx43 by RhoA/ROCK signaling. • We reveal the relationship between RhoA/ROCK and Cx43 in the activation of NF-κB.

  11. Blocking connexin43 expression caused abnormal zebrafish posterior somites development%Connexin43基因表达下调导致斑马鱼胚胎后部体节发育异常

    Institute of Scientific and Technical Information of China (English)

    刘东

    2011-01-01

    目的:验证斑马鱼胚胎体节的发育是否受到间隙连接蛋白connexin43(cx43)基因表达调控.方法:利用吗啉修饰的反义寡核苷酸(morpholino antisense oligos)下调cx43基因的表达;单克隆抗体CH1进行全胚胎的免疫荧光来标记体节;全胚胎的TUNEL实验检测细胞凋亡.结果:研究发现cx43基因表达下调的斑马鱼胚胎呈现为尾部向侧下弯曲,并且后部体节排列紊乱.进一步的研究证实cx43下调诱导了后部体节细胞的异常凋亡.结论:cx43与斑马鱼后部体节的正常发育有关.

  12. Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells

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    Pollok, Simone; Pfeiffer, Ann-Catherine; Lobmann, Ralf; Wright, Catherine S; Moll, Ingrid; Martin, Patricia E M; Johanna M Brandner

    2010-01-01

    Abstract During early wound healing (WH) events Connexin 43 (Cx43) is down-regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down-regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its t...

  13. Thrombocytosis and immunohistochemical expression of connexin 43 at diagnosis predict survival in advanced non-small-cell lung cancer treated with cisplatin-based chemotherapy.

    Science.gov (United States)

    Du, Gangjun; Yang, Yiming; Yang, Yingming; Zhang, Yaping; Sun, Ting; Liu, Weijie; Wang, Yingying; Li, Jiahuan; Zhang, Houyun

    2013-04-01

    Patients with advanced non-small-cell lung cancer (NSCLC) have poor survival, and platinum-based chemotherapy agents are the standard first-line chemotherapy agents for advanced NSCLC. This study aimed to identify predictive factors associated with the response to chemotherapy and survival in 258 patients with advanced NSCLC treated with platinum-based chemotherapy. Stage IIIA-IV NSCLC patients diagnosed in Kaifeng second people's hospital (Henan, China) between March 2002 and September 2011 were retrospectively reviewed. All of the patients had received platinum-based chemotherapy, and patients were followed up to date of death or last follow-up to obtain data of response to chemotherapy and survival. Potential prognostic factors such as gender, age, tumor size, tumor type, histologic stage, anemia, calcium levels, ECOG performance status (PS), thrombocytosis, TTF-1, p63, and connexin 43 were analyzed. Response to chemotherapy, overall survival (OS) and progression-free survival (PFS) were calculated by the Kaplan-Meier method and Cox regression model. A univariate analysis indicated that thrombocytosis and connexin 43 were found to be significant prognostic factors (p thrombocytosis was associated with increased mortality and resistance to chemotherapy in chemotherapy responders. In addition, all 21 patients of the 5-year OS were from chemotherapy responders with connexin 43 ≥ +2 or non-thrombocytosis. Thrombocytosis and connexin 43 absence may be reliable surrogate markers for the prediction of chemotherapy response and prognosis for patients with advanced NSCLC, and assessment of these factors may identify a population of patients with advanced NSCLC that is likely to have a prolonged life expectancy.

  14. TLR-Activated Gap Junction Channels Protect Mice against Bacterial Infection through Extracellular UDP Release.

    Science.gov (United States)

    Qin, Juliang; Zhang, Guangxu; Zhang, Xiaoyu; Tan, Binghe; Lv, Zhangsheng; Liu, Mingyao; Ren, Hua; Qian, Min; Du, Bing

    2016-02-15

    Extracellular UDP (eUDP), released as a danger signal by stressed or apoptotic cells, plays an important role in a series of physiological processes. Although the mechanism of eUDP release in apoptotic cells has been well defined, how the eUDP is released in innate immune responses remains unknown. In this study, we demonstrated that UDP was released in both Escherichia coli-infected mice and LPS- or Pam3CSK4-treated macrophages. Also, LPS-induced UDP release could be significantly blocked by selective TLR4 inhibitor Atractylenolide I and selective gap junction inhibitors carbenoxolone and flufenamic acid (FFA), suggesting the key role of TLR signaling and gap junction channels in this process. Meanwhile, eUDP protected mice from peritonitis by reducing invaded bacteria that could be rescued by MRS2578 (selective P2Y6 receptor inhibitor) and FFA. Then, connexin 43, as one of the gap junction proteins, was found to be clearly increased by LPS in a dose- and time-dependent manner. Furthermore, if we blocked LPS-induced ERK signaling by U0126, the expression of connexin 43 and UDP release was also inhibited dramatically. In addition, UDP-induced MCP-1 secretion was significantly reduced by MRS2578, FFA, and P2Y6 mutation. Accordingly, pretreating mice with U0126 and Gap26 increased invaded bacteria and aggravated mice death. Taken together, our study reveals an internal relationship between danger signals and TLR signaling in innate immune responses, which suggests a potential therapeutic significance of gap junction channel-mediated UDP release in infectious diseases. Copyright © 2016 by The American Association of Immunologists, Inc.

  15. Histopathological analysis of neonatal mouse hearts with connexin43 gene defects%Connexin43基因缺陷新生小鼠心脏组织病理学研究

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    谢利剑; 黄国英; 赵晓晴; 沈媛; 周国民

    2005-01-01

    目的观察不同程度Connexin43基因缺陷对新生小鼠心脏发育的影响.方法采用Cx43基因敲除(Cx43 KO)和CMV43CT两种Cx43基因缺陷的小鼠模型,聚合酶链反应鉴定基因型.然后将新生小鼠心脏分离固定,HE染色观察心脏的形态结构.普通C57BL6/SJ小鼠作为对照.结果所有11只纯合型Cx43 KO小鼠生后1 d内均死亡,心脏表现为严重右室流出道梗阻.在20只纯合型CMV43CT新生小鼠中,有12只生后2 d内死亡,其心脏不仅表现有右室流出道梗阻,还出现了房间隔缺损、室间隔缺损等其他心脏畸形;而另外8只未见心脏异常.所有杂合型Cx43 KO和CMV43CT基因缺陷的小鼠生后均存活,心脏病理学检测未见异常.结论 Cx43基因缺陷与心脏发育异常有密切的关系,但不同类型和不同程度的Cx43基因功能缺失对心脏发育的影响也不尽相同.

  16. Connexin43- and Pannexin-Based Channels in Neuroinflammation and Cerebral Neuropathies

    Directory of Open Access Journals (Sweden)

    Denis Sarrouilhe

    2017-10-01

    Full Text Available Connexins (Cx are largely represented in the central nervous system (CNS with 11 Cx isoforms forming intercellular channels. Moreover, in the CNS, Cx43 can form hemichannels (HCs at non-junctional membrane as does the related channel-forming Pannexin1 (Panx1 and Panx2. Opening of Panx1 channels and Cx43 HCs appears to be involved in inflammation and has been documented in various CNS pathologies. Over recent years, evidence has accumulated supporting a link between inflammation and cerebral neuropathies (migraine, Alzheimer’s disease (AD, Parkinson’s disease (PD, major depressive disorder, autism spectrum disorder (ASD, epilepsy, schizophrenia, bipolar disorder. Involvement of Panx channels and Cx43 HCs has been also proposed in pathophysiology of neurological diseases and psychiatric disorders. Other studies showed that following inflammatory injury of the CNS, Panx1 activators are released and prolonged opening of Panx1 channels triggers neuronal death. In neuropsychiatric diseases, comorbidities are frequently present and can aggravate the symptoms and make therapeutic management more complex. The high comorbidity between some neuropathies can be partially understood by the fact that these diseases share a common etiology involving inflammatory pathways and Panx1 channels or Cx43 HCs. Thus, anti-inflammatory therapy opens perspectives of targets for new treatments and could have real potential in controlling a cerebral neuropathy and some of its comorbidities. The purpose of this mini review is to provide information of our knowledge on the link between Cx43- and Panx-based channels, inflammation and cerebral neuropathies.

  17. Dynamic alterations of connexin43, matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 during ventricular fibrillation in canine.

    Science.gov (United States)

    Wang, Jing; Li, Jing-sha; Liu, Hong-zhen; Yi, Shao-lei; Su, Guo-ying; Zhang, Yun; Zhong, Jing-quan

    2014-06-01

    The aim of this study is to investigate the dynamic alterations of cardiac connexin 43 (Cx43), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the setting of different ventricular fibrillation (VF) duration. In this study, thirty-two dogs were randomly divided into sham control group, 8-min VF group, 12-min VF group, and 30-min VF group. Cx43 and phosphorylated Cx43 (p-Cx43) in tissues were detected by western blot and immunofluorescence analysis. MMP-2 and TIMP-2 were detected by western blot and immunohistochemistry analysis. The results showed that Cx43 levels in three VF groups were significantly decreased compared with sham control group. p-Cx43 levels in 12-min and 30-min VF groups were significantly reduced compared with sham control group. The ratio of p-Cx43/Cx43 was also decreased in VF groups. Compared with sham controls, no significant difference was observed between the sham control group and 8-min VF group in MMP-2 level, but MMP-2 level increased in 12-min and 30-min VF groups. The ratios of MMP-2/TIMP-2 were higher in VF groups, and were correlated with the duration of VF. A remarkable correlation was observed between the ratio of p-Cx43/Cx43 and MMP-2/TIMP-2 (r = -0.93, P MMP-2 and TIMP-2 may contribute to the initiation and/or persistence of VF. Maneuvers managed to modulate Cx43 level or normalize the balance of MMP-2/TIMP-2 are promising to ameliorate prognosis of VF.

  18. Lentivirus-mediated RNAi knockdown of the gap junction protein, Cx43, attenuates the development of vascular restenosis following balloon injury.

    Science.gov (United States)

    Han, Xiao-Jian; Chen, Min; Hong, Tao; Zhu, Ling-Yu; He, Dan; Feng, Jiu-Geng; Jiang, Li-Ping

    2015-04-01

    Percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] has been developed into a mature interventional treatment for atherosclerotic cardiovascular disease. However, the long-term therapeutic effect is compromised by the high incidence of vascular restenosis following angioplasty, and the underlying mechanisms of vascular restenosis have not yet been fully elucidated. In the present study, we investigated the role of the gap junction (GJ) protein, connexin 43 (Cx43), in the development of vascular restenosis. To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty injury. At 0, 7, 14 and 2 days following balloon injury, the arteries were removed, and the intimal/medial area of the vessels was measured to evaluate the degree of restenosis. We found that the intimal area gradually increased following balloon injury. Intimal hyperplasia and restenosis were particularly evident at 14 and 28 days after injury. In addition, the mRNA and protein expression of Cx43 was temporarily decreased at 7 days, and subsequently increased at 14 and 28 days following balloon injury, as shown by RT-PCR and western blot analysis. To determine the involvement of Cx43 in vascular restenosis, the lentivirus vector expressing shRNA targeting Cx43, Cx43-RNAi-LV, was used to silence Cx43 in the rat carotid arteries. The knockdown of Cx43 effectively attenuated the development of intimal hyperplasia and vascular restenosis following balloon injury. Thus, our data indicate the vital role of the GJ protein, Cx43, in the development of vascular restenosis, and provide new insight into the pathogenesis of vascular restenosis. Cx43 may prove to be a novel potential pharmacological target for the prevention of vascular restenosis following PCI.

  19. Researches on Regulation Mechanism of Connexin 43 Expression in Reproductive System by Sexual Hormones%性激素对生殖系统Cx43表达调控机制的研究进展

    Institute of Scientific and Technical Information of China (English)

    孙馥箐; 段华; 汪沙

    2015-01-01

    由连接蛋白43 (connexin 43,Cx43)构成的细胞间间隙连接(gap junction,GJ)是介导细胞间直接的物质、能量交换及电、化学信号耦合的重要通路,其可保证细胞间功能活动的协调一致,在生殖系统细胞发育、分化及成熟等生理过程及功能活动中发挥非常重要的调控作用.性激素可在转录及翻译等层面调节生殖系统细胞Cx43的结构及表达,一方面通过核受体基因组机制调节Cx43基因的转录,另一方面通过非基因组快速信号传导通路机制调节Cx43的磷酸化水平,共同影响细胞间隙连接通讯(gapjunction intracellular communication,GJIC),进一步干预生殖系统细胞的病理、生理过程及功能活动.近年来,性激素在心血管系统病生理状态下对连接蛋白调节的变化及机制研究较为成熟,而在子宫、卵巢等生殖器官中,对性激素通过调节连接蛋白及间隙连接进而影响细胞间信息流通的研究较少,其作用机制并不清晰.故本文结合近年来文献,综述性激素对生殖系统细胞Cx43表达及GJIC的调控机制的研究进展,旨在为今后深入地研究提供可行的思路.%Gap junction (G J) composed of connexin 43 (Cx43) is a kind of cell-cell junction which significantly mediates the direct interchange of metabolites,energy and electrochemical signals from one cell to another.GJ ensures the coordination of intercellular activities and plays a vital role in the regulation of cell development,differentiation,maturation and functional activities in the reproductive system.Sexual hormones can influence the pathophysiological processes and functional activities of the reproductive cells by regulating gap junction intracellular communication (GJIC),which depends on the expression of Cx43.On one hand,sexual hormones regulate Cx43 gene transcription via the nuclear receptor mechanism,on the other hand,sexual hormones regulate Cx43 phosphorylation level through the signal

  20. Estimation of the effective intercellular diffusion coefficient in cell monolayers coupled by gap junctions

    DEFF Research Database (Denmark)

    Olesen, Niels Erik; Hofgaard, Johannes P; von Holstein-Rathlou, Niels-Henrik;

    2012-01-01

    A recently developed dye-based assay to study gap junction permeability is analysed. The assay is based on electroporation of dye into a large number of connexin 43 expressing cells, grown to confluency on electrically conductive slides. The subsequent intercellular spread of dye to non-electroporated...

  1. Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

    Institute of Scientific and Technical Information of China (English)

    Wei ZHAO; Zhong Xiang LIN; Zhi Qian ZHANG

    2004-01-01

    To examine the role of gap junctions in cell senescence,the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore,cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis. p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin (10 mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis,elevation of p53 expression,loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.

  2. Bioengineering a Single-Protein Junction.

    Science.gov (United States)

    Ruiz, Marta P; Aragones, Albert C; Camarero, Nuria; Vilhena, J G; Ortega, Maria; Zotti, Linda Angela; Perez, Ruben; Cuevas, Juan Carlos; Gorostiza, Pau; Díez-Pérez, Ismael

    2017-10-05

    Bioelectronics moves towards designing nanoscale electronic platforms that allow in vivo determinations. Such devices require interfacing complex biomolecular moieties as the sensing units to an electronic platform for signal transduction. Inevitably, a systematic design goes through a bottom-up understanding of the structurally related electrical signatures of the biomolecular circuit, which will ultimately lead us to tailor its electrical properties. Toward this aim, we show here the first example of bioengineered charge transport in a single-protein electrical contact. The results reveal that a single point-site mutation at the docking hydrophobic patch of a Cu-Azurin causes minor structural distortion of the protein blue Cu site and a dramatic change in the charge transport regime of the single-protein contact, which goes from the classical Cu-mediated 2-step transport in this system to a direct coherent tunneling. Our extensive spectroscopic studies and molecular-dynamics simulations show that the proteins' folding structures are preserved in the single-protein junction. The DFT-computed frontier orbital of the relevant protein segments suggests that the Cu center participation in each protein variant accounts for the different observed charge transport behavior. This work is a direct evidence of charge transport control in a protein backbone through external mutagenesis and a unique nanoscale platform to study structurally related biological electron transfer.

  3. 连续性血液净化对肺泡上皮细胞Connexin43的影响%Effects of continuous blood purification on Connexin43 in human alveolar epi-thelial cells

    Institute of Scientific and Technical Information of China (English)

    杨溢; 甘华; 李正荣; 文以君; 王喜超

    2009-01-01

    AIM: To investigate the effect of continuous blood purification (CBP) on the expression of Connexin43 (Cx43) in human alveolar epithelial cells (AECs) induced by the serum of patients with severe acute pancreatitis (SAP) accompanied with acute lung injury (ALI). METHODS: Fasting serum of healthy volunteers and serum of patients with SAP and ALI at pre-CBP, 6-hour-CBP and 20-hour-CBP were collected, Immunofluores-cence staining and RT-PCR were used to detect the expression of Cx43 and Cx43 mRNA in AECs separately cultured by the serum from different groups for 48 h in vitro. ELISA was used to detect the level of serum TNF-α in each group. RESULTS: Cx43 posi-tive staining in each patient group was significantly less than that in healthy control group. With the increase of the treatment time, the positive staining gradually increased. The relative expression of Cx43 mRNA in pre-CBP group(0.08±0.01) was significantly leas than that in healthy control group (0.57±0.02) (P<0.01). With the increase of the treatment time, the expression gradually increased in 6-hoar-CBP group(0.23±0.02) and 20-hour-CBP group(0.36±0.02) (P<0.01). The level of serum TNF-α in pre-CBP group (59.43±4.50) ng/L was significantly higher than that in healthy control group(16.06±3.68) ng/L(P<0.01). With the increase of the treatment time, the level gradually decreased in 6-hour-CBP group (41.16±3.49) ng/L and 20-hour-CBP group (34.65±3.22) ng/L (P<0.01). The expression of Cx43 mRNA was negatively correlated with the level of TNF-α. CONCLUSION: The decrease of Cx43 in AECs plays a part in the process of SAP patients with secondary ALI. CBP up-regulates the expression of Cx43 by removing TNF-α and thus protects the respiratory function.%目的:研究连续性血液净化(CBP)对重症急性胰腺炎(SAP)伴急性肺损伤(ALI)患者血清诱导的人肺泡上皮细胞(AECs)间隙连接蛋白Connexin43(Cx43)表达的影响.方法:采集健康志愿者清晨空腹及SAP伴ALI患者CBP

  4. [Gap junctions: A new therapeutic target in major depressive disorder?].

    Science.gov (United States)

    Sarrouilhe, D; Dejean, C

    2015-11-01

    Major depressive disorder is a multifactorial chronic and debilitating mood disease with high lifetime prevalence and is associated with excess mortality, especially from cardiovascular diseases and through suicide. The treatments of this disease with tricyclic antidepressants and monoamine oxidase inhibitors are poorly tolerated and those that selectively target serotonin and norepinephrine re-uptake are not effective in all patients, showing the need to find new therapeutic targets. Post-mortem studies of brains from patients with major depressive disorders described a reduced expression of the gap junction-forming membrane proteins connexin 30 and connexin 43 in the prefrontal cortex and the locus coeruleus. The use of chronic unpredictable stress, a rodent model of depression, suggests that astrocytic gap junction dysfunction contributes to the pathophysiology of major depressive disorder. Chronic treatments of rats with fluoxetine and of rat cultured cortical astrocytes with amitriptyline support the hypothesis that the upregulation of gap junctional intercellular communication between brain astrocytes could be a novel mechanism for the therapeutic effect of antidepressants. In conclusion, astrocytic gap junctions are emerging as a new potential therapeutic target for the treatment of patients with major depressive disorder.

  5. [Dynamics of local expression of connexin-43 and basic fibroblast growth factor receptors in patients with skin and soft-tissue infections against the background of diabetes mellitus type II].

    Science.gov (United States)

    Vinnik, Iu S; Salmina, A B; Tepliakova, O V; Drobushevskaia, A I; Malinovskaia, N A; Pozhilenkova, E A; Morgun, A V; Gitlina, A G

    2014-01-01

    Clinical results of wound healing dynamics were studied in 60 patients with soft-tissue infection against the background of diabetes mellitus type II. At the same time the study considered indices of intercellular contacts protein tissue expression such as connexin 43 (Cx43) and basic fibroblast growth factor receptors (bFGFR). The basic therapy of biopsy material of wound borders was applied. The reduction of bFGFR expression and the minor growth of Cx43 expression were observed. The pain syndrome proceeded for a long time and there were signs of perifocal inflammation, retard wound healing with granulation tissue. The application of combined method of ozone therapy which included autohemotherapy with ozone and an external management of wound by ozone-oxygen mixture facilitated to considerable shortening of inflammatory phase and regeneration. It was associated with increased Cx43 expression (in 1.9 times) in comparison with initial level and bFGFR was enlarged in 1.7 times to eighth day of postoperative period.

  6. HPV16 E6 Controls the Gap Junction Protein Cx43 in Cervical Tumour Cells

    Directory of Open Access Journals (Sweden)

    Peng Sun

    2015-10-01

    Full Text Available Human papillomavirus type 16 (HPV16 causes a range of cancers including cervical and head and neck cancers. HPV E6 oncoprotein binds the cell polarity regulator hDlg (human homologue of Drosophila Discs Large. Previously we showed in vitro, and now in vivo, that hDlg also binds Connexin 43 (Cx43, a major component of gap junctions that mediate intercellular transfer of small molecules. In HPV16-positive non-tumour cervical epithelial cells (W12G Cx43 localised to the plasma membrane, while in W12T tumour cells derived from these, it relocated with hDlg into the cytoplasm. We now provide evidence that E6 regulates this cytoplasmic pool of Cx43. E6 siRNA depletion in W12T cells resulted in restoration of Cx43 and hDlg trafficking to the cell membrane. In C33a HPV-negative cervical tumour cells expressing HPV16 or 18 E6, Cx43 was located primarily in the cytoplasm, but mutation of the 18E6 C-terminal hDlg binding motif resulted in redistribution of Cx43 to the membrane. The data indicate for the first time that increased cytoplasmic E6 levels associated with malignant progression alter Cx43 trafficking and recycling to the membrane and the E6/hDlg interaction may be involved. This suggests a novel E6-associated mechanism for changes in Cx43 trafficking in cervical tumour cells.

  7. Altered detrusor gap junction communications induce storage symptoms in bladder inflammation: a mouse cyclophosphamide-induced model of cystitis.

    Science.gov (United States)

    Okinami, Takeshi; Imamura, Masaaki; Nishikawa, Nobuyuki; Negoro, Hiromitsu; Sugino, Yoshio; Yoshimura, Koji; Kanematsu, Akihiro; Hashitani, Hikaru; Ogawa, Osamu

    2014-01-01

    Lower urinary tract symptoms (LUTS) include storage, voiding and post-micturition symptoms, featuring many urological diseases. Storage symptoms are the most frequent among these and associated with overactive bladder and non-bacterial bladder inflammation such as interstitial cystitis/bladder pain syndrome (IC/BPS). Gap junction, a key regulator of hyperactive conditions in the bladder, has been reported to be involved in pathological bladder inflammation. Here we report involvement of gap junction in the etiology of storage symptoms in bladder inflammation. In this study, cyclophosphamide-induced cystitis was adapted as a model of bladder inflammation. Cyclophosphamide-treated mice showed typical storage symptoms including increased urinary frequency and reduced bladder capacity, with concurrent up-regulation of connexin 43 (GJA1), one of the major gap junction proteins in the bladder. In isometric tension study, bladder smooth muscle strips taken from the treated mice showed more pronounced spontaneous contraction than controls, which was attenuated by carbenoxolone, a gap junction inhibitor. In voiding behavior studies, the storage symptoms in the treated mice characterized by frequent voiding were alleviated by 18α-glycyrrhetinic acid, another gap junction inhibitor. These results demonstrate that cyclophosphamide-induced mouse model of cystitis shows clinical storage symptoms related with bladder inflammation and that gap junction in the bladder may be a key molecule of these storage symptoms. Therefore, gap junction in the bladder might be an alternative therapeutic target for storage symptoms in bladder inflammation.

  8. Altered detrusor gap junction communications induce storage symptoms in bladder inflammation: a mouse cyclophosphamide-induced model of cystitis.

    Directory of Open Access Journals (Sweden)

    Takeshi Okinami

    Full Text Available Lower urinary tract symptoms (LUTS include storage, voiding and post-micturition symptoms, featuring many urological diseases. Storage symptoms are the most frequent among these and associated with overactive bladder and non-bacterial bladder inflammation such as interstitial cystitis/bladder pain syndrome (IC/BPS. Gap junction, a key regulator of hyperactive conditions in the bladder, has been reported to be involved in pathological bladder inflammation. Here we report involvement of gap junction in the etiology of storage symptoms in bladder inflammation. In this study, cyclophosphamide-induced cystitis was adapted as a model of bladder inflammation. Cyclophosphamide-treated mice showed typical storage symptoms including increased urinary frequency and reduced bladder capacity, with concurrent up-regulation of connexin 43 (GJA1, one of the major gap junction proteins in the bladder. In isometric tension study, bladder smooth muscle strips taken from the treated mice showed more pronounced spontaneous contraction than controls, which was attenuated by carbenoxolone, a gap junction inhibitor. In voiding behavior studies, the storage symptoms in the treated mice characterized by frequent voiding were alleviated by 18α-glycyrrhetinic acid, another gap junction inhibitor. These results demonstrate that cyclophosphamide-induced mouse model of cystitis shows clinical storage symptoms related with bladder inflammation and that gap junction in the bladder may be a key molecule of these storage symptoms. Therefore, gap junction in the bladder might be an alternative therapeutic target for storage symptoms in bladder inflammation.

  9. Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters

    Science.gov (United States)

    Stains, Joseph P.; Lecanda, Fernando; Screen, Joanne; Towler, Dwight A.; Civitelli, Roberto

    2003-01-01

    Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.

  10. The ouabain-sensitive isoform of Na+-pump regulates vascular gap junctions via interaction with the Na+/Ca2+-exchanger in membrane microdomain

    DEFF Research Database (Denmark)

    Matchkov, Vladimir; Nilsson, Holger; Aalkjær, Christian

    leading to increases in [Ca2+]i in discrete areas near the plasma membrane. This suggests close association of these transport proteins in microdomains. Using PCR and co-immunoprecipitation we aimed to test this hypothesis in SMCs from mesenteric small arteries and in A7r5 cell line. Intercellular...... electrical coupling was evaluated in functional studies. SMCs were electrically uncoupled when the ouabain-sensitive Na+-pump was inhibited by 10 mM ouabain. Inhibition of the Na+/Ca2+-exchanger with 1 mM SEA0400 also uncoupled the SMCs. Depletion of [Na+]i and clamping [Ca2+]i at low levels prevented...... leading to local [Ca2+]i transients near the membrane which block the closely associated connexin-43 containing gap junctions....

  11. Ultrastructure of junction areas between neurons and astrocytes in rat supraoptic nuclei

    Institute of Scientific and Technical Information of China (English)

    Li Duan; Hua Yuan; Chang-Jun Su; Ying-Ying Liu; Zhi-Ren Rao

    2004-01-01

    AIM: To determine the ultrastructure of junction areas between neurons and astrocytes of supraoptic nuclei in rats orally administered 30 g/L NaCl solution for 5 days.METHODS: The anti-connexin (CX) 43 and anti-CX32 double immunoelectromicroscopic labeled method, and anti-Fos or anti-glial fibrillary acidic protein (GFAP) immunohistochemistry were used to detect changes in the junctional area between neurons and astrocytes in supraoptic nuclei of 5 rats after 30 g/L NaCL solution was given for 5days.RESULTS: A heterotypic connexin32/connexin43 gap junction (HGJ) between neurons and astrocytes (AS) in rat supraoptic nuclei was observed, which was characterized by the thickening and dark staining of cytomembranes with a narrow cleft between them. The number of HGJs and Fos like immunoreactive (-LI) cells was significantly increased following hyperosmotic stimuli, that is, the rats were administered 30 g/L NaCl solution orally or 90 g/L NaCl solution intravenously. HGJs could be blocked with carbenoxolone (CBX), a gap junction blocker, and the number of Fos-LI neurons was significantly decreased compared with that in rats without CBX injection, while Fos-LI ASs were not affected.CONCLUSION: HGJ may be a rapid adaptive signal structure between neurons and ASs in response to stimulation.

  12. Connexin-47 and connexin-32 in gap junctions of oligodendrocyte somata, myelin sheaths, paranodal loops and Schmidt-Lanterman incisures: implications for ionic homeostasis and potassium siphoning.

    Science.gov (United States)

    Kamasawa, N; Sik, A; Morita, M; Yasumura, T; Davidson, K G V; Nagy, J I; Rash, J E

    2005-01-01

    The subcellular distributions and co-associations of the gap junction-forming proteins connexin 47 and connexin 32 were investigated in oligodendrocytes of adult mouse and rat CNS. By confocal immunofluorescence light microscopy, abundant connexin 47 was co-localized with astrocytic connexin 43 on oligodendrocyte somata, and along myelinated fibers, whereas connexin 32 without connexin 47 was co-localized with contactin-associated protein (caspr) in paranodes. By thin-section transmission electron microscopy, connexin 47 immunolabeling was on the oligodendrocyte side of gap junctions between oligodendrocyte somata and astrocytes. By freeze-fracture replica immunogold labeling, large gap junctions between oligodendrocyte somata and astrocyte processes contained much more connexin 47 than connexin 32. Along surfaces of internodal myelin, connexin 47 was several times as abundant as connexin 32, and in the smallest gap junctions, often occurred without connexin 32. In contrast, connexin 32 was localized without connexin 47 in newly-described autologous gap junctions in Schmidt-Lanterman incisures and between paranodal loops bordering nodes of Ranvier. Thus, connexin 47 in adult rodent CNS is the most abundant connexin in most heterologous oligodendrocyte-to-astrocyte gap junctions, whereas connexin 32 is the predominant if not sole connexin in autologous ("reflexive") oligodendrocyte gap junctions. These results clarify the locations and connexin compositions of heterologous and autologous oligodendrocyte gap junctions, identify autologous gap junctions at paranodes as potential sites for modulating paranodal electrical properties, and reveal connexin 47-containing and connexin 32-containing gap junctions as conduits for long-distance intracellular and intercellular movement of ions and associated osmotic water. The autologous gap junctions may regulate paranodal electrical properties during saltatory conduction. Acting in series and in parallel, autologous and

  13. Adipocytes in both brown and white adipose tissue of adult mice are functionally connected via gap junctions: implications for Chagas disease.

    Science.gov (United States)

    Burke, Shoshana; Nagajyothi, Fnu; Thi, Mia M; Hanani, Menachem; Scherer, Philipp E; Tanowitz, Herbert B; Spray, David C

    2014-11-01

    Adipose tissue serves as a host reservoir for the protozoan Trypanosoma cruzi, the causative organism in Chagas disease. Gap junctions interconnect cells of most tissues, serving to synchronize cell activities including secretion in glandular tissue, and we have previously demonstrated that gap junctions are altered in various tissues and cells infected with T. cruzi. Herein, we examined the gap junction protein connexin 43 (Cx43) expression in infected adipose tissues. Adipose tissue is the largest endocrine organ of the body and is also involved in other physiological functions. In mammals, it is primarily composed of white adipocytes. Although gap junctions are a prominent feature of brown adipocytes, they have not been explored extensively in white adipocytes, especially in the setting of infection. Thus, we examined functional coupling in both white and brown adipocytes in mice. Injection of electrical current or the dye Lucifer Yellow into adipocytes within fat tissue spread to adjacent cells, which was reduced by treatment with agents known to block gap junctions. Moreover, Cx43 was detected in both brown and white fat tissue. At thirty and ninety days post-infection, Cx43 was downregulated in brown adipocytes and upregulated in white adipocytes. Gap junction-mediated intercellular communication likely contributes to hormone secretion and other functions in white adipose tissue and to nonshivering thermogenesis in brown fat, and modulation of the coupling by T. cruzi infection is expected to impact these functions. Copyright © 2014. Published by Elsevier Masson SAS.

  14. Regulatory effects of simvastatin on osteoblast proliferation, differentiation and connexin 43 expression%辛伐他汀对成骨细胞增殖分化及连接蛋白43表达的调控

    Institute of Scientific and Technical Information of China (English)

    王国亮; 蔡湘波; 李文壮; 罗胜明; 陈泽雁; 陈戈胜

    2014-01-01

    BACKGROUND:The effects and molecular mechanism of simvastatin on the proliferation and differentiation of osteoblasts remain unclear. Especial y, we do not know much about the effects of connexin 43. OBJECTIVE:To evaluate the effects of simvastatin on the proliferation and differentiation of osteoblasts and the regulatory effect of simvastatin on the expression of osteogenic genes and connexin 43. METHODS:Newborn Sprague-Dawley rats were chosen and the cranium digestion method was used to culture osteoblasts. The different concentrations of simvastatin (0.062 5, 0.125, 0.25, 0.5 and 1.0μmol/L) were used to deal with osteoblasts. The proliferative effect of simvastatin on osteoblasts was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The effect of simvastatin on osteoblast differentiation was measured with alkaline phosphatase activities. The mRNA and protein expression of osteogenic genes and connexin 43 were measured by real time quantitative RT-PCR and western blot assay. RESULTS AND CONCLUSION:There were no significant differences in absorbance values of simvastatin groups at 3 days (P>0.05). However, at 4 and 5 days, absorbance values were lower in the simvastatin groups than those in the control group (P  目的:探讨辛伐他汀对成骨细胞增殖分化及成骨基因和连接蛋白43表达的调控作用。  方法:选取新生SD大鼠,采用颅盖骨消化法培养成骨细胞。采用不同浓度辛伐他汀(0.0625,0.125,0.25,0.5和1.0μmol/L)处理成骨细胞,MTT检测辛伐他汀对成骨细胞增殖作用的影响;碱性磷酸酶活性检测辛伐他汀对成骨细胞分化作用的影响;实时定量RT-PCR和免疫印迹检测细胞成骨基因和间隙连接蛋白43 mRNA和蛋白的表达。  结果与结论:细胞培养第3天,辛伐他汀组各浓度组MTT吸光度值比较差异无显著性意义(P >0.05);但是培养第4天和第5天,辛伐他汀各浓度组MTT

  15. Endocytosis and Recycling of Tight Junction Proteins in Inflammation

    Directory of Open Access Journals (Sweden)

    Markus Utech

    2010-01-01

    Full Text Available A critical function of the epithelial lining is to form a barrier that separates luminal contents from the underlying interstitium. This barrier function is primarily regulated by the apical junctional complex (AJC consisting of tight junctions (TJs and adherens junctions (AJs and is compromised under inflammatory conditions. In intestinal epithelial cells, proinflammatory cytokines, for example, interferon-gamma (IFN-γ, induce internalization of TJ proteins by endocytosis. Endocytosed TJ proteins are passed into early and recycling endosomes, suggesting the involvement of recycling of internalized TJ proteins. This review summarizes mechanisms by which TJ proteins under inflammatory conditions are internalized in intestinal epithelial cells and point out comparable mechanism in nonintestinal epithelial cells.

  16. Effect of the gap junction blocker 1-heptanol on chondrogenic differentiation of mouse bone marrow mesenchymal stem cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Liu Ouyang; Yukun Zhang; Shuhua Yang; Shunan Ye; Weihua Xu

    2009-01-01

    Objective:To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesencbymal stem cells(MSCs) following induction by GDF-5. Methods:MSCs were isohted from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5(100 ng/ml), with or without 1-heptanol(2.5 μ mol/L). The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay. Type Ⅱ collagen mRNA and protein were examined by RT-PCR and immunocytochernistry respectively, and the sulfate glycosaminoglycan was assessed by Alcian blue dye staining. Connexin43(Cx43) protein was examined by western blotting. Results:GDF-5 induced proliferation and chondrogenic differentiation of MSCs. While 1-heptanol treatment had no effect on this proliferation, it inhibited the expression of both type Ⅱ collagen mRNA and protein. The Alcian blue staining revealed that 1-heptanol also inhibited the deposition of the typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol had no effect on the expression of Cx43. Conclusion:Tbese results suggest that mouse bone marrow MSCs can be differentiated into a chondrogenic phenotype by GDF-5 administration in vitro. While the gap junction blocker, 1-heptanol, did not reduce gap junction Cx43, these intercellular communication pathways clearly played an important functional role in GDF-5-induced cartilage differentiation.

  17. Increased Connexin 43 Expression Improves the Migratory and Proliferative Ability of H9c2 Cells by Wnt-3a Overexpression

    Institute of Scientific and Technical Information of China (English)

    Xiaoyu LIU; Wen LIU; Ling YANG; Beili XIA; Jinyan LI; Ji ZUO; Xiaotian LI

    2007-01-01

    The change of connexin 43 (Cx43) expression and the biological behaviors of Cx43 in rat heart cell line H9c2, expressing Wnt-3a (wingless-type MMTV integration site family, member 3A), were evaluated in the present study. Plasmid pcDNA3.1/Wnt-3a was constructed and transferred into H9c2 cells.The cell model Wnt-3a+-H9c2 steadily expressing Wnt-3a was obtained. Compared with H9c2 and pcDNA3.1-H9c2 cells, the expression of Cx43 in Wnt-3a+-H9c2 cells was clearly increased, the proliferation of Wnt-3a+-H9c2 cells was significantly changed, and cell migration abilities were also improved (P<0.05).In comparison with H9c2 and pcDNA3.1-H9c2 cells, the G2 phase of the cell cycle increased by 11% in Wnt-3a+-H9c2 cells. Thus, Wnt-3a overexpression is associated with an increase in Cx43 expression and altered migratory and proliferative activity in H9c2 cells. Cx43 might be one of the downstream target genes regulated by Wnt-3a.

  18. Transforming growth factor-β stimulates human ovarian cancer cell migration by up-regulating connexin43 expression via Smad2/3 signaling.

