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6 results on '"MESH : CHO Cells"'

1. CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

Author

Georges Bismuth, Mark G.H. Scott, Catherine Labbé-Jullié, Lamia Achour, Hamasseh Shirvani, Stefano Marullo, Alain Thuret, Marullo, Stefano, Institut Cochin (UMR_S567 / UMR 8104), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Université Paris Descartes - Paris 5 (UPD5) - Institut National de la Santé et de la Recherche Médicale (INSERM) - Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH : Hela Cells, Chemokine receptor CCR5, viruses, Receptor expression, MESH: Antigens, CD4, Intracellular Space, MESH : Intracellular Space, MESH: Cricetinae, Plasma protein binding, MESH: Receptors, CCR5, Endoplasmic Reticulum, Biochemistry, MESH: Cell Compartmentation, MESH : Protein Transport, MESH: Cricetulus, Cell–cell interaction, Cricetinae, MESH : CHO Cells, MESH: Animals, Receptor, Cells, Cultured, MESH : Endoplasmic Reticulum, biology, MESH : Cricetinae, virus diseases, MESH : Protein Binding, MESH : Receptors, CCR5, Hematology, Cell biology, Protein Transport, Antigens, Surface, CD4 Antigens, MESH: Intracellular Space, MESH : Antigens, Surface, Intracellular, MESH: Cells, Cultured, Protein Binding, MESH: Protein Transport, Receptors, CCR5, MESH: Antigens, Surface, MESH : Cricetulus, Immunology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, CHO Cells, Article, Cricetulus, MESH: CHO Cells, MESH: Endoplasmic Reticulum, MESH : Cells, Cultured, MESH: Protein Binding, Animals, Humans, MESH : Cell Compartmentation, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, MESH: Humans, Endoplasmic reticulum, MESH : Humans, Cell Biology, Cell Compartmentation, MESH: Hela Cells, biology.protein, MESH : Antigens, CD4, MESH : Animals, CC chemokine receptors, HeLa Cells
Abstract

International audience; The association of CD4, a glycoprotein involved in T-cell development and antigen recognition, and CC chemokine receptor 5 (CCR5), a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for HIV. We observed that the majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relatively low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration-dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell-surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cellular distribution of CXCR4, the other HIV coreceptor. These results reveal a previously unappreciated role of CD4, which contributes to regulating CCR5 export to the plasma membrane.

Published
2009
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2. Lysosomal serine protease CLN2 regulates tumor necrosis factor-alpha-mediated apoptosis in a Bid-dependent manner

Author

Nathalie Andrieu-Abadie, Virginie Garcia, Hélène Autefage, Nicole Therville, Thierry Levade, Marie-Françoise Altié, Catherine Caillaud, Virginie Albinet, Hortense Berges, Marie-Laure Nicolau, Institut de médecine moléculaire de Rangueil ( I2MR ), Université Toulouse III - Paul Sabatier ( UPS ), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-IFR150-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Institut Cochin ( UMR_S567 / UMR 8104 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Biochimie [Purpan], Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-CHU Toulouse [Toulouse]-Institut Fédératif de Biologie (IFB) - Hôpital Purpan, Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]-Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse], Institut de médecine moléculaire de Rangueil (I2MR), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées- Institut Fédératif de Recherche Bio-médicale Institution (IFR150)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Cochin (UMR_S567 / UMR 8104), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie [CHU Toulouse], Institut Fédératif de Biologie (IFB), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Pôle Biologie [CHU Toulouse], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), and Simon, Marie Francoise

Subjects
Small interfering RNA, MESH : Cytochromes c, MESH: Neurons, MESH: Cricetinae, Apoptosis, MESH: Caspase 9, Biochemistry, Aminopeptidases, MESH: Recombinant Proteins, MESH: Cricetulus, Cricetinae, MESH: Caspase 3, MESH : CHO Cells, MESH : Catalysis, MESH: Animals, MESH : Caspase 3, MESH: Endopeptidases, Caspase, Neurons, MESH : Tumor Necrosis Factor-alpha, biology, Tripeptidyl-Peptidase 1, MESH : Caspase 9, Caspase 3, Mechanisms of Signal Transduction, MESH : Cricetinae, Cytochromes c, Transfection, MESH: Cytochromes c, Caspase 9, Recombinant Proteins, MESH : Endopeptidases, Cell biology, Tumor necrosis factor alpha, BH3 Interacting Domain Death Agonist Protein, Programmed cell death, Proteases, MESH : Recombinant Proteins, MESH : Cricetulus, MESH: BH3 Interacting Domain Death Agonist Protein, CHO Cells, Catalysis, MESH : Neurons, Cricetulus, MESH: CHO Cells, Endopeptidases, MESH : Fibroblasts, Animals, Humans, MESH : BH3 Interacting Domain Death Agonist Protein, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases, Molecular Biology, Serine protease, MESH: Humans, Tumor Necrosis Factor-alpha, MESH: Apoptosis, MESH : Humans, Cell Biology, Fibroblasts, MESH : Lysosomes, MESH: Catalysis, Molecular biology, MESH: Fibroblasts, MESH: Tumor Necrosis Factor-alpha, biology.protein, MESH : Animals, Serine Proteases, Lysosomes, MESH : Apoptosis, MESH: Lysosomes
Abstract