    Science.gov (United States)

    Qiu, Xin; Cheng, Jung-Chien; Zhao, Jianfang; Chang, Hsun-Ming; Leung, Peter C K

    2015-10-01

    Reduced connexin43 (Cx43) expression is frequently detected in different types of human cancer. Cx43 has been shown to regulate cancer cell migration in a cell-type dependent manner. In both primary and recurrent human ovarian cancer, overexpression of TGF-β ligand and its receptors have been detected. TGF-β can regulate Cx43 expression in other cell types and stimulate human ovarian cancer cell migration. However, whether Cx43 can be regulated by TGF-β and is involved in TGF-β-stimulated cell migration in human ovarian cancer cells remain unknown. In this study, we demonstrate that TGF-β up-regulates Cx43 in two human ovarian cancer cell lines, SKOV3 and OVCAR4. The stimulatory effect of TGF-β on Cx43 expression is blocked by inhibition of TGF-β receptor. Treatment with TGF-β activates Smad2 and Smad3 signaling pathways in both ovarian cancer cell lines. In addition, siRNA-mediated knockdown of Smad2 or Smd3 abolishes TGF-β-induced up-regulation of Cx43 expression. Moreover, knockdown of Cx43 attenuates TGF-β-stimulated cell migration. This study demonstrates an important role for Cx43 in mediating the effects of TGF-β on human ovarian cancer cell migration.

  19. Functional expression of gap junction gene Cx43 and the myogenic differentiation of rhabdomyosarcoma cells

    Institute of Scientific and Technical Information of China (English)

    林仲翔; 张志谦; 韩亚玲; C.C.G.Naus; K.R.Yu; H.Holtzer

    1995-01-01

    Rhabdomyosarcoma (RD) cells express low levels of the gap junction protein connexin 43 (Cx43), and its mRNA, and display very weak gap junctional intercellular communication (GJIC) as detected by Cx43 immunofluorescence, slot-blot and dye-transfer methods. These cells grow rapidly and show aberrant and incomplete myogenic differentiation. To investigate the role of gap junctions in these cells, the expression of Cx43 with relation to cell growth and myogenic differentiation in RD single-cell subclones-RDL3 and RDL6 is studied. The subclone RDL3 grows slowly and displays better myogenic differentiation. The expression of Cx43, its mRNA and the GJIC in RDL3 is comparable to that in normal myoblasts. Another subclone RDL6 which grows rapidly, but is poorly differentiated, expresses very low levels of Cx43 and its mRNA, and very weak GJIC. By using the calcium phosphate precipitate transfection technique, a full-length cDNA-encoding Cx43 and a pSV2neo have been introduced into the RDL6 cells. Several stably

  20. Testosterone regulates tight junction proteins and influences prostatic autoimmune responses.

    Science.gov (United States)

    Meng, Jing; Mostaghel, Elahe A; Vakar-Lopez, Funda; Montgomery, Bruce; True, Larry; Nelson, Peter S

    2011-06-01

    Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry, and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an autoimmune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology.

  1. Transitions of protein traffic from cardiac ER to junctional SR

    OpenAIRE

    Sleiman, Naama H.; McFarland, Timothy P.; Jones, Larry R.; Cala, Steven E.

    2015-01-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appe...

  2. Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro

    Science.gov (United States)

    Eugenín, Eliseo A.; González, Hernán E.; Sánchez, Helmuth A.; Brañes, María C.; Sáez, Juan C.

    2007-01-01

    Connexin43 (Cx43), a gap junction protein subunit, has been previously detected in Kupffer cells (KCs) during liver inflammation, however, KCs phagocytose cell debris that may include Cx43 protein, which could explain the detection of Cx43 in KCs. We determined that KCs express Cx43 and form gap junctions both in vivo and in vitro. In liver sections of animals treated with LPS, Cx43 was detected at ED2+ cells interfaces, indicating formation of GJ between KCs in vivo. In vitro, unstimulated KCs cultures did not form functional GJs, and expressed low levels of Cx43 that showed a diffuse intracellular distribution. In contrast, KCs treated with LPS plus IFN-γ, expressed a greater amount of Cx43 at both the, protein and mRNA levels, and showed Cx43 at cell-cell contacts associated with higher dye coupling. In conclusion, activation of KCs in vivo or in vitro resulted in enhanced Cx43 expression levels and formation of GJ that might play relevant roles during liver inflammation. PMID:17900549

  3. Benzalkonium chloride suppresses rabbit corneal endothelium intercellular gap junction communication.

    Directory of Open Access Journals (Sweden)

    Zhenhao Zhang

    Full Text Available Gap junction intercellular communication (GJIC plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.Thirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM. Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43 and tight junction zonula occludens-1 (ZO-1 gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP. Primary rabbit corneal endothelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT was used to assess GJIC activity.Topical administration of BAK (0.05%, 0.1% dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.The dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to

  4. Inhibition of gap junction intercellular communication is involved in silica nanoparticles-induced H9c2 cardiomyocytes apoptosis via the mitochondrial pathway

    Directory of Open Access Journals (Sweden)

    Du ZJ

    2017-03-01

    was correlated with abnormal expressions of the proteins involved in the mitochondrial pathway-related apoptosis such as Bcl-2/Bax, cytochrome C, Caspase-9, and Caspase-3. Taken together, our results provide the first evidence that SNPs-decreased GJIC promotes apoptosis in cardiomyocytes via the mitochondrial pathway. In addition, downregulation of GJIC by SNPs in cardiomyocytes is mediated through downregulation of Cx43 and upregulation of P-Cx43. These results suggest that in rat cardiomyocytes cell line, GJIC plays a protective role in SNPs-induced apoptosis and that GJIC may be one of the targets for SNPs-induced biological effects. Keywords: silica nanoparticles, cardiotoxicity, connexin 43, cell death, gap junction

  5. Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells.

    Science.gov (United States)

    Pollok, Simone; Pfeiffer, Ann-Catherine; Lobmann, Ralf; Wright, Catherine S; Moll, Ingrid; Martin, Patricia E M; Brandner, Johanna M

    2011-04-01

    During early wound healing (WH) events Connexin 43 (Cx43) is down-regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down-regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non-diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non-diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non-diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non-diabetic cells. The cause of the discrepancy between diabetic and non-diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368-phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.

  6. Impact of the controlled release of a connexin 43 peptide on corneal wound closure in an STZ model of type I diabetes.

    Directory of Open Access Journals (Sweden)

    Keith Moore

    Full Text Available The alpha-carboxy terminus 1 (αCT1 peptide is a synthetically produced mimetic modified from the DDLEI C-terminus sequence of connexin 43 (Cx43. Previous research using various wound healing models have found promising therapeutic effects when applying the drug, resulting in increased wound healing rates and reduced scarring. Previous data suggested a rapid metabolism rate in vitro, creating an interest in long term release. Using a streptozotocin (STZ type I diabetic rat model with a surgically induced corneal injury, we delivered αCT1 both directly, in a pluronic gel solution, and in a sustained system, using polymeric alginate-poly-l-ornithine (A-PLO microcapsules (MC. Fluorescent staining of wound area over a 5 day period indicated a significant increase in wound closure rates for both αCT1 and αCT1 MC treated groups, withαCT1 MC groups showing the most rapid wound closure overall. Analysis of inflammatory reaction to the treatment groups indicated significantly lower levels of both Interferon Inducible T-Cell Alpha Chemoattractant (ITAC and Tumor Necrosis Factor Alpha (TNFα markers using confocal quantification and ELISA assays. Additional analysis examining genes selected from the EMT pathway using RT-PCR and Western blotting suggested αCT1 modification of Transforming Growth Factor Beta 2 (TGFβ2, Keratin 8 (Krt8, Estrogen Receptor 1 (Esr1, and Glucose Transporter 4 (Glut4 over a 14 day period. Combined, this data indicated a possible suppression of the inflammatory response by αCT1, leading to increased wound healing rates.

  7. Alteration in synaptic junction proteins following traumatic brain injury.

    Science.gov (United States)

    Merlo, Lucia; Cimino, Francesco; Angileri, Filippo Flavio; La Torre, Domenico; Conti, Alfredo; Cardali, Salvatore Massimiliano; Saija, Antonella; Germanò, Antonino

    2014-08-15

    Extensive research and scientific efforts have been focused on the elucidation of the pathobiology of cellular and axonal damage following traumatic brain injury (TBI). Conversely, few studies have specifically addressed the issue of synaptic dysfunction. Synaptic junction proteins may be involved in post-TBI alterations, leading to synaptic loss or disrupted plasticity. A Synapse Protein Database on synapse ontology identified 109 domains implicated in synaptic activities and over 5000 proteins, but few of these demonstrated to play a role in the synaptic dysfunction after TBI. These proteins are involved in neuroplasticity and neuromodulation and, most importantly, may be used as novel neuronal markers of TBI for specific intervention.

  8. Implanted neural progenitor cells regulate glial reaction to brain injury and establish gap junctions with host glial cells.

    Science.gov (United States)

    Talaverón, Rocío; Matarredona, Esperanza R; de la Cruz, Rosa R; Macías, David; Gálvez, Victoria; Pastor, Angel M

    2014-04-01

    Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant-induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC-grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants.

  9. Diabetes Increases Cryoinjury Size with Associated Effects on Cx43 Gap Junction Function and Phosphorylation in the Mouse Heart

    Directory of Open Access Journals (Sweden)

    Joseph A. Palatinus

    2016-01-01

    Full Text Available Diabetic patients develop larger myocardial infarctions and have an increased risk of death following a heart attack. The poor response to myocardial injury in the diabetic heart is likely related to the many metabolic derangements from diabetes that create a poor substrate in general for wound healing, response to injury and infection. Studies in rodents have implicated a role for the gap junction protein connexin 43 (Cx43 in regulating the injury response in diabetic skin wounds. In this study, we sought to determine whether diabetes alters Cx43 molecular interactions or intracellular communication in the cryoinjured STZ type I diabetic mouse heart. We found that epicardial cryoinjury size is increased in diabetic mice and this increase is prevented by preinjury insulin administration. Consistent with these findings, we found that intercellular coupling via gap junctions is decreased after insulin administration in diabetic and nondiabetic mice. This decrease in coupling is associated with a concomitant increase in phosphorylation of Cx43 at serine 368, a residue known to decrease channel conductance. Taken together, our results suggest that insulin regulates both gap junction-mediated intercellular communication and injury propagation in the mouse heart.

  10. Cell junction proteins within the cochlea:A review of recent research

    Institute of Scientific and Technical Information of China (English)

    Bo Wang; Bohua Hu; Shiming Yang

    2015-01-01

    Cell—cell junctions in the cochlea are highly complex and well organized. The role of these junctions is to maintain structural and functional integrity of the cochlea. In this review, we describe classification of cell junction-associated proteins identified within the cochlea and provide a brief overview of the function of these proteins in adherent junctions, gap junctions and tight junctions. Copyright © 2016, PLA General Hospital Department of Otolaryngology Head and Neck Surgery. Production and hosting by Elsevier (Singapore) Pte Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

  11. Tumor necrosis factor-mediated downregulation of spinal astrocytic connexin43 leads to increased glutamatergic neurotransmission and neuropathic pain in mice.

    Science.gov (United States)

    Morioka, Norimitsu; Zhang, Fang Fang; Nakamura, Yoki; Kitamura, Tomoya; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2015-10-01

    Spinal cord astrocytes are critical in the maintenance of neuropathic pain. Connexin 43 (Cx43) expressed on spinal dorsal horn astrocytes modulates synaptic neurotransmission, but its role in nociceptive transduction has yet to be fully elaborated. In mice, Cx43 is mainly expressed in astrocytes, not neurons or microglia, in the spinal dorsal horn. Hind paw mechanical hypersensitivity was observed beginning 3days after partial sciatic nerve ligation (PSNL), but a persistent downregulation of astrocytic Cx43 in ipsilateral lumbar spinal dorsal horn was not observed until 7days post-PSNL, suggesting that Cx43 downregulation mediates the maintenance and not the initiation of nerve injury-induced hypersensitivity. Downregulation of Cx43 expression by intrathecal treatment with Cx43 siRNA also induced mechanical hypersensitivity. Conversely, restoring Cx43 by an adenovirus vector expressing Cx43 (Ad-Cx43) ameliorated PSNL-induced mechanical hypersensitivity. The sensitized state following PSNL is likely maintained by dysfunctional glutamatergic neurotransmission, as Cx43 siRNA-induced mechanical hypersensitivity was attenuated with intrathecal treatment of glutamate receptor antagonists MK801 and CNQX, but not neurokinin-1 receptor antagonist CP96345 or the Ca(2+) channel subunit α2δ1 blocker gabapentin. The source of this dysfunctional glutamatergic neurotransmission is likely decreased clearance of glutamate from the synapse rather than increased glutamate release into the synapse. Astrocytic expression of glutamate transporter GLT-1, but not GLAST, and activity of glutamate transport were markedly decreased in mice intrathecally injected with Cx43-targeting siRNA but not non-targeting siRNA. Glutamate release from spinal synaptosomes prepared from mice treated with either Cx43-targeting siRNA or non-targeting siRNA was unchanged. Intrathecal injection of Ad-Cx43 in PSNL mice restored astrocytic GLT-1 expression. The cytokine tumor necrosis factor (TNF) has been

  12. 间隙连接蛋白Connexin43对力刺激下人牙周膜细胞成骨相关转录因子表达的影响%Effect of Connexin43 on Mechanical Tension-stimulated Osteogenic Transcription Factors of Human Periodontal Ligament Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    李盛楠; 张华菁; 霍波; 张丁

    2016-01-01

    目的 研究间隙连接蛋白Connexin43 (Cx43)在牵张力刺激下对牙周膜成纤维细胞成骨相关转录因子表达的影响.方法 对体外培养的人牙周膜成纤维细胞(hPDLFs)施加变形量为5%的牵张力,在受力1、2、4、8、24 h后检测成骨相关转录因子Osterix、RUNX2及Cx43 mRNA的表达.分别采用间隙连接的阻断剂18α-GA和基因沉没Cx43的方法阻断Cx43后观察Osteix、RUNX2 mRNA表达的变化.结果 对hPDLFs施加牵张力时,hPDLFs内的Cx43、Osterix和RUNX2mRNA水平随加力时间增加表达均明显增强(p均<O.05),在24h达到最高值.分别阻断间隙连接通道和抑制间隙连接蛋白Cx43,结果显示Osterix和RUNX2 mRNA表达水平均受到明显抑制(P均<O.05).结论 力刺激下间隙连接蛋白Cx43及成骨转录因子Osterix和RUNX2在hPDLFs中的表达呈现时间依赖性增加.间隙连接蛋白Cx43可能参与了这两种成骨转录因子表达的调节.

  13. The role of spinal interleukin-1β and astrocyte connexin 43 in the development of mirror-image pain in an inflammatory pain model.

    Science.gov (United States)

    Choi, Hoon-Seong; Roh, Dae-Hyun; Yoon, Seo-Yeon; Kwon, Soon-Gu; Choi, Sheu-Ran; Kang, Suk-Yun; Moon, Ji-Young; Han, Ho-Jae; Kim, Hyun-Woo; Beitz, Alvin J; Lee, Jang-Hern

    2016-10-20

    Although we have recently demonstrated that carrageenan-induced inflammation upregulates the expression of spinal interleukin (IL)-1β, which inhibits spinal astrocyte activation and results in the delayed development of Mirror-Image Pain (MIP), little is known regarding the mechanisms that underlie how spinal IL-1β inhibits the astrocyte activation. In this study, we examined the effect of spinal IL-1β on astrocyte gap junctions (GJ) and the development of MIP. Following unilateral carrageenan (CA) injection, mechanical allodynia (MA) was evaluated at various time points. Immunohistochemistry and Western blot analysis were used to determine changes in the expression of GFAP and connexins (Cx) in the spinal cord dorsal horn. Carrageenan rats showed a delayed onset of contralateral MA, which mimicked the temporal expression pattern of spinal Cx43 (an astrocyte gap junctional protein) and GFAP. Intrathecal administration of an interleukin-1 receptor antagonist (IL-1ra) twice-a-day on post-carrageenan injection days 0 to 3 caused a significant increase in contralateral MA and spinal Cx43 and GFAP expression. In addition, co-administration of IL-1β with IL-1ra blocked the IL-1ra-induced increase in contralateral MA and the upregulated expression of spinal Cx43 and GFAP. Finally, co-administration of carbenoxolone (CBX; a GJ decoupler) or Gap26 (a specific Cx43 mimetic blocking peptide) with IL-1ra significantly blocked the IL-1ra-induced early development of contralateral MA and the associated upregulation of spinal Cx43 and GFAP expression. These results demonstrate that spinal IL-1β suppresses Cx43 expression and astrocyte activation during the early phase of CA-induced inflammation resulting in the delayed onset of contralateral MA. These findings imply that spinal IL-1β can inhibit astrocyte activation and regulate the time of induction of contralateral MA through modulation of spinal Cx43 expression.

  14. Rad54 protein promotes branch migration of Holliday junctions.

    Science.gov (United States)

    Bugreev, Dmitry V; Mazina, Olga M; Mazin, Alexander V

    2006-08-03

    Homologous recombination has a crucial function in the repair of DNA double-strand breaks and in faithful chromosome segregation. The mechanism of homologous recombination involves the search for homology and invasion of the ends of a broken DNA molecule into homologous duplex DNA to form a cross-stranded structure, a Holliday junction (HJ). A HJ is able to undergo branch migration along DNA, generating increasing or decreasing lengths of heteroduplex. In both prokaryotes and eukaryotes, the physical evidence for HJs, the key intermediate in homologous recombination, was provided by electron microscopy. In bacteria there are specialized enzymes that promote branch migration of HJs. However, in eukaryotes the identity of homologous recombination branch-migration protein(s) has remained elusive. Here we show that Rad54, a Swi2/Snf2 protein, binds HJ-like structures with high specificity and promotes their bidirectional branch migration in an ATPase-dependent manner. The activity seemed to be conserved in human and yeast Rad54 orthologues. In vitro, Rad54 has been shown to stimulate DNA pairing of Rad51, a key homologous recombination protein. However, genetic data indicate that Rad54 protein might also act at later stages of homologous recombination, after Rad51 (ref. 13). Novel DNA branch-migration activity is fully consistent with this late homologous recombination function of Rad54 protein.

  15. Gap junction proteins are key drivers of endocrine function.

    Science.gov (United States)

    Meda, Paolo

    2017-03-08

    It has long been known that the main secretory cells of exocrine and endocrine glands are connected by gap junctions, made by a variety of connexin species that ensure their electrical and metabolic coupling. Experiments in culture systems and animal models have since provided increasing evidence that connexin signaling contributes to control the biosynthesis and release of secretory products, as well as to the life and death of secretory cells. More recently, genetic studies have further provided the first lines of evidence that connexins also control the function of human glands, which are central to the pathogenesis of major endocrine diseases. Here, we summarize the recent information gathered on connexin signaling in these systems, since the last reviews on the topic, with particular regard to the pancreatic beta cells which produce insulin, and the renal cells which produce renin. These cells are keys to the development of various forms of diabetes and hypertension, respectively, and combine to account for the exploding, worldwide prevalence of the metabolic syndrome. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Upregulation of Connexin 43 Expression Via C-Jun N-Terminal Kinase Signaling in Prion Disease.

    Science.gov (United States)

    Lee, Geon-Hwi; Jang, Byungki; Choi, Hong-Seok; Kim, Hee-Jun; Park, Jeong-Ho; Jeon, Yong-Chul; Carp, Richard I; Kim, Yong-Sun; Choi, Eun-Kyoung

    2016-01-01

    Prion infection leads to neuronal cell death, glial cell activation, and the accumulation of misfolded prion proteins. However, the altered cellular environments in animals with prion diseases are poorly understood. In the central nervous system, cells connect the cytoplasm of adjacent cells via connexin (Cx)-assembled gap junction channels to allow the direct exchange of small molecules, including ions, neurotransmitters, and signaling molecules, which regulate the activities of the connected cells. Here, we investigate the role of Cx43 in the pathogenesis of prion diseases. Upregulated Cx43 expression, which was dependent on c-Jun N-Terminal Kinase (JNK)/c-Jun signaling cascades, was found in prion-affected brain tissues and hippocampal neuronal cells. Scrapie infection-induced Cx43 formed aggregated plaques within the cytoplasmic compartments at the cell-cell interfaces. The ethidium bromide (EtBr) uptake assay and scrape-loading dye transfer assay demonstrated that increased Cx43 has functional consequences for the activity of Cx43 hemichannels. Interestingly, blockade of PrPSc accumulation reduced Cx43 expression through the inhibition of JNK signaling, indicating that PrPSc accumulation may be directly involved in JNK activation-mediated Cx43 upregulation. Overall, our findings describe a scrapie infection-mediated novel regulatory signaling pathway of Cx43 expression and may suggest a role for Cx43 in the pathogenesis of prion diseases.

  17. Gap junction proteins in the light-damaged albino rat.

    Science.gov (United States)

    Guo, Cindy X; Tran, Henry; Green, Colin R; Danesh-Meyer, Helen V; Acosta, Monica L

    2014-01-01

    Changes in connexin expression are associated with many pathological conditions seen in animal models and in humans. We hypothesized that gap junctions are important mediators in tissue dysfunction and injury processes in the retina, and therefore, we investigated the pattern of connexin protein expression in the light-damaged albino rat eye. Adult Sprague-Dawley rats were exposed to intense light for 24 h. The animals were euthanized, and ocular tissue was harvested at 0 h, 6 h, 24 h, 48 h, and 7 days after light damage. The tissues were processed for immunohistochemistry and western blotting to analyze the expression of the gap junction proteins in the light-damaged condition compared to the non-light-damaged condition. Cell death was detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) technique. Intense light exposure caused increased TUNEL labeling of photoreceptor cells. Immunocytochemistry revealed that connexin 36 (Cx36) was significantly increased in the inner plexiform layer and Cx45 was significantly decreased in the light-damaged retina. The pattern of Cx36 and Cx45 labeling returned to normal 7 days after light damage. Cx43 significantly increased in the RPE and the choroid in the light-damaged tissue, and decreased but not significantly in the retina. This elevated Cx43 expression in the choroid colocalized with markers of nitration-related oxidative stress (nitrotyrosine) and inflammation (CD45 and ionized calcium-binding adaptor molecule-1) in the choroid. The results suggest that connexins are regulated differently in the retina than in the choroid in response to photoreceptor damage. Changes in connexins, including Cx36, Cx43, and Cx45, may contribute to the damage process. Specifically, Cx43 was associated with inflammatory damage. Therefore, connexins may be candidate targets for treatment for ameliorating disease progression.

  18. Biphasic Effect of Basic Fibroblast Growth Factor on Anterior Pituitary Folliculostellate TtT/GF Cell Coupling, and Connexin 43 Expression and Phosphorylation.

    Science.gov (United States)

    Vitale, M L; Barry, A

    2015-10-01

    Basic fibroblast growth factor (bFGF) is a mitogenic and differentiating cytokine. In the anterior pituitary, folliculostellate (FS) cells constitute the major source of bFGF. bFGF affects endocrine cell proliferation and secretion in the anterior pituitary. In addition, bFGF increases its own expression by acting directly on FS cells. FS cell Cx43-mediated gap junction intercellular communication allows the establishment of an intrapituitary network for the transmission of information. In the present study, we assessed how bFGF regulates FS cell coupling. Time course studies were carried out on the FS cell line TtT/GF. Short-term bFGF treatment induced a transient cell uncoupling and the phosphorylation in Ser368 of membrane-bound Cx43 without modifying Cx43 levels. We demonstrated the involvement of the protein kinase C (PKC) isoform α in the phosphorylation of Cx43 in S368. Moreover, we showed that bFGF induced PKCα activation by stimulating its expression, phosphorylation and association with the plasma membrane. The long-term incubation with bFGF increased TtT/GF cell coupling, total Cx43 levels and Cx43 accumulation at the cell membrane of cytoplasmic projections. The Cx43 level increase was a result of the stimulation of Cx43 gene transcription as mediated by the extracellular-regulated kinase 1/2 signalling pathway. Taken together, the data show that bFGF modulates TtT/GF cell coupling by activating different pathways that lead to opposite effects on Cx43 phosphorylation and expression depending on the duration of the exposure of the cells to bFGF. A short-term bFGF exposure reduces cell-to-cell communication as a mean of desynchronising FS cells. By contrast, long-term exposure to bFGF enhances cell-to-cell communication and facilitates coordination among FS cells. © 2015 British Society for Neuroendocrinology.

  19. Reduced expression of adherens and gap junction proteins can have a fundamental role in the development of heart failure following cardiac hypertrophy in rats.

    Science.gov (United States)

    dos Santos, Daniele O; Blefari, Valdecir; Prado, Fernanda P; Silva, Carlos A; Fazan, Rubens; Salgado, Helio C; Ramos, Simone G; Prado, Cibele M

    2016-02-01

    Hypertension causes cardiac hypertrophy, cardiac dysfunction and heart failure (HF). The mechanisms implicated in the transition from compensated to decompensated cardiac hypertrophy are not fully understood. This study was aimed to investigate whether alterations in the expression of intercalated disk proteins could contribute to the transition of compensated cardiac hypertrophy to dilated heart development that culminates in HF. Male rats were submitted to abdominal aortic constriction and at 90 days post surgery (dps), three groups were observed: sham-operated animals (controls), animals with hypertrophic hearts (HH) and animals with hypertrophic + dilated hearts (HD). Blood pressure was evaluated. The hearts were collected and Western blot and immunofluorescence were performed to desmoglein-2, desmocollin-2, N-cadherin, plakoglobin, Bcatenin, and connexin-43. Cardiac systolic function was evaluated using the Vevo 2100 ultrasound system. Data were considered significant when p b 0.05. Seventy percent of the animals presented with HH and 30% were HD at 90 dps. The blood pressure increased in both groups. The amount of desmoglein-2 and desmocollin-2 expression was increased in both groups and no difference was observed in either group. The expression of N-cadherin, plakoglobin and B-catenin increased in the HHgroup and decreased in the HDgroup; and connexin-43 decreased only in theHDgroup. Therewas no difference between the ejection fraction and fractional shortening at 30 and 60 dps; however, they were decreased in the HD group at 90 dps. We found that while some proteins have increased expression accompanied by the increase in the cell volume associated with preserved systolic cardiac function in theHHgroup, these same proteins had decreased expression evenwithout significant reduction in the cell volume associated with decreased systolic cardiac function in HD group. The increased expression of desmoglein-2 and desmocollin-2 in both the HH and HD groups could

  20. Intestinal epithelial barrier function and tight junction proteins with heat and exercise

    DEFF Research Database (Denmark)

    Dokladny, Karol; Zuhl, Micah N; Moseley, Pope L

    2016-01-01

    (passive hyperthermia) heat stress on tight junction barrier function in in vitro and in vivo (animals and humans) models. Our secondary focus is to review changes in tight junction proteins in response to exercise or hyperthermic conditions. Finally, we discuss some pharmacological or nutritional...... interventions that may affect the cellular mechanisms involved in maintaining homeostasis of the intestinal epithelial tight junction barrier during heat stress or exercise....

  1. Inhibition of gap junction intercellular communication is involved in silica nanoparticles-induced H9c2 cardiomyocytes apoptosis via the mitochondrial pathway.

    Science.gov (United States)

    Du, Zhong-Jun; Cui, Guan-Qun; Zhang, Juan; Liu, Xiao-Mei; Zhang, Zhi-Hu; Jia, Qiang; Ng, Jack C; Peng, Cheng; Bo, Cun-Xiang; Shao, Hua

    2017-01-01

    Gap junction intercellular communication (GJIC) between cardiomyocytes is essential for synchronous heart contraction and relies on connexin-containing channels. Connexin 43 (Cx43) is a major component involved in GJIC in heart tissue, and its abnormal expression is closely associated with various cardiac diseases. Silica nanoparticles (SNPs) are known to induce cardiovascular toxicity. However, the mechanisms through which GJIC plays a role in cardiomyocytes apoptosis induced by SNPs remain unknown. The aim of the present study is to determine whether SNPs-decreased GJIC promotes apoptosis in rat cardiomyocytes cell line (H9c2 cells) via the mitochondrial pathway using CCK-8 Kit, scrape-loading dye transfer technique, Annexin V/PI double-staining assays, and Western blot analysis. The results showed that SNPs elicited cytotoxicity in H9c2 cells in a time- and concentration-dependent manner. SNPs also reduced GJIC in H9c2 cells in a concentration-dependent manner through downregulation of Cx43 and upregulation of P-Cx43. Inhibition of gap junctions by gap junction blocker carbenoxolone disodium resulted in decreased survival and increased apoptosis, whereas enhancement of the gap junctions by retinoic acid led to enhanced survival but decreased apoptosis. Furthermore, SNPs-induced apoptosis through the disrupted functional gap junction was correlated with abnormal expressions of the proteins involved in the mitochondrial pathway-related apoptosis such as Bcl-2/Bax, cytochrome C, Caspase-9, and Caspase-3. Taken together, our results provide the first evidence that SNPs-decreased GJIC promotes apoptosis in cardiomyocytes via the mitochondrial pathway. In addition, downregulation of GJIC by SNPs in cardiomyocytes is mediated through downregulation of Cx43 and upregulation of P-Cx43. These results suggest that in rat cardiomyocytes cell line, GJIC plays a protective role in SNPs-induced apoptosis and that GJIC may be one of the targets for SNPs-induced biological

  2. 异氟烷对心肌动作电位及缝隙连接蛋白Cx43的作用%Effects of Isoflurane on Action Potential of Heart and Expression of Myocardial Connexin 43

    Institute of Scientific and Technical Information of China (English)

    谷宇; 高鸿; 杨烨

    2012-01-01

    目的:观察异氟烷对心肌动作电位及缝隙连接蛋白Cx43表达的影响.方法:健康家兔40只,采用Langendorff离体心脏平衡灌注15min后,随机分为5组:I0.65组,用0.65MAC异氟烷的K-H液灌注;I0,65H用0.65MAC异氟烷加0.5mmol/L庚醇的K-H液灌注;I1.3组,用1.3MAC异氟烷的K-H液灌注;H0.5组,用庚醇0.5mmol/L的K-H液灌注;S组,用K-H液灌注.观察记录灌流15min时(基础值)和给药15min时的心率(HR)及心内膜心肌(Endo)、中层心肌(M)、心外膜心肌(Epi)的动作电位时程(APD)和振幅(APA),灌注完毕取左心室心肌组织,用免疫组织化学检测心肌Cx43蛋白表达.结果:I0.65组心肌动作电位无明显影响,I1.3组APD显著延长(P<0.05),H0.5组HR显著减慢(P<0.05),APD显著延长(P<0.05),k0.65H组给药后有发生心律失常甚至心电活动停止现象;Cx43蛋白表达I0.65H组较H0.5组表达少(P<0.05),H0.5组较I0.65、I1.3组表达少(P<0.05),I1.3组较I0.65组、S组表达少(P<0.05).结论:异氟烷与庚醇对心脏动作电位和缝隙连接蛋白Cx43可能有着相似的作用,延长动作单位时程,减少Cx43蛋白表达,改变Cx43蛋白分布;异氟烷可能通过阻滞缝隙连接,对心脏动作电位产生作用.%Objective: To observe the effects of isoflurane on action potential ( AP) of heart and the expression of myocardial connexin43 (Cx43). Methods: Forty isolated rabbit hearts were perfused for 15 minutes with modified Krebs-Hensleit buffer using Langendorff device. Then, all the hearts were randomly divided into five groups (re =8 in each) : group I0.65, group I0.65H , group 1.3, group t0. 5 and group S, in which hearts were perfused with Kreb-Hensleit buffer containing 0. 65 MAC isoflurane, 0.65 MAC isoflurane plus 0.5 mmol/L heptanol, 1.3 MAC isoflurane, 0.5 mmol/L heptanol and Kreb-Hensleit buffer only respectively. Heart rates ( HR) , AP course ( APD) and AP amplitude ( APA) of endocardium ( Endo) , mid-layer myocardium ( M ) and

  3. Altered expression of epithelial junctional proteins in atopic asthma: Possible role in inflammation

    NARCIS (Netherlands)

    W.I. de Boer (Pim); H.S. Sharma (Hari); S.M. Baelemans (Sophia); H.C. Hoogsteden (Henk); B.N.M. Lambrecht (Bart); G.J. Braunstahl (Gert-Jan)

    2008-01-01

    textabstractEpithelial cells form a tight barrier against environmental stimuli via tight junctions (TJs) and adherence junctions (AJs). Defects in TJ and AJ proteins may cause changes in epithelial morphology and integrity and potentially lead to faster trafficking of inflammatory cells through the

  4. Transitions of protein traffic from cardiac ER to junctional SR.

    Science.gov (United States)

    Sleiman, Naama H; McFarland, Timothy P; Jones, Larry R; Cala, Steven E

    2015-04-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca(2+) release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12-24h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog, demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQ-DsRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed ((del)TRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog and (del)TRD were therefore able to elucidate areas of ER-SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER-SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic.

  5. Transitions of protein traffic from cardiac ER to junctional SR

    Science.gov (United States)

    Sleiman, Naama H.; McFarland, Timothy P.; Jones, Larry R.; Cala, Steven E.

    2015-01-01

    The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca2+ release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12-24 h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24-48 h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog, demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQD-sRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed (delTRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog and del TRD were therefore able to elucidate areas of ER-SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER-SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic. PMID:25640161

  6. Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor.

    Science.gov (United States)

    Cherian, Priscilla P; Cheng, Benxu; Gu, Sumin; Sprague, Eugene; Bonewald, Lynda F; Jiang, Jean X

    2003-10-31

    Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components

  7. [Remodeling of cardiac gap junctions and arrhythmias].

    Science.gov (United States)

    Yu, Zhi-Bin; Sheng, Juan-Juan

    2011-12-25

    In the heart, gap junctions mediate electrical and chemical coupling between adjacent cardiomyocytes, forming the cell-to-cell pathways for orderly spread of the wave of electrical excitation responsible for a functional syncytium. Three principal connexins are expressed in cardiomyocytes, connexin 43 (CX43), CX40, and CX45. CX43 predominates in ventricular muscle cells. Most of the gap junctions, assembled from CX43, are located at the intercalated discs, often with larger junctional plaques at the disc periphery. The gap junctions are rarely distributed to the sides of the cardiomyocyte. The ischemia-reperfusion, cardiac hypertrophy, heart failure, hypercholesterolemia, and diabetes mellitus induce gap junction remodeling. The gap junction remodeling induced by above-mentioned diseases shows similar characteristics, including down-regulation of CX43, reduction in gap junction plaque size, increased heterogeneity and lateralization of gap junction distribution, and dephosphorylation of CX43. The elevated angiotensin II concentration in local myocardium may play an important role in the gap junction remodeling. The down-regulation of CX43 and lateralization of gap junction distribution alter anisotropic spread of the impulse of ventricular myocardium. The dephosphorylation of CX43 not only reduces electrical conductance, but also decreases permeability of chemicals between cardiomyocytes. The lateralization of gap junctions may increase the number of hemichannels formed by CX43. The opening of hemichannels induces ATP efflux and Na(+) influx, which forms a delayed after-depolarization. The gap junction remodeling in pathological condition produces arrhythmia substrate in the ventricles. In this review, the current knowledge on the relationship between the remodeling of cardiac gap junctions and arrhythmias were summarized.

  8. Do cell junction protein mutations cause an airway phenotype in mice or humans?

    Science.gov (United States)

    Chang, Eugene H; Pezzulo, Alejandro A; Zabner, Joseph

    2011-08-01

    Cell junction proteins connect epithelial cells to each other and to the basement membrane. Genetic mutations of these proteins can cause alterations in some epithelia leading to varied phenotypes such as deafness, renal disease, skin disorders, and cancer. This review examines if genetic mutations in these proteins affect the function of lung airway epithelia. We review cell junction proteins with examples of disease mutation phenotypes in humans and in mouse knockout models. We also review which of these genes are expressed in airway epithelium by microarray expression profiling and immunocytochemistry. Last, we present a comprehensive literature review to find the lung phenotype when cell junction and adhesion genes are mutated or subject to targeted deletion. We found that in murine models, targeted deletion of cell junction and adhesion genes rarely result in a lung phenotype. Moreover, mutations in these genes in humans have no obvious lung phenotype. Our research suggests that simply because a cell junction or adhesion protein is expressed in an organ does not imply that it will exhibit a drastic phenotype when mutated. One explanation is that because a functioning lung is critical to survival, redundancy in the system is expected. Therefore mutations in a single gene might be compensated by a related function of a similar gene product. Further studies in human and animal models will help us understand the overlap in the function of cell junction gene products. Finally, it is possible that the human lung phenotype is subtle and has not yet been described.

  9. Tight junction-associated MARVEL proteins marveld3, tricellulin, and occludin have distinct but overlapping functions.

    Science.gov (United States)

    Raleigh, David R; Marchiando, Amanda M; Zhang, Yong; Shen, Le; Sasaki, Hiroyuki; Wang, Yingmin; Long, Manyuan; Turner, Jerrold R

    2010-04-01

    In vitro studies have demonstrated that occludin and tricellulin are important for tight junction barrier function, but in vivo data suggest that loss of these proteins can be overcome. The presence of a heretofore unknown, yet related, protein could explain these observations. Here, we report marvelD3, a novel tight junction protein that, like occludin and tricellulin, contains a conserved four-transmembrane MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain. Phylogenetic tree reconstruction; analysis of RNA and protein tissue distribution; immunofluorescent and electron microscopic examination of subcellular localization; characterization of intracellular trafficking, protein interactions, dynamic behavior, and siRNA knockdown effects; and description of remodeling after in vivo immune activation show that marvelD3, occludin, and tricellulin have distinct but overlapping functions at the tight junction. Although marvelD3 is able to partially compensate for occludin or tricellulin loss, it cannot fully restore function. We conclude that marvelD3, occludin, and tricellulin define the tight junction-associated MARVEL protein family. The data further suggest that these proteins are best considered as a group with both redundant and unique contributions to epithelial function and tight junction regulation.

  10. Caveolin-1 modulates cardiac gap junction homeostasis and arrhythmogenecity by regulating cSrc tyrosine kinase.

    Science.gov (United States)

    Yang, Kai-Chien; Rutledge, Cody A; Mao, Mao; Bakhshi, Farnaz R; Xie, An; Liu, Hong; Bonini, Marcelo G; Patel, Hemal H; Minshall, Richard D; Dudley, Samuel C

    2014-08-01

    Genome-wide association studies have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear. Using adult Cav1(-/-) mice, we revealed a marked reduction in the left ventricular conduction velocity in the absence of myocardial Cav1, which is accompanied with increased inducibility of ventricular arrhythmias. Further studies demonstrated that loss of Cav1 leads to the activation of cSrc tyrosine kinase, resulting in the downregulation of connexin 43 and subsequent electric abnormalities. Pharmacological inhibition of cSrc mitigates connexin 43 downregulation, slowed conduction, and arrhythmia inducibility in Cav1(-/-) animals. Using a transgenic mouse model with cardiac-specific overexpression of angiotensin-converting enzyme (ACE8/8), we demonstrated that, on enhanced cardiac renin-angiotensin system activity, Cav1 dissociated from cSrc because of increased Cav1 S-nitrosation at Cys(156), leading to cSrc activation, connexin 43 reduction, impaired gap junction function, and subsequent increase in the propensity for ventricular arrhythmias and sudden cardiac death. Renin-angiotensin system-induced Cav1 S-nitrosation was associated with increased Cav1-endothelial nitric oxide synthase binding in response to increased mitochondrial reactive oxidative species generation. The present studies reveal the critical role of Cav1 in modulating cSrc activation, gap junction remodeling, and ventricular arrhythmias. These data provide a mechanistic explanation for the observed genetic link between Cav1 and cardiac arrhythmias in humans and suggest that targeted regulation of Cav1 may reduce arrhythmic risk in cardiac diseases associated with renin-angiotensin system activation. © 2014 American Heart Association, Inc.

  11. TMEM43 mutation p.S358L alters intercalated disc protein expression and reduces conduction velocity in arrhythmogenic right ventricular cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Vinayakumar Siragam

    Full Text Available Arrhythmogenic right ventricular cardiomyopathy (ARVC is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43 gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM, immunofluorescence (IF, and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1 expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43 phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.

  12. Red paprika (Capsicum annuum L.) and its main carotenoids, capsanthin and β-carotene, prevent hydrogen peroxide-induced inhibition of gap-junction intercellular communication.

    Science.gov (United States)

    Kim, Ji-Sun; Lee, Woo-Moon; Rhee, Han Cheol; Kim, Suna

    2016-07-25

    This study was conducted to investigate the protective effect of red paprika extract (RPE) and its main carotenoids, namely, capsanthin (CST) and β-carotene (BCT), on the H2O2-induced inhibition of gap-junction intercellular communication (GJIC) in WB-F344 rat liver epithelial cells (WB cells). We found that pre-treatment with RPE, CST and BCT protected WB cells from H2O2-induced inhibition of GJIC. RPE, CST and BCT not only recovered connexin 43 (Cx43) mRNA expression but also prevented phosphorylation of Cx43 protein by H2O2 treatment. RPE attenuated the phosphorylation of ERK, p38 and JNK, whereas pre-treatment with CST and BCT only attenuated the phosphorylation of ERK and p38 and did not affect JNK in H2O2-treated WB cells. RPE, CST and BCT significantly suppressed the formation of reactive oxygen species (ROS) in H2O2-treated cells compared to untreated WB cells. These results suggest that dietary intake of red paprika might be helpful for lowering the risk of diseases caused by oxidative stress.

  13. The ouabain-sensitive isoform of Na+-pump regulates vascular gap junctions via interaction with the Na+/Ca2+-exchanger in membrane microdomain

    DEFF Research Database (Denmark)

    Matchkov, Vladimir; Nilsson, Holger; Aalkjær, Christian

    Ouabain, an inhibitor of the Na+-pump, has been shown to inhibit intercellular communication. We have recently shown that gap junctions between vascular smooth muscle cells (SMCs) are regulated through an interaction between a ouabain-sensitive isoform of the Na+-pump and the Na+/Ca2+-exchanger...... electrical coupling was evaluated in functional studies. SMCs were electrically uncoupled when the ouabain-sensitive Na+-pump was inhibited by 10 mM ouabain. Inhibition of the Na+/Ca2+-exchanger with 1 mM SEA0400 also uncoupled the SMCs. Depletion of [Na+]i and clamping [Ca2+]i at low levels prevented......+-exchanger-1 and connexin-43. The α3 Na+-pump subunit was not associated with these proteins but co-immunoprecipitated with caveolin-1. Based on these experiments we suggest that α2 Na+ -pump subunit is involved in regulation of the intercellular communication via interaction with the Na+/Ca2+-exchanger-1...

  14. Improvement of cardiac function and reversal of gap junction remodeling by Neuregulin-1β in volume-overloaded rats with heart failure

    Institute of Scientific and Technical Information of China (English)

    Xue-Hui Wang; Xiao-Zhen Zhuo; Ya-Juan Ni; Min Gong; Ting-Zhong Wang; Qun Lu; Ai-Qun Ma

    2012-01-01

    Objective We performed experiments using Neuregulin-1β (NRG-1β) treatment to determine a mechanism for the protective role derived from its beneficial effects by remodeling gap junctions (GJs) during heart failure (HF). Methods Rat models of HF were established by aortocaval fistula. Forty-eight rats were divided randomly into the HF (HF, n = 16), NRG-1β treatment (NRG, n = 16), and sham operation (S, n = 16) group. The rats in the NRG group were administered NRG-1β (10 μg/kg per day) for 7 days via the tail vein, whereas the other groups were injected with the same doses of saline. Twelve weeks after operation, Connexin 43 (Cx43) expression in single myocytes obtained from the left ventricle was determined by immunocytochemistry. Total protein was extracted from frozen left ventricular tissues for immunoblotting assay, and the ultrastructure of myocytes was observed by transmission electron microscopy. Results Compared with the HF group, the cardiac function of rats in the NRG group was markedly improved, irregular distribution and deceased Cx43 expression were relieved. The ultrastructure of myocytes was seriously damaged in HF rats, and NRG-1β reduced these pathological damages. Conclusions Short-term NRG-1β treatment can rescue pump failure in experimental models of volume overload-induced HF, which is related to the recovery of GJs structure and the improvement of Cx43 expression.

  15. Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion.

    Science.gov (United States)

    Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Gentilini, María Virginia; Velásquez, Lis Noelia; Fossati, Carlos Alberto; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

    2015-10-12

    Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-β, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage.

  16. A membrane fusion protein αSNAP is a novel regulator of epithelial apical junctions.

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    Nayden G Naydenov

    Full Text Available Tight junctions (TJs and adherens junctions (AJs are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF attachment protein alpha (αSNAP, regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins.

  17. Direct Exposure to Ethanol Disrupts Junctional Cell-Cell Contact and Hippo-YAP Signaling in HL-1 Murine Atrial Cardiomyocytes.

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    Kanako Noritake

    Full Text Available Direct exposure of cardiomyocytes to ethanol causes cardiac damage such as cardiac arrythmias and apoptotic cell death. Cardiomyocytes are connected to each other through intercalated disks (ID, which are composed of a gap junction (GJ, adherens junction, and desmosome. Changes in the content as well as the subcellular localization of connexin43 (Cx43, the main component of the cardiac GJ, are reportedly involved in cardiac arrythmias and subsequent damage. Recently, the hippo-YAP signaling pathway, which links cellular physical status to cell proliferation, differentiation, and apoptosis, has been implicated in cardiac homeostasis under physiological as well as pathological conditions. This study was conducted to explore the possible involvement of junctional intercellular communication, mechanotransduction through cytoskeletal organization, and the hippo-YAP pathway in cardiac damage caused by direct exposure to ethanol. HL-1 murine atrial cardiac cells were used since these cells retain cardiac phenotypes through ID formation and subsequent synchronous contraction. Cells were exposed to 0.5-2% ethanol; significant apoptotic cell death was observed after exposure to 2% ethanol for 48 hours. A decrease in Cx43 levels was already observed after 3 hours exposure to 2% ethanol, suggesting a rapid degradation of this protein. Upon exposure to ethanol, Cx43 translocated into lysosomes. Cellular cytoskeletal organization was also dysregulated by ethanol, as demonstrated by the disruption of myofibrils and intermediate filaments. Coinciding with the loss of cell-cell adherence, decreased phosphorylation of YAP, a hippo pathway effector, was also observed in ethanol-treated cells. Taken together, the results provide evidence that cells exposed directly to ethanol show 1 impaired cell-cell adherence/communication, 2 decreased cellular mechanotransduction by the cytoskeleton, and 3 a suppressed hippo-YAP pathway. Suppression of hippo-YAP pathway

  18. Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions.

    Science.gov (United States)

    Lechuga, Susana; Baranwal, Somesh; Ivanov, Andrei I

    2015-05-01

    Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis.

  19. Neuroinflammation leads to region-dependent alterations in astrocyte gap junction communication and hemichannel activity.

    Science.gov (United States)

    Karpuk, Nikolay; Burkovetskaya, Maria; Fritz, Teresa; Angle, Amanda; Kielian, Tammy

    2011-01-12

    Inflammation attenuates gap junction (GJ) communication in cultured astrocytes. Here we used a well-characterized model of experimental brain abscess as a tool to query effects of the CNS inflammatory milieu on astrocyte GJ communication and electrophysiological properties. Whole-cell patch-clamp recordings were performed on green fluorescent protein (GFP)-positive astrocytes in acute brain slices from glial fibrillary acidic protein-GFP mice at 3 or 7 d after Staphylococcus aureus infection in the striatum. Astrocyte GJ communication was significantly attenuated in regions immediately surrounding the abscess margins and progressively increased to levels typical of uninfected brain with increasing distance from the abscess proper. Conversely, astrocytes bordering the abscess demonstrated hemichannel activity as evident by enhanced ethidium bromide (EtBr) uptake that could be blocked by several pharmacological inhibitors, including the connexin 43 (Cx43) mimetic peptide Gap26, carbenoxolone, the pannexin1 (Panx1) mimetic peptide (10)Panx1, and probenecid. However, hemichannel opening was transient with astrocytic EtBr uptake observed near the abscess at day 3 but not day 7 after infection. The region-dependent pattern of hemichannel activity at day 3 directly correlated with increases in Cx43, Cx30, Panx1, and glutamate transporter expression (glial L-glutamate transporter and L-glutamate/L-aspartate transporter) along the abscess margins. Changes in astrocyte resting membrane potential and input conductance correlated with the observed changes in GJ communication and hemichannel activity. Collectively, these findings indicate that astrocyte coupling and electrical properties are most dramatically affected near the primary inflammatory site and reveal an opposing relationship between the open states of GJ channels versus hemichannels during acute infection. This relationship may extend to other CNS diseases typified with an inflammatory component.

  20. Gap junction hemichannels contribute to the generation of diarrhoea during infectious enteric disease.

    Science.gov (United States)

    Guttman, Julian Andrew; Lin, Ann En-Ju; Li, Yuling; Bechberger, John; Naus, Christian C; Vogl, A Wayne; Finlay, B Brett

    2010-02-01

    The attaching and effacing (A/E) pathogens enterohaemorrhagic Escherichia coli, enteropathogenic E coli and Citrobacter rodentium colonise intestinal tracts, attach to enterocytes, collapse infected cell microvilli and alter numerous host cell processes during infection. Enterocyte alterations result in numerous small molecules being released from host cells that likely contribute to diarrhoeal phenotypes observed during these infections. One possible route for small molecules to be released from intestinal cells may be through functional gap junction hemichannels. Here we examine the involvement of these hemichannels during the diarrhoeal disease caused by A/E pathogens in vivo. Mice were infected with the diarrhoea-causing murine A/E pathogen C rodentium for 7 days. Connexin43 (Cx43) protein levels and immunolocalization in the colon were initially used to determine alterations during A/E bacterial infections in vivo. Connexin mimetic peptides and connexin permeable tracer molecules were used to gage the presence and function of unpaired connexin hemichannels. The role of Cx43 in diarrhoea generation was assessed by comparing infections of wild-type mice to Cx43 mutant mice and determining the water abundance in the colonic luminal material. We demonstrate that Cx43 protein levels are increased in colonocytes during in vivo A/E bacterial infections, resulting in functionally open connexon hemichannels in apical membranes of infected cells. moreover, infected Cx43 +/- mice do not suffer from diarrhoeal disease. This study provides the first evidence that functional connexon hemichannels can occur in the intestine and are a novel molecular mechanism of water release during infectious diarrhoea.

  1. S-diclofenac Protects against Doxorubicin-Induced Cardiomyopathy in Mice via Ameliorating Cardiac Gap Junction Remodeling

    Science.gov (United States)

    Zhang, Huili; Zhang, Alian; Guo, Changfa; Shi, Chunzhi; Zhang, Yang; Liu, Qing; Sparatore, Anna; Wang, Changqian

    2011-01-01

    Hydrogen sulfide (H2S), as a novel gaseous mediator, plays important roles in mammalian cardiovascular tissues. In the present study, we investigated the cardioprotective effect of S-diclofenac (2-[(2,6-dichlorophenyl)amino] benzeneacetic acid 4-(3H-1,2,dithiol-3-thione-5-yl)phenyl ester), a novel H2S-releasing derivative of diclofenac, in a murine model of doxorubicin-induced cardiomyopathy. After a single dose injection of doxorubicin (15 mg/kg, i.p.), male C57BL/6J mice were given daily treatment of S-diclofenac (25 and 50 µmol/kg, i.p.), diclofenac (25 and 50 µmol/kg, i.p.), NaHS (50 µmol/kg, i.p.), or same volume of vehicle. The cardioprotective effect of S-diclofenac was observed after 14 days. It showed that S-diclofenac, but not diclofenac, dose-dependently inhibited the doxorubicin-induced downregulation of cardiac gap junction proteins (connexin 43 and connexin 45) and thus reversed the remodeling of gap junctions in hearts. It also dose-dependently suppressed doxorubicin-induced activation of JNK in hearts. Furthermore, S-diclofenac produced a dose-dependent anti-inflammatory and anti-oxidative effect in this model. As a result, S-diclofenac significantly attenuated doxorubicin-related cardiac injury and cardiac dysfunction, and improved the survival rate of mice with doxorubicin-induced cardiomyopathy. These effects of S-diclofenac were mimicked in large part by NaHS. Therefore, we propose that H2S released from S-diclofenac in vivo contributes to the protective effect in doxorubicin-induced cardiomyopathy. These data also provide evidence for a critical role of H2S in the pathogenesis of doxorubicin-induced cardiomyopathy. PMID:22039489

  2. S-diclofenac protects against doxorubicin-induced cardiomyopathy in mice via ameliorating cardiac gap junction remodeling.

    Directory of Open Access Journals (Sweden)

    Huili Zhang

    Full Text Available Hydrogen sulfide (H(2S, as a novel gaseous mediator, plays important roles in mammalian cardiovascular tissues. In the present study, we investigated the cardioprotective effect of S-diclofenac (2-[(2,6-dichlorophenylamino] benzeneacetic acid 4-(3H-1,2,dithiol-3-thione-5-ylphenyl ester, a novel H(2S-releasing derivative of diclofenac, in a murine model of doxorubicin-induced cardiomyopathy. After a single dose injection of doxorubicin (15 mg/kg, i.p., male C57BL/6J mice were given daily treatment of S-diclofenac (25 and 50 µmol/kg, i.p., diclofenac (25 and 50 µmol/kg, i.p., NaHS (50 µmol/kg, i.p., or same volume of vehicle. The cardioprotective effect of S-diclofenac was observed after 14 days. It showed that S-diclofenac, but not diclofenac, dose-dependently inhibited the doxorubicin-induced downregulation of cardiac gap junction proteins (connexin 43 and connexin 45 and thus reversed the remodeling of gap junctions in hearts. It also dose-dependently suppressed doxorubicin-induced activation of JNK in hearts. Furthermore, S-diclofenac produced a dose-dependent anti-inflammatory and anti-oxidative effect in this model. As a result, S-diclofenac significantly attenuated doxorubicin-related cardiac injury and cardiac dysfunction, and improved the survival rate of mice with doxorubicin-induced cardiomyopathy. These effects of S-diclofenac were mimicked in large part by NaHS. Therefore, we propose that H(2S released from S-diclofenac in vivo contributes to the protective effect in doxorubicin-induced cardiomyopathy. These data also provide evidence for a critical role of H(2S in the pathogenesis of doxorubicin-induced cardiomyopathy.

  3. Autophagy and gap junctional intercellular communication inhibition are involved in cadmium-induced apoptosis in rat liver cells

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Hui [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Zhuo, Liling [College of Life Science, Zaozhuang University, Zaozhuang, Shandong, 277160 (China); Han, Tao; Hu, Di; Yang, Xiaokang; Wang, Yi; Yuan, Yan; Gu, Jianhong; Bian, Jianchun; Liu, Xuezhong [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China); Liu, Zongping, E-mail: liuzongping@yzu.edu.cn [College of Veterinary Medicine, Yangzhou University, and Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, 225009 (China)

    2015-04-17

    Cadmium (Cd) is known to induce hepatotoxicity, yet the underlying mechanism of how this occurs is not fully understood. In this study, Cd-induced apoptosis was demonstrated in rat liver cells (BRL 3A) with apoptotic nuclear morphological changes and a decrease in cell index (CI) in a time- and concentration-dependent manner. The role of gap junctional intercellular communication (GJIC) and autophagy in Cd-induced apoptosis was investigated. Cd significantly induced GJIC inhibition as well as downregulation of connexin 43 (Cx43). The prototypical gap junction blocker carbenoxolone disodium (CBX) exacerbated the Cd-induced decrease in CI. Cd treatment was also found to cause autophagy, with an increase in mRNA expression of autophagy-related genes Atg-5, Atg-7, Beclin-1, and microtubule-associated protein light chain 3 (LC3) conversion from cytosolic LC3-I to membrane-bound LC3-II. The autophagic inducer rapamycin (RAP) prevented the Cd-induced CI decrease, while the autophagic inhibitor chloroquine (CQ) caused a further reduction in CI. In addition, CBX promoted Cd-induced autophagy, as well as changes in expression of Atg-5, Atg-7, Beclin-1 and LC3. CQ was found to block the Cd-induced decrease in Cx43 and GJIC inhibition, whereas RAP had opposite effect. These results demonstrate that autophagy plays a protective role during Cd-induced apoptosis in BRL 3A cells during 6 h of experiment, while autophagy exacerbates Cd-induced GJIC inhibition which has a negative effect on cellular fate. - Highlights: • GJIC and autophagy is crucial for biological processes. • Cd exposure causes GJIC inhibition and autophagy increase in BRL 3A cells. • Autophagy protects Cd induced BRL 3A cells apoptosis at an early stage. • Autophagy exacerbates Cd-induced GJIC inhibition. • GJIC plays an important role in autophagy induced cell death or survival.

  4. Protein tyrosine phosphatase σ targets apical junction complex proteins in the intestine and regulates epithelial permeability.

    Science.gov (United States)

    Murchie, Ryan; Guo, Cong-Hui; Persaud, Avinash; Muise, Aleixo; Rotin, Daniela

    2014-01-14

    Protein tyrosine phosphatase (PTP)σ (PTPRS) was shown previously to be associated with susceptibility to inflammatory bowel disease (IBD). PTPσ(-/-) mice exhibit an IBD-like phenotype in the intestine and show increased susceptibility to acute models of murine colitis. However, the function of PTPσ in the intestine is uncharacterized. Here, we show an intestinal epithelial barrier defect in the PTPσ(-/-) mouse, demonstrated by a decrease in transepithelial resistance and a leaky intestinal epithelium that was determined by in vivo tracer analysis. Increased tyrosine phosphorylation was observed at the plasma membrane of epithelial cells lining the crypts of the small bowel and colon of the PTPσ(-/-) mouse, suggesting the presence of PTPσ substrates in these regions. Using mass spectrometry, we identified several putative PTPσ intestinal substrates that were hyper-tyrosine-phosphorylated in the PTPσ(-/-) mice relative to wild type. Among these were proteins that form or regulate the apical junction complex, including ezrin. We show that ezrin binds to and is dephosphorylated by PTPσ in vitro, suggesting it is a direct PTPσ substrate, and identified ezrin-Y353/Y145 as important sites targeted by PTPσ. Moreover, subcellular localization of the ezrin phosphomimetic Y353E or Y145 mutants were disrupted in colonic Caco-2 cells, similar to ezrin mislocalization in the colon of PTPσ(-/-) mice following induction of colitis. Our results suggest that PTPσ is a positive regulator of intestinal epithelial barrier, which mediates its effects by modulating epithelial cell adhesion through targeting of apical junction complex-associated proteins (including ezrin), a process impaired in IBD.

  5. Morphological adaptation and protein modulation of myotendinous junction following moderate aerobic training.

    Science.gov (United States)

    Curzi, Davide; Baldassarri, Valentina; De Matteis, Rita; Salamanna, Francesca; Bolotta, Alessandra; Frizziero, Antonio; Fini, Milena; Marini, Marina; Falcieri, Elisabetta

    2015-04-01

    Myotendinous junction is the muscle-tendon interface through which the contractile force can be transferred from myofibrils to the tendon extracellular matrix. At the ultrastructural level, aerobic training can modify the distal myotendinous junction of rat gastrocnemius, increasing the contact area between tissues. The aim of this work is to investigate the correlation between morphological changes and protein modulation of the myotendinous junction following moderate training. For this reason, talin, vinculin and type IV collagen amount and spatial distribution were investigated by immunohistochemistry and confocal microscopy. The images were then digitally analyzed by evaluating fluorescence intensity. Morphometric analysis revealed a significant increased thickening of muscle basal lamina in the trained group (53.1 ± 0.4 nm) with respect to the control group (43.9 ± 0.3 nm), and morphological observation showed the presence of an electron-dense area in the exercised muscles, close to the myotendinous junction. Protein concentrations appeared significantly increased in the trained group (talin +22.2%; vinculin +22.8% and type IV collagen +11.8%) with respect to the control group. Therefore, our findings suggest that moderate aerobic training induces/causes morphological changes at the myotendinous junction, correlated to the synthesis of structural proteins of the muscular basal lamina and of the cytoskeleton.

  6. Side-stream smoking reduces intestinal inflammation and increases expression of tight junction proteins

    Institute of Scientific and Technical Information of China (English)

    Hui Wang; Jun-Xing Zhao; Nan Hu; Jun Ren; Min Du; Mei-Jun Zhu

    2012-01-01

    AIM:To investigate the effect of side-stream smoking on gut microflora composition,intestinal inflammation and expression of tight junction proteins.METHODS:C57BL/6 mice were exposed to side-stream cigarette smoking for one hour daily over eight weeks.Cecal contents were collected for microbial composition analysis.Large intestine was collected for immunoblotting and quantitative reverse transcriptase polymerase chain reaction analyses of the inflammatory pathway and tight junction proteins.RESULTS:Side-stream smoking induced significant changes in the gut microbiota with increased mouse intestinal bacteria,Clostridium but decreased Fermicutes (Lactoccoci and Ruminococcus),Enterobacteriaceae family and Segmented filamentous baceteria compared to the control mice.Meanwhile,side-stream smoking inhibited the nuclear factor-κB pathway with reduced phosphorylation of p65 and IκBα,accompanied with unchanged mRNA expression of tumor necrosis factor-α or interleukin-6.The contents of tight junction proteins,claudin3 and ZO2 were up-regulated in the large intestine of mice exposed side-stream smoking.In addition,side-stream smoking increased c-Jun N-terminal kinase and p38 MAPK kinase signaling,while inhibiting AMP-activated protein kinase in the large intestine.CONCLUSION:Side-stream smoking altered gut microflora composition and reduced the inflammatory response,which was associated with increased expression of tight junction proteins.

  7. Gap junctional communication in osteocytes is amplified by low intensity vibrations in vitro.

    Directory of Open Access Journals (Sweden)

    Gunes Uzer

    Full Text Available The physical mechanism by which cells sense high-frequency mechanical signals of small magnitude is unknown. During exposure to vibrations, cell populations within a bone are subjected not only to acceleratory motions but also to fluid shear as a result of fluid-cell interactions. We explored displacements of the cell nucleus during exposure to vibrations with a finite element (FE model and tested in vitro whether vibrations can affect osteocyte communication independent of fluid shear. Osteocyte like MLO-Y4 cells were subjected to vibrations at acceleration magnitudes of 0.15 g and 1 g and frequencies of 30 Hz and 100 Hz. Gap junctional intracellular communication (GJIC in response to these four individual vibration regimes was investigated. The FE model demonstrated that vibration induced dynamic accelerations caused larger relative nuclear displacement than fluid shear. Across the four regimes, vibrations significantly increased GJIC between osteocytes by 25%. Enhanced GJIC was independent of vibration induced fluid shear; there were no differences in GJIC between the four different vibration regimes even though differences in fluid shear generated by the four regimes varied 23-fold. Vibration induced increases in GJIC were not associated with altered connexin 43 (Cx43 mRNA or protein levels, but were dependent on Akt activation. Combined, the in silico and in vitro experiments suggest that externally applied vibrations caused nuclear motions and that large differences in fluid shear did not influence nuclear motion (<1% or GJIC, perhaps indicating that vibration induced nuclear motions may directly increase GJIC. Whether the increase in GJIC is instrumental in modulating anabolic and anti-catabolic processes associated with the application of vibrations remains to be determined.

  8. Linoleic Acid Permeabilizes Gastric Epithelial Cells by Increasing Connexin43 Levels in the Cell Membrane Via a GPR40- and Akt-Dependent Mechanism

    Science.gov (United States)

    Puebla, Carlos; Cisterna, Bruno A.; Salas, Daniela P.; Delgado-López, Fernando; Lampe, Paul D.; Sáez, Juan C.

    2016-01-01

    Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintain the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt. PMID:26869446

  9. Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism.

    Science.gov (United States)

    Puebla, Carlos; Cisterna, Bruno A; Salas, Daniela P; Delgado-López, Fernando; Lampe, Paul D; Sáez, Juan C

    2016-05-01

    Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.

  10. Roles of neuro-exocytotic proteins at the neuromuscular junction

    NARCIS (Netherlands)

    Sons-Michel, Michèle S.

    2011-01-01

    The aim of the studies described in the thesis was to elucidate the roles of several neuro-exocytotic proteins at the motor nerve terminal in neuromuscular synaptic transmission, making use of genetic knockout (KO) mice, each missing one (or more) neuro-exocytotic proteins. In addition, it was

  11. The relationship between gap junctional remodeling and human atrial fibrillation

    Institute of Scientific and Technical Information of China (English)

    李大强; 冯义柏; 张会琴

    2004-01-01

    @@ Atrial fibrillation (AF) is currently the most common cardiac tachyarrhythmia in clinical practice. AF has a tendency to become more persistent over time. Progression of an underlying disease is one explanation. Another possible explanation is electrical, structural, and gap junctional remodeling of the atrium by repetitive induction of AF.1 The expression level and distribution of it have close relation with the conduction velocity of electrical activation in the atrium. The aim of the present study was to investigate the alternations of the expression and distribution of (connexin 40, Cx 40) and (connexin 43, Cx 43) in the right atrial appendages of the patients with AF by laser confocal scanning microscopy and Western blot technique.

  12. West Nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins.

    Directory of Open Access Journals (Sweden)

    Zaikun Xu

    Full Text Available West Nile virus (WNV is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion.

  13. Characterization and significance of adhesion and junction-related proteins in mouse ovarian follicles.

    Science.gov (United States)

    Mora, Jocelyn M; Fenwick, Mark A; Castle, Laura; Baithun, Marianne; Ryder, Timothy A; Mobberley, Margaret; Carzaniga, Raffaella; Franks, Stephen; Hardy, Kate

    2012-05-01

    In the ovary, initiation of follicle growth is marked by cuboidalization of flattened granulosa cells (GCs). The regulation and cell biology of this shape change remains poorly understood. We propose that characterization of intercellular junctions and associated proteins is key to identifying as yet unknown regulators of this important transition. As GCs are conventionally described as epithelial cells, this study used mouse ovaries and isolated follicles to investigate epithelial junctional complexes (tight junctions [TJ], adherens junctions [AJ], and desmosomes) and associated molecules, as well as classic epithelial markers, by quantitative PCR and immunofluorescence. These junctions were further characterized using ultrastructural, calcium depletion and biotin tracer studies. Junctions observed by transmission electron microscopy between GCs and between GCs and oocyte were identified as AJs by expression of N-cadherin and nectin 2 and by the lack of TJ and desmosome-associated proteins. Follicles were also permeable to biotin, confirming a lack of functional TJs. Surprisingly, GCs lacked all epithelial markers analyzed, including E-cadherin, cytokeratin 8, and zonula occludens (ZO)-1alpha+. Furthermore, vimentin was expressed by GCs, suggesting a more mesenchymal phenotype. Under calcium-free conditions, small follicles maintained oocyte-GC contact, confirming the importance of calcium-independent nectin at this stage. However, in primary and multilayered follicles, lack of calcium resulted in loss of contact between GCs and oocyte, showing that nectin alone cannot maintain attachment between these two cell types. Lack of classic markers suggests that GCs are not epithelial. Identification of AJs during GC cuboidalization highlights the importance of AJs in regulating initiation of follicle growth.

  14. Targeting Holliday junctions by origin DNA-binding protein of herpes simplex virus type 1.

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    Moiseeva, E D; Bazhulina, N P; Gursky, Y G; Grokhovsky, S L; Surovaya, A N; Gursky, G V

    2017-03-01

    In the present paper, the interactions of the origin binding protein (OBP) of herpes simplex virus type 1 (HSV1) with synthetic four-way Holliday junctions (HJs) were studied using electrophoresis mobility shift assay and the FRET method and compared with the interactions of the protein with duplex and single-stranded DNAs. It has been found that OBP exhibits a strong preference for binding to four-way and three-way DNA junctions and possesses much lower affinities to duplex and single-stranded DNAs. The protein forms three types of complexes with HJs. It forms complexes I and II which are reminiscent of the tetramer and octamer complexes with four-way junction of HJ-specific protein RuvA of Escherichia coli. The binding approaches saturation level when two OBP dimers are bound per junction. In the presence of Mg(2+) ions (≥2 mM) OBP also interacts with HJ in the stacked arm form (complex III). In the presence of 5 mM ATP and 10 mM Mg(2+) ions OBP catalyzes processing of the HJ in which one of the annealed oligonucleotides has a 3'-terminal tail containing 20 unpaired thymine residues. The observed preference of OBP for binding to the four-way DNA junctions provides a basis for suggestion that OBP induces large DNA structural changes upon binding to Box I and Box II sites in OriS. These changes involve the bending and partial melting of the DNA at A+T-rich spacer and also include the formation of HJ containing Box I and Box II inverted repeats and flanking DNA sequences.

  15. Interaction of HMG proteins and H1 with hybrid PNA-DNA junctions.

    Science.gov (United States)

    Totsingan, Filbert; Bell, Anthony J

    2013-11-01

    The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein-binding surface of an immobilized four-way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ-PNA1 and 4WJ-PNA3 ). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two-state equilibrium between: (i) an open-x state favored by absence of Mg(2+), low salt, and protein binding, and (ii) a compact stacked-x state favored by Mg(2+). 4WJ-PNA3, like J1, shifts readily from an open to stacked conformation in the presence of Mg(+2), while 4WJ-PNA1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ-PNA3 with nearly identical affinity (K(D)s) and 4WJ-PNA1 with two-fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.

  16. 白藜芦醇上调心肌缝隙连接蛋白43的表达降低大鼠心肌梗死后心律失常%Resveratrol exerts anti-arrhythmic effects in rats with myocardial infarction through up-regulation of cardiac connexin43 expression

    Institute of Scientific and Technical Information of China (English)

    王光宇; 毕亚光; 叶婷婷; 张庆勇; 魏盟

    2016-01-01

    stimulation was performed in vivo electrophysiological studies .Myocardial infarct size was detected by Masson’s staining.The distribution of connexin43(Cx43) and P368Cx43 were detected by immuno-histochemical staining methods .The Cx43,P368Cx43 and PKCβ2 expressions were measured using western blot . Results Compared with MI group ,resveratrol significantly reduced the incidence rate of induced ventricular arrhythmias (87.5%vs.37.5%,P<0.05),and the ventricular fibrillation threshold was significantly higher [(3.2±0.3)V vs.(5.2±0.8)V,P<0.05].Compared with the sham group,the expression of Cx43 within the infracted myocardium was significantly lower (P<0.05),while the expressions of P368Cx43 and PKCβ2 were greatly higher ( P<0.05 ) .Cx43 levels increased in myocardial infarction rats which received resveratrol ( P<0.05).Conclusion Resveratrol could effectively improve the cardiac electrophysiological properties in MI rats and increase myocardial expression of Cx 43 protein, and could prevent the occurrence of ventricular arrhythmias.

  17. Niflumic acid relaxes mesenteric small artery through downregulating connexin 43 expression in smooth muscle cells from spontaneously hypertensive rat%尼氟灭酸对自发性高血压大鼠肠系膜微小动脉平滑肌细胞连接蛋白43的下调作用

    Institute of Scientific and Technical Information of China (English)

    刘卫东; 张雯; 司军强; 马克涛; 李丽

    2014-01-01

    目的 探讨尼氟灭酸(niflumic acid,NFA)对自发性高血压大鼠(SHR)肠系膜微小动脉平滑肌细胞缝隙连接中连接蛋白43(connexin43,Cx43)表达量的影响.方法 通过尾动脉无创血压测定Wistar大鼠和SHR的血压值.应用压力肌动图检测不同浓度NFA对Wistar大鼠和SHR肠系膜微小动脉舒缩功能的影响.Western blot检测Wistar大鼠和SHR肠系膜动脉Cx43表达的差异.Western blot检测不同浓度NFA处理原代培养的SHR肠系膜微小动脉平滑肌细胞Cx43的蛋白表达水平.结果 苯肾上腺素(phenylephrine,PE)可以引起肠系膜微小动脉血管预收缩至(193±13.5) μm,而3×10-4mol/L的NFA可以使其舒张至初始水平(275±17.1) μm,且Wistar大鼠的舒张反应显著高于SHR,差异有统计学意义(F=55.47,P<0.05,n=6).SHR肠系膜动脉血管一级分支和三级分支Cx43蛋白的表达水平分别高于Wistar大鼠相应血管分支,且肠系膜三级分支血管Cx43蛋白的表达水平高于一级分支,差异有统计学意义(F=1 014.43,P<0.01).不同浓度的NFA处理的肠系膜微小动脉培养的平滑肌细胞,其Cx43蛋白的表达水平均低于对照,且呈浓度依赖性,差异有统计学意义(F=1 480.20,P<0.01).结论 Cx43可能介导了SHR肠系膜微小动脉平滑肌细胞间的通讯,从而影响血管的舒缩反应.而NFA可以降低平滑肌细胞Cx43蛋白的表达,并在一定程度上舒张血管.%Objective To explore the impact of niflumic acid (NFA) on connexin 43 (Cx43)expression in smooth muscle cells of mesenteric small artery from spontaneously hypertensive rats (SHR).Methods Blood pressure of Wistar rats and spontaneously hypertensive rats (SHR) was measured by the tail cuff method.Relaxation and contraction of mesenteric small artery from Wistar rat and SHR were evaluated by pressure myograph system under various concentrations of NFA.Protein Cx43 expression on primary cultured mesenteric smooth muscle cells from Wistar rats and SHR was

  18. Grainy head promotes expression of septate junction proteins and influences epithelial morphogenesis.

    Science.gov (United States)

    Narasimha, Maithreyi; Uv, Anne; Krejci, Alena; Brown, Nicholas H; Bray, Sarah J

    2008-03-15

    Transcription factors of the Grainy head (Grh) family are required in epithelia to generate the impermeable apical layer that protects against the external environment. This function is conserved in vertebrates and invertebrates, despite the differing molecular composition of the protective barrier. Epithelial cells also have junctions that create a paracellular diffusion barrier (tight or septate junctions). To examine whether Grh has a role in regulating such characteristics, we used an epidermal layer in the Drosophila embryo that has no endogenous Grh and lacks septate junctions, the amnioserosa. Expression of Grh in the amnioserosa caused severe defects in dorsal closure, a process similar to wound closure, and induced robust expression of the septate junction proteins Coracle, Fasciclin 3 and Sinuous. Grh-binding sites are present within the genes encoding these proteins, consistent with them being direct targets. Removal of Grh from imaginal disc cells caused a reduction in Fasciclin 3 and Coracle levels, suggesting that Grh normally fine tunes their epithelial expression and hence contributes to barrier properties. The fact that ectopic Grh arrests dorsal closure also suggests that this dynamic process relies on epithelia having distinct adhesive properties conferred by differential deployment of Grh.