International audience; Apoptosis is a highly organized, energy-dependent program by which multicellular organisms eliminate damaged, superfluous, and potentially harmful cells. Although caspases are the most prominent group of proteases involved in the apoptotic process, the role of lysosomes has only recently been unmasked. This study investigated the role of the lysosomal serine protease CLN2 in apoptosis. We report that cells isolated from patients affected with late infantile neuronal ceroid lipofuscinosis (LINCL) having a deficient activity of CLN2 are resistant to the toxic effect of death ligands such as tumor necrosis factor (TNF), CD95 ligand, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not to receptor-independent stress agents. CLN2-deficient cells exhibited a defect in TNF-induced Bid cleavage, release of cytochrome c, and caspase-9 and -3 activation. Moreover, extracts from CLN2-overexpressing cells or a CLN2 recombinant protein were able to catalyze the in vitro cleavage of Bid. Noteworthy, correction of the lysosomal enzyme defect of LINCL fibroblasts using a medium enriched in CLN2 protein enabled restoration of TNF-induced Bid and caspase-3 processing and toxicity. Conversely, transfection of CLN2-corrected cells with small interfering RNA targeting Bid abrogated TNF-induced cell death. Altogether, our study demonstrates that genetic deletion of the lysosomal serine protease CLN2 and the subsequent loss of its catalytic function confer resistance to TNF in non-neuronal somatic cells, indicating that CLN2 plays a yet unsuspected role in TNF-induced cell death.

Published
2009
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3. Asp218 participates with Asp213 to bind a Ca2+ atom into the S1 subsite of aminopeptidase A: a key element for substrate specificity

Author

Bernard Maigret, Xavier Iturrioz, Mayumi Okada, Nicolas Inguimbert, Catherine Llorens-Cortes, Bernard P. Roques, Cédric Claperon, Raphael Rozenfeld, Neuropeptides centraux et régulations hydrique et cardiovasculaire, Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Labex MemoLife, Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de Pharmacologie Chimique et Génétique (UPCG - UMR_S 640/UMR 8151), Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut des sciences du Médicament -Toxicologie - Chimie - Environnement (IFR71), Institut de Recherche pour le Développement (IRD)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Knowledge representation, reasonning (ORPAILLEUR), INRIA Lorraine, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire Lorrain de Recherche en Informatique et ses Applications (LORIA), Institut National de Recherche en Informatique et en Automatique (Inria)-Université Henri Poincaré - Nancy 1 (UHP)-Université Nancy 2-Institut National Polytechnique de Lorraine (INPL)-Centre National de la Recherche Scientifique (CNRS)-Université Henri Poincaré - Nancy 1 (UHP)-Université Nancy 2-Institut National Polytechnique de Lorraine (INPL)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre interdisciplinaire de recherche en biologie (CIRB), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Labex MemoLife, Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut des sciences du Médicament -Toxicologie - Chimie - Environnement (IFR71), Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC), Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Lorraine (INPL)-Université Nancy 2-Université Henri Poincaré - Nancy 1 (UHP)-Institut National de Recherche en Informatique et en Automatique (Inria)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Lorraine (INPL)-Université Nancy 2-Université Henri Poincaré - Nancy 1 (UHP), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Collège de France ( CdF ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Unité de Pharmacologie Chimique et Génétique ( UPCG - UMR_S 640/UMR 8151 ), Centre National de la Recherche Scientifique ( CNRS ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut des sciences du Médicament -Toxicologie - Chimie - Environnement ( IFR71 ), Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL ( ENSCP ) -Centre National de la Recherche Scientifique ( CNRS ) -Institut de Recherche pour le Développement ( IRD ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL ( ENSCP ) -Centre National de la Recherche Scientifique ( CNRS ) -Institut de Recherche pour le Développement ( IRD ) -Université Paris Descartes - Paris 5 ( UPD5 ), Knowledge representation, reasonning ( ORPAILLEUR ), Institut National de Recherche en Informatique et en Automatique ( Inria ) -Institut National de Recherche en Informatique et en Automatique ( Inria ) -Laboratoire Lorrain de Recherche en Informatique et ses Applications ( LORIA ), Institut National de Recherche en Informatique et en Automatique ( Inria ) -Université Henri Poincaré - Nancy 1 ( UHP ) -Université Nancy 2-Institut National Polytechnique de Lorraine ( INPL ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Henri Poincaré - Nancy 1 ( UHP ) -Université Nancy 2-Institut National Polytechnique de Lorraine ( INPL ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire Lorrain de Recherche en Informatique et ses Applications (LORIA), Institut National de Recherche en Informatique et en Automatique (Inria)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Iturrioz, Xavier, and Llorens Cortes, Catherine