  19. Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

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    Balda Maria S

    2009-12-01

    Full Text Available Abstract Background Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction. Results Here we identify a new transmembrane component of tight junctions: MarvelD3, a four-span transmembrane protein. Its predicted transmembrane helices form a Marvel (MAL and related proteins for vesicle traffic and membrane link domain, a structural motif originally discovered in proteins involved in membrane apposition and fusion events, such as the tight junction proteins occludin and tricellulin. In mammals, MarvelD3 is expressed as two alternatively spliced isoforms. Both isoforms exhibit a broad tissue distribution and are expressed by different types of epithelial as well as endothelial cells. MarvelD3 co-localises with occludin at tight junctions in intestinal and corneal epithelial cells. RNA interference experiments in Caco-2 cells indicate that normal MarvelD3 expression is not required for the formation of functional tight junctions but depletion results in monolayers with increased transepithelial electrical resistance. Conclusions Our data indicate that MarvelD3 is a third member of the tight junction-associated occludin family of transmembrane proteins. Similar to occludin, normal expression of MarvelD3 is not essential for the formation of functional tight junctions. However, MarvelD3 functions as a determinant of epithelial paracellular permeability properties.

  20. Gap junction proteins: master regulators of the planarian stem cell response to tissue maintenance and injury.

    Science.gov (United States)

    Peiris, T Harshani; Oviedo, Néstor J

    2013-01-01

    Gap junction (GJ) proteins are crucial mediators of cell-cell communication during embryogenesis, tissue regeneration and disease. GJ proteins form plasma membrane channels that facilitate passage of small molecules across cells and modulate signaling pathways and cellular behavior in different tissues. These properties have been conserved throughout evolution, and in most invertebrates GJ proteins are known as innexins. Despite their critical relevance for physiology and disease, the mechanisms by which GJ proteins modulate cell behavior are poorly understood. This review summarizes findings from recent work that uses planarian flatworms as a paradigm to analyze GJ proteins in the complexity of the whole organism. The planarian model allows access to a large pool of adult somatic stem cells (known as neoblasts) that support physiological cell turnover and tissue regeneration. Innexin proteins are present in planarians and play a fundamental role in controlling neoblast behavior. We discuss the possibility that GJ proteins participate as cellular sensors that inform neoblasts about local and systemic physiological demands. We believe that functional analyses of GJ proteins will bring a complementary perspective to studies that focus on the temporal expression of genes. Finally, integrating functional studies along with molecular genetics and epigenetic approaches would expand our understanding of cellular regulation in vivo and greatly enhance the possibilities for rationally modulating stem cell behavior in their natural environment. This article is part of a Special Issue entitled: The communicating junctions, roles and dysfunctions. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Engineered Cx26 variants established functional heterotypic Cx26/Cx43 and Cx26/Cx40 gap junction channels.

    Science.gov (United States)

    Karademir, Levent B; Aoyama, Hiroshi; Yue, Benny; Chen, Honghong; Bai, Donglin

    2016-05-15

    Gap junction (GJ) channels mediate direct intercellular communication and are composed of two docked hemichannels (connexin oligomers). It is well documented that the docking and formation of GJs are possible only between compatible hemichannels (or connexins). The mechanisms of heterotypic docking compatibility are not fully clear. We aligned the protein sequences of docking-compatible and -incompatible connexins with that of connexin26 (Cx26). We found that two docking hydrogen bond (HB)-forming residues on the second extracellular domain (E2) of Cx26 and their equivalent residues are well conserved within docking-compatible connexins, but different between docking-incompatible connexins. Replacing one or both of these residues of Cx26 into the corresponding residues in the docking incompatible connexins (K168V, N176H or K168V-N176H) increased the formation of morphological and functional heterotypic GJs with connexin43 (Cx43) or connexin40 (Cx40), indicating that these two residues are important for docking incompatibility between Cx26 and these connexins. Our homology structure models predict that both HBs and hydrophobic interactions at the E2 docking interface are important docking mechanisms in heterotypic Cx26 K168V-N176H/Cx43 GJs and probably other docking compatible connexins. Revealing the key residues and mechanisms of heterotypic docking compatibility will assist us in understanding why these putative docking residues are hotspots of disease-linked mutants. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  2. The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila.

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    Assia Hijazi

    Full Text Available BACKGROUND: Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes. METHODOLOGY/PRINCIPAL FINDINGS: In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component. CONCLUSION/SIGNIFICANCE: We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.

  3. SynProt: A Comprehensive Database for Proteins of the Detergent-Resistant Synaptic Junctions Fraction

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    Rainer ePielot

    2012-06-01

    Full Text Available Chemical synapses are highly specialized cell-cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse and some human proteins, which mainly have been manually extracted from twelve proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed. We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design.

  4. Resolution of Holliday junction recombination intermediates by wild-type and mutant IntDOT proteins.

    Science.gov (United States)

    Kim, Seyeun; Gardner, Jeffrey F

    2011-03-01

    CTnDOT encodes an integrase that is a member of the tyrosine recombinase family. The recombination reaction proceeds by sequential sets of genetic exchanges between the attDOT site in CTnDOT and an attB site in the chromosome. The exchanges are separated by 7 base pairs in each site. Unlike most tyrosine recombinases, IntDOT exchanges sites that contain different DNA sequences between the exchange sites to generate Holliday junctions (HJs) that contain mismatched bases. We demonstrate that IntDOT resolves synthetic HJs in vitro. Holliday junctions that contain identical sequences between the exchange sites are resolved into both substrates and products, while HJs that contain mismatches are resolved only to substrates. This result implies that resolution of HJs to products requires the formation of a higher-order nucleoprotein complex with natural sites containing IntDOT. We also found that proteins with substitutions of residues (V95, K94, and K96) in a putative alpha helix at the junction of the N and CB domains (coupler region) were defective in resolving HJs. Mutational analysis of charged residues in the coupler and the N terminus of the protein did not provide evidence for a charge interaction between the regions of the protein. V95 may participate in a hydrophobic interaction with another region of IntDOT.

  5. Effect of salvianolate on intestinal epithelium tight junction protein zonula occludens protein 1 in cirrhotic rats

    Institute of Scientific and Technical Information of China (English)

    Dan-Hong Yang; Zai-Yuan Ye; Yuan-Jun Xie; Xu-Jun He; Wen-Juan Xu; Wei-Ming Zhou

    2012-01-01

    AIM:To study the effect of salvianolate on tight junctions (TJs) and zonula occludens protein 1 (ZO-1) in small intestinal mucosa of cirrhotic rats.METHODS:Cirrhosis was induced using carbon tetrachloride.Rats were randomly divided into the untreated group,low-dose salvianolate (12 mg/kg) treatment group,medium-dose salvianolate (24 mg/kg) treatment group,and high-dose salvianolate (48 mg/kg) treatment group,and were treated for 2 wk.Another 10 healthy rats served as the normal control group.Histological changes in liver tissue samples were observed under a light microscope.We evaluated morphologic indices of ileal mucosa including intestinal villi width and thickness of mucosa and intestinal wall using a pathological image analysis system.Ultrastructural changes in small intestinal mucosa were investigated in the five groups using transmission electron microscopy.The changes in ZO-1 expression,a tight junction protein,were analyzed by immunocytochemistry.The staining index was calculated as the product of the staining intensity score and the proportion of positive cells.RESULTS:In the untreated group,hepatocytes showed a disordered arrangement,fatty degeneration was extensive,swelling was obvious,and disorganized lobules were divided by collagen fibers in hepatic tissue,which were partly improved in the salvianolate treated groups.In the untreated group,abundant lymphocytes infiltrated the fibrous tissue with proliferation of bile ducts,and collagen fibers gradually decreased and damaged hepatic lobules were partly repaired following salvianolate treatment.Compared with the untreated group,no differences in intestinal villi width between the five groups were observed.The villi height as well as mucosa and intestinal wall thickness gradually thickened with salvianolate treatment and were significantly shorter in the untreated group compared with those in the salvianolate treatment groups and normal group (P < 0.01).The number of microvilli decreased and showed

  6. Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication

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    Christ G.J.

    2000-01-01

    Full Text Available Connexin43 (Cx43 is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of a1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM on equivalent submaximal (»75% of the phenylephrine maximum aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 µM, prostaglandin F2a (PGF2a; 1 µM and endothelin-1 (ET-1; 20 nM. Statistical analysis revealed that 200 µM and 500 µM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 ± 6% (N = 26 rings to 57 ± 7% (N = 26 rings and 34.9 ± 6% (N = 13 rings, respectively (P<0.05, and for PGF2a from a control response of 75 ± 10% (N = 16 rings to 52 ± 8% (N = 19 rings and 25.9 ± 6% (N = 18 rings, respectively (P<0.05. In contrast, 200 µM and 500 µM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 ± 5.0% for the control response (N = 38 rings, 59 ± 6.0% in the presence of 200 µM heptanol (N = 17 rings, and 70 ± 6.0% in the presence of 500 µM heptanol (N = 23 rings (P<0.13. Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 ± 5% (21 rings; P<0.01; further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then

  7. Cx43基因shRNA慢病毒载体的构建及其对大鼠心肌细胞Cx43基因的作用%Construction of a lentiviral vector for RNA interference targeting rat cardiac myocytes connexin 43 gene and its effect

    Institute of Scientific and Technical Information of China (English)

    陈立; 钟国强; 涂荣会; 黎庆捷; 何艳

    2013-01-01

    目的 构建缝隙连接蛋白(Cx)43基因shRNA慢病毒载体,并检测其对大鼠心肌细胞Cx43基因的作用.方法 针对Cx43基因序列,设计RNA干扰靶点序列,合成靶序列的双链DNA,接入pGCL-GFP载体,挑选阳性克隆行PCR鉴定及测序.用pHelper1.0和pHelper2.0质粒转染293T细胞,包装产生具备感染能力的慢病毒.以293T细胞中绿色荧光蛋白的细胞数量计算病毒滴度;以最适感染复数感染大鼠心肌细胞,通过荧光显微镜观察感染效率;应用real-time PCR和Western blot法检测大鼠心肌细胞.Cx43 mRNA及Cx43蛋白表达,并评价其抑制效果.结果 经PCR鉴定和测序证实,Cx43慢病毒载体构建正确,其病毒滴度为8×108 TU/mL、转染效率为82.59%.荧光定量real-time PCR法检测Cx43 mRNA的抑制率为96.10%,Western blot法检测Cx43蛋白的抑制率为77.16%.结论 成功构建了Cx43基因shRNA慢病毒载体,其能显著抑制大鼠心肌细胞Cx43基因的表达.%Objective To construct a lentiviral RNAi vector and to detect its effect on connexin 43 (Cx43) gene in rat cardiac myocytes.Methods Short hairpin RNA (shRNA) sequence targeting rat cardiac myocytes Cx43 gene was designed.After synthesis and annealing,the double-stranded oligonucleotides (ds oligo) were connecting to pGC-LV vectors.The positive clones were selected and conducted by PCR identification and sequencing.Then,The viral particles were gen erated by cotransfection of 293T cells with the pGC-lv-Cx43 and two packaging vector (pHelper1.0,pHelper2.0),and the virus titer was determined by counting the number of GFP positive cells.After transfection of lentiviral vector into rat cardiac myocytes with multiplicity of infection (MOI),the level of Cx43 mRNA in rat cardiac myocytes was determined by realtime PCR and the level of Cx43 protein was determined by Western blot assay.The inhibitory effect was evaluated.Results The construction of Cx43 lentiviral vector was confirmed by PCR identification

  8. Gap Junction Intercellular Communication in Bone Marrow Failure

    Science.gov (United States)

    2012-10-01

    necessary for radial migration in the neocortex . Nature 448:901-907. 21. Katbamna, B., Jelaso, A.M., and Ide, C.F. 2004. Connexin 43 expression in glial...proliferation in the developing neocortex . Neuron 43:647-661. 26. Xu, X., Francis, R., Wei, C.J., Linask, K.L., and Lo, C.W. 2006. Connexin 43-mediated

  9. The tight junction protein ZO-2 blocks cell cycle progression and inhibits cyclin D1 expression.

    Science.gov (United States)

    Gonzalez-Mariscal, Lorenza; Tapia, Rocio; Huerta, Miriam; Lopez-Bayghen, Esther

    2009-05-01

    ZO-2 is an adaptor protein of the tight junction that belongs to the MAGUK protein family. ZO-2 is a dual localization protein that in sparse cultures is present at the cell borders and the nuclei, whereas in confluent cultures it is concentrated at the cell boundaries. Here we have studied whether ZO-2 is able to regulate the expression of cyclin D1 (CD1) and cell proliferation. We have demonstrated that ZO-2 negatively regulates CD1 transcription by interacting with c-Myc at an E box present in CD1 promoter. We have further found that ZO-2 transfection into epithelial MDCK cells triggers a diminished expression of CD1 protein and decreases the rate of cell proliferation in a wound-healing assay.

  10. Dendrobium chrysotoxum Lindl. Alleviates Diabetic Retinopathy by Preventing Retinal Inflammation and Tight Junction Protein Decrease

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    Zengyang Yu

    2015-01-01

    Full Text Available Diabetic retinopathy (DR is a serious complication of diabetes mellitus. This study aimed to observe the alleviation of the ethanol extract of Dendrobium chrysotoxum Lindl. (DC, a traditional Chinese herbal medicine, on DR and its engaged mechanism. After DC (30 or 300 mg/kg was orally administrated, the breakdown of blood retinal barrier (BRB in streptozotocin- (STZ- induced diabetic rats was attenuated by DC. Decreased retinal mRNA expression of tight junction proteins (including occludin and claudin-1 in diabetic rats was also reversed by DC. Western blot analysis and retinal immunofluorescence staining results further confirmed that DC reversed the decreased expression of occludin and claudin-1 proteins in diabetic rats. DC reduced the increased retinal mRNA expressions of intercellular adhesion molecule-1 (ICAM-1, tumor necrosis factor α (TNFα, interleukin- (IL- 6, and IL-1β in diabetic rats. In addition, DC alleviated the increased 1 and phosphorylated p65, IκB, and IκB kinase (IKK in diabetic rats. DC also reduced the increased serum levels of TNFα, interferon-γ (IFN-γ, IL-6, IL-1β, IL-8, IL-12, IL-2, IL-3, and IL-10 in diabetic rats. Therefore, DC can alleviate DR by inhibiting retinal inflammation and preventing the decrease of tight junction proteins, such as occludin and claudin-1.

  11. Petri Net-Based Model of Helicobacter pylori Mediated Disruption of Tight Junction Proteins in Stomach Lining during Gastric Carcinoma

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    Anam Naz

    2017-09-01

    Full Text Available Tight junctions help prevent the passage of digestive enzymes and microorganisms through the space between adjacent epithelial cells lining. However, Helicobacter pylori encoded virulence factors negatively regulate these tight junctions and contribute to dysfunction of gastric mucosa. Here, we have predicted the regulation of important tight junction proteins, such as Zonula occludens-1, Claudin-2 and Connexin32 in the presence of pathogenic proteins. Molecular events such as post translational modifications and crosstalk between phosphorylation, O-glycosylation, palmitoylation and methylation are explored which may compromise the integrity of these tight junction proteins. Furthermore, the signaling pathways disrupted by dysregulated kinases, proteins and post-translational modifications are reviewed to design an abstracted computational model showing the situation-dependent dynamic behaviors of these biological processes and entities. A qualitative hybrid Petri Net model is therefore constructed showing the altered host pathways in the presence of virulence factor cytotoxin-associated gene A, leading to the disruption of tight junction proteins. The model is qualitative logic-based, which does not depend on any kinetic parameter and quantitative data and depends on knowledge derived from experiments. The designed model provides insights into the tight junction disruption and disease progression. Model is then verified by the available experimental data, nevertheless formal in vitro experimentation is a promising way to ensure its validation. The major findings propose that H. pylori activated kinases are responsible to trigger specific post translational modifications within tight junction proteins, at specific sites. These modifications may favor alterations in gastric barrier and provide a route to bacterial invasion into host cells.

  12. Connexins in the early development of the African clawed frog Xenopus laevis (Amphibia: The role of the connexin43 carboxyl terminal tail in the establishment of the dorso-ventral axis

    Directory of Open Access Journals (Sweden)

    Jaime Cofre

    2007-03-01

    Full Text Available Connexins are a family of related proteins identified in vertebrate forming gap junctions, which mediate cell-to-cell communication in early embryos, with an important role in establishing embryonic asymmetry and ‘communication compartments’. By in situ hybridization, immunocytochemistry, reverse transcriptase PCR (RT-PCR and western blotting we show that a Cx43-like molecule is present in oocytes and embryos of the African clawed frog Xenopus laevis, with specific localization in the animal-vegetal axis. This specific distribution is suggestive for an important role for this protein in the establishment of the dorso-ventral axis. Antisense RNA and antibodies directed against rat carboxyl terminal tail of the Cx43 (CT-Cx43 and injected in 1-cell stage Xenopus embryos, induced pronounced alterations in nervous system development, with a severe ventralization phenotype. Coherently, the overexpression of CT-Cx43 produced a dorsalization of the embryos. In antisense treated embryos, the expression of the beta-catenin gene is eliminated from the Nieuwkoop center, the pattern expression of the Chordin, Xnot and Xbra is modified, with no effect in expression of the Goosecoid gene. In CT-Cx43 mRNA treated embryos the pattern of expression of the beta-catenin, Chordin, Goosecoid, Xnot and engrailed-2 genes is modified. The expression of beta-catenin is increased in the Nieuwkoop center, the expression pattern of Chordin and Goosecoid is expanded to the posterior neural plate and engrailed-2 presents ectopic expression in the ventral region. Taken together our data suggest a role for CT-Cx43 as a maternal determinant with a critical function in the formation of the dorso-ventral axis in Xenopus laevis. The Cx43 may be one of the earliest markers of the dorso-ventral axis in these embryos and could possibly be acting through regionalization of factors responsible for the establishment of this axis.

  13. Variable conformation of GAP junctions linking bone cells: a transmission electron microscopic study of linear, stacked linear, curvilinear, oval, and annular junctions.

    Science.gov (United States)

    Shapiro, F

    1997-10-01

    There is a marked variability in the conformation of bone cell gap junctions in newborn murine cortical bone as defined by transmission electron microscopy (TEM). Studies were done in newborn BALB/c mouse and Sprague-Dawley rat femurs and tibias. Femoral and tibial cortices were dissected into 1 mm3 fragments and prepared in standardized fashion using modified Karnovsky fixation, 7.5% EDTA decalcification, 1% osmium tetroxide-sym collidine buffer with 1% lanthanum nitrate postfixation, Epon resin, 60 nm sections, lead citrate/uranyl acetate staining, and examination at 60 kV. Previous TEM descriptions of bone junctions have, with rare exceptions, noted only isolated linear or mildly curvilinear structures. In this study we noted gap junctional shapes on thin-section TEM preparations of osteoblasts and osteocytes to be extremely variable and complex encompassing linear, curvilinear, stacked linear, oval, and annular conformations. Multiple observations revealed linear gap junctions linking surface osteoblast cell bodies; linear, curvilinear, stacked linear, and oval junctions linking osteoblast processes in osteoid; linear and curvilinear junctions where cell processes joined with osteocyte cell bodies and each of the five conformations linking osteocyte processes within canaliculi. The annular junctions were found within osteoblast and osteocyte cytoplasm and in osteocyte cell processes within canaliculi. The annular junctions are intracellular, degenerating structures which appear as ultrastructural markers of gap junction involution. The more complex shapes reported here must be considered in (1) interpreting quantitative studies using freeze-fracture replicas, thin sections, and confocal microscopy immunolabeled junction connexin-43 components and (2) assessing gap junction biogenesis and turnover. 3-D reconstruction of bone junctions will enhance our understanding of these complex conformations.

  14. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein.

    Science.gov (United States)

    Marsh, Andrew; Casey-Green, Katherine; Probert, Fay; Withall, David; Mitchell, Daniel A; Dilly, Suzanne J; James, Sean; Dimitri, Wade; Ladwa, Sweta R; Taylor, Paul C; Singer, Donald R J

    2016-01-01

    Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively 'regulating' connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and

  15. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein.

    Directory of Open Access Journals (Sweden)

    Andrew Marsh

    Full Text Available Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29. Further analysis indicated the binding site to be for the N-terminal domain putatively 'regulating' connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the

  16. Tight Junction Proteins Claudin-1 and Occludin Are Important for Cutaneous Wound Healing.

    Science.gov (United States)

    Volksdorf, Thomas; Heilmann, Janina; Eming, Sabine A; Schawjinski, Kathrin; Zorn-Kruppa, Michaela; Ueck, Christopher; Vidal-Y-Sy, Sabine; Windhorst, Sabine; Jücker, Manfred; Moll, Ingrid; Brandner, Johanna M

    2017-06-01

    Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  17. Activation of Holliday junction recognizing protein involved in the chromosomal stability and immortality of cancer cells.

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    Kato, Tatsuya; Sato, Nagato; Hayama, Satoshi; Yamabuki, Takumi; Ito, Tomoo; Miyamoto, Masaki; Kondo, Satoshi; Nakamura, Yusuke; Daigo, Yataro

    2007-09-15

    We identified a novel gene HJURP (Holliday junction-recognizing protein) whose activation seemed to play a pivotal role in the immortality of cancer cells. HJURP was considered a possible downstream target for ataxia telangiectasia mutated signaling, and its expression was increased by DNA double-strand breaks (DSB). HJURP was involved in the homologous recombination pathway in the DSB repair process through interaction with hMSH5 and NBS1, which is a part of the MRN protein complex. HJURP formed nuclear foci in cells at S phase and those subjected to DNA damage. In vitro assays implied that HJURP bound directly to the Holliday junction and rDNA arrays. Treatment of cancer cells with small interfering RNA (siRNA) against HJURP caused abnormal chromosomal fusions and led to genomic instability and senescence. In addition, HJURP overexpression was observed in a majority of lung cancers and was associated with poor prognosis as well. We suggest that HJURP is an indispensable factor for chromosomal stability in immortalized cancer cells and is a potential novel therapeutic target for the development of anticancer drugs.

  18. EMP-1 is a junctional protein in a liver stem cell line and in the liver.

    Science.gov (United States)

    Lee, Hsuan-Shu; Sherley, James L; Chen, Jeremy J W; Chiu, Chien-Chang; Chiou, Ling-Ling; Liang, Ja-Der; Yang, Pan-Chyr; Huang, Guan-Tarn; Sheu, Jin-Chuan

    2005-09-09

    In an attempt to discover cell markers for liver stem cells, a cDNA microarray analysis was carried out to compare the gene expression profiles between an adult liver stem cell line, Lig-8, and mature hepatocytes. Several genes in the categories of extracellular matrix, cell membrane, cell adhesion, transcription factor, signal molecule, transporter, and metabolic enzyme were shown to be differentially expressed in Lig-8 cells. Among them, epithelial membrane protein (EMP)-1 has been previously implicated with stem cell phenotypes. Antiserum to EMP-1 was produced to localize its expression. On monolayers of Lig-8 cells, EMP-1 was expressed along the intercellular border. In the liver harboring proliferating oval cells, the liver progenitors, EMP-1 was localized as ribbon bands, a staining pattern for epithelial junctions, all the way through bile duct epithelia, oval cell ductules, and into peri-hepatocytic regions. These peri-hepatocytic regions were proved to be bile canaliculi by co-localization of EMP-1 and dipeptidyl peptidase IV, an enzyme located on bile canaliculi. This report is the first to indicate EMP-1 to be a junctional protein in the liver.

  19. Tight junction protein claudin 4 in gastric carcinoma and its relation to lymphangiogenic activity.

    Science.gov (United States)

    Shareef, Mohamed Moustafa; Radi, Dina Mohammed Adel; Eid, Asmaa Mustafa Mohammed

    2015-01-01

    Gastric cancer is the second most common cause of cancer-related death worldwide. Claudins are a family of tight junction proteins that are biologically relevant in many cancer progression steps. This study aimed to investigate the expression of the intestinal claudin (claudin 4) in gastric carcinoma and to evaluate its relation to the different clinicopathologic prognostic parameters, especially lymphangiogenesis (production of new lymphatic vessels, measured by lymphovascular density (LVD)) and lymphovascular invasion (LVI). Fifty-five gastric carcinoma specimens were immunohistochemically stained for claudin 4 and D2-40 (for detection of lymphatic vessel endothelium). High expression of claudin 4 was detected in 26 of 55 (47.3%) cases. Low expression of claudin 4 was related to poorly differentiated type (p=0.001), non-intestinal (diffuse) type (p=0.001), deeper tumour invasion (pgastric carcinoma and lost in poorly differentiated diffuse type. So, claudin 4 may be used as one of the differentiating markers between the two major types of gastric carcinoma (intestinal vs. diffuse). LVD and LVI were related to higher incidence of lymph node metastasis and therefore could be used as predictive markers for lymph node metastasis in limited specimens during early gastric carcinoma to determine the need for more invasive surgery. Low expression of claudin 4 was related to lymphangiogenesis. This may shed light on the relation of tight junction protein expression and lymphangiogenesis. Copyright © 2015 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.

  20. Cardioprotective effect of connexin 43 expression regulated by PI3K/Akt on hydrogen sulfide postconditioning in isolated ischemic and reperfused rat hearts%PI3K/Akt调节线粒体Cx43蛋白表达在H2S后处理离体大鼠心肌中的保护作用

    Institute of Scientific and Technical Information of China (English)

    张可璇; 毛洪雅; 孟雪; 惠夏; 张邓新; 季永

    2013-01-01

    Aim To investigate whether the PBK/Akt signaling pathway regulates connexin 43 expression to protect isolated rat hearts against ischemia/reperfusion ( I/R ) injury in hydrogen sulfide postconditioning. Methods Hearts of 56 male SD rats were isolated and linked to the Langendorff apparatus. They were randomly divided into 4 groups( n = 14): ischemia reper-fusion group ( I/R ), PBK/Akt signaling pathway inhibitor LY294002 group ( LY ), hydrogen sulfide post-conditioning group ( NP ), and hydrogen sulfide with to all the nuclei counted was used as apoptotic index ( AI ). The expression of total Cx43 and phosphoryla-tion Cx43 in mitochondria were determined with Western blot analysis at the end of reperfusion. Results No differences in baseline hemodynamics were observed a-mong the experimental groups ( P > 0. 05 ). After reperfusion, compared with I/R group, NP group had better hemodynamics; the myocardial infarct size and cardiocyte apoptotic index were much lower ( P < 0. 05 ); the expression of tCx43 ( total connexin 43 , tCx43 ) and pCx43 ( phosphorylated connexin 43, pCx43 ) in mitochondria increased significantly. How- LY294002 group( N + L ). The heart rate ( HR ) , the left ventricular diastolic pressure ( LVEDP ), the left ventricular developed pressure ( LVDP ), the maximum rate of increase or decrease of left ventricular pressure ( ± dp/dtmax ) were recorded at 20 min of equilibrium at 30 min of reperfusion and at the end of reperfusion, respectively. Myocardial infarct size was measured by triphenyltet tetrazolium chloride ( TTC ) staining. Myocardial TUNEL staining was determined by in situ cell death detection kit. The ratio of TUNEL positive nuclei ever , LY294002 abolished the cardioprotection offered by hydrogen sulfide postconditioning and the increase in tCx43 and pCx43 expression in mitochondria. Conclusion The PI3K/Akt pathway upregulates connexin 43 expression to protect isolated rat hearts against is-chemia/reperfusion ( I/R ) injury in

  1. A Gap Junction Protein, Inx2, Modulates Calcium Flux to Specify Border Cell Fate during Drosophila oogenesis

    Science.gov (United States)

    Ghosh, Ritabrata; Deshpande, Girish

    2017-01-01

    Intercellular communication mediated by gap junction (GJ) proteins is indispensable during embryogenesis, tissue regeneration and wound healing. Here we report functional analysis of a gap junction protein, Innexin 2 (Inx2), in cell type specification during Drosophila oogenesis. Our data reveal a novel involvement of Inx2 in the specification of Border Cells (BCs), a migratory cell type, whose identity is determined by the cell autonomous STAT activity. We show that Inx2 influences BC fate specification by modulating STAT activity via Domeless receptor endocytosis. Furthermore, detailed experimental analysis has uncovered that Inx2 also regulates a calcium flux that transmits across the follicle cells. We propose that Inx2 mediated calcium flux in the follicle cells stimulates endocytosis by altering Dynamin (Shibire) distribution which is in turn critical for careful calibration of STAT activation and, thus for BC specification. Together our data provide unprecedented molecular insights into how gap junction proteins can regulate cell-type specification. PMID:28114410

  2. Gap junction remodeling and cardiac arrhythmogenesis in a murine model of oculodentodigital dysplasia.

    Science.gov (United States)

    Kalcheva, Nellie; Qu, Jiaxiang; Sandeep, Nefthi; Garcia, Luis; Zhang, Jie; Wang, Zhiyong; Lampe, Paul D; Suadicani, Sylvia O; Spray, David C; Fishman, Glenn I

    2007-12-18

    Gap junction channels are required for normal cardiac impulse propagation, and gap junction remodeling is associated with enhanced arrhythmic risk. Oculodentodigital dysplasia (ODDD) is a multisystem syndrome due to mutations in the connexin43 (Cx43) gap junction channel gene. To determine the effects of a human connexin channelopathy on cardiac electrophysiology and arrhythmogenesis, we generated a murine model of ODDD by introducing the disease-causing I130T mutant allele into the mouse genome. Cx43 abundance was markedly reduced in mutant hearts with preferential loss of phosphorylated forms that interfered with trafficking and assembly of gap junctions in the junctional membrane. Dual whole-cell patch-clamp studies showed significantly lower junctional conductance between neonatal cell pairs from mutant hearts, and optical mapping of isolated-perfused hearts with voltage-sensitive dyes demonstrated significant slowing of conduction velocity. Programmed electrical stimulation revealed a markedly increased susceptibility to spontaneous and inducible ventricular tachyarrhythmias. In summary, our data demonstrate that the I130T mutation interferes with Cx43 posttranslational processing, resulting in diminished cell-cell coupling, slowing of impulse propagation, and a proarrhythmic substrate.

  3. Dysfunction of mitochondria and deformed gap junctions in the heart of IL-18-deficient mice.

    Science.gov (United States)

    Li, Wen; Jin, Denan; Hata, Masaki; Takai, Shinji; Yamanishi, Kyosuke; Shen, Weili; El-Darawish, Yosif; Yamanishi, Hiromichi; Okamura, Haruki

    2016-08-01

    Interleukin-18 (IL-18) was discovered as an interferon-γ-inducing factor and has been regarded as a proinflammatory cytokine. However, IL-18 is ubiquitously expressed both in immune/inflammatory cells and in nonimmune cells, and its biological roles have not been sufficiently elucidated. Here, we demonstrate that IL-18-deficient [IL-18 knockout (KO)] mice have heart abnormalities that may be related to impaired autophagy. In endurance running tests, IL-18KO mice ran significantly shorter distances compared with wild-type (WT) mice. Echocardiographs indicated disability in the systolic and diastolic functions of the IL-18KO mouse heart. Immunostaining of connexin 43 showed heterogeneous localization of gap junctions in the lateral membranes of the IL-18KO cardiac myocytes. Western blotting analysis revealed decreased phosphorylated connexin 43 in the IL-18KO heart. Electron microscopy revealed unusual localization of intercalated disks, swollen or damaged mitochondria, and broad, indistinct Z-lines in the IL-18KO heart. In accordance with the morphological observation, mitochondrial respiratory function, including that of complexes I and IV, was impaired, and production of reactive oxygen species was augmented in IL-18KO hearts. Notably, levels of LC3-II were markedly lower in the IL-18KO hearts than in WT hearts. In the culture of cardiac myocytes of IL-18KO neonates, exogenous IL-18 upregulated LC3-II and increased the number of intact mitochondria with high mitochondrial membrane potential. These results indicated that IL-18 has roles apart from those as a proinflammatory cytokine in cardiac myocytes and suggested that IL-18 contributes to the homeostatic maintenance of mitochondrial function and gap-junction turnover in cardiac myocytes, possibly by upregulating autophagy.

  4. Protein preconcentration using nanofractures generated by nanoparticle-assisted electric breakdown at junction gaps.

    Directory of Open Access Journals (Sweden)

    Chun-Ping Jen

    Full Text Available Sample preconcentration is an important step that increases the accuracy of subsequent detection, especially for samples with extremely low concentrations. Due to the overlapping of electrical double layers in the nanofluidic channel, the concentration polarization effect can be generated by applying an electric field. Therefore, a nonlinear electrokinetic flow is induced, which results in the fast accumulation of proteins in front of the induced ionic depletion zone, the so-called exclusion-enrichment effect. Nanofractures were created in this work to preconcentrate proteins via the exclusion-enrichment effect. The protein sample was driven by electroosmotic flow and accumulated at a specific location. The preconcentration chip for proteins was fabricated using simple standard soft lithography with a polydimethylsiloxane replica. Nanofractures were formed by utilizing nanoparticle-assisted electric breakdown. The proposed method for nanofracture formation that utilizes nanoparticle deposition at the junction gap between microchannels greatly decreases the required electric breakdown voltage. The experimental results indicate that a protein sample with an extremely low concentration of 1 nM was concentrated to 1.5×10(4-fold in 60 min using the proposed chip.

  5. Effect of FCCP on tight junction permeability and cellular distribution of ZO-1 protein in epithelial (MDCK) cells.

    Science.gov (United States)

    Li, C X; Poznansky, M J

    1990-12-14

    The effect of the uncoupler of oxidative phosphorylation, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), on the tight junction of Madin-Darby canine kidney cells was examined. FCCP induced an abrupt decrease in the transepithelial electrical resistance of the confluent monolayers over a period of 20 s. When FCCP was withdrawn from the incubation medium, the monolayer resistance recovered to close to the original level in less than 2 h. Staining of the tight junction-associated protein ZO-1 showed that the changes in transepithelial electrical resistance were accompanied by a diffusing of the protein away from cell peripheries and a reconcentration to the tight junction areas following resistance recovery. Intracellular pH was decreased by FCCP on a similar time-scale with no obvious changes in ATP levels over this time-course. These data suggest that the uncoupler FCCP has a profound effect on tight junction permeability and cellular distribution of the tight junction protein ZO-1 in the epithelial cells and that it probably acts by breaking down proton gradients and altering intracellular pH.

  6. The epithelial membrane protein 1 is a novel tight junction protein of the blood-brain barrier.

    Science.gov (United States)

    Bangsow, Thorsten; Baumann, Ewa; Bangsow, Carmen; Jaeger, Martina H; Pelzer, Bernhard; Gruhn, Petra; Wolf, Sabine; von Melchner, Harald; Stanimirovic, Danica B

    2008-06-01

    In the central nervous system, a constant microenvironment required for neuronal cell activity is maintained by the blood-brain barrier (BBB). The BBB is formed by the brain microvascular endothelial cells (BMEC), which are sealed by tight junctions (TJ). To identify genes that are differentially expressed in BMEC compared with peripheral endothelial cells, we constructed a subtractive cDNA library from porcine BMEC (pBMEC) and aortic endothelial cells (AOEC). Screening the library for differentially expressed genes yielded 26 BMEC-specific transcripts, such as solute carrier family 35 member F2 (SLC35F2), ADP-ribosylation factor-like 5B (ARL5B), TSC22 domain family member 1 (TSC22D1), integral membrane protein 2A (ITM2A), and epithelial membrane protein 1 (EMP1). In this study, we show that EMP1 transcript is enriched in pBMEC compared with brain tissue and that EMP1 protein colocalizes with the TJ protein occludin in mouse BMEC by coimmunoprecipitation and in rat brain vessels by immunohistochemistry. Epithelial membrane protein 1 expression was transiently induced in laser-capture microdissected rat brain vessels after a 20-min global cerebral ischemia, in parallel with the loss of occludin immunoreactivity. The study identifies EMP1 as a novel TJ-associated protein of the BBB and suggests its potential role in the regulation of the BBB function in cerebral ischemia.

  7. Expression of gap junction proteins connexins 26, 30, and 43 in Dupuytren's disease.

    Science.gov (United States)

    Holzer, Lukas A; Cör, Andrej; Holzer, Gerold

    2014-02-01

    Dupuytren's disease (DD) is a benign fibroproliferative process of the palmar aponeurosis showing similarities to wound healing. Communication of cells involved in wound healing is mediated by the composition of gap junction (GJ) proteins. We investigated the expression of 3 GJ proteins, connexins 26, 30, and 43 (Cx26, Cx30, and Cx43) in DD. Fragments of Dupuytren's tissue from 31 patients (mean age 56 (30-76) years, 24 male) were analyzed immunohistochemically and compared to control tissue for expression of the GJ proteins Cx26, Cx30, and Cx43 and also alfa-smooth muscle actin (α-SMA). 14 of 31 samples could be attributed to the involutional phase (α-SMA positive) whereas 17 samples had to be considered cords in the residual phase (α-SMA negative). Expression of Cx26 and Cx43 was seen in 12 of the 14 samples from the involutional phase, and Cx30 was seen in 7 of these. Only 4 of the 17 samples from the residual phase showed any Cx, and there was none in the controls. The high expression of GJ proteins Cx26, Cx30, and Cx43 in α-SMA positive myofibroblast-rich nodules, which are characteristic of the active involutional phase of DD, suggests that connexins could be a novel treatment target for the treatment of DD.

  8. Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus).

    Science.gov (United States)

    Lavrov, Igor; Fox, Lyle; Shen, Jun; Han, Yingchun; Cheng, Jianguo

    2016-01-01

    Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32), connexin 36 (Cx36), connexin 37 (Cx37), and connexin 43 (Cx43). Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols) substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.

  9. Gap Junctions Contribute to the Regulation of Walking-Like Activity in the Adult Mudpuppy (Necturus Maculatus.

    Directory of Open Access Journals (Sweden)

    Igor Lavrov

    Full Text Available Although gap junctions are widely expressed in the developing central nervous system, the role of electrical coupling of neurons and glial cells via gap junctions in the spinal cord in adults is largely unknown. We investigated whether gap junctions are expressed in the mature spinal cord of the mudpuppy and tested the effects of applying gap junction blocker on the walking-like activity induced by NMDA or glutamate in an in vitro mudpuppy preparation. We found that glial and neural cells in the mudpuppy spinal cord expressed different types of connexins that include connexin 32 (Cx32, connexin 36 (Cx36, connexin 37 (Cx37, and connexin 43 (Cx43. Application of a battery of gap junction blockers from three different structural classes (carbenexolone, flufenamic acid, and long chain alcohols substantially and consistently altered the locomotor-like activity in a dose-dependent manner. In contrast, these blockers did not significantly change the amplitude of the dorsal root reflex, indicating that gap junction blockers did not inhibit neuronal excitability nonselectively in the spinal cord. Taken together, these results suggest that gap junctions play a significant modulatory role in the spinal neural networks responsible for the generation of walking-like activity in the adult mudpuppy.

  10. Gap junction intercellular communication: a review of a potential platform to modulate craniofacial tissue engineering.

    Science.gov (United States)

    Rossello, Ricardo A; Kohn, David H

    2009-02-01

    Defects in craniofacial tissues, resulting from trauma, congenital abnormalities, oncologic resection or progressive deforming diseases, may result in aesthetic deformity, pain and reduced function. Restoring the structure, function and aesthetics of craniofacial tissues represents a substantial clinical problem in need of new solutions. More biologically-interactive biomaterials could potentially improve the treatment of craniofacial defects, and an understanding of developmental processes may help identify strategies and materials that can be used in tissue engineering. One such strategy that can potentially advance tissue engineering is cell-cell communication. Gap junction intercellular communication is the most direct way of achieving such signaling. Gap junction communication through connexin-mediated junctions, in particular connexin 43 (Cx43), plays a major role bone development. Given the important role of Cx43 in controlling development and differentiation, especially in bone cells, controlling the expression of Cx43 may provide control over cell-to-cell communication and may help overcome some of the challenges in craniofacial tissue engineering. Following a review of gap junctions in bone cells, the ability to enhance cell-cell communication and osteogenic differentiation via control of gap junctions is discussed, as is the potential utility of this approach in craniofacial tissue engineering. Copyright 2008 Wiley Periodicals, Inc.

  11. Eya1 protein phosphatase regulates tight junction formation in lung distal epithelium.

    Science.gov (United States)

    El-Hashash, Ahmed H K; Turcatel, Gianluca; Varma, Saaket; Berika, Mohamed; Al Alam, Denise; Warburton, David

    2012-09-01

    Little is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity, such as aPKCζ. We recently reported that Eya1 phosphatase functions through aPKCζ-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKCζ phosphorylation levels, aPKCζ-mediated TJ protein phosphorylation and Notch1-Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wild-type Eya1 or partially inhibiting aPKCζ in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1(-/-) lung epithelial TJ defects. These findings uncover novel functions for the Eya1-aPKCζ-Notch1-Cdc42 pathway as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area.

  12. 星形胶质细胞的缝隙连接蛋白43参与冷冻伤后脑水肿的形成∗%Astrocytic connexin 43 is involved in development of cryoinjury-induced cerebral edema

    Institute of Scientific and Technical Information of China (English)

    章科娜; 丁悦敏; 杜月光; 龚婉; 张雄

    2016-01-01

    AIM: To discuss the change of astrocytic connexin 43 (Cx43) expression in cerebral edema and the potential roles of Cx43 in the development of cerebral edema. METHODS:Cryoinjury-induced cerebral edema model was established in the right parietal lobe cortex in the rats. The animals were divided into sham group, cryoinjury-induced cerebral edema model group, and cryoinjury-induced cerebral edema model injected with carbenoxolone ( or octanol ) group. Dry and wet weight method was used to measure the content of brain water. Methanamide method was used to evalu-ate the permeability of blood-brain barrier ( BBB) after cryoinjury. HE staining showed the physiological changes in injured brain cortex. The protein expression of Cx43 was detected by Western blot and immunohistochemical staining. RESULTS:The brain water content in the injured cortex of rats was increased and cerebral edema developed to the peak at 24 h after cryoinjury. The permeability of BBB was increased in the injured cortex of the rats, and the area where BBB was damaged was larger than injured area. HE staining showed that many cells were dead in the central area of cryoinjury, and at the same time cerebral edema was appeared obviously in the surrounding area of injured brain cortex. The protein expression of Cx43 was up-regulated in the surrounding area of injured brain cortex, while Cx43 expression decreased in the injured cor-tex at different periods after cryoinjury. Both the content of brain water in the injured cortex and the permeability of BBB at 24 h after cryoinjury were reduced after injecting carbenoxolone or octanol intraperitoneally to block Cx43. CONCLU-SION:The cerebral edema up-regulates the expression of Cx43 and also strengthens its function. Once Cx43 is blocked, the degree of cerebral edema in rats is attenuated.%目的::探讨脑水肿后星形胶质细胞缝隙连接蛋白43(Cx43)的表达及其在脑水肿的发生发展过程中所起的作用。方法:采用颅骨外液

  13. Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Mate Suzanne E

    2012-09-01

    Full Text Available Abstract Background Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ. We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs. Methods Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle. Results Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle. Conclusion EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs.

  14. L. plantarum prevents Enteroinvasive Escherichia coli-induced tight junction proteins changes in intestinal epithelial cells

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    Hang Xiaomin

    2009-03-01

    Full Text Available Abstract Background It is increasingly recognized that Lactobacillus plantarum (L. plantarum has the ability to protect against Enteropathogenic Escherichia coli (EPEC-induced damage of the epithelial monolayer barrier function by preventing changes in host cell morphology, attaching/effacing (A/E lesion formation, monolayer resistance, and macromolecular permeability. However, the cellular mechanism involved in this protective effect still remained to be clarified. Methods This study was to investigate the effect of L. plantarum on the changes of Caco-2 cells responding to Enteroinvasive Escherichia coli (EIEC, the permeability of cell monolayer and the transmissivity of dextran, and the distribution and expression of the tight junction (TJ proteins, such as Claudin-1, Occludin, JAM-1 and ZO-1 were examined when infected with EIEC or adhesived of L. plantarum after infection by confocal laser scanning microscopy (CLSM, immunohistochemistry and Western blotting, the cytoskeleton protein F-actin were observed with FITC-phalloidin. Results This study demonstrated that the transepithelial electrical resistance (TER step down and dextran integrated intensity (DII step up with time after infected with EIEC, but after treating with L. plantarum, the changes of TER and DII were improved as compared with EIEC group. L. plantarum prevented the damage of expression and rearrangement of Claudin-1, Occludin, JAM-1 and ZO-1 proteins induced by EIEC, and could ameliorate the injury of cytoskeleton protein F-actin infected with EIEC. Conclusion L. plantarum exerted a protective effect against the damage to integrity of Caco-2 monolayer cells and the structure and distribution of TJ proteins by EIEC infection.

  15. Proteins in load-bearing junctions: the histidine-rich metal-binding protein of mussel byssus.

    Science.gov (United States)

    Zhao, Hua; Waite, J Herbert

    2006-11-28

    Building complex load-bearing scaffolds depends on effective ways of joining functionally different biomacromolecules. The junction between collagen fibers and foamlike adhesive plaques in mussel byssus is robust despite the strikingly dissimilar connected structures. mcfp-4, the matrix protein from this junction, and its presecreted form from the foot tissue of Mytilus californianus were isolated and characterized. mcfp-4 has a mass of approximately 93 kDa as determined by MALDI-TOF mass spectrometry. Its composition is dominated by histidine (22 mol %), but levels of lysine, arginine, and aspartate are also significant. A small amount of 3,4-dihydroxyphenyl-l-alanine (2 mol %) can be detected by amino acid analysis and redox cycling assays. The cDNA-deduced sequence of mcfp-4 reveals multiple variants with highly repetitive internal structures, including approximately 36 tandemly repeated His-rich decapeptides (e.g., HVHTHRVLHK) in the N-terminal half and 16 somewhat more degenerate aspartate-rich undecapeptides (e.g., DDHVNDIAQTA) in the C-terminal half. Incubation of a synthetic peptide based on the His-rich decapeptide with Fe3+, Co2+, Ni2+, Zn2+, and Cu2+ indicates that only Cu is strongly bound. MALDI-TOF mass spectrometry of the peptide modified with diethyl pyrocarbonate before and after Cu binding suggests that histidine residues dominate Cu binding. In contrast, the aspartate-rich undecapeptides preferentially bind Ca2+. mcfp-4 is strategically positioned to function as a macromolecular bifunctional linker by using metal ions to couple its own His-rich domains to the His-rich termini of the preCOLs. Ca2+ may mediate coupling of the C-terminus to other calcium-binding plaque proteins.

  16. Lymphocytes accelerate epithelial tight junction assembly: role of AMP-activated protein kinase (AMPK.

    Directory of Open Access Journals (Sweden)

    Xiao Xiao Tang

    Full Text Available The tight junctions (TJs, characteristically located at the apicolateral borders of adjacent epithelial cells, are required for the proper formation of epithelial cell polarity as well as for sustaining the mucosal barrier to the external environment. The observation that lymphocytes are recruited by epithelial cells to the sites of infection [1] suggests that they may play a role in the modulation of epithelial barrier function and thus contribute to host defense. To test the ability of lymphocytes to modulate tight junction assembly in epithelial cells, we set up a lymphocyte-epithelial cell co-culture system, in which Madin-Darby canine kidney (MDCK cells, a well-established model cell line for studying epithelial TJ assembly [2], were co-cultured with mouse lymphocytes to mimic an infection state. In a typical calcium switch experiment, the TJ assembly in co-culture was found to be accelerated compared to that in MDCK cells alone. This accelaration was found to be mediated by AMP-activated protein kinase (AMPK. AMPK activation was independent of changes in cellular ATP levels but it was found to be activated by the pro-inflammatory cytokine TNF-alpha. Forced suppression of AMPK, either with a chemical inhibitor or by knockdown, abrogated the accelerating effect of lymphocytes on TJ formation. Similar results were also observed in a co-culture with lymphocytes and Calu-3 human airway epithelial cells, suggesting that the activation of AMPK may be a general mechanism underlying lymphocyte-accelerated TJ assembly in different epithelia. These results suggest that signals from lymphocytes, such as cytokines, facilitate TJ assembly in epithelial cells via the activation of AMPK.

  17. The Werner and Bloom syndrome proteins help resolve replication blockage by converting (regressed) holliday junctions to functional replication forks.

    Science.gov (United States)

    Machwe, Amrita; Karale, Rajashree; Xu, Xioahua; Liu, Yilun; Orren, David K

    2011-08-16

    Cells cope with blockage of replication fork progression in a manner that allows DNA synthesis to be completed and genomic instability minimized. Models for resolution of blocked replication involve fork regression to form Holliday junction structures. The human RecQ helicases WRN and BLM (deficient in Werner and Bloom syndromes, respectively) are critical for maintaining genomic stability and thought to function in accurate resolution of replication blockage. Consistent with this notion, WRN and BLM localize to sites of blocked replication after certain DNA-damaging treatments and exhibit enhanced activity on replication and recombination intermediates. Here we examine the actions of WRN and BLM on a special Holliday junction substrate reflective of a regressed replication fork. Our results demonstrate that, in reactions requiring ATP hydrolysis, both WRN and BLM convert this Holliday junction substrate primarily to a four-stranded replication fork structure, suggesting they target the Holliday junction to initiate branch migration. In agreement, the Holliday junction binding protein RuvA inhibits the WRN- and BLM-mediated conversion reactions. Importantly, this conversion product is suitable for replication with its leading daughter strand readily extended by DNA polymerases. Furthermore, binding to and conversion of this Holliday junction are optimal at low MgCl(2) concentrations, suggesting that WRN and BLM preferentially act on the square planar (open) conformation of Holliday junctions. Our findings suggest that, subsequent to fork regression events, WRN and/or BLM could re-establish functional replication forks to help overcome fork blockage. Such a function is highly consistent with phenotypes associated with WRN- and BLM-deficient cells.

  18. Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation.

    Science.gov (United States)

    Nighot, Prashant K; Hu, Chien-An Andy; Ma, Thomas Y

    2015-03-13

    Autophagy is an intracellular degradation pathway and is considered to be an essential cell survival mechanism. Defects in autophagy are implicated in many pathological processes, including inflammatory bowel disease. Among the innate defense mechanisms of intestinal mucosa, a defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in the causation and progression of inflammatory bowel disease by allowing increased antigenic permeation. The cross-talk between autophagy and the TJ barrier has not yet been described. In this study, we present the novel finding that autophagy enhances TJ barrier function in Caco-2 intestinal epithelial cells. Nutrient starvation-induced autophagy significantly increased transepithelial electrical resistance and reduced the ratio of sodium/chloride paracellular permeability. Nutrient starvation reduced the paracellular permeability of small-sized urea but not larger molecules. The role of autophagy in the modulation of paracellular permeability was confirmed by pharmacological induction as well as pharmacological and genetic inhibition of autophagy. Consistent with the autophagy-induced reduction in paracellular permeability, a marked decrease in the level of the cation-selective, pore-forming TJ protein claudin-2 was observed after cell starvation. Starvation reduced the membrane presence of claudin-2 and increased its cytoplasmic, lysosomal localization. Therefore, our data show that autophagy selectively reduces epithelial TJ permeability of ions and small molecules by lysosomal degradation of the TJ protein claudin-2.

  19. The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

    Science.gov (United States)

    Rossi, Matthew J; Mazina, Olga M; Bugreev, Dmitry V; Mazin, Alexander V

    2011-04-19

    The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.

  20. Adaptive evolution of tight junction protein claudin-14 in echolocating whales.

    Science.gov (United States)

    Xu, Huihui; Liu, Yang; He, Guimei; Rossiter, Stephen J; Zhang, Shuyi

    2013-11-10

    Toothed whales and bats have independently evolved specialized ultrasonic hearing for echolocation. Recent findings have suggested that several genes including Prestin, Tmc1, Pjvk and KCNQ4 appear to have undergone molecular adaptations associated with the evolution of this ultrasonic hearing in mammals. Here we studied the hearing gene Cldn14, which encodes the claudin-14 protein and is a member of tight junction proteins that functions in the organ of Corti in the inner ear to maintain a cationic gradient between endolymph and perilymph. Particular mutations in human claudin-14 give rise to non-syndromic deafness, suggesting an essential role in hearing. Our results uncovered two bursts of positive selection, one in the ancestral branch of all toothed whales and a second in the branch leading to the delphinid, phocoenid and ziphiid whales. These two branches are the same as those previously reported to show positive selection in the Prestin gene. Furthermore, as with Prestin, the estimated hearing frequencies of whales significantly correlate with numbers of branch-wise non-synonymous substitutions in Cldn14, but not with synonymous changes. However, in contrast to Prestin, we found no evidence of positive selection in bats. Our findings from Cldn14, and comparisons with Prestin, strongly implicate multiple loci in the acquisition of echolocation in cetaceans, but also highlight possible differences in the evolutionary route to echolocation taken by whales and bats. © 2013.

  1. Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells.

    Science.gov (United States)

    Jaramillo, Blanca Estela; Ponce, Arturo; Moreno, Jacqueline; Betanzos, Abigail; Huerta, Miriam; Lopez-Bayghen, Esther; Gonzalez-Mariscal, Lorenza

    2004-07-01

    ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

  2. Alterations in junctional proteins, inflammatory mediators and extracellular matrix molecules in eosinophilic esophagitis.

    Science.gov (United States)

    Abdulnour-Nakhoul, Solange M; Al-Tawil, Youhanna; Gyftopoulos, Alex A; Brown, Karen L; Hansen, Molly; Butcher, Kathy F; Eidelwein, Alexandra P; Noel, Robert A; Rabon, Edd; Posta, Allison; Nakhoul, Nazih L

    2013-08-01

    Eosinophilic esophagitis (EoE), an inflammatory atopic disease of the esophagus, causes massive eosinophil infiltration, basal cell hyperplasia, and sub-epithelial fibrosis. To elucidate cellular and molecular factors involved in esophageal tissue damage and remodeling, we examined pinch biopsies from EoE and normal pediatric patients. An inflammation gene array confirmed that eotaxin-3, its receptor CCR3 and interleukins IL-13 and IL-5 were upregulated. An extracellular matrix (ECM) gene array revealed upregulation of CD44 & CD54, and of ECM proteases (ADAMTS1 & MMP14). A cytokine antibody array showed a marked decrease in IL-1α and IL-1 receptor antagonist and an increase in eotaxin-2 and epidermal growth factor. Western analysis indicated reduced expression of intercellular junction proteins, E-cadherin and claudin-1 and increased expression of occludin and vimentin. We have identified a number of novel genes and proteins whose expression is altered in EoE. These findings provide new insights into the molecular mechanisms of the disease.

  3. Gap junction communication involved in brain protection following focal ischemia and reperfusion in rats

    Institute of Scientific and Technical Information of China (English)

    Fei Wang; Jian Hai; Yuhong Jing

    2009-01-01

    BACKGROUND: Studies have suggested that gap junctions not only modulate the fate of the neocortex, but are also involved in maintaining homeostasis in the mature brain. However, the neuroprotective effects of gap junction communication following brain ischemic injury remain poorly understood. OBJECTIVE: To investigate the neuroprotective effects and possible mechanisms of gap junction communication following focal ischemia and reperfusion. DESIGN, TIME AND SETTING: A randomized, controlled, animal experiment was performed at the School of Basic Medical Sciences of Lanzhou University between June 2007 and May 2008.MATERIALS: Rabbit polyclonal anti-connexin 43 (Cx43) and gap junction blocking agent octanol were purchased from Sigma, USA;mouse monoclonal anti-rat glial fibrillary acidic protein (GFAP) was provided by Santa Cruz, USA;mouse monoclonal anti-rat CD11b was produced by Abcam, England. METHODS: A total of 52 adult, male, Sprague Dawley rats were randomly assigned to three groups: sham-operated (n=12), vehicle control (n=20), and octanol-treated (n=20). Brain ischemia and reperfusion were induced by transient middle cerebral artery occlusion (MCAO) in vehicle control and octanol-treated groups, while no MCAO was administered to the sham-operated group. In the octanol-treated group, 5 mmol/kg octanol was dissolved in dimethyl sulfoxide (0.005% v/v) and was intraperitoneally injected 30 minutes prior to ischemic onset. Sham-operated and vehicle groups received equivalent volumes of dimethyl sulfoxide. MAIN OUTCOME MEASURES: Infarct volumes in ipsilateral striatum after MCAO were measured using cresyl violet dye;GFAP, CD11b, and Cx43 expression in the ipsilateral striatum following MCAO were detected by immunohistochemistry;Western blot analysis was employed to determine Cx43 and GFAP expression. RESULTS: At 1 and 3 days following MCAO and reperfusion, ipsilateral striatum infarct volumes in the octanol group were significantly greater than in the vehicle group

  4. EMP-induced alterations of tight junction protein expression and disruption of the blood-brain barrier.

    Science.gov (United States)

    Ding, Gui-Rong; Qiu, Lian-Bo; Wang, Xiao-Wu; Li, Kang-Chu; Zhou, Yong-Chun; Zhou, Yan; Zhang, Jie; Zhou, Jia-Xing; Li, Yu-Rong; Guo, Guo-Zhen

    2010-07-15

    The blood-brain barrier (BBB) is critical to maintain cerebral homeostasis. In this study, we examined the effects of exposure to electromagnetic pulse (EMP) on the functional integrity of BBB and, on the localization and expression of tight junction (TJ) proteins (occludin and ZO-1) in rats. Animals were sham or whole-body exposed to EMP at 200 kV/m for 400 pulses. The permeability of BBB in rat cerebral cortex was examined by using Evans Blue (EB) and lanthanum nitrate as vascular tracers. The localization and expression of TJ proteins were assessed by western blot and immunofluorescence analysis, respectively. The data indicated that EMP exposure caused: (i) increased permeability of BBB, and (ii) altered localization as well as decreased levels of TJ protein ZO-1. These results suggested that the alteration of ZO-1 may play an important role in the disruption of tight junctions, which may lead to dysfunction of BBB after EMP exposure.

  5. Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins.

    Science.gov (United States)

    Markov, Alexander G; Falchuk, Evgeny L; Kruglova, Natalia M; Rybalchenko, Oksana V; Fromm, Michael; Amasheh, Salah

    2014-11-01

    Claudin tight junction proteins have been identified to primarily determine intestinal epithelial barrier properties. While functional contribution of single claudins has been characterized in detail, information on the interplay with secretory mechanisms in native intestinal epithelium is scarce. Therefore, effects of cholera toxin and theophylline on rat colon were analyzed, including detection of sealing claudins. Tissue specimens were stripped off submucosal tissue layers and mounted in Ussing chambers, and short-circuit current (ISC) and transepithelial resistance (TER) were recorded. In parallel, expression and localization of claudins was analyzed and histological studies were performed employing hematoxylin-eosin staining and light and electron microscopy. Theophylline induced a strong increase of ISC in colon tissue specimens. In parallel, a decrease of TER was observed. In contrast, cholera toxin did not induce a significant increase of ISC, whereas an increase of TER was detected after 120 min. Western blots of membrane fractions revealed an increase of claudin-3 and -4 after incubation with cholera toxin, and theophylline induced an increase of claudin-4. In accordance, confocal laser-scanning microscopy exhibited increased signals of claudin-3 and -4 after incubation with cholera toxin, and increased signals of claudin-4 after incubation with theophylline, within tight junction complexes. Morphological analyses revealed no general changes of tight junction complexes, but intercellular spaces were markedly widened after incubation with cholera toxin and theophylline. We conclude that cholera toxin and theophylline have different effects on sealing tight junction proteins in native colon preparations, which may synergistically contribute to transport functions, in vitro.

  6. Acidic bile salts modulate the squamous epithelial barrier function by modulating tight junction proteins.

    Science.gov (United States)

    Chen, Xin; Oshima, Tadayuki; Tomita, Toshihiko; Fukui, Hirokazu; Watari, Jiro; Matsumoto, Takayuki; Miwa, Hiroto

    2011-08-01

    Experimental models for esophageal epithelium in vitro either suffer from poor differentiation or complicated culture systems. An air-liquid interface system with normal human bronchial epithelial cells can serve as a model of esophageal-like squamous epithelial cell layers. Here, we explore the influence of bile acids on barrier function and tight junction (TJ) proteins. The cells were treated with taurocholic acid (TCA), glycocholic acid (GCA), or deoxycholic acid (DCA) at different pH values, or with pepsin. Barrier function was measured by transepithelial electrical resistance (TEER) and the diffusion of paracellular tracers (permeability). The expression of TJ proteins, including claudin-1 and claudin-4, was examined by Western blotting of 1% Nonidet P-40-soluble and -insoluble fractions. TCA and GCA dose-dependently decreased TEER and increased paracellular permeability at pH 3 after 1 h. TCA (4 mM) or GCA (4 mM) did not change TEER and permeability at pH 7.4 or pH 4. The combination of TCA and GCA at pH 3 significantly decreased TEER and increased permeability at lower concentrations (2 mM). Pepsin (4 mg/ml, pH 3) did not have any effect on barrier function. DCA significantly decreased the TEER and increased permeability at pH 6, a weakly acidic condition. TCA (4 mM) and GCA (4 mM) significantly decreased the insoluble fractions of claudin-1 and claudin-4 at pH 3. In conclusion, acidic bile salts disrupted the squamous epithelial barrier function partly by modulating the amounts of claudin-1 and claudin-4. These results provide new insights for understanding the role of TJ proteins in esophagitis.

  7. A role for tight junction-associated MARVEL proteins in larval sea lamprey (Petromyzon marinus) osmoregulation.

    Science.gov (United States)

    Kolosov, Dennis; Bui, Phuong; Donini, Andrew; Wilkie, Mike P; Kelly, Scott P

    2017-08-10

    This study reports on tight junction-associated MARVEL proteins of larval sea lamprey (Petromyzon marinus) and their potential role in ammocoete osmoregulation. Two Occludin isoforms (designated Ocln and Ocln-a) and a tricellulin (Tric) were identified. Transcripts encoding ocln, ocln-a, and tric were broadly expressed in larval lamprey, with greatest abundance of ocln in gut, liver and kidney, ocln-a in the gill and skin, and tric in the kidney. Ocln and Ocln-a resolved as ∼63 kDa and ∼35 kDa MW proteins respectively while Tric resolved as a ∼50 kDa protein. Ocln immunolocalized to the gill vasculature and in gill mucous cells while Ocln-a localized to the gill pouch and gill epithelium. Both Ocln and Ocln-a localized in the nephron, the epidermis and the luminal side of the gut. In branchial tissue, Tric exhibited punctate localization, consistent with its presence at regions of tricellular contact. Following ion-poor water (IPW) acclimation of ammocoetes, serum [Na(+)] and [Cl(-)] reduced, but not [Ca(++)], and carcass moisture content increased. In association, Ocln abundance increased in skin and kidney, but reduced in gill of IPW-acclimated ammocoetes while Ocln-a abundance reduced in the kidney only. Tric abundance increased in the gill. Region-specific alterations in ocln, ocln-a and tric mRNA abundance was also observed in the gut. Data support a role for Ocln, Ocln-a and Tric in the osmoregulatory strategies of a basal vertebrate. © 2017. Published by The Company of Biologists Ltd.

  8. Pharmacological enhancement of cardiac gap junction coupling prevents arrhythmias in canine LQT2 model.

    Science.gov (United States)

    Quan, Xiao-Qing; Bai, Rong; Lu, Jia-Gao; Patel, Chinmay; Liu, Nian; Ruan, Yanfei; Chen, Bo-Di; Ruan, Lei; Zhang, Cun-Tai

    2009-01-01

    Gap junctions contribute to the transmural heterogeneity of repolarization in the normal heart and under conditions of prolonged QT interval in the diseased heart. This study examined whether enhancing of gap junction coupling can reduce transmural dispersion of repolarization (TDR) and prevent torsade de pointes (TdP) in a canine LQT2 model. Canine left ventricular wedge preparations were perfused with delayed rectifier potassium current (IKr) blocker d-sotalol to mimic LQT2 and the antiarrhythmic peptide 10 (AAP10) was used as a gap junction coupling enhancer. As compared with the control group, the LQT2 group had significantly augmented TDR and higher incidence of TdP associated with increased nonphosphorylated connexin 43 (Cx43). AAP10 prevented augmentation of TDR and induction of TdP while rescuing Cx43 phosphorylation. There was no significant change in the quantity and spatial distribution of Cx43. These data indicate that gap junction enhancer AAP10 can prevent augmentation of TDR and suppress TdP by preventing dephosphorylation of Cx43 in a LQT2 model.

  9. Characterization of the structural and protein recognition properties of hybrid PNA-DNA four-way junctions.

    Science.gov (United States)

    Iverson, Douglas; Serrano, Crystal; Brahan, Ann Marie; Shams, Arik; Totsingan, Filbert; Bell, Anthony J

    2015-12-01

    The objective of this study is to evaluate the structure and protein recognition properties of hybrid four-way junctions (4WJs) composed of DNA and peptide nucleic acid (PNA) strands. We compare a classic immobile DNA junction, J1, vs. six PNA-DNA junctions, including a number with blunt DNA ends and multiple PNA strands. Circular dichroism (CD) analysis reveals that hybrid 4WJs are composed of helices that possess structures intermediate between A- and B-form DNA, the apparent level of A-form structure correlates with the PNA content. The structure of hybrids that contain one PNA strand is sensitive to Mg(+2). For these constructs, the apparent B-form structure and conformational stability (Tm) increase in high Mg(+2). The blunt-ended junction, b4WJ-PNA3, possesses the highest B-form CD signals and Tm (40.1 °C) values vs. all hybrids and J1. Protein recognition studies are carried out using the recombinant DNA-binding protein, HMGB1b. HMGB1b binds the blunt ended single-PNA hybrids, b4WJ-PNA1 and b4WJ-PNA3, with high affinity. HMGB1b binds the multi-PNA hybrids, 4WJ-PNA1,3 and b4WJ-PNA1,3, but does not form stable protein-nucleic acid complexes. Protein interactions with hybrid 4WJs are influenced by the ratio of A- to B-form helices: hybrids with helices composed of higher levels of B-form structure preferentially associate with HMGB1b.

  10. Role of calcium and vesicle-docking proteins in remobilising dormant neuromuscular junctions in desert frogs.

    Science.gov (United States)

    Lavidis, Nickolas A; Hudson, Nicholas J; Choy, Peng T; Lehnert, Sigrid A; Franklin, Craig E

    2008-01-01

    Despite prolonged immobility the desert frog, Cyclorana alboguttata, suffers little impairment in muscle function. To determine compensatory mechanisms at neuromuscular junctions, transmitter release was examined along primary terminals in C. alboguttata iliofibularis muscle. Using extracellular recording we found the amplitudes of evoked endplate currents were significantly smaller in dormant frogs. In active frogs we identified two negatively sloping proximal-distal gradients of transmitter frequency and quantal content; a shallow proximal-distal gradient with low probability of transmitter release (0.6). During aestivation, only a shallow gradient was identified. The high probability release sites in control frogs were inhibited during aestivation by a mechanism that could be reversed by (1) increasing the extracellular calcium concentration, and (2) increasing the frequency of stimulation. This suggests that transmitter vesicles are available during aestivation but not released. We quantified expression of messenger RNA transcripts coding for the transmitter vesicle-docking proteins synaptotagmin 1, syntaxin 1B and UNC-13. All three were rare transcripts maintained at control values during aestivation. Neuromuscular remobilisation after dormancy in C. alboguttata is more likely a product of rapidly reversible physiologic mechanisms than reorganisations of the neuromuscular transcriptome.

  11. Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein b-5

    Institute of Scientific and Technical Information of China (English)

    XIA; Jiahui; (夏家辉); ZHENG; Duo; (郑多),; TANG; Dongsheng; (唐冬生); DAI; Heping; (戴和平); PAN; Qian; (潘乾); LONG; Zhigao; (龙志高); LIAO; Xiaodong; (廖晓东)

    2001-01-01

    By homologous EST searching and nested PCR a new human gene GJB5 encoding gap junction protein b-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.

  12. Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein b-5

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    By homologous EST searching and nested PCR a new human gene GJB5encoding gap junction protein b-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.

  13. Interleukin-6, desmosome and tight junction protein expression levels in reflux esophagitis-affected mucosa

    Institute of Scientific and Technical Information of China (English)

    Fei-Yue Li; Yan Li

    2009-01-01

    AIM: To investigate the correlation between the expression levels of interleukin (IL)-6 and proteins in tight junctions (TJs) in the esophageal mucosa of rats modeling different types of reflux esophagitis (RE), and the ability of aluminum phosphate to protect against RE-induced mucosal damage via these proteins. METHODS: Male SPF Wistar rats aged 56 d were divided randomly into acid RE, alkaline RE, mixed RE, and control groups. Various surgical procedures were performed to establish rat models of acid RE. At 14 d after the procedure, some of the rats started aluminum phosphate treatment. Transmission electron microscopy (TEM) was used to observe the morphological features of TJs and desmosomes in the esophageal epithelium. Immunohistochemical methods and Western blotting were used to measure expression of claudin 1, occludin, ZO-1, JAM-1, DSG-1 and IL-6; reverse transcription polymerase chain reaction (RTPCR) was used to measure expression of mRNA of claudin 1, occludin, ZO-1, JAM-1, DSG-1 and IL-6. RESULTS: At day 14 after the procedures, an RE model was established in all subsequently sacrificed rats of groups A, B and C. By both gross and microscopic observation, the mucosa was damaged and thickened as the disease progressed. With TEM observation, a widened intercellular space was noticed, with significantly fewer desmosomes. Immunohistochemistry showed significantly higher levels of all proteins in all RE models compared to control rats at 3 d after operation (65.5% ± 25.6% vs 20.5% ± 2.1%, P 0.05, treated vs untreated, respectively). These levels increased in the rat with alkaline RE, and this increase was accompanied by continued hyperplasia in comparison with controls (85.5% ± 25.6% vs 20.5% ± 2.1%, P < 0.05, respectively). Furthermore, the expression of TJ proteins was not correlated significantly with that of IL-6 in this group. CONCLUSION: These findings indicate that TJ proteins are highly expressed as an early molecular event involved in RE

  14. Analysis of the distribution and expression of claudin-1 tight junction protein in the oral cavity.

    Science.gov (United States)

    Ouban, Abderrahman; Ahmed, Atif

    2015-07-01

    Claudins are the main sealing proteins of the intercellular tight junctions and play an important role in cancer cell progression and dissemination. The authors have previously shown that overexpression of claudin-1 is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior and with advanced stage disease in oral squamous cell carcinomas (OSCCs). Our goal in this study was to examine claudin-1 expression in a tissue microarray of OSCCs taken from multiple sites within the oral cavity. This study examined and compared the expression of claudin-1 by immunohistochemistry in 60 tissue samples (49 OSCCs and 10 cases of non-neoplastic tissue, single core per case) were analyzed for claudin-1 expression by immunohistochemistry. The tumors included SCCs from the tongue (n=28), the cheek (n=9), gingival (n=4), lip (n=3), and oral cavity (n=5). Nonmalignant normal oral mucosa from the tongue (unmatched cases, n=2). Cancer adjacent tissue samples were taken from the tongue (n=6), gingival (n=2), and palate (n=1). This study demonstrates the expression of claudin-1 protein across a sample of OSCCs originating from multiple locations in the oral cavity. The highest expression of claudin-1 was observed in well-differentiated OSCCs, whereas poorly differentiated OSCCs exhibited mostly negative staining for claudin-1. In addition, we hereby report differential pattern of expression among tumors of different sites within the oral cavity, and between benign and cancerous samples. Our understanding of the exact function and role of claudin-1 in tumorigenesis is expanding exponentially.

  15. Volatile Anesthetics Influence Blood-Brain Barrier Integrity by Modulation of Tight Junction Protein Expression in Traumatic Brain Injury

    OpenAIRE

    Thal, Serge C.; Clara Luh; Eva-Verena Schaible; Ralph Timaru-Kast; Jana Hedrich; Luhmann, Heiko J.; Kristin Engelhard; Zehendner, Christoph M.

    2012-01-01

    Disruption of the blood-brain barrier (BBB) results in cerebral edema formation, which is a major cause for high mortalityrnafter traumatic brain injury (TBI). As anesthetic care is mandatory in patients suffering from severe TBI it may be importantrnto elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ) such as zonularnoccludens-1 (ZO-1) and claudin-5 (cl5) play a central role for BBB stability. First, the influence of the volatile anesthet...