Subjects
Models, Molecular, MESH : Glutamyl Aminopeptidase, MESH : Glutamates, MESH: Amino Acids, [SDV]Life Sciences [q-bio], Angiotensin III, monozinc aminopeptidase, MESH : Aspartic Acid, MESH: Cricetinae, MESH: Amino Acid Sequence, Biochemistry, MESH: Aspartic Acid, Substrate Specificity, MESH : Sulfhydryl Compounds, Mice, chemistry.chemical_compound, Glutamates, MESH: Cricetulus, Cricetinae, [CHIM] Chemical Sciences, MESH : CHO Cells, MESH: Animals, Amino Acids, Enzyme Inhibitors, Site-directed mutagenesis, inhibitor design, [SDV.MHEP.EM] Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, [PHYS]Physics [physics], MESH: Glutamyl Aminopeptidase, 0303 health sciences, MESH : Substrate Specificity, MESH: Kinetics, Hydrogen bond, MESH : Amino Acid Sequence, 030302 biochemistry & molecular biology, MESH : Cricetinae, Life Sciences, [ SDV.MHEP.EM ] Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Phosphonate, MESH : Mutagenesis, Site-Directed, molecular modelling, [SDV] Life Sciences [q-bio], MESH: Mutagenesis, Site-Directed, Ectodomain, MESH: Enzyme Inhibitors, MESH: Calcium, Pharmacophore, MESH : Kinetics, site-directed mutagenesis, MESH : Phosphonic Acids, MESH: Models, Molecular, calcium-binding site, hypertension, Stereochemistry, MESH : Models, Molecular, education, MESH : Cricetulus, Organophosphonates, MESH: Phosphonic Acids, CHO Cells, Glutamyl Aminopeptidase, 03 medical and health sciences, Cricetulus, MESH: Glutamates, site directed mutagenesis, MESH: CHO Cells, MESH : Mice, Animals, [CHIM]Chemical Sciences, Amino Acid Sequence, Sulfhydryl Compounds, Carboxylate, calcium binding site, MESH : Calcium, MESH: Sulfhydryl Compounds, Molecular Biology, MESH: Mice, 030304 developmental biology, Aspartic Acid, MESH : Enzyme Inhibitors, Binding Sites, molecular modeling, Rational design, Cell Biology, Kinetics, chemistry, MESH: Binding Sites, Mutagenesis, Site-Directed, Calcium, MESH : Amino Acids, MESH: Substrate Specificity, MESH : Animals, MESH : Binding Sites
Abstract

International audience; APA (aminopeptidase A; EC 3.4.11.7) is a membrane-bound zinc metallopeptidase, also activated by Ca(2+), involved in the formation of brain angiotensin III, which exerts a tonic stimulatory action on the central control of blood pressure in hypertensive animals. In the present study, in the three-dimensional model of the ectodomain of mouse APA, we docked the specific APA inhibitor glutamate phosphonate, in the presence of Ca(2+). The model showed the presence of one Ca(2+) atom in an hydrophilic pocket corresponding to the S1 subsite in which the lateral chain of the inhibitor is pointing. In this pocket, the Ca(2+) atom was hexaco-ordinated with the acidic side chains of Asp(213) and Asp(218), the carbonyl group of Glu(215) and three water molecules, one of them being engaged in a hydrogen bond with the negatively charged carboxylate side chain of the inhibitor. Mutagenic replacement of Asp(213) and Asp(218) with a conservative residue maintained the ability of mutated APAs to be activated by Ca(2+). However, the replacement by a non-conservative residue abolished this property, demonstrating the crucial role of these residues in Ca(2+) binding. We also showed the involvement of these residues in the strict specificity of APA in the presence of Ca(2+) for N-terminal acidic residues from substrates or inhibitors, since mutagenic replacement of Asp(213) and Asp(218) induced a decrease of the inhibitory potencies of inhibitors homologous with acidic residues. Finally, this led to the rational design of a new potent APA inhibitor, NI926 (K(i)=70 nM), which allowed us to precisely localize Asp(213) at the entrance and Asp(218) at the bottom of the S1 subsite. Taken together, these data provide new insight into the organization and functional role of the APA S1 subsite and will allow the design of pharmacophore of the inhibitor, helpful for the development of a new generation of APA inhibitors as central-acting antihypertensive agents.