  16. Gap junction connexins in female reproductive organs: implications for women's reproductive health.

    Science.gov (United States)

    Winterhager, Elke; Kidder, Gerald M

    2015-01-01

    Connexins comprise a family of ~20 proteins that form intercellular membrane channels (gap junction channels) providing a direct route for metabolites and signalling molecules to pass between cells. This review provides a critical analysis of the evidence for essential roles of individual connexins in female reproductive function, highlighting implications for women's reproductive health. No systematic review has been carried out. Published literature from the past 35 years was surveyed for research related to connexin involvement in development and function of the female reproductive system. Because of the demonstrated utility of genetic manipulation for elucidating connexin functions in various organs, much of the cited information comes from research with genetically modified mice. In some cases, a distinction is drawn between connexin functions clearly related to the formation of gap junction channels and those possibly linked to non-channel roles. Based on work with mice, several connexins are known to be required for female reproductive functions. Loss of connexin43 (CX43) causes an oocyte deficiency, and follicles lacking or expressing less CX43 in granulosa cells exhibit reduced growth, impairing fertility. CX43 is also expressed in human cumulus cells and, in the context of IVF, has been correlated with pregnancy outcome, suggesting that this connexin may be a determinant of oocyte and embryo quality in women. Loss of CX37, which exclusively connects oocytes with granulosa cells in the mouse, caused oocytes to cease growing without acquiring meiotic competence. Blocking of CX26 channels in the uterine epithelium disrupted implantation whereas loss or reduction of CX43 expression in the uterine stroma impaired decidualization and vascularization in mouse and human. Several connexins are important in placentation and, in the human, CX43 is a key regulator of the fusogenic pathway from the cytotrophoblast to the syncytiotrophoblast, ensuring placental growth

  17. Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling

    Science.gov (United States)

    Adams, Ralph; Burnley, Rebecca J.; Valenzano, Chiara R.; Qureshi, Omar; Doyle, Carl; Lumb, Simon; del Carmen Lopez, Maria; Griffin, Robert; McMillan, David; Taylor, Richard D.; Meier, Chris; Mori, Prashant; Griffin, Laura M.; Wernery, Ulrich; Kinne, Jörg; Rapecki, Stephen; Baker, Terry S.; Lawson, Alastair D. G.; Wright, Michael; Ettorre, Anna

    2017-01-01

    Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the “junctional epitope” nature of VHH6, a camelid single domain antibody recognizing the IL-6–gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions. PMID:28134246

  18. Cdc42-dependent Modulation of Tight Junctions and Membrane Protein Traffic in Polarized Madin-Darby Canine Kidney Cells

    Science.gov (United States)

    Rojas, Raul; Ruiz, Wily G.; Leung, Som-Ming; Jou, Tzuu-Shuh; Apodaca, Gerard

    2001-01-01

    Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity. PMID:11514615

  19. Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells

    NARCIS (Netherlands)

    Soares, Ana Rosa; Martins-Marques, Tania; Ribeiro-Rodrigues, Teresa; Ferreira, Joao Vasco; Catarino, Steve; Pinho, Maria Joao; Zuzarte, Monica; Anjo, Sandra Isabel; Manadas, Bruno; Sluijter, Joost P. G.; Pereira, Paulo; Girao, Henrique

    2015-01-01

    Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, m

  20. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption

    NARCIS (Netherlands)

    Simon, DB; Lu, Y; Choate, KA; Velazquez, H; Al-Sabban, E; Praga, M; Casari, C; Bettinelli, A; Colussi, C; Rodriguez-Soriano, J; McCredie, D; Milford, D; Sanjad, S; Lifton, RP

    1999-01-01

    Epithelia permit selective and regulated flux from apical to basolateral surfaces by transcellular passage through cells or paracellular flux between cells. Tight junctions constitute the barrier to paracellular conductance; however, Little is known about the specific molecules that mediate paracell

  1. The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret [Department of Nephrology, Hannover Medical School, Hannover D-30625 (Germany); Kusch, Angelika [Department of Nephrology and Intensive Care Medicine, Charite Campus Virchow-Klinikum, Berlin D-13353 (Germany); Korenbaum, Elena; Haller, Hermann [Department of Nephrology, Hannover Medical School, Hannover D-30625 (Germany); Dumler, Inna, E-mail: dumler.inna@mh-hannover.de [Department of Nephrology, Hannover Medical School, Hannover D-30625 (Germany)

    2011-07-08

    Highlights: {yields} The tight junction protein ZO-2 associates with Jak1 in vascular smooth muscle cells via ZO-2 N-terminal fragment. {yields} Jak1 mediates ZO-2 tyrosine phosphorylation and ZO-2 localization to the sites of homotypic intercellular contacts. {yields} The urokinase receptor uPAR regulates ZO-2/Jak1 functional association. {yields} The ZO-2/Jak1/uPAR signaling complex is required for vascular smooth muscle cells functional network formation. -- Abstract: Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC), little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.

  2. Cochlear implantation effect on deaf children with gap junction protein beta 2 gene mutation

    Institute of Scientific and Technical Information of China (English)

    KONG Ying; LIU Sha; WANG Su-ju; Li Shu-jing; LIANG Shuang

    2013-01-01

    Background The popularization and promotion of gene diagnosis technology makes it possible to detect deafness genes for children with congenital hearing impairment,and the proportion of gap junction protein beta 2 (GJB2) gene mutations in cochlear implant patients is 26.5% We did follow-up evaluation on auditory rehabilitation effect for all 31 deaf children with GJB2 gene mutation after cochlear implantation to provide a reference for such patients.Methods Application of “the genetic deafness gene chip detection kit” and “gene complete sequence analysis” were applied to conduct detection on common genetic deafness gene mutation hotspots of the hearing impaired children with cochlear implantation.To conduct auditory rehabilitation effect evaluation on all 31 cases of patients with GJB2 genetic deafness after 3,6 and 12 months of the operation respectively.The single factor repeated measure analysis of variance (ANOVA) was applied to analysis whether there were significant difference among the results of initial consonant of a Chinese syllable recognition at 3 different stages after the operation,the results of vowel of a Chinese syllable recognition at 3 different stages after the operation,and the results of two-syllable recognition at 3 different stages after the operation.Results The 235delC is the high-incidence mutational site in 31 cases of patients with GJB2 genetic deafness,and the total detection rate is up to 90.3% (28/31).There were significant differences in the initial consonant and the vowel of a Chinese syllable recognition rate,and the two-syllable recognition rates at 3,6,and 12 months after the operation (P<0.01).Conclusion Cochlear implantation is a safe and effective measure for auditory reconstruction,enabling patients with GJB2 hereditary severe sensorineural deafness to achieve auditory speech recognition effectively.

  3. The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells.

    Science.gov (United States)

    Torres-Flores, Jesús M; Silva-Ayala, Daniela; Espinoza, Marco A; López, Susana; Arias, Carlos F

    2015-01-15

    Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.

  4. Glucocorticoids upregulates transepithelial electrical resistance and expression of tight junction-related protein in human trabecular meshwork cells

    Institute of Scientific and Technical Information of China (English)

    ZHUO Ye-hong; HUANG Ya-lin; WEI Yan-tao; LING Yun-lan; LIN Ming-kai; GE Jian

    2005-01-01

    @@ The trabecular meshwork is located at the anterior chamber angle, and is the main route for the outflow of aqueous humor. It is composed of perforated sheets of collagen and elastic tissue covered by trabecular meshwork (TM) cells, forming a filter with decreasing pore size as the canal of Schlemm is approached. TM cells have some endothelial properties, such as the presence of intercellular junctional complexes, particularly tight junctions (TJs). TJs form paracellular seals between adjacent cells and act as fences that segregate protein (and partially lipid) components of the apical and basolateral plasma membrane domains. Under the electron microscope, TJs appear as a series of discrete contacts between the lateral membranes of adjacent cells.

  5. Enhanced Protein Production in Escherichia coli by Optimization of Cloning Scars at the Vector-Coding Sequence Junction

    DEFF Research Database (Denmark)

    Mirzadeh, Kiavash; Martinez, Virginia; Toddo, Stephen

    2015-01-01

    Protein production in Escherichia coli is a fundamental activity for a large fraction of academic, pharmaceutical, and industrial research laboratories. Maximum production is usually sought, as this reduces costs and facilitates downstream purification steps. Frustratingly, many coding sequences...... are poorly expressed even when they are codon-optimized and expressed from vectors with powerful genetic elements. In this study, we show that poor expression can be caused by certain nucleotide sequences (e.g., cloning scars) at the junction between the vector and the coding sequence. Since these sequences...... lie between the Shine-Dalgarno sequence and the start codon, they are an integral part of the translation initiation region. To identify the most optimal sequences, we devised a simple and inexpensive PCR-based step that generates sequence variants at the vector-coding sequence junction...

  6. Gap junction reduction in cardiomyocytes following transforming growth factor-β treatment and Trypanosoma cruzi infection

    Directory of Open Access Journals (Sweden)

    Mariana C Waghabi

    2009-12-01

    Full Text Available Gap junction connexin-43 (Cx43 molecules are responsible for electrical impulse conduction in the heart and are affected by transforming growth factor-β (TGF-β. This cytokine increases during Trypanosoma cruzi infection, modulating fibrosis and the parasite cell cycle. We studied Cx43 expression in cardiomyocytes exposed or not to TGF-β T. cruzi, or SB-431542, an inhibitor of TGF-β receptor type I (ALK-5. Cx43 expression was also examined in hearts with dilated cardiopathy from chronic Chagas disease patients, in which TGF-β signalling had been shown previously to be highly activated. We demonstrated that TGF-β treatment induced disorganised gap junctions in non-infected cardiomyocytes, leading to a punctate, diffuse and non-uniform Cx43 staining. A similar pattern was detected in T. cruzi-infected cardiomyocytes concomitant with high TGF-β secretion. Both results were reversed if the cells were incubated with SB-431542. Similar tests were performed using human chronic chagasic patients and we confirmed a down-regulation of Cx43 expression, an altered distribution of plaques in the heart and a significant reduction in the number and length of Cx43 plaques, which correlated negatively with cardiomegaly. We conclude that elevated TGF-β levels during T. cruzi infection promote heart fibrosis and disorganise gap junctions, possibly contributing to abnormal impulse conduction and arrhythmia that characterise severe cardiopathy in Chagas disease.

  7. Gap junction reduction in cardiomyocytes following transforming growth factor-beta treatment and Trypanosoma cruzi infection.

    Science.gov (United States)

    Waghabi, Mariana C; Coutinho-Silva, Robson; Feige, Jean-Jacques; Higuchi, Maria de Lourdes; Becker, David; Burnstock, Geoffrey; Araújo-Jorge, Tânia C de

    2009-12-01

    Gap junction connexin-43 (Cx43) molecules are responsible for electrical impulse conduction in the heart and are affected by transforming growth factor-beta (TGF-beta). This cytokine increases during Trypanosoma cruzi infection, modulating fibrosis and the parasite cell cycle. We studied Cx43 expression in cardiomyocytes exposed or not to TGF-beta T. cruzi, or SB-431542, an inhibitor of TGF-beta receptor type I (ALK-5). Cx43 expression was also examined in hearts with dilated cardiopathy from chronic Chagas disease patients, in which TGF-beta signalling had been shown previously to be highly activated. We demonstrated that TGF-beta treatment induced disorganised gap junctions in non-infected cardiomyocytes, leading to a punctate, diffuse and non-uniform Cx43 staining. A similar pattern was detected in T. cruzi-infected cardiomyocytes concomitant with high TGF-beta secretion. Both results were reversed if the cells were incubated with SB-431542. Similar tests were performed using human chronic chagasic patients and we confirmed a down-regulation of Cx43 expression, an altered distribution of plaques in the heart and a significant reduction in the number and length of Cx43 plaques, which correlated negatively with cardiomegaly. We conclude that elevated TGF-beta levels during T. cruzi infection promote heart fibrosis and disorganise gap junctions, possibly contributing to abnormal impulse conduction and arrhythmia that characterise severe cardiopathy in Chagas disease.

  8. Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas.

    Science.gov (United States)

    Wu, Jie; Gao, Ming; Rice, Stephen G; Tsang, Candy; Beggs, John; Turner, Dharshaun; Li, Guohui; Yang, Bo; Xia, Kunkun; Gao, Fenfei; Qiu, Shenfeng; Liu, Qiang; Kerrigan, John F

    2016-06-01

    Human hypothalamic hamartoma (HH) is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36) and connexin-43 (Cx43) in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA) neurons observed with whole-cell patch-clamp recordings from the same patient's HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients.

  9. Gap Junctions Contribute to Ictal/Interictal Genesis in Human Hypothalamic Hamartomas

    Directory of Open Access Journals (Sweden)

    Jie Wu

    2016-06-01

    Full Text Available Human hypothalamic hamartoma (HH is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36 and connexin-43 (Cx43 in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA neurons observed with whole-cell patch-clamp recordings from the same patient's HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients.

  10. Expression of TM4SF10, a Claudin/EMP/PMP22 family cell junction protein, during mouse kidney development and podocyte differentiation.

    Science.gov (United States)

    Bruggeman, Leslie A; Martinka, Scott; Simske, Jeffrey S

    2007-02-01

    Cell junctions in the nephron are highly specialized to perform specific and distinct filtration and reabsorption functions. The mature kidney forms complex cell junctions including slit diaphragms that prevent the passage of serum proteins into the filtrate, and tubule cell junctions that regulate specific paracellular ion reuptake. We have investigated the expression of TM4SF10 (Trans-Membrane tetra(4)-Span Family 10) in mouse kidneys. TM4SF10 is the vertebrate orthologue of Caenorhabditis elegans VAB-9, a tetraspan adherens junction protein in the PMP22/EMP/Claudin family of proteins. We found that TM4SF10 localizes at the basal-most region of podocyte precursors before the capillary loop stage, at some tubule precursors, and at the ureteric bud junction with S-shaped bodies. Overall expression of TM4SF10 peaked at postnatal day 4 and was virtually absent in adult kidneys. The very limited expression of TM4SF10 protein that persisted into adulthood was restricted to a few tubule segments but remained localized to the basal region of lateral membranes. In undifferentiated cultured podocytes, TM4SF10 localized to the perinuclear region and translocated to the cell membrane after Cadherin appearance at cell-cell contacts. TM4SF10 colocalized with ZO1 and p120ctn in undifferentiated confluent podocytes and also colocalized with the tips of actin filaments at cell contacts. Upon differentiation of cultured podocytes, TM4SF10 protein disappeared from cell contacts and expression ceased. These results suggest that TM4SF10 functions during differentiation of podocytes and may participate in the maturation of cell junctions from simple adherens junctions to elaborate slit diaphragms. TM4SF10 may define a new class of Claudin-like proteins that function during junctional development.

  11. The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio)

    Science.gov (United States)

    Kwong, Raymond W. M.; Kumai, Yusuke; Perry, Steve F.

    2013-01-01

    Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (Ku) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H+-ATPase-rich cells or Na+/K+-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca2+-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased. PMID:23967101

  12. The role of aquaporin and tight junction proteins in the regulation of water movement in larval zebrafish (Danio rerio.

    Directory of Open Access Journals (Sweden)

    Raymond W M Kwong

    Full Text Available Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio. We observed that the half-time for saturation of water influx (K(u was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM. Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H⁺-ATPase-rich cells or Na⁺/K⁺-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca²⁺-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP; the paracellular routes may become significant when the paracellular permeability is increased.

  13. Autophagy and tight junction proteins in the intestine and intestinal diseases

    Directory of Open Access Journals (Sweden)

    Chien-An A. Hu

    2015-09-01

    Full Text Available The intestinal epithelium (IE forms an indispensible barrier and interface between the intestinal interstitium and the luminal environment. The IE regulates water, ion and nutrient transport while providing a barrier against toxins, pathogens (bacteria, fungi and virus and antigens. The apical intercellular tight junctions (TJ are responsible for the paracellular barrier function and regulate trans-epithelial flux of ions and solutes between adjacent cells. Increased intestinal permeability caused by defects in the IE TJ barrier is considered an important pathogenic factor for the development of intestinal inflammation, diarrhea and malnutrition in humans and animals. In fact, defects in the IE TJ barrier allow increased antigenic penetration, resulting in an amplified inflammatory response in inflammatory bowel disease (IBD, necrotizing enterocolitis and ischemia-reperfusion injury. Conversely, the beneficial enhancement of the intestinal TJ barrier has been shown to resolve intestinal inflammation and apoptosis in both animal models of IBD and human IBD. Autophagy (self-eating mechanism is an intracellular lysosome-dependent degradation and recycling pathway essential for cell survival and homeostasis. Dysregulated autophagy has been shown to be directly associated with many pathological processes, including IBD. Importantly, the crosstalk between IE TJ and autophagy has been revealed recently. We showed that autophagy enhanced IE TJ barrier function by increasing transepithelial resistance and reducing the paracellular permeability of small solutes and ions, which is, in part, by targeting claudin-2, a cation-selective, pore-forming, transmembrane TJ protein, for lysosome (autophagy-mediated degradation. Interestingly, previous studies have shown that the inflamed intestinal mucosa in patients with active IBD has increased claudin-2 expression. In addition, inflammatory cytokines (for example, tumor necrosis factor-α, interleukin-6

  14. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    Science.gov (United States)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  15. Human articular chondrocytes express multiple gap junction proteins: differential expression of connexins in normal and osteoarthritic cartilage.

    Science.gov (United States)

    Mayan, Maria D; Carpintero-Fernandez, Paula; Gago-Fuentes, Raquel; Martinez-de-Ilarduya, Oskar; Wang, Hong-Zhang; Valiunas, Virginijus; Brink, Peter; Blanco, Francisco J

    2013-04-01

    Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  16. Butyrate Enhances the Intestinal Barrier by Facilitating Tight Junction Assembly via Activation of AMP-Activated Protein Kinase in Caco-2 Cell Monolayers12

    Science.gov (United States)

    Peng, Luying; Li, Zhong-Rong; Green, Robert S.; Holzman, Ian R.; Lin, Jing

    2009-01-01

    Butyrate, one of the SCFA, promotes the development of the intestinal barrier. However, the molecular mechanisms underlying the butyrate regulation of the intestinal barrier are unknown. To test the hypothesis that the effect of butyrate on the intestinal barrier is mediated by the regulation of the assembly of tight junctions involving the activation of the AMP-activated protein kinase (AMPK), we determined the effect of butyrate on the intestinal barrier by measuring the transepithelial electrical resistance (TER) and inulin permeability in a Caco-2 cell monolayer model. We further used a calcium switch assay to study the assembly of epithelial tight junctions and determined the effect of butyrate on the assembly of epithelial tight junctions and AMPK activity. We demonstrated that the butyrate treatment increased AMPK activity and accelerated the assembly of tight junctions as shown by the reorganization of tight junction proteins, as well as the development of TER. AMPK activity was also upregulated by butyrate during calcium switch-induced tight junction assembly. Compound C, a specific AMPK inhibitor, inhibited the butyrate-induced activation of AMPK. The facilitating effect of butyrate on the increases in TER in standard culture media, as well as after calcium switch, was abolished by compound C. We conclude that butyrate enhances the intestinal barrier by regulating the assembly of tight junctions. This dynamic process is mediated by the activation of AMPK. These results suggest an intriguing link between SCFA and the intracellular energy sensor for the development of the intestinal barrier. PMID:19625695

  17. Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers.

    Science.gov (United States)

    Peng, Luying; Li, Zhong-Rong; Green, Robert S; Holzman, Ian R; Lin, Jing

    2009-09-01

    Butyrate, one of the SCFA, promotes the development of the intestinal barrier. However, the molecular mechanisms underlying the butyrate regulation of the intestinal barrier are unknown. To test the hypothesis that the effect of butyrate on the intestinal barrier is mediated by the regulation of the assembly of tight junctions involving the activation of the AMP-activated protein kinase (AMPK), we determined the effect of butyrate on the intestinal barrier by measuring the transepithelial electrical resistance (TER) and inulin permeability in a Caco-2 cell monolayer model. We further used a calcium switch assay to study the assembly of epithelial tight junctions and determined the effect of butyrate on the assembly of epithelial tight junctions and AMPK activity. We demonstrated that the butyrate treatment increased AMPK activity and accelerated the assembly of tight junctions as shown by the reorganization of tight junction proteins, as well as the development of TER. AMPK activity was also upregulated by butyrate during calcium switch-induced tight junction assembly. Compound C, a specific AMPK inhibitor, inhibited the butyrate-induced activation of AMPK. The facilitating effect of butyrate on the increases in TER in standard culture media, as well as after calcium switch, was abolished by compound C. We conclude that butyrate enhances the intestinal barrier by regulating the assembly of tight junctions. This dynamic process is mediated by the activation of AMPK. These results suggest an intriguing link between SCFA and the intracellular energy sensor for the development of the intestinal barrier.

  18. Basolateral junction proteins regulate competition for the follicle stem cell niche in the Drosophila ovary.

    Science.gov (United States)

    Kronen, Maria R; Schoenfelder, Kevin P; Klein, Allon M; Nystul, Todd G

    2014-01-01

    Epithelial stem cells are routinely lost or damaged during adult life and must therefore be replaced to maintain homeostasis. Recent studies indicate that stem cell replacement occurs through neutral competition in many types of epithelial tissues, but little is known about the factors that determine competitive outcome. The epithelial follicle stem cells (FSCs) in the Drosophila ovary are regularly lost and replaced during normal homeostasis, and we show that FSC replacement conforms to a model of neutral competition. In addition, we found that FSCs mutant for the basolateral junction genes, lethal giant larvae (lgl) or discs large (dlg), undergo a biased competition for niche occupancy characterized by increased invasion of neighboring FSCs and reduced loss. Interestingly, FSCs mutant for a third basolateral junction gene, scribble (scrib), do not exhibit biased competition, suggesting that Lgl and Dlg regulate niche competition through a Scrib-independent process. Lastly, we found that FSCs have a unique cell polarity characterized by broadly distributed adherens junctions and the lack of a mature apical domain. Collectively, these observations indicate that Lgl and Dlg promote the differentiation of FSC progeny to a state in which they are less prone to invade the neighboring niche. In addition, we demonstrate that the neutral drift model can be adapted to quantify non-neutral behavior of mutant clones.

  19. Basolateral junction proteins regulate competition for the follicle stem cell niche in the Drosophila ovary.

    Directory of Open Access Journals (Sweden)

    Maria R Kronen

    Full Text Available Epithelial stem cells are routinely lost or damaged during adult life and must therefore be replaced to maintain homeostasis. Recent studies indicate that stem cell replacement occurs through neutral competition in many types of epithelial tissues, but little is known about the factors that determine competitive outcome. The epithelial follicle stem cells (FSCs in the Drosophila ovary are regularly lost and replaced during normal homeostasis, and we show that FSC replacement conforms to a model of neutral competition. In addition, we found that FSCs mutant for the basolateral junction genes, lethal giant larvae (lgl or discs large (dlg, undergo a biased competition for niche occupancy characterized by increased invasion of neighboring FSCs and reduced loss. Interestingly, FSCs mutant for a third basolateral junction gene, scribble (scrib, do not exhibit biased competition, suggesting that Lgl and Dlg regulate niche competition through a Scrib-independent process. Lastly, we found that FSCs have a unique cell polarity characterized by broadly distributed adherens junctions and the lack of a mature apical domain. Collectively, these observations indicate that Lgl and Dlg promote the differentiation of FSC progeny to a state in which they are less prone to invade the neighboring niche. In addition, we demonstrate that the neutral drift model can be adapted to quantify non-neutral behavior of mutant clones.

  20. Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway.

    Science.gov (United States)

    Sivagurunathan, Suganya; Palanisamy, Karthikka; Arunachalam, Jayamuruga Pandian; Chidambaram, Subbulakshmi

    2017-03-01

    PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3β in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/β in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/β signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.

  1. Dipyridamole increases gap junction coupling in bovine GM-7373 aortic endothelial cells by a cAMP-protein kinase A dependent pathway.

    Science.gov (United States)

    Begandt, D; Bintig, W; Oberheide, K; Schlie, S; Ngezahayo, A

    2010-02-01

    The scrape-loading/dye transfer technique was applied on the bovine aortic endothelial cell line GM-7373 to analyze the effects of the antithrombolytic drug dipyridamole on gap junction coupling in endothelial cells. We found that a cell treatment for 24 h with dipyridamole in therapeutically relevant concentrations (1-100 microM) increased gap junction coupling in a dose dependent manner. Similar to dipyridamole, forskolin as well as 8-Br-cAMP increased the gap junction coupling, while dibutyryl-cGMP (db-cGMP) did not affect the gap junction coupling of the GM-7373 endothelial cells. In parallel, a pharmacological inhibition of protein kinase A (PKA) with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89), antagonised the action of dipyridamole on gap junction coupling. We propose that the observed dipyridamole induced increase in gap junction coupling in endothelial cells is related to a cAMP-PKA dependent phosphorylation pathway. The report shows that gap junction coupling in endothelial cells is a suitable therapeutic target for treatment of cardiovascular diseases.

  2. Evolution and cell physiology. 4. Why invent yet another protein complex to build junctions in epithelial cells?

    Science.gov (United States)

    Le Bivic, André

    2013-12-15

    The formation of the first epithelium was an essential step for animal evolution, since it has allowed coordination of the behavior of a cell layer and creation of a selective barrier between the internal medium and the outside world. The possibility of coupling the cells in a single layer has allowed morphogenetic events, such as tube formation, or gastrulation, to form more complex animal morphologies. The invention of sealed junctions between cells has allowed, on the other hand, creation of an asymmetry of nutrients or salts between the apical and the basal side of the epithelial layer. Creation of an internal medium has led to homeostasis, allowing the evolution of more complex physiological functions and the emergence of sophisticated animal shapes. During evolution, the origins of the first animals coincided with the invention of several protein complexes, including true cadherins and the polarity protein complexes. How these complexes regulate formation of the apicolateral border and the adherens junctions is still not fully understood. This review focuses on the role of these apical polarity complexes and, in particular, the Crumbs complex, which is essential for proper organization of epithelial layers from Drosophila to humans.

  3. The zinc finger protein Zfr1p is localized specifically to conjugation junction and required for sexual development in Tetrahymena thermophila.

    Directory of Open Access Journals (Sweden)

    Jing Xu

    Full Text Available Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell.

  4. Methamphetamine compromises gap junctional communication in astrocytes and neurons.

    Science.gov (United States)

    Castellano, Paul; Nwagbo, Chisom; Martinez, Luis R; Eugenin, Eliseo A

    2016-05-01

    Methamphetamine (meth) is a central nervous system (CNS) stimulant that results in psychological and physical dependency. The long-term effects of meth within the CNS include neuronal plasticity changes, blood-brain barrier compromise, inflammation, electrical dysfunction, neuronal/glial toxicity, and an increased risk to infectious diseases including HIV. Most of the reported meth effects in the CNS are related to dysregulation of chemical synapses by altering the release and uptake of neurotransmitters, especially dopamine, norepinephrine, and epinephrine. However, little is known about the effects of meth on connexin (Cx) containing channels, such as gap junctions (GJ) and hemichannels (HC). We examined the effects of meth on Cx expression, function, and its role in NeuroAIDS. We found that meth altered Cx expression and localization, decreased GJ communication between neurons and astrocytes, and induced the opening of Cx43/Cx36 HC. Furthermore, we found that these changes in GJ and HC induced by meth treatment were mediated by activation of dopamine receptors, suggesting that dysregulation of dopamine signaling induced by meth is essential for GJ and HC compromise. Meth-induced changes in GJ and HC contributed to amplified CNS toxicity by dysregulating glutamate metabolism and increasing the susceptibility of neurons and astrocytes to bystander apoptosis induced by HIV. Together, our results indicate that connexin containing channels, GJ and HC, are essential in the pathogenesis of meth and increase the sensitivity of the CNS to HIV CNS disease. Methamphetamine (meth) is an extremely addictive central nervous system stimulant. Meth reduced gap junctional (GJ) communication by inducing internalization of connexin-43 (Cx43) in astrocytes and reducing expression of Cx36 in neurons by a mechanism involving activation of dopamine receptors (see cartoon). Meth-induced changes in Cx containing channels increased extracellular levels of glutamate and resulted in higher

  5. Peroxisome proliferator-activated receptor gamma promotes exogenous mesenchymal stem cells to express connexin 43:its role and mechanism%过氧化物酶体增殖物激活受体γ促进外源性骨髓间充质干细胞表达Cx43的作用及机制

    Institute of Scientific and Technical Information of China (English)

    晏平; 侯婧瑛; 郑韶欣; 龙会宝; 周长青; 郭天柱; 伍权华; 钟婷婷; 王彤

    2016-01-01

    BACKGROUND:Piglitazone, aperoxisome proliferator-activated receptor γ(PPAR-γ) agonist, has been demonstrated topromote survivalandcardiac differentiation ofexogenous bone marrow mesenchymal stem celsto improvecardiacfunction.In this study, we attempted to investigate whether pioglitazone couldinduce cardiac differentiation of endogenous bone marrow mesenchymal stem celsandimprove cardiacfunction, andmeanwhile, probed into the relevant mechanisms. OBJECTIVE:To compare the therapeutic efficacy ofpioglitazone combined with bone marrow mesenchymal stem cel transplantation, pioglitazone alone and phosphate buffer solution(PBS)and to investigatetherelevant mechanisms. METHODS:ThirtySprague-Dawley ratswith myocardial infarctioninducedby ligation of the left anterior descending coronary artery were randomized intocombined group (combination of bone marrow mesenchymal stem cels and pioglitazone), pioglitazone group andPBSgroup. Two weeks later, PKH26-labeled bone marrow mesenchymal stem cels inPBSorPBSalone wereinjected into the local infarct zone in the combinedgroup andthe other twogroups, respectively. Pioglitazone (3 mg/kg/d) was given by the oral gavage in the combinedand pioglitazone groups forcontinuous2weeks after cels transplantation. At 2weeks after treatment, cardiac functions were evaluated. In addition, expressions of PPAR-γ, connexin 43 and relative factors in transforming growth factor-β1/SMAD signaling pathway were examined in different areas of the left ventricle from each harvested heart. RESULTS AND CONCLUSION:There were no differences in the baseline parameters of cardiac function between the two groups.Twoweeksafter treatment, left ventricular end-diastolic diameter, left ventricular end-systolic diameter and left ventricular ejection fraction were significantlyimprovedin the combined groupcompared with the other two groups; the expression of PPAR-γ was significantly increased in different zones of the left ventriclein the combined andpioglitazone

  6. Mechanism of Holliday junction resolution by the human GEN1 protein.

    Science.gov (United States)

    Rass, Ulrich; Compton, Sarah A; Matos, Joao; Singleton, Martin R; Ip, Stephen C Y; Blanco, Miguel G; Griffith, Jack D; West, Stephen C

    2010-07-15

    Holliday junction (HJ) resolution is essential for chromosome segregation at meiosis and the repair of stalled/collapsed replication forks in mitotic cells. All organisms possess nucleases that promote HJ resolution by the introduction of symmetrically related nicks in two strands at, or close to, the junction point. GEN1, a member of the Rad2/XPG nuclease family, was isolated recently from human cells and shown to promote HJ resolution in vitro and in vivo. Here, we provide the first biochemical/structural characterization of GEN1, showing that, like the Escherichia coli HJ resolvase RuvC, it binds specifically to HJs and resolves them by a dual incision mechanism in which nicks are introduced in the pair of continuous (noncrossing) strands within the lifetime of the GEN1-HJ complex. In contrast to RuvC, but like other Rad2/XPG family members such as FEN1, GEN1 is a monomeric 5'-flap endonuclease. However, the unique feature of GEN1 that distinguishes it from other Rad2/XPG nucleases is its ability to dimerize on HJs. This functional adaptation provides the two symmetrically aligned active sites required for HJ resolution.

  7. The tight junction protein ZO-2 associates with Jun, Fos and C/EBP transcription factors in epithelial cells.

    Science.gov (United States)

    Betanzos, Abigail; Huerta, Miriam; Lopez-Bayghen, Esther; Azuara, Elisa; Amerena, José; González-Mariscal, Lorenza

    2004-01-01

    ZO-2 is a membrane-associated guanylate kinase (MAGUK) protein present at the tight junction (TJ) of epithelial cells. While confluent monolayers have ZO-2 at their cellular borders, sparse cultures conspicuously show ZO-2 at the nuclei. To study the role of nuclear ZO-2, we tested by pull-down assays and gel shift analysis the interaction between ZO-2 GST fusion proteins and different transcription factors. We identified the existence of a specific interaction of ZO-2 with Fos, Jun and C/EBP (CCAAT/enhancer binding protein). To analyze if this association is present "in vivo", we performed immunoprecipitation and immunolocalization experiments, which revealed an interaction of ZO-2 with Jun, Fos and C/EBP not only at the nucleus but also at the TJ region. To test if the association of ZO-2 with AP-1 (activator protein-1) modulates gene transcription, we performed reporter gene assays employing chloramphenicol acetyltransferase (CAT) constructs with promoters under the control of AP-1 sites. We observed that the co-transfected ZO-2 down-regulates CAT expression in a dose-dependent manner. Since ZO-2 is a multidomain protein, we proceeded to determine which region of the molecule is responsible for the modulation of gene expression, and observed that both the amino and the carboxyl domains are capable of inhibiting gene transcription.

  8. Molecular characterisation of the nucleocapsid protein gene, glycoprotein gene and gene junctions of rhabdovirus 903/87, a novel fish pathogenic rhabdovirus

    DEFF Research Database (Denmark)

    Johansson, Tove; Nylund, S.; Olesen, Niels Jørgen

    2001-01-01

    The sequences of the nucleocapsid and glycoprotein genes and the gene junctions of the fish pathogenic rhabdovirus 903/87 were determined from cDNA and PCR clones. The mRNA of the nucleocapsid is most likely 1492 nucleotides long and encodes a protein of 426 amino acids, whereas the mRNA of the g......The sequences of the nucleocapsid and glycoprotein genes and the gene junctions of the fish pathogenic rhabdovirus 903/87 were determined from cDNA and PCR clones. The mRNA of the nucleocapsid is most likely 1492 nucleotides long and encodes a protein of 426 amino acids, whereas the m...

  9. Chlorogenic acid decreases intestinal permeability and increases expression of intestinal tight junction proteins in weaned rats challenged with LPS.

    Directory of Open Access Journals (Sweden)

    Zheng Ruan

    Full Text Available Chlorogenic acid, a natural phenolic acid present in fruits and plants, provides beneficial effects for human health. The objectives of this study were to investigate whether chlorogenic acid (CHA could improve the intestinal barrier integrity for weaned rats with lipopolysaccharide (LPS challenge. Thirty-two weaned male Sprague Dawley rats (21 ± 1 d of age; 62.26 ± 2.73 g were selected and randomly allotted to four treatments, including weaned rat control, LPS-challenged and chlorogenic acid (CHA supplemented group (orally 20 mg/kg and 50 mg/kg body. Dietary supplementation with CHA decreased (P<0.05 the concentrations of urea and albumin in the serum, compared to the LPS-challenged group. The levels of IFN-γ and TNF-α were lower (P<0.05 in the jejunal and colon of weaned rats receiving CHA supplementation, in comparison with the control group. CHA supplementation increased (P<0.05 villus height and the ratio of villus height to crypt depth in the jejunal and ileal mucosae under condictions of LPS challenge. CHA supplementation decreased (P<0.05 intestinal permeability, which was indicated by the ratio of lactulose to mannitol and serum DAO activity, when compared to weaned rats with LPS challenge. Immunohistochemical analysis of tight junction proteins revealed that ZO-1 and occludin protein abundances in the jejunum and colon were increased (P<0.05 by CHA supplementation. Additionally, results of immunoblot analysis revealed that the amount of occludin in the colon was also increased (P<0.05 in CHA-supplemented rats. In conclusion, CHA decreases intestinal permeability and increases intestinal expression of tight junction proteins in weaned rats challenged with LPS.

  10. Tight junction protein Par6 interacts with an evolutionarily conserved region in the amino terminus of PALS1/stardust.

    Science.gov (United States)

    Wang, Qian; Hurd, Toby W; Margolis, Ben

    2004-07-16

    Tight junctions are the structures in mammalian epithelial cells that separate the apical and basolateral membranes and may also be important in the establishment of cell polarity. Two evolutionarily conserved multiprotein complexes, Crumbs-PALS1 (Stardust)-PATJ and Cdc42-Par6-Par3-atypical protein kinase C, have been implicated in the assembly of tight junctions and in polarization of Drosophila melanogaster epithelia. These two complexes have been linked physically and functionally by an interaction between PALS1 and Par6. Here we identify an evolutionarily conserved region in the amino terminus of PALS1 as the Par6 binding site and identify valine and aspartic acid residues in this region as essential for interacting with the PDZ domain of Par6. We have also characterized, in more detail, the amino terminus of Drosophila Stardust and demonstrate that the interaction mechanism between Stardust and Drosophila Par6 is evolutionarily conserved. Par6 interferes with PATJ in binding PALS1, and these two interactions do not appear to function synergistically. Taken together, these results define the molecular mechanisms linking two conserved polarity complexes.

  11. Microfluidic chips with multi-junctions: an advanced tool in recovering proteins from inclusion bodies.

    Science.gov (United States)

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2015-01-01

    Active recombinant proteins are used for studying the biological functions of genes and for the development of therapeutic drugs. Overexpression of recombinant proteins in bacteria often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. Protein refolding is an important process for obtaining active recombinant proteins from inclusion bodies. However, the conventional refolding method of dialysis or dilution is time-consuming and recovered active protein yields are often low, and a cumbersome trial-and-error process is required to achieve success. To circumvent these difficulties, we used controllable diffusion through laminar flow in microchannels to regulate the denaturant concentration. This method largely aims at reducing protein aggregation during the refolding procedure. This Commentary introduces the principles of the protein refolding method using microfluidic chips and the advantage of our results as a tool for rapid and efficient recovery of active recombinant proteins from inclusion bodies.

  12. Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia

    Science.gov (United States)

    Longchamp, Alban; Allagnat, Florent; Alonso, Florian; Kuppler, Christopher; Dubuis, Céline; Ozaki, Charles-Keith; Mitchell, James R.; Berceli, Scott; Corpataux, Jean-Marc

    2015-01-01

    Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment. PMID:26398895

  13. Alpha-catenin-Dependent Recruitment of the Centrosomal Protein CAP350 to Adherens Junctions Allows Epithelial Cells to Acquire a Columnar Shape

    Science.gov (United States)

    Zurbano, Angel; Formstecher, Etienne; Martinez-Morales, Juan R.; Bornens, Michel; Rios, Rosa M.

    2015-01-01

    Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis. PMID:25764135

  14. Telomeric protein TRF2 protects Holliday junctions with telomeric arms from displacement by the Werner syndrome helicase.

    Science.gov (United States)

    Nora, Gerald J; Buncher, Noah A; Opresko, Patricia L

    2010-07-01

    WRN protein loss causes Werner syndrome (WS), which is characterized by premature aging as well as genomic and telomeric instability. WRN prevents telomere loss, but the telomeric protein complex must regulate WRN activities to prevent aberrant telomere processing. Telomere-binding TRF2 protein inhibits telomere t-loop deletion by blocking Holliday junction (HJ) resolvase cleavage activity, but whether TRF2 also modulates HJ displacement at t-loops is unknown. In this study, we used multiplex fluorophore imaging to track the fate of individual strands of HJ substrates. We report the novel finding that TRF2 inhibits WRN helicase strand displacement of HJs with telomeric repeats in duplex arms, but unwinding of HJs with a telomeric center or lacking telomeric sequence is unaffected. These data, together with results using TRF2 fragments and TRF2 HJ binding assays, indicate that both the TRF2 B- and Myb domains are required to inhibit WRN HJ activity. We propose a novel model whereby simultaneous binding of the TRF2 B-domain to the HJ core and the Myb domain to telomeric arms promote and stabilize HJs in a stacked arm conformation that is unfavorable for unwinding. Our biochemical study provides a mechanistic basis for the cellular findings that TRF2 regulates WRN activity at telomeres.

  15. PLEKHA7 is an adherens junction protein with a tissue distribution and subcellular localization distinct from ZO-1 and E-cadherin.

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    Pamela Pulimeno

    Full Text Available The pleckstrin-homology-domain-containing protein PLEKHA7 was recently identified as a protein linking the E-cadherin-p120 ctn complex to the microtubule cytoskeleton. Here we characterize the expression, tissue distribution and subcellular localization of PLEKHA7 by immunoblotting, immunofluorescence microscopy, immunoelectron microscopy, and northern blotting in mammalian tissues. Anti-PLEKHA7 antibodies label the junctional regions of cultured kidney epithelial cells by immunofluorescence microscopy, and major polypeptides of M(r approximately 135 kDa and approximately 145 kDa by immunoblotting of lysates of cells and tissues. Two PLEKHA7 transcripts ( approximately 5.5 kb and approximately 6.5 kb are detected in epithelial tissues. PLEKHA7 is detected at epithelial junctions in sections of kidney, liver, pancreas, intestine, retina, and cornea, and its tissue distribution and subcellular localization are distinct from ZO-1. For example, PLEKHA7 is not detected within kidney glomeruli. Similarly to E-cadherin, p120 ctn, beta-catenin and alpha-catenin, PLEKHA7 is concentrated in the apical junctional belt, but unlike these adherens junction markers, and similarly to afadin, PLEKHA7 is not localized along the lateral region of polarized epithelial cells. Immunoelectron microscopy definitively establishes that PLEKHA7 is localized at the adherens junctions in colonic epithelial cells, at a mean distance of 28 nm from the plasma membrane. In summary, we show that PLEKHA7 is a cytoplasmic component of the epithelial adherens junction belt, with a subcellular localization and tissue distribution that is distinct from that of ZO-1 and most AJ proteins, and we provide the first description of its distribution and localization in several tissues.

  16. Expression of intestinal mucosa tight junctions claudin proteins and mRNA in patients with irritable bowel syndrome

    Institute of Scientific and Technical Information of China (English)

    KONG Wu-ming; GONG Jun; DONG Lei; LU Xiao-lan; XU Jun-rong

    2007-01-01

    Objective:To investigate the changes of intestinal mucosa tight junctions(TJs)claudin-1,-3,-4 proteins and mRNA changes in patients with irritable bowel syndrome(IBS)and to elucidate their possible roles in the changes of bowel evacuation habit and formation.Methods:Claudin-1,-3,-4 proteins and mRNA were evaluated in intestinal mucosa in control group,D-IBS(diarrhea IBS)group and C-IBS (constipation IBS)group with immunohistochemical assay and Realtime-PCR.Results:It was observed that claudin-1,-3,-4 proteins were localized in the membranes of epithelial cells along the entire length of the plasma membrane including the apical end of the epithelial cells.The claudins were concentrated at the site of TJs only.Claudin-1,-3,-4 mRNA and claudin-1 protein in small intestinal mucosa and colonal mucous in D-IBS group were significantly downregulated(P<0.05).Claudin-1,-3,-4 mRNA and proteins in small intestinal mucosa and co1onal mucous in C-IBS group were significantly upregulated(P<0.05).There was no significant difference in the expression of claudin-3 protein in both small intestinal mucosa and colonal mucous between D-IBS group and control group(P>0.05).Similarly,no significantly different expression of claudin-4 protein in colonal mucous in D-IBS group was found compared with control group(P>0.05).Otherwise,the expression of claudin-4 protein in small intestinal mucosa decreased in D-IBS group(P<0.05).Conclusion:Claudin-1,-3,-4 may play a potential important role in the changes of bowel evacuation habit and formation in patients with IBS.It is not due to the localization changes of claudin proteins in TJ,but may be caused by the quantitative changes of the expression of TJ proteins and mRNA.

  17. Nonmechanical Roles of Dystrophin and Associated Proteins in Exercise, Neuromuscular Junctions, and Brains

    Directory of Open Access Journals (Sweden)

    Bailey Nichols

    2015-07-01

    Full Text Available Dystrophin-glycoprotein complex (DGC is an important structural unit in skeletal muscle that connects the cytoskeleton (f-actin of a muscle fiber to the extracellular matrix (ECM. Several muscular dystrophies, such as Duchenne muscular dystrophy, Becker muscular dystrophy, congenital muscular dystrophies (dystroglycanopathies, and limb-girdle muscular dystrophies (sarcoglycanopathies, are caused by mutations in the different DGC components. Although many early studies indicated DGC plays a crucial mechanical role in maintaining the structural integrity of skeletal muscle, recent studies identified novel roles of DGC. Beyond a mechanical role, these DGC members play important signaling roles and act as a scaffold for various signaling pathways. For example, neuronal nitric oxide synthase (nNOS, which is localized at the muscle membrane by DGC members (dystrophin and syntrophins, plays an important role in the regulation of the blood flow during exercise. DGC also plays important roles at the neuromuscular junction (NMJ and in the brain. In this review, we will focus on recently identified roles of DGC particularly in exercise and the brain.

  18. Tight Junction Proteins and Oxidative Stress in Heavy Metals-Induced Nephrotoxicity

    Directory of Open Access Journals (Sweden)

    José L. Reyes

    2013-01-01

    Full Text Available Kidney is a target organ for heavy metals. They accumulate in several segments of the nephron and cause profound alterations in morphology and function. Acute intoxication frequently causes acute renal failure. The effects of chronic exposure have not been fully disclosed. In recent years increasing awareness of the consequences of their presence in the kidney has evolved. In this review we focus on the alterations induced by heavy metals on the intercellular junctions of the kidney. We describe that in addition to the proximal tubule, which has been recognized as the main site of accumulation and injury, other segments of the nephron, such as glomeruli, vessels, and distal nephron, show also deleterious effects. We also emphasize the participation of oxidative stress as a relevant component of the renal damage induced by heavy metals and the beneficial effect that some antioxidant drugs, such as vitamin A (all-trans-retinoic acid and vitamin E (α-tocopherol, depict on the morphological and functional alterations induced by heavy metals.

  19. Specific deletion of AMP-activated protein kinase (α1AMPK in murine oocytes alters junctional protein expression and mitochondrial physiology.

    Directory of Open Access Journals (Sweden)

    Michael J Bertoldo

    Full Text Available Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK, an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues. Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.

  20. The tight junction component protein, claudin-4, is expressed by enteric neurons in the rat distal colon.

    Science.gov (United States)

    Karaki, Shin-ichiro; Kaji, Izumi; Otomo, Yasuko; Tazoe, Hideaki; Kuwahara, Atsukazu

    2007-11-27

    The expression of a tight junction (TJ) component protein, claudin-4, in the enteric neurons was investigated in the rat distal colon by immunohistochemistry and RT-PCR. Claudin-4 immunoreactivity was detected in almost all neurofilament-positive enteric neurons both of the submucosal and the myenteric plexuses, and both of the cell bodies and the neurofibers. The immunoreactivity of enteric neurons for claudin-4 was divided into two types: strongly and weakly positive neurons. Especially in the myenteric plexus, the stained neurons were classified by Dogiel's morphological classification of enteric neurons. The strongly stained claudin-4 positive neurons show Dogiel type II morphology, while the weakly stained claudin-4 positive neurons show Dogiel type I morphology. These immunohistochemical data were supported by mRNA expression in the muscle plus submucosa preparation containing the submucosal and myenteric plexuses, as well as mucosa preparation. The physiological function of claudin-4 expressed on enteric neurons is unclear up to now. It is however suggested that claudin-4 expressed on enteric neurons might play roles for the neural activity, for example as insulation between neurofibers. In conclusion, the present study clearly shows that claudin-4 is expressed by enteric neurons. This is the first evidence that the neuron itself expresses the TJ component protein, claudin-4, in the nervous system.

  1. Polycystin-2 activity is controlled by transcriptional coactivator with PDZ binding motif and PALS1-associated tight junction protein.

    Science.gov (United States)

    Duning, Kerstin; Rosenbusch, Deike; Schlüter, Marc A; Tian, Yuemin; Kunzelmann, Karl; Meyer, Nina; Schulze, Ulf; Markoff, Arseni; Pavenstädt, Hermann; Weide, Thomas

    2010-10-29

    Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic cause of kidney failure, characterized by the development of renal cysts. ADPKD is caused by mutations of the polycystin-1 (PC1) or polycystin-2 (PC2) genes. PC2 encodes a Ca(2+)-permeable cation channel, and its dysfunction has been implicated in cyst development. The transcriptional coactivator with PDZ binding motif (TAZ) is required for the integrity of renal cilia. Its absence results in the development of renal cysts in a knock-out mouse model. TAZ directly interacts with PC2, and it has been suggested that another yet unidentified PDZ domain protein may be involved in the TAZ/PC2 interaction. Here we describe a novel interaction of TAZ with the multi-PDZ-containing PALS1-associated tight junction protein (PATJ). TAZ interacts with both the N-terminal PDZ domains 1-3 and the C-terminal PDZ domains 8-10 of PATJ, suggesting two distinct TAZ binding domains. We also show that the C terminus of PC2 strongly interacts with PDZ domains 8-10 and to a weaker extent with PDZ domains 1-3 of PATJ. Finally, we demonstrate that both TAZ and PATJ impair PC2 channel activity when co-expressed with PC2 in oocytes of Xenopus laevis. These results implicate TAZ and PATJ as novel regulatory elements of the PC2 channel and might thus be involved in ADPKD pathology.

  2. Hypoxia/Aglycemia-induced endothelial barrier dysfunction and tight junction protein downregulation can be ameliorated by citicoline.

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    Xiaotang Ma

    Full Text Available This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs and mouse brain microvascular endothelial cells (bEnd.3s. The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1 and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs.

  3. Hypoxia/Aglycemia-induced endothelial barrier dysfunction and tight junction protein downregulation can be ameliorated by citicoline.

    Science.gov (United States)

    Ma, Xiaotang; Zhang, Huiting; Pan, Qunwen; Zhao, Yuhui; Chen, Ji; Zhao, Bin; Chen, Yanfang

    2013-01-01

    This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs.

  4. Inhibition of gap junction communication at ectopic Eph/ephrin boundaries underlies craniofrontonasal syndrome.

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    Alice Davy

    2006-10-01

    Full Text Available Mutations in X-linked ephrin-B1 in humans cause craniofrontonasal syndrome (CFNS, a disease that affects female patients more severely than males. Sorting of ephrin-B1-positive and -negative cells following X-inactivation has been observed in ephrin-B1(+/- mice; however, the mechanisms by which mosaic ephrin-B1 expression leads to cell sorting and phenotypic defects remain unknown. Here we show that ephrin-B1(+/- mice exhibit calvarial defects, a phenotype autonomous to neural crest cells that correlates with cell sorting. We have traced the causes of calvarial defects to impaired differentiation of osteogenic precursors. We show that gap junction communication (GJC is inhibited at ectopic ephrin boundaries and that ephrin-B1 interacts with connexin43 and regulates its distribution. Moreover, we provide genetic evidence that GJC is implicated in the calvarial defects observed in ephrin-B1(+/- embryos. Our results uncover a novel role for Eph/ephrins in regulating GJC in vivo and suggest that the pleiotropic defects seen in CFNS patients are due to improper regulation of GJC in affected tissues.

  5. Effects of reperfusion arrhythmias and myocardial connexin 43 by Compound Astragalus Mixture Nourishing Heart in a rat model of acute myocardial ischemic reperfusion injury%复方黄芪养心合剂对大鼠缺血再灌注心肌缝隙连接蛋白43的影响

    Institute of Scientific and Technical Information of China (English)

    陈启兰; 龚一萍; 祝光礼; 齐国安; 赫小龙; 任兴昌; 王骋

    2013-01-01

    目的:观察复方黄芪养心合剂对SD大鼠缺血再灌注心肌细胞缝隙连接蛋白43(Cx43)分布的改变和表达的影响.方法:应用复方黄芪养心合剂按每天14g/kg剂量、琥珀酸美托洛尔缓释片按每天(MSSRT) 9.5mg/kg剂量灌胃2周后,对SD大鼠行冠状动脉左前降支结扎30min后再灌注60min造成心肌缺血再灌注(I/R)损伤模型,记录Ⅱ导联心电图,采用免疫组织化学法(IHC)观察心肌细胞Cx43分布的改变;运用Image Pro Plus 6.0图像分析软件对Cx43的表达进行半定量分析.结果:Cx43平均光密度(AOD)I/R组心肌内膜下缺血区Cx43的AOD显著低于假手术组(P<0.01),且MSSRT组、复方组较1/R组升高(P<0.05).结论:对冠状动脉左前降支结扎术后再灌注SD大鼠,复方黄芪养心合剂对心肌细胞Cx43分布的改变有改善作用,有促进心肌内膜下缺血区域Cx43表达的作用,其作用与MSSRT相近.%Objective: To evaluate the effects of Compound Astragalus Mixture Nourishing Heart (CAMNH) antiarrhythmia and myocardial connexin 43 (Cx43) in a rat model of acute myocardial ischemic reperfusion injury. Methods: Myocardial infarction (MI) was induced by ligating left anterior descending coronary artery (LAD) for 30 minutes, followed by reperfusion of 60 minutes in rats. Rats were treated with CAMNH (14g·kg-1·d-1) or metoprolol succinate sustained-release tablets (MSSRT, 9.5mg·kg-1d-1) for 14 days before MI. The distribution of myocardial Cx43 was observed by Immunohistochemistry (IHC), and the expression of myocardial Cx43, which were represented by average optical density (AOD), was measured by Image Pro Plus 6.0. Results: The expression of myocardial Cx43 in infarction region was significantly reduced, disordered and even all disappearing, the expression of myocardial Cx43 from infarction region into ischemic region and normal region had a gradual recovery of transitional variability. Ischemia/reperfusion(I/R) group vs sham-operated (SO

  6. 温胆汤对地卓西平马来酸盐诱发精神分裂症模型大鼠脑组织中Cx43 mRNA表达的影响%Effect of Wendan Decoction on Connexin 43 mRNA Expression of in Rat Brain Tissue with Schizophrenia Induced by Dizocilpine Maleate

    Institute of Scientific and Technical Information of China (English)

    付艳丽; 万红娇; 朱金华; 马广强

    2012-01-01

    目的:探讨温胆汤对精神分裂症模型大鼠脑组织中神经胶质细胞连接蛋白(Cx43) mRNA的影响.方法:将健康的SD大鼠50只随机分为5组:正常组(A)、模型组(B)、温胆汤高、中、低剂量组(C,D,E组,剂量为生药40,20,10 g·kg-1).A,B组生理盐水ig,C,D,E组温胆汤ig,1次/d,21 d后,按照0.6 mg· kg -1一次性ip地卓西平马来酸盐(MK-801)建立拟精神分裂症模型,然后行为学观察3d,处死大鼠.应用逆转录聚合酶链式反应(RT-PCR)法,检测大鼠Cx43 mRNA的表达量.结果:模型组Cx43 mRNA的表达量低于正常组,有显著性意义(P<0.05);温胆汤3个剂量组Cx43 mRNA的表达量明显高于模型组,有显著性意义(P<0.05).结论:温胆汤能够增强精神分裂症模型大鼠脑组织中Cx43 mRNA的表达.%Objective; To study the effect of Wendan decoction on connexin 43 ( Cx43 ) mRNA expression in brain tissue of rats with schizophrenia. Method; Fifty SD rats were divided five groups by random, including normal group ( A ) , model group ( B ) , Wendan decoction high-dosage group ( C ) , medium-dosage group (D) and low-dosage group (E) , respectively. The A and B groups were given normal saline and the C, D, E groups were given Wendan decoction (40, 20, 10 g - kg-1 ) every day, which lasted for 21 days. Dizocilpine maleate ( MK-801 ) 0. 6 mg - kg was ip given to establishthe rat schizophrenic model. Then rat behaviors were observed for 3 days all rats were sacrificed. Then RT-PCR method was used to detect Cx43 mRNA expression in the nervecells of the every group rats. Result; The Cx43 mRNA expression of group B was lower than that of group A ( P < 0. 05) . And the Cx43 mRNA expression of group C , D and E was rose compared with that of group B ( P < 0. 05 ) . Conclusion; Wendan decoction can reinforce the expression of Cx43 mRNA in the brain tissue of rats with schizophrenia.

  7. When proteome meets genome: the alpha helix and the beta strand of proteins are eschewed by mRNA splice junctions and may define the minimal indivisible modules of protein architecture

    Indian Academy of Sciences (India)

    Sailen Barik

    2004-09-01

    The significance of the intron-exon structure of genes is a mystery. As eukaryotic proteins are made up of modular functional domains, each exon was suspected to encode some form of module; however, the definition of a module remained vague. Comparison of pre-mRNA splice junctions with the three-dimensional architecture of its protein product from different eukaryotes revealed that the junctions were far less likely to occur inside the -helices and -strands of proteins than within the more flexible linker regions (‘turns’ and ‘loops’) connecting them. The splice junctions were equally distributed in the different types of linkers and throughout the linker sequence, although a slight preference for the central region of the linker was observed. The avoidance of the -helix and the -strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units of a protein and represent the minimal modules in the evolution of protein structure. These results should find use in comparative genomics, designing of cloning strategies, and in the mutual verification of genome sequences with protein structures.

  8. A novel mutation of gap junction protein β 1 gene in X-linked Charcot-Marie-Tooth disease.

    Science.gov (United States)

    Chen, Sheng Dong; Li, Zheng Xi; Guan, Yang Tai; Zhou, Xia Jun; Jiang, Jian Ming; Hao, Yong

    2011-06-01

    In this study we report a novel mutation in the gap junction protein beta 1 (GJB1) gene of a Chinese X-linked Charcot-Marie-Tooth disease (CMTX1) family, which has specific electrophysiological characteristics. Twenty members in the family were studied by clinical neurological examination and GJB1 gene mutation analysis, and 3 patients were studied electrophysiologically. The proband and his mother also underwent sural nerve biopsy. All patients have the CMT phenotype, except for 2 asymptomatic carriers. Electrophysiological examinations showed non-uniform slowing of motor conduction velocities and partial motor conduction blocks and temporal dispersion. Sural nerve biopsy confirmed a predominantly demyelinating neuropathy, and an Asn2Lys mutation in the amino-terminal domain was found in 9 members of this family, but not in 25 normal controls in the family. This family represents a novel mutation in the GJB1 form of CMTX1. The mutation in the amino-terminus has an impact on the electrophysiological characteristics of the disease. Copyright © 2011 Wiley Periodicals, Inc.

  9. The role of gap junctions and mechanical loading on mineral formation in a collagen-I scaffold seeded with osteoprogenitor cells.

    Science.gov (United States)

    Damaraju, Swathi; Matyas, John R; Rancourt, Derrick E; Duncan, Neil A

    2015-05-01

    Fracture nonunions represent one of many large bone defects where current treatment strategies fall short in restoring both form and function of the injured tissue. In this case, the use of a tissue-engineered scaffold for promoting bone healing offers an accessible and easy-to-manipulate environment for studying bone formation processes in vitro. We have previously shown that mechanical prestimulation using confined compression of differentiating osteoblasts results in an increase in mineralization formed in a 3D collagen-I scaffold. This study builds on this knowledge by evaluating the short and long-term effects of blocking gap junction-mediated intercellular communication among osteogenic cells on their effectiveness to mineralize collagen-I scaffolds in vitro, and in the presence and absence of mechanical stimulation. In this study, confined compression was applied in conjunction with octanol (a general communication blocker) or 18-α-glycerrhetinic acid (AGA, a specific gap junction blocker) using a modified FlexCell plate to collagen-I scaffolds seeded with murine embryonic stem cells stimulated toward osteoblast differentiation using beta-glycerol phosphate. The activity, presence, and expression of osteoblast cadherin, connexin-43, as well as various pluripotent and osteogenic markers were examined at 5-30 days of differentiation. Fluorescence recovery after photobleaching, immunofluorescence, viability, histology assessments, and reverse-transcriptase polymerase chain reaction assessments revealed that inhibiting communication in this scaffold altered the lineage and function of differentiating osteoblasts. In particular, treatment with communication inhibitors caused reduced mineralization in the matrix, and dissociation between connexin-43 and integrin α5β1. This dissociation was not restored even after long-term recovery. Thus, in order for this scaffold to be considered as an alternative strategy for the repair of large bone defects, cell

  10. Cell culture model predicts human disease: Altered expression of junction proteins and matrix metalloproteinases in cervical dysplasia

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    Kivi Niina

    2012-08-01

    Full Text Available Abstract Background Cervical cancer is necessarily caused by human papillomaviruses, which encode three oncogenes manifesting their functions by interfering with a number of cellular proteins and pathways: the E5, E6, and E7 proteins. We have earlier found in our microarray studies that the E5 oncogene crucially affects the expression of cellular genes involved in adhesion and motility of epithelial cells. Methods In order to biologically validate our previous experimental findings we performed immunohistochemical staining of a representative set of tissue samples from different grades of high-risk human papillomavirus associated cervical disease as well as normal squamous and columnar cervical epithelium. Three-dimensional collagen raft cultures established from E5-expressing and control epithelial cells were also examined. The expression of p16, matrix metalloproteinase (MMP -7, MMP-16, cytokeratin (CK 8/18, laminin, E-cadherin and beta-catenin was studied. Results In agreement with our previous microarray studies, we found intense staining for E-cadherin and beta-catenin in adherens junctions even in high-grade cervical lesions. Staining for MMP-16 was increased in severe disease as well. No significant change in staining for MMP-7 and cytokeratin 8/18 along with the grade of cervical squamous epithelial disease was observed. Conclusions Here we have confirmed, using tissue material from human papillomavirus associated lesions, some of the cellular gene expression modifications that we earlier reported in an experimental system studying specifically the E5 oncogene of papillomaviruses. These findings were partially surprising in the context of cervical carcinogenesis and emphasize that the complexity of carcinogenesis is not yet fully understood. Microarray approaches provide a wide overwiev of gene expression in experimental settings, which may yield biologically valid biomarkers for disease diagnostics, prognosis, and follow-up.

  11. Volatile anesthetics influence blood-brain barrier integrity by modulation of tight junction protein expression in traumatic brain injury.

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    Serge C Thal

    Full Text Available Disruption of the blood-brain barrier (BBB results in cerebral edema formation, which is a major cause for high mortality after traumatic brain injury (TBI. As anesthetic care is mandatory in patients suffering from severe TBI it may be important to elucidate the effect of different anesthetics on cerebral edema formation. Tight junction proteins (TJ such as zonula occludens-1 (ZO-1 and claudin-5 (cl5 play a central role for BBB stability. First, the influence of the volatile anesthetics sevoflurane and isoflurane on in-vitro BBB integrity was investigated by quantification of the electrical resistance (TEER in murine brain endothelial monolayers and neurovascular co-cultures of the BBB. Secondly brain edema and TJ expression of ZO-1 and cl5 were measured in-vivo after exposure towards volatile anesthetics in native mice and after controlled cortical impact (CCI. In in-vitro endothelial monocultures, both anesthetics significantly reduced TEER within 24 hours after exposure. In BBB co-cultures mimicking the neurovascular unit (NVU volatile anesthetics had no impact on TEER. In healthy mice, anesthesia did not influence brain water content and TJ expression, while 24 hours after CCI brain water content increased significantly stronger with isoflurane compared to sevoflurane. In line with the brain edema data, ZO-1 expression was significantly higher in sevoflurane compared to isoflurane exposed CCI animals. Immunohistochemical analyses revealed disruption of ZO-1 at the cerebrovascular level, while cl5 was less affected in the pericontusional area. The study demonstrates that anesthetics influence brain edema formation after experimental TBI. This effect may be attributed to modulation of BBB permeability by differential TJ protein expression. Therefore, selection of anesthetics may influence the barrier function and introduce a strong bias in experimental research on pathophysiology of BBB dysfunction. Future research is required to investigate

  12. Structure of Yeast OSBP-Related Protein Osh1 Reveals Key Determinants for Lipid Transport and Protein Targeting at the Nucleus-Vacuole Junction.

    Science.gov (United States)

    Manik, Mohammad Kawsar; Yang, Huiseon; Tong, Junsen; Im, Young Jun

    2017-03-10

    Yeast Osh1 belongs to the oxysterol-binding protein (OSBP) family of proteins and contains multiple targeting modules optimized for lipid transport at the nucleus-vacuole junction (NVJ). The key determinants for NVJ targeting and the role of Osh1 at NVJs have remained elusive because of unknown lipid specificities. In this study, we determined the structures of the ankyrin repeat domain (ANK), and OSBP-related domain (ORD) of Osh1, in complex with Nvj1 and ergosterol, respectively. The Osh1 ANK forms a unique bi-lobed structure that recognizes a cytosolic helical segment of Nvj1. We discovered that Osh1 ORD binds ergosterol and phosphatidylinositol 4-phosphate PI(4)P in a competitive manner, suggesting counter-transport function of the two lipids. Ergosterol is bound to the hydrophobic pocket in a head-down orientation, and the structure of the PI(4)P-binding site in Osh1 is well conserved. Our results suggest that Osh1 performs non-vesicular transport of ergosterol and PI(4)P at the NVJ.

  13. Gap Junctions

    Science.gov (United States)

    Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L.; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

    2013-01-01

    Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1981-2035, 2012. PMID:23723031

  14. Gap junctions.

    Science.gov (United States)

    Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

    2012-07-01

    Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1853-1872, 2012.

  15. Interfering amino terminal peptides and functional implications for heteromeric gap junction formation

    Directory of Open Access Journals (Sweden)

    Richard David Veenstra

    2013-05-01

    Full Text Available Connexin43 (Cx43 is widely expressed in many different tissues of the human body. In cells of some organs, Cx43 is co-expressed with other connexins (Cx, including Cx46 and Cx50 in lens, Cx40 in atrium, Purkinje fibers, and the blood vessel wall, Cx45 in heart, and Cx37 in the ovary. Interactions with the co-expressed connexins may have profound functional implications. The abilities of Cx37, Cx45, Cx46, and Cx50 to function in heteromeric gap junction combinations with Cx43 are well documented. Different studies disagree regarding the ability of Cx43 and Cx40 to produce functional heteromeric gap junctions with each other. We review previous studies regarding the heteromeric interactions of Cx43. The possibility of negative functional interactions between the cytoplasmic pore-forming amino terminal (NT domains of these connexins was assessed using pentameric connexin sequence-specific NT domain (iNT peptides applied to cells expressing homomeric Cx40, Cx37, Cx45, Cx46, and Cx50 gap junctions. A Cx43 iNT peptide corresponding to amino acids 9 to 13 (Ac-KLLDK-NH2 specifically inhibited the electrical coupling of Cx40 gap junctions in a transjunctional (Vj voltage-dependent manner without affecting the function of homologous Cx37, Cx46, Cx50, and Cx45 gap junctions. A Cx40 iNT (Ac-EFLEE-OH peptide counteracted the Vj-dependent block of Cx40 gap junctions, whereas a similarly charged Cx50 iNT (Ac-EEVNE-OH peptide did not, suggesting that these NT domain interactions are not solely based on electrostatics. These data are consistent with functional Cx43 heteromeric gap junction formation with Cx37, Cx45, Cx46, and Cx50 and suggest that Cx40 uniquely experiences functional suppressive interactions with a Cx43 NT domain sequence. These findings present unique functional implications about the heteromeric interactions between Cx43 and Cx40 that may influence cardiac conduction in atrial myocardium and the specialized conduction system.

  16. Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis).

    Science.gov (United States)

    Yamamoto, Yoji; Yoshizaki, Goro; Takeuchi, Toshio; Soyano, Kiyoshi; Patiño, Reynaldo

    2008-02-01

    Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18alpha-glycyrrhetinic acid (alpha-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20beta-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption.

  17. Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva.

    Science.gov (United States)

    Nakayama, Yohei; Kobayashi, Ryoki; Matsui, Sari; Matsumura, Hiroyoshi; Iwai, Yasunobu; Noda, Keisuke; Yamazaki, Mizuho; Kurita-Ochiai, Tomoko; Yoshimura, Atsutoshi; Shinomura, Tamayuki; Ganss, Bernhard; Ogata, Yorimasa

    2017-07-01

    The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.

  18. Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA.

    Science.gov (United States)

    Hedglin, Mark; Aitha, Mahesh; Benkovic, Stephen J

    2017-07-11

    In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient Förster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.

  19. Effect of inhibiting connexin 43 expression on the expression of rat glial cell growth factors in prolactinomas%抑制缝隙连接蛋白43表达对大鼠催乳素腺瘤中胶质细胞生长因子表达的影响

    Institute of Scientific and Technical Information of China (English)

    张亚菊; 王海涛; 张冉; 靳峰

    2016-01-01

    Objective To investigate the effect of inhibiting connexin 43 (Cx43) expression on the expression of rat glial cell growth factor in prolactinomas.Methods Forty-eight female rats were randomly assigned to normal, simple estradiol, estradiol + carbenoxolone, and simple carbenoxolone groups by the completely randomized method (n =12 in each group).A model of rat prolactinoma was induced by estradiol.The normal group did not accept drug treatment;the simple estradiol group was injected estradiol intramuscularly (3 000 mg/kg, once a week, for 13 weeks);10 weeks after injection of estradiol, the estradiol + carbenoxolone group injected carbenoxolone via subarachnoid (once a week, for 3 weeks);and the simple carbenoxolone group only injected carbenoxolone for 3 weeks.The changes of body mass of pituitary and histomorphology were observed.Western blot was used to detect the expression levels of Cx43 and glial growth factor (GGF).Results The pituitary weights of the normal, simple estradiol, estradiol + carbenoxolone and simple carbenoxolone groups were 51.0 ±0.4 mg, 93.3 ± 1.0 mg, 52.7 ±2.1 mg and 68.5 ± 1.3 mg, respectively.There were significant differences (F =786.73, P < 0.01);the relative expression quantities of Cx43 were 8.0 ± 2.2% , 34.7 ± 4.0,.9.3 ± 3.1% , and 15.3 ± 3.8% ,respectively, and the relative expression quantities of GGF were 17.7 ± 5.2%, 46.9 ± 1.4%, 15.8 ± 3.6%, and 20.0 ±2.3%, respectively.There were significant differences (F =1051.51.P <0.01;F =806.58, P < 0.01).The pituitary histopathological morphology of the estradiol + carbenoxolone group was close to the normal group and the simple carbenoxolone group.Conclusions Inhibition of pituitary Cx43 expression may inhibit the GGF expression, thereby inhibiting the growth of pituitary prolactinomas in rats.%目的 探讨在催乳素腺瘤中,抑制缝隙连接蛋白43(Cx43)的表达对大鼠胶质细胞生长因子表达的影响.方法 采用

  20. 缝隙连接蛋白43在偏头痛大鼠三叉神经颈髓复合体的表达及其对痛觉超敏的影响%Connexin43 expression in trigeminocervical complex and its effect on allodynia in rats with migraine

    Institute of Scientific and Technical Information of China (English)

    武茜; 黄琳; 郭永涛; 荣蓉; 史兆春; 燕兰云; 万琪

    2013-01-01

    Objective To investigate the efficacy of connexin43 (Cx43) in trigeminocervical complex on allodynia in rat models of migraine.Methods SD rats were randomly divided into 9 groups (n=6):blank group,sham-operated group,saline group,improved inflammatory soup for 3 d group,improved inflammatory soup for 7 d group,carbenoxolone (CBX) prevention group,CBX prevention control group,CBX treatment group and CBX treatment control group.Improved inflammatory soup was used to stimulate the dual matter adjacent to the superior sagittal sinus of rats repeatedly to induce migraine models in the later 7 groups.The pain threshold of periorbital skin was measured by Von-Frey hairs.On the 11th postoperative day,the spinal cord was removed for detection of Cx43 expression in the trigeminocervical complex by immunofluorescence.Results Von-Frey hairs study showed that:the pain threshold of improved inflammatory soup groups had a daily drop; rats in the improved inflammatory soup for 3 d group suffered allodynia,while those in the CBX prevention group did not; the pain threshold in the CBX treatment group ascended after using CBX.Immunofluorescence of nucleus and Cx43 showed that:the mean optical density (OD) of improved inflammatory soup for 3 d group and for 7 d group significantly incresed (P<0.05); nucleus and Cx43 abundantly expressed in lamina Ⅰ and Ⅱ of dorsal horn in the improved inflammatory soup for 3 d group and lamina Ⅰ-Ⅳ of dorsal horn in improved inflammatory soup for 7 d group; significant difference was noted on the mean OD values between of CBX prevention/treatment groups and their control groups (P<0.05).Conclusion Repeatedly infusing improved inflammatory soup to dual matter can effectively induce allodynia in rat models of migraine and Cx43 abundantly expresses in trigeminocervical complex after being given the soup; allodynia in migraine model of rats can be prevented and relieved by intraperitoneal administrating of CBX,which suggests that Cx43 may play

  1. Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

    Science.gov (United States)

    Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

    2014-10-01

    To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.

  2. Influence of drugs on gap junctions in glioma cell lines and primary astrocytes in vitro

    Directory of Open Access Journals (Sweden)

    Zahra eMoinfar

    2014-05-01

    Full Text Available Gap junctions (GJs are hemichannels on cell membrane. Once they are intercellulary connected to the neighboring cells, they build a functional syncytium which allows rapid transfer of ions and molecules between cells. This characteristic makes GJs a potential modulator in proliferation, migration and development of the cells. So far, several types of GJs are recognized on different brain cells as well as in glioma. Astrocytes, as one of the major cells that maintain neuronal homeostasis, express different types of GJs that let them communicate with neurons, oligodendrocytes and endothelial cells of the blood brain barrier; however, the main GJ in astrocytes is connexin 43. There are different cerebral diseases in which astrocyte GJs might play a role. Several drugs have been reported to modulate gap junctional communication in the brain which can consequently have beneficial or detrimental effects on the course of treatment in certain diseases. However, the exact cellular mechanism behind those pharmaceutical efficacies on GJs is not well-understood. Accordingly, how specific drugs would affect GJs and what some consequent specific brain diseases would be are the interests of the authors of this chapter. We would focus on pharmaceutical effects on GJs on astrocytes in specific diseases where GJs could possibly play a role including: 1 migraine and a novel therapy for migraine with aura, 2 neuroautoimmune diseases and immunomodulatory drugs in the treatment of demyelinating diseases of the central nervous system such as multiple sclerosis, 3 glioma and antineoplastic and anti-inflammatory agents that are used in treating brain tumors and 4 epilepsy and anticonvulsants that are widely used for seizures therapy. All of the above-mentioned therapeutic categories can possibly affect GJs expression of astrocytes and the role is discussed in the upcoming chapter.

  3. Poly-L-arginine-Induced internalization of tight junction proteins increases the paracellular permeability of the Caco-2 cell monolayer to hydrophilic macromolecules.

    Science.gov (United States)

    Yamaki, Tsutomu; Ohtake, Kazuo; Ichikawa, Keiko; Uchida, Masaki; Uchida, Hiroyuki; Oshima, Shinji; Ohshima, Shinji; Juni, Kazuhiko; Kobayashi, Jun; Morimoto, Yasunori; Natsume, Hideshi

    2013-01-01

    We investigated whether poly-L-arginine (PLA) enhances the paracellular permeability of the Caco-2 monolayer to hydrophilic macromolecules and clarified the disposition of tight junction (TJ) proteins. The transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran (FD-4) permeation were determined after treatment with PLA. TJ proteins were visualized using immunofluorescence microscopy after PLA exposure and depletion, and their expression levels were determined. The barrier function of TJs was also evaluated by measuring the alterations in the TEER and in the localization of TJ proteins. PLA induced an increase in hydrophilic macromolecule, FD-4, permeation through Caco-2 cell monolayers and a decrease in the TEER in a concentration-dependent manner, without any significant impact on the cell viability. This increased paracellular permeability induced by PLA was found to be internalized of claudin-4, ZO-1, tricellulin and mainly occludin from cell-cell junction to the subcellular space. ZO-1 appeared to play an important role in the reconstitution of TJ strand structures following PLA depletion. These results indicate that the PLA led to the internalization of TJ proteins to the subcellular space, subsequently increasing the permeability of the Caco-2 cell monolayer to FD-4 via a paracellular route.

  4. Pharmacological modulation of gap junction function with the novel compound rotigaptide: a promising new principle for prevention of arrhythmias.

    Science.gov (United States)

    Kjølbye, Anne Louise; Haugan, Ketil; Hennan, James K; Petersen, Jørgen S

    2007-10-01

    Existing anti-arrhythmic therapy is hampered by lack of efficacy and unacceptable side effects. Thus, ventricular tachycardia and fibrillation remains the strongest predictor of in-hospital mortality in patients with myocardial infarction. In atrial fibrillation, rhythm control with conventional ion channel blockers provide no therapeutic benefit relative to rate control. Several lines of research indicate that impaired gap junctional cell-to-cell coupling between neighbouring cardiomyocytes is critical for the development of cardiac re-entry arrhythmias. Rotigaptide is the first drug that has been developed to prevent arrhythmias by re-establishing gap junctional intercellular communication. During conditions with acute cardiac ischaemia, rotigaptide effectively prevents induction of both ventricular and atrial tachyarrhythmia. Moreover, rotigaptide effectively prevents ischaemia reperfusion arrhythmias. At the cellular level, rotigaptide inhibits ischaemia-induced dephosphorylation of Ser297 and Ser368, which is considered important for the gating of connexin43 gap junction channels. No drug-related toxicity has been demonstrated at plasma concentrations 77,000 times above therapeutic concentrations. In rats and dogs, rotigaptide reduces infarct size following myocardial infarction. A series of phase I trials has been completed in which rotigaptide has been administered intravenously to ~200 healthy persons. No drug-related side effects have been demonstrated in healthy human beings. Clinical safety, tolerability and efficacy in patients with heart disease are being evaluated in ongoing clinical trials. Rotigaptide represents a pioneering pharmacological principle with a highly favourable preclinical and clinical safety profile, which makes this molecule a promising drug candidate for the prevention of cardiac arrhythmias.