Published
2008
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4. Erk5 controls Slug expression and keratinocyte activation during wound healing

Author

Pierre Savagner, Valérie Arnoux, Annie L'Helgoualc'h, Mayssaa Nassour, Robert A. Hipskind, Genotypes et Phenotypes Tumoraux, Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Ligue Regionale contre le Cancer (Languedoc-Roussillon), Groupement des Entreprises Francaises dans la Lutte contre le Cancer, the Fondation de France and the Association pour la Recherche sur le Cancer (VA and RAH)., Université Montpellier 1 ( UM1 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Institut de Génétique Moléculaire de Montpellier ( IGMM ), Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Le Ster, Yves, and Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.MHEP.PHY] Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], MESH : Wound Healing, MESH : Promoter Regions, Genetic, MESH : RNA, Small Interfering, migration, [ SDV.CAN ] Life Sciences [q-bio]/Cancer, Mice, MESH : Phosphorylation, MESH : Cytoskeleton, 0302 clinical medicine, Desmosome organization, MESH: Cricetulus, MESH : CHO Cells, MESH: Animals, Cytoskeleton, Mitogen-Activated Protein Kinase 1, 0303 health sciences, Mitogen-Activated Protein Kinase 3, MESH : Gene Expression Regulation, MESH : Cricetinae, Keratinocyte activation, MESH: Transcription Factors, Cell biology, ErbB Receptors, 030220 oncology & carcinogenesis, MESH: Mitogen-Activated Protein Kinase 7, MESH: Mitogen-Activated Protein Kinase 3, MESH: Mitogen-Activated Protein Kinase 1, Slug, MESH : Cricetulus, MESH: Receptor, Epidermal Growth Factor, MESH: Cell Adhesion, 03 medical and health sciences, [SDV.CAN] Life Sciences [q-bio]/Cancer, MESH: CHO Cells, MESH: Promoter Regions, Genetic, Humans, Cell adhesion, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, EGF, MESH: Humans, Epidermal Growth Factor, MESH: Phosphorylation, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, HaCaT, MESH : Humans, MAPK, MESH : Epithelium, MESH: Wound Healing, MESH: Epithelium, Wound healing, [ SDV.MHEP.DERM ] Life Sciences [q-bio]/Human health and pathology/Dermatology, [SDV.MHEP.DERM]Life Sciences [q-bio]/Human health and pathology/Dermatology, Transcription Factors, Keratinocytes, MESH: Epidermal Growth Factor, Cytoskeleton organization, MESH : Transcription Factors, MESH: Cricetinae, Epithelium, MESH : Receptor, Epidermal Growth Factor, Cricetinae, MESH: RNA, Small Interfering, Phosphorylation, RNA, Small Interfering, Keratinocyte migration, Promoter Regions, Genetic, MESH : Epidermal Growth Factor, MESH : Mitogen-Activated Protein Kinase 1, biology, MESH : Mitogen-Activated Protein Kinase 3, ERK1/2, MESH : Keratinocytes, [ SDV.MHEP.PHY ] Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Articles, MESH: Gene Expression Regulation, MESH: Keratinocytes, MESH : Mitogen-Activated Protein Kinase 7, Motility, [SDV.CAN]Life Sciences [q-bio]/Cancer, CHO Cells, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Cricetulus, MESH : Mice, Cell Adhesion, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Cytoskeleton, [SDV.MHEP.PHY]Life Sciences [q-bio]/Human health and pathology/Tissues and Organs [q-bio.TO], Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, MESH: Mice, Mitogen-Activated Protein Kinase 7, 030304 developmental biology, Wound Healing, Cell Biology, [SDV.MHEP.DERM] Life Sciences [q-bio]/Human health and pathology/Dermatology, biology.organism_classification, MESH : Cell Adhesion, Gene Expression Regulation, Cancer research, Snail Family Transcription Factors, MESH : Animals
Abstract

International audience; Reepithelialization during cutaneous wound healing involves numerous signals that result in basal keratinocyte activation, spreading