  5. Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet

    Science.gov (United States)

    Wilkens, Mirja R.; Breves, Gerhard; Langeheine, Marion; Brehm, Ralph; Muscher-Banse, Alexandra S.

    2016-01-01

    Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability. PMID:27120348

  6. Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet.

    Directory of Open Access Journals (Sweden)

    Kristin Elfers

    Full Text Available Diets fed to ruminants should contain nitrogen (N as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ and adherens junction (AJ proteins in intestinal epithelia were examined on mRNA level by qPCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability.

  7. PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Haiying; Cui, Yazhou; Luan, Jing [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China); Zhang, Xiumei [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Li, Chengzhi; Zhou, Xiaoyan; Shi, Liang [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China); Wang, Huaxin [Shandong University of Traditional Chinese Medicine, Ji' an, Shandong (China); Han, Jinxiang, E-mail: jxhan9888@aliyun.com [School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Ji' nan, Shandong (China); Key Laboratory for Rare Disease Research of Shandong Province, Key Laboratory for Biotech Drugs of the Ministry of Health, Shandong Medical Biotechnological Center, Shandong Academy of Medical Sciences, Ji' nan, Shandong (China)

    2016-02-12

    Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit the level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.

  8. Gap junction proteins in the blood-brain barrier control nutrient-dependent reactivation of Drosophila neural stem cells.

    Science.gov (United States)

    Spéder, Pauline; Brand, Andrea H

    2014-08-11

    Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. Here we show that gap junctions in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. We propose that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion.

  9. Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain.

    Science.gov (United States)

    Mullier, Amandine; Bouret, Sebastien G; Prevot, Vincent; Dehouck, Bénédicte

    2010-04-01

    The median eminence is one of the seven so-called circumventricular organs. It is located in the basal hypothalamus, ventral to the third ventricle and adjacent to the arcuate nucleus. This structure characteristically contains a rich capillary plexus and features a fenestrated endothelium, making it a direct target of blood-borne molecules. The median eminence also contains highly specialized ependymal cells called tanycytes, which line the floor of the third ventricle. It has been hypothesized that one of the functions of these cells is to create a barrier that prevents substances in the portal capillary spaces from entering the brain. In this paper, we utilize immunohistochemistry to study the expression of tight junction proteins in the cells that compose the median eminence in adult mice. Our results indicate that tanycytes of the median eminence express occludin, ZO-1, and claudin 1 and 5, but not claudin 3. Remarkably, these molecules are organized as a continuous belt around the cell bodies of the tanycytes that line the ventral part of the third ventricle. In contrast, the tanycytes at the periphery of the arcuate nucleus do not express claudin 1 and instead exhibit a disorganized expression pattern of occludin, ZO-1, and claudin 5. Consistent with these observations, permeability studies using peripheral or central injections of Evans blue dye show that only the tanycytes of the median eminence are joined at their apices by functional tight junctions, whereas tanycytes located at the level of the arcuate nucleus form a permeable layer. In conclusion, this study reveals a unique expression pattern of tight junction proteins in hypothalamic tanycytes, which yields new insights into their barrier properties.

  10. The stardust family protein MPP7 forms a tripartite complex with LIN7 and DLG1 that regulates the stability and localization of DLG1 to cell junctions.

    Science.gov (United States)

    Bohl, Joanna; Brimer, Nicole; Lyons, Charles; Vande Pol, Scott B

    2007-03-30

    MPP7, a previously uncharacterized member of the p55 Stardust family of membrane-associated guanylate kinase (MAGUK) proteins, was found in a tripartite complex with DLG1 and LIN7A or LIN7C. MPP7 dimerizes with all three LIN7 family members (LIN7A, -B, and -C) through interaction of the single L27 domain of LIN7 with the carboxyl-terminal L27 domain of MPP7, thereby stabilizing both proteins. The dimer of MPP7 with LIN7A or LIN7C associates with DLG1 through an interaction requiring the amino-terminal L27 domain of MPP7. The amino-terminal L27 domain of MPP7 is not sufficient for interaction with DLG1 but interacts efficiently only if MPP7 is in a complex with LIN7A or -C. Thus the specificity of interaction of DLG1 with the LIN7-MPP7 complex is determined by L27 interactions with both MPP7 and LIN7. The tripartite complex forms in a ratio of 1:1:1 and localizes to epithelial adherens junctions in a manner dependent upon MPP7. Expression of MPP7 stabilizes DLG1 in an insoluble compartment. Expression of MPP7 deleted of the PDZ or Src homology 3 domain redistributes MPP7, DLG1, and LIN7 out of adherens junctions and into the soluble cytoplasmic fraction without changing the localization of E-cadherin. Thus, the stability and localization of DLG1 to cell-cell junctions are complex functions determined by the expression and association of particular Stardust family members together with particular LIN7 family members.

  11. Selective permeability of gap junction channels.

    Science.gov (United States)

    Goldberg, Gary S; Valiunas, Virginijus; Brink, Peter R

    2004-03-23

    Gap junctions mediate the transfer of small cytoplasmic molecules between adjacent cells. A family of gap junction proteins exist that form channels with unique properties, and differ in their ability to mediate the transfer of specific molecules. Mutations in a number of individual gap junction proteins, called connexins, cause specific human diseases. Therefore, it is important to understand how gap junctions selectively move molecules between cells. Rules that dictate the ability of a molecule to travel through gap junction channels are complex. In addition to molecular weight and size, the ability of a solute to transverse these channels depends on its net charge, shape, and interactions with specific connexins that constitute gap junctions in particular cells. This review presents some data and interpretations pertaining to mechanisms that govern the differential transfer of signals through gap junction channels.

  12. Transforming growth factor-β3 regulates cell junction restructuring via MAPK-mediated mRNA destabilization and Smad-dependent protein degradation of junctional adhesion molecule B (JAM-B).

    Science.gov (United States)

    Zhang, Xu; Lui, Wing-Yee

    2015-06-01

    Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the blood-testis barrier (BTB) as well as between Sertoli and germ cells at the apical ectoplasmic specializations (ES) in the testis. The expression of JAM-B is tightly regulated to modulate the passage of spermatocytes across the BTB as well as the release of mature spermatozoa from the seminiferous epithelium. Transforming growth factor beta (TGF-β) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the effects of TGF-β3 on the expression of JAM-B as well as the underlying mechanisms on how TGF-β3 regulates JAM-B expression to facilitate the disassembly of the BTB and apical ES. Our results revealed that TGF-β3 suppresses JAM-B at post-transcriptional and post-translational levels. Inhibitor, siRNA knockdown and co-immunoprecipitation have shown that TGF-β3 induces JAM-B protein degradation via ubiquitin-proteasome pathway. Immunofluorescence staining further confirmed that blockage of ubiquitin-proteasome pathway could abrogate TGF-β3-induced loss of JAM-B at the cell-cell interface. siRNA knockdown and immunofluorescence staining also demonstrated that activation of Smad signaling is required for TGF-β3-induced JAM-B protein degradation. In addition, TGF-β3 reduces JAM-B mRNA levels, at least in part, via post-transcriptional regulation. mRNA stability assay has confirmed that TGF-β3 promotes the degradation of JAM-B transcript and TGF-β3-mediated mRNA destabilization requires the activation of ERK1/2 and p54 JNK signal cascades. Taken together, TGF-β3 significantly downregulates JAM-B expression via post-transcriptional and post-translational modulation and results in the disruption of BTB and apical ES.

  13. Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein. GP32 Single-Stranded DNA-Binding Protein and Ternary Complex with Pseudo-Y Junction DNA

    Energy Technology Data Exchange (ETDEWEB)

    Hinerman, Jennifer M. [Univ. of Toledo, OH (United States); Dignam, J. David [Univ. of Toledo, OH (United States); Mueser, Timothy C. [Univ. of Toledo, OH (United States)

    2012-04-05

    The bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Moreover, fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596–18607).

  14. Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall.

    Science.gov (United States)

    Blank, Fabian; Wehrli, Marc; Lehmann, Andrea; Baum, Oliver; Gehr, Peter; von Garnier, Christophe; Rothen-Rutishauser, Barbara M

    2011-01-01

    The human airway epithelium serves as structural and functional barrier against inhaled particulate antigen. Previously, we demonstrated in an in vitro epithelial barrier model that monocyte derived dendritic cells (MDDC) and monocyte derived macrophages (MDM) take up particulate antigen by building a trans-epithelial interacting network. Although the epithelial tight junction (TJ) belt was penetrated by processes of MDDC and MDM, the integrity of the epithelium was not affected. These results brought up two main questions: (1) Do MDM and MDDC exchange particles? (2) Are those cells expressing TJ proteins, which are believed to interact with the TJ belt of the epithelium to preserve the epithelial integrity? The expression of TJ and adherens junction (AJ) mRNA and proteins in MDM and MDDC monocultures was determined by RT-PCR, and immunofluorescence, respectively. Particle uptake and exchange was quantified by flow cytometry and laser scanning microscopy in co-cultures of MDM and MDDC exposed to polystyrene particles (1 μm in diameter). MDM and MDDC constantly expressed TJ and AJ mRNA and proteins. Flow cytometry analysis of MDM and MDDC co-cultures showed increased particle uptake in MDDC while MDM lost particles over time. Quantitative analysis revealed significantly higher particle uptake by MDDC in co-cultures of epithelial cells with MDM and MDDC present, compared to co-cultures containing only epithelial cells and MDDC. We conclude from these findings that MDM and MDDC express TJ and AJ proteins which could help to preserve the epithelial integrity during particle uptake and exchange across the lung epithelium.

  15. Engineered holliday junctions as single-molecule reporters for protein-DNA interactions with application to a MerR-family regulator.

    Science.gov (United States)

    Sarkar, Susanta K; Andoy, Nesha May; Benítez, Jaime J; Chen, Peng R; Kong, Jason S; He, Chuan; Chen, Peng

    2007-10-17

    Protein-DNA interactions are essential for gene maintenance, replication, and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here, we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs. Because the effects of protein binding are converted to the changes in the structure and dynamics of HJs, protein-DNA interactions that involve small structural changes of DNA can be studied. We apply this method to investigate how the MerR-family regulator PbrR691 interacts with DNA for transcriptional regulation. Both apo- and holo-PbrR691 bind the stacked conformers of the engineered HJ, change their structures, constrain their conformational distributions, alter the kinetics, and shift the equilibrium of their structural dynamics. The information obtained maps the potential energy surfaces of HJ before and after PbrR691 binding and reveals the protein actions that force DNA structural changes for transcriptional regulation. The ability of PbrR691 to bind both HJ conformers and still allow HJ structural dynamics also informs about its conformational flexibility that may have significance for its regulatory function. This method of using engineered HJs offers quantification of the changes both in structure and in dynamics of DNA upon protein binding and thus provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.

  16. Antiproliferative Action of Conjugated Linoleic Acid on Human MCF-7 Breast Cancer Cells Mediated by Enhancement of Gap Junctional Intercellular Communication through Inactivation of NF-κB

    Directory of Open Access Journals (Sweden)

    Md. Abdur Rakib

    2013-01-01

    Full Text Available The major conjugated linoleic acid (CLA isomers, c9,t11-CLA and t10,c12-CLA, have anticancer effects; however, the exact mechanisms underlying these effects are unknown. Evidence suggests that reversal of reduced gap junctional intercellular communication (GJIC in cancer cells inhibits cell growth and induces cell death. Hence, we determined that CLA isomers enhance GJIC in human MCF-7 breast cancer cells and investigated the underlying molecular mechanisms. The CLA isomers significantly enhanced GJIC of MCF-7 cells at 40 μM concentration, whereas CLA inhibited cell growth and induced caspase-dependent apoptosis. CLA increased connexin43 (Cx43 expression both at the transcriptional and translational levels. CLA inhibited nuclear factor-κB (NF-κB activity and enhanced reactive oxygen species (ROS generation. No significant difference was observed in the efficacy of c9,t11-CLA and t10,c12-CLA. These results suggest that the anticancer effect of CLA is associated with upregulation of GJIC mediated by enhanced Cx43 expression through inactivation of NF-κB and generation of ROS in MCF-7 cells.

  17. Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo

    Science.gov (United States)

    Véliz, Loreto P.; González, Francisco G.; Duling, Brian R.; Sáez, Juan C.; Boric, Mauricio P.

    2008-01-01

    To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e−/−). In the cheek pouch, topical tumor necrosis factor-α (TNF-α; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-α-glycyrrhetinic acid (AGA; 75 μM) or 18-β-glycyrrhetinic acid (50 μM) abolished the TNF-α-induced increase in LAV and LPV; carbenoxolone (75 μM) or oleamide (100 μM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 μM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-α stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-α reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-α (0.5 μg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-α stimulation did not increase LAV or LPV in Cx43e−/− mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes. PMID:18599597

  18. Applying 3D-FRAP microscopy to analyse gap junction-dependent shuttling of small antisense RNAs between cardiomyocytes.

    Science.gov (United States)

    Lemcke, Heiko; Peukert, Janine; Voronina, Natalia; Skorska, Anna; Steinhoff, Gustav; David, Robert

    2016-09-01

    Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Chronic administration of dietary grape seed extract increases colonic expression of gut tight junction protein occludin and reduces fecal calprotectin: a secondary analysis of healthy Wistar Furth rats.

    Science.gov (United States)

    Goodrich, Katheryn M; Fundaro, Gabrielle; Griffin, Laura E; Grant, Ar'quette; Hulver, Matthew W; Ponder, Monica A; Neilson, Andrew P

    2012-10-01

    Animal studies have demonstrated the potential of grape seed extract (GSE) to prevent metabolic syndrome, obesity, and type 2 diabetes. Recently, metabolic endotoxemia induced by bacterial endotoxins produced in the colon has emerged as a possible factor in the etiology of metabolic syndrome. Improving colonic barrier function may control endotoxemia by reducing endotoxin uptake. However, the impact of GSE on colonic barrier integrity and endotoxin uptake has not been evaluated. We performed a secondary analysis of samples collected from a chronic GSE feeding study with pharmacokinetic end points to examine potential modulation of biomarkers of colonic integrity and endotoxin uptake. We hypothesized that a secondary analysis would indicate that chronic GSE administration increases colonic expression of intestinal tight junction proteins and reduces circulating endotoxin levels, even in the absence of an obesity-promoting stimulus. Wistar Furth rats were administered drinking water containing 0.1% GSE for 21 days. Grape seed extract significantly increased the expression of gut junction protein occludin in the proximal colon and reduced fecal levels of the neutrophil protein calprotectin, compared with control. Grape seed extract did not significantly reduce serum or fecal endotoxin levels compared with control, although the variability in serum levels was widely increased by GSE. These data suggest that the improvement of gut barrier integrity and potential modulation of endotoxemia warrant investigation as a possible mechanism by which GSE prevents metabolic syndrome and associated diseases. Further investigation of this mechanism in high-fat feeding metabolic syndrome and obesity models is therefore justified. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Exon Junction Complexes Show a Distributional Bias toward Alternatively Spliced mRNAs and against mRNAs Coding for Ribosomal Proteins

    Directory of Open Access Journals (Sweden)

    Christian Hauer

    2016-08-01

    Full Text Available The exon junction complex (EJC connects spliced mRNAs to posttranscriptional processes including RNA localization, transport, and regulated degradation. Here, we provide a comprehensive analysis of bona fide EJC binding sites across the transcriptome including all four RNA binding EJC components eIF4A3, BTZ, UPF3B, and RNPS1. Integration of these data sets permits definition of high-confidence EJC deposition sites as well as assessment of whether EJC heterogeneity drives alternative nonsense-mediated mRNA decay pathways. Notably, BTZ (MLN51 or CASC3 emerges as the EJC subunit that is almost exclusively bound to sites 20–24 nucleotides upstream of exon-exon junctions, hence defining EJC positions. By contrast, eIF4A3, UPF3B, and RNPS1 display additional RNA binding sites suggesting accompanying non-EJC functions. Finally, our data show that EJCs are largely distributed across spliced RNAs in an orthodox fashion, with two notable exceptions: an EJC deposition bias in favor of alternatively spliced transcripts and against the mRNAs that encode ribosomal proteins.

  1. Relationship of gelatinases-tight junction proteins and blood-brain barrier permeability in the early stage of cerebral ischemia and reperfusion

    Institute of Scientific and Technical Information of China (English)

    Haolin Xin; Wenzhao Liang; Jing Mang; Lina Lin; Na Guo; Feng Zhang; Zhongxin Xu

    2012-01-01

    Gelatinases matrix metalloproteinase-2 and matrix metalloproteinase-9 have been shown to mediate claudin-5 and occludin degradation, and play an important regulatory role in blood-brain barrier permeability. This study established a rat model of 1.5-hour middle cerebral artery occlusion with reperfusion. Protein expression levels of claudin-5 and occludin gradually decreased in the early stage of reperfusion, which corresponded to the increase of the gelatinolytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. In addition, rats that received treatment with matrix metalloproteinase inhibitor N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpenthanoyl]-L- tryptophan methylamide (GM6001) showed a significant reduction in Evans blue leakage and an inhibition of claudin-5 and occludin protein degradation in striatal tissue. These data indicate that matrix metalloproteinase-2 and matrix metalloproteinase-9-mediated claudin-5 and occludin degradation is an important reason for blood-brain barrier leakage in the early stage of reperfusion. The leakage of the blood-brain barrier was present due to gelatinases-mediated degradation of claudin-5 and occludin proteins. We hypothesized that the timely closure of the structural component of the blood-brain barrier (tight junction proteins) is of importance.

  2. Localization of the tight junction protein gene TJP1 to human chromosome 15q13, distal to the Prader-Willi/Angelman region, and to mouse chromosome 7

    Energy Technology Data Exchange (ETDEWEB)

    Mohandas, T.K. [Darthmouth-Hitchcock Medical Center, Lebanon, NH (United States); Chen, X.N.; Korenberg, J.R. [UCLA School of Medicine, Los Angeles, CA (United States)] [and others

    1995-12-10

    The gene encoding the tight junction (zonula occludens) protein, TJP1, was mapped to human chromosome 15q13 by fluorescence in situ hybridization (FISH) using a cDNA probe. The Jackson Laboratory backcross DNA panel derived from the cross (C57BL/6JEi X SPRET/Ei) F1 females X SPRET/Ei males was used to map the mouse Tjp1 to chromosome 7 near position 30 on the Chromosome Committee Map, a region with conserved homology to human chromosome 15q13. FISH studies on metaphases from patients with the Prader-Willi (PWS) or the Angelman syndrome (AS) showed that TJP1 maps close but distal to the PWS/AS chromosome region. 13 refs., 2 figs.

  3. The gene for human gap junction protein connexin37 (GJA4) maps to chromosome 1p35.1, in the vicinity of D1S195

    Energy Technology Data Exchange (ETDEWEB)

    Van Camp, G.; Coucke, P.; Willems, P.J. [Univ. of Antwerp (Belgium)] [and others

    1995-11-20

    Gap junctions are plasma membrane structures containing channels that allow the exchange of small molecules between cells. Each hemichannel is an oligomer of six subunit proteins called connexins. The formation of intercellular channels is possible through interaction with connexins in the plasma membrane of adjacent cells. Gapjunction channels allow the passage of different molecules up to 1 kDa, such as ions, many second messengers, and small metabolites. Connexins are numbered according to their molecular mass in kilodaltons, calculated from the gene sequences. They are found in the vast majority of cell types and facilitate intercellular communication between cells. Connexins are encoded by a family of homologous genes with highly conserved extracellular and transmembrane domains, whereas the cytoplasmic regions are specific for each subtype. All connexin genes described up to now contain no introns in the coding region. 17 refs., 1 fig.

  4. Deficiency of angulin-2/ILDR1, a tricellular tight junction-associated membrane protein, causes deafness with cochlear hair cell degeneration in mice.

    Science.gov (United States)

    Higashi, Tomohito; Katsuno, Tatsuya; Kitajiri, Shin-Ichiro; Furuse, Mikio

    2015-01-01

    Tricellular tight junctions seal the extracellular spaces of tricellular contacts, where the vertices of three epithelial cells meet, and are required for the establishment of a strong barrier function of the epithelial cellular sheet. Angulins and tricellulin are known as specific protein components of tricellular tight junctions, where angulins recruit tricellulin. Mutations in the genes encoding angulin-2/ILDR1 and tricellulin have been reported to cause human hereditary deafness DFNB42 and DFNB49, respectively. To investigate the pathogenesis of DFNB42, we analyzed mice with a targeted disruption of Ildr1, which encodes angulin-2/ILDR1. Ildr1 null mice exhibited profound deafness. Hair cells in the cochlea of Ildr1 null mice develop normally, but begin to degenerate by two weeks after birth. Tricellulin localization at tricellular contacts of the organ of Corti in the cochlea was retained in Ildr1 null mice, but its distribution along the depth of tricellular contacts was affected. Interestingly, compensatory tricellular contact localization of angulin-1/LSR was observed in the organ of Corti in Ildr1 null mice although it was hardly detected in the organ of Corti in wild-type mice. The onset of hair cell degeneration in Ildr1 null mice was earlier than that in the reported Tric mutant mice, which mimic one of the tricellulin mutations in DFNB49 deafness. These results indicate that the angulin-2/ILDR1 deficiency causes the postnatal degenerative loss of hair cells in the cochlea, leading to human deafness DFNB42. Our data also suggest that angulin family proteins have distinct functions in addition to their common roles of tricellulin recruitment and that the function of angulin-2/ILDR1 for hearing cannot be substituted by angulin-1/LSR.

  5. Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells.

    Science.gov (United States)

    Kojima, Takashi; Takasawa, Akira; Kyuno, Daisuke; Ito, Tatsuya; Yamaguchi, Hiroshi; Hirata, Koichi; Tsujiwaki, Mitsuhiro; Murata, Masaki; Tanaka, Satoshi; Sawada, Norimasa

    2011-10-01

    The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-β treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Arecoline induced disruption of expression and localization of the tight junctional protein ZO-1 is dependent on the HER 2 expression in human endometrial Ishikawa cells.

    Science.gov (United States)

    Giri, Sarbani; Poindexter, Kevin M; Sundar, Shyam N; Firestone, Gary L

    2010-07-06

    Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells. Using the human Ishikawa endometrial cancer cell line, we investigated the effects of arecoline on expression, localization and functional connections between the ZO-1 tight junction protein and the HER2 EGF receptor family member. Treatment of Ishikawa cells with arecoline coordinately down-regulated expression of both ZO-1 and HER2 protein and transcripts in a dose dependent manner. Biochemical fractionation of cells as well as indirect immunofluorescence revealed that arecoline disrupted the localization of ZO-1 to the junctional complex at the cell periphery. Compared to control transfected cells, ectopic expression of exogenous HER2 prevented the arecoline mediated down-regulation of ZO-1 expression and restored the localization of ZO-1 to the cell periphery. Furthermore, treatment with dexamethasone, a synthetic glucocorticoid reported to up-regulate expression of HER2 in Ishikawa cells, precluded arecoline from down-regulating ZO-1 expression and disrupting ZO-1 localization. Arecoline is known to induce precancerous lesions and cancer in the oral cavity of betel nut users. The arecoline down-regulation of ZO-1 expression and subcellular distribution suggests that arecoline potentially

  7. Arecoline induced disruption of expression and localization of the tight junctional protein ZO-1 is dependent on the HER 2 expression in human endometrial Ishikawa cells

    Directory of Open Access Journals (Sweden)

    Sundar Shyam N

    2010-07-01

    Full Text Available Abstract Background Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells. Results Using the human Ishikawa endometrial cancer cell line, we investigated the effects of arecoline on expression, localization and functional connections between the ZO-1 tight junction protein and the HER2 EGF receptor family member. Treatment of Ishikawa cells with arecoline coordinately down-regulated expression of both ZO-1 and HER2 protein and transcripts in a dose dependent manner. Biochemical fractionation of cells as well as indirect immunofluorescence revealed that arecoline disrupted the localization of ZO-1 to the junctional complex at the cell periphery. Compared to control transfected cells, ectopic expression of exogenous HER2 prevented the arecoline mediated down-regulation of ZO-1 expression and restored the localization of ZO-1 to the cell periphery. Furthermore, treatment with dexamethasone, a synthetic glucocorticoid reported to up-regulate expression of HER2 in Ishikawa cells, precluded arecoline from down-regulating ZO-1 expression and disrupting ZO-1 localization. Conclusion Arecoline is known to induce precancerous lesions and cancer in the oral cavity of betel nut users. The arecoline down-regulation of ZO-1 expression and

  8. Identification of a novel mutation of the gene for gap junction protein α3 (GJA3) in a Chinese family with congenital cataract.

    Science.gov (United States)

    Hu, Ying; Gao, Lin; Feng, Yali; Yang, Tao; Huang, Shangzhi; Shao, Zhengbo; Yuan, Huiping

    2014-07-01

    Cataract, defined as any opacity of the crystallin lens, can be divided into early onset (congenital or infantile) and age-related. It is the leading cause of visual disability in children, and mutations in many genes have currently been linked with this disorder. In the present study, we identified a genetic defect in a Chinese family with congenital cataract. Genomic DNA was extracted from the venous blood of the family and 100 normal controls. To screen for the disease-causing mutation, we sequenced eight candidate genes, and to predict the functional consequences of the mutation, a structural model of the protein was developed using the Protein Data Bank and PyMOL 1.1r1. We found a novel variant (c.163 A > G transition) in the gene for gap junction protein α3, or the connexin46 gene. This mutation resulted in the substitution of a highly conserved asparagine at codon 55 by aspartic acid (p.N55D). There were no nucleotide polymorphisms in the other candidate genes sequenced.

  9. Calcium-Dependent Protein Kinase in Ginger Binds with Importin-α through Its Junction Domain for Nuclear Localization, and Further Interacts with NAC Transcription Factor

    Science.gov (United States)

    Vivek, Padmanabhan Jayanthi; Resmi, Mohankumar Saraladevi; Sreekumar, Sweda; Sivakumar, K. C.; Tuteja, Narendra; Soniya, Eppurathu Vasudevan

    2017-01-01

    Calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ elevations in plant cells regulating the gene expression linked with various cellular processes like stress response, growth and development, metabolism, and cytoskeleton dynamics. Ginger is an extensively used spice due to its unique flavor and immense medicinal value. The two major threats that interfere with the large scale production of ginger are the salinity and drought stress. ZoCDPK1 (Zingiber officinale Calcium-dependent protein kinase 1) is a salinity and drought-inducible CDPK gene isolated from ginger and undergoes dynamic subcellular localization during stress conditions. ZoCDPK1, with signature features of a typical Ca2+ regulated kinase, also possesses a bipartite nuclear localization sequence (NLS) in its junction domain (JD). A striking feature in ZoCDPK1 is the rare occurrence of a coupling between the NLS in JD and consensus sequences in regulatory domain. Here, we further identified its nature of nuclear localization and its interaction partners. In the homology model generated for ZoCDPK1, the regulatory domain mimics the crystal structure of the regulatory domain in Arabidopsis CDPK1. Molecular docking simulation of importin (ZoIMPα), an important protein involved in nuclear translocation, into the NLS of ZoCDPK1 was well-visualized. Furthermore, the direct interaction of ZoCDPK1 and ZoIMPα proteins was studied by the yeast 2-hybrid (Y2H) system, which confirmed that junction domain (JD) is an important interaction module required for ZoCDPK1 and ZoIMPα binding. The probable interacting partners of ZoCDPK1 were also identified using Y2H experiment. Of the 10 different stress-related interacting partners identified for ZoCDPK1, NAC transcription factor (TF) needs special mention, especially in the context of ZoCDPK1 function. The interaction between ZoCDPK1 and NAC TF, in fact, corroborate with the results of gene expression and over-expression studies of ZoCDPK1. Hence

  10. Mutation Analysis of Gap Junction Protein Beta 1 and Genotype-Phenotype Correlation in X-linked Charcot-Marie-Tooth Disease in Chinese Patients.

    Science.gov (United States)

    Sun, Bo; Chen, Zhao-Hui; Ling, Li; Li, Yi-Fan; Liu, Li-Zhi; Yang, Fei; Huang, Xu-Sheng

    2016-05-05

    Among patients with Charcot-Marie-Tooth disease (CMT), the X-linked variant (CMTX) caused by gap junction protein beta 1 (GJB1) gene mutation is the second most frequent type, accounting for approximately 90% of all CMTX. More than 400 mutations have been identified in the GJB1 gene that encodes connexin 32 (CX32). CX32 is thought to form gap junctions that promote the diffusion pathway between cells. GJB1 mutations interfere with the formation of the functional channel and impair the maintenance of peripheral myelin, and novel mutations are continually discovered. We included 79 unrelated patients clinically diagnosed with CMT at the Department of Neurology of the Chinese People's Liberation Army General Hospital from December 20, 2012, to December 31, 2015. Clinical examination, nerve conduction studies, and molecular and bioinformatics analyses were performed to identify patients with CMTX1. Nine GJB1 mutations (c.283G>A, c.77C>T, c.643C>T, c.515C>T, c.191G>A, c.610C>T, c.490C>T, c.491G>A, and c.44G>A) were discovered in nine patients. Median motor nerve conduction velocities of all nine patients were T, c.191G>A, and c.610C>T, were revealed and bioinformatics analyses indicated high pathogenicity. The three novel missense mutations within the GJB1 gene broaden the mutational diversity of CMT1X. Molecular analysis of family members and bioinformatics analyses of the afflicted patients confirmed the pathogenicity of these mutations.

  11. Mutation Analysis of Gap Junction Protein Beta 1 and Genotype-Phenotype Correlation in X-linked Charcot-Marie-Tooth Disease in Chinese Patients

    Institute of Scientific and Technical Information of China (English)

    Bo Sun; Zhao-Hui Chen; Li Ling; Yi-Fan Li; Li-Zhi Liu; Fei Yang; Xu-Sheng Huang

    2016-01-01

    Background:Among patients with Charcot-Marie-Tooth disease (CMT),the X-linked variant (CMTX) caused by gap junction protein beta 1 (GJB1) gene mutation is the second most frequent type,accounting for approximately 90% of all CMTX.More than 400 mutations have been identified in the GJB1 gene that encodes connexin 32 (CX32).CX32 is thought to form gap junctions that promote the diffusion pathway between cells.GJB1 mutations interfere with the formation of the functional channel and impair the maintenance of peripheral myelin,and novel mutations are continually discovered.Methods:We included 79 unrelated patients clinically diagnosed with CMT at the Department of Neurology of the Chinese People's Liberation Army General Hospital from December 20,2012,to December 31,2015.Clinical examination,nerve conduction studies,and molecular and bioinformatics analyses were performed to identify patients with CMTX 1.Results:Nine GJB1 mutations (c.283G>A,c.77C>T,c.643C>T,c.515C>T,c.191G>A,c.610C>T,c.490C>T,c.491G>A,and c.44G>A) were discovered in nine patients.Median motor nerve conduction velocities of all nine patients were < 38 m/s,resembling CMT Type 1.Three novel mutations,c.643C>T,c.191G>A,and c.610C>T,were revealed and bioinformatics analyses indicated high pathogenicity.Conclusions:The three novel missense mutations within the GJB1 gene broaden the mutational diversity of CMT 1 X.Molecular analysis of family members and bioinformatics analyses of the afflicted patients confirmed the pathogenicity of these mutations.

  12. The protein kinase A pathway contributes to Hg2+-induced alterations in phosphorylation and subcellular distribution of occludin associated with increased tight junction permeability of salivary epithelial cell monolayers.

    Science.gov (United States)

    Kawedia, Jitesh D; Jiang, Mengmeng; Kulkarni, Amit; Waechter, Holly E; Matlin, Karl S; Pauletti, Giovanni M; Menon, Anil G

    2008-09-01

    Hg(2+) is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg(2+) affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg(2+) decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg(2+)-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg(2+) treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg(2+)-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögren's syndrome, and possibly other disorders of fluid transport.

  13. Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein.

    Science.gov (United States)

    Takahashi, K; Nakanishi, H; Miyahara, M; Mandai, K; Satoh, K; Satoh, A; Nishioka, H; Aoki, J; Nomoto, A; Mizoguchi, A; Takai, Y

    1999-05-03

    We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").

  14. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4.

    Science.gov (United States)

    Kominsky, Scott L; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G; Weitzman, Sigmund A; Argani, Pedram; Sukumar, Saraswati

    2004-05-01

    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy.

  15. Expressions of tight junction proteins Occludin and Claudin-1 are under the circadian control in the mouse large intestine: implications in intestinal permeability and susceptibility to colitis.

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    Oh-oka Kyoko

    Full Text Available BACKGROUND & AIMS: The circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine. METHODS: The expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2(m/m. In addition, the susceptibility to dextran sodium sulfate (DSS-induced colitis was compared between wild-type mice and mPer2(m/m mice. RESULTS: The mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2(m/m mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2(m/m mice. mPer2(m/m mice were more resistant to the colonic injury induced by DSS than wild-type mice. CONCLUSIONS: Occludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.

  16. The EhCPADH112 complex of Entamoeba histolytica interacts with tight junction proteins occludin and claudin-1 to produce epithelial damage.

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    Abigail Betanzos

    Full Text Available Entamoeba histolytica, the protozoan responsible for human amoebiasis, causes between 30,000 and 100,000 deaths per year worldwide. Amoebiasis is characterized by intestinal epithelial damage provoking severe diarrhea. However, the molecular mechanisms by which this protozoan causes epithelial damage are poorly understood. Here, we studied the initial molecular interactions between the E. histolytica EhCPADH112 virulence complex and epithelial MDCK and Caco-2 cells. By confocal microscopy, we discovered that after contact with trophozoites or trophozoite extracts (TE, EhCPADH112 and proteins forming this complex (EhCP112 and EhADH112 co-localize with occludin and claudin-1 at tight junctions (TJ. Immunoprecipitation assays revealed interaction between EhCPADH112 and occludin, claudin-1, ZO-1 and ZO-2. Overlay assays confirmed an interaction of EhCP112 and EhADH112 with occludin and claudin-1, whereas only EhADH112 interacted also with ZO-2. We observed degradation of all mentioned TJ proteins after incubation with TE. Importantly, inhibiting proteolytic activity or blocking the complex with a specific antibody not only prevented TJ protein degradation but also epithelial barrier disruption. Furthermore, we discovered that TE treatment induces autophagy and apoptosis in MDCK cells that could contribute to the observed barrier disruption. Our results suggest a model in which epithelial damage caused by E. histolytica is initiated by the interaction of EhCP112 and EhADH112 with TJ proteins followed by their degradation. Disruption of TJs then induces increased paracellular permeability, thus facilitating the entry of more proteases and other parasite molecules leading eventually to tissue destruction.

  17. Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion.

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    Sébastien Besteiro

    2009-02-01

    Full Text Available One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1 and the neck of the rhoptries (for RON2/RON4/RON5 proteins, have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

  18. Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice.

    Science.gov (United States)

    Oguro, K; Jover, T; Tanaka, H; Lin, Y; Kojima, T; Oguro, N; Grooms, S Y; Bennett, M V; Zukin, R S

    2001-10-01

    Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cell-specific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbumin-positive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/-) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cell-specific patterns of global ischemia-induced neuronal death.

  19. All-trans retinoic acid enhances bystander effect of suicide gene therapy in the treatment of breast cancer.

    Science.gov (United States)

    Kong, Heng; Liu, Xia; Yang, Liucheng; Qi, Ke; Zhang, Haoyun; Zhang, Jingwen; Huang, Zonghai; Wang, Hongxian

    2016-03-01

    All-trans retinoic acid (ATRA) has been shown to enhance the expression of connexin 43 (Cx43) and the bystander effect (BSE) in suicide gene therapy. These in turn improve effects of suicide gene therapies for several tumor types. However, whether ATRA can improve BSE remains unclear in suicide gene therapy for breast cancer. In the present study, MCF-7, human breast cancer cells were treated with ATRA in combination with a VEGFP-TK/CD gene suicide system developed by our group. We found that this combination enhances the efficiency of cell killing and apoptosis of breast cancer by strengthening the BSE in vitro. ATRA also promotes gap junction intercellular communication (GJIC) in MCF-7 cells by upregulation of the connexin 43 mRNA and protein in MCF-7 cells. These results indicate that enhancement of GJIC by ATRA in suicide gene system might serve as an attractive and cost-effective strategy of therapy for breast cancer cells.

  20. Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2014-01-15

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P < 0.05) at 4 days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P < 0.05) but CX43 protein increased (P < 0.05) at 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P < 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia. - Highlights: • DMBA increases Cx37 and Cx43 expression prior to follicle loss. • During follicle loss both Cx37 and Cx43 expressions are reduced. • CX43 protein is absent in follicle remnants lacking an oocyte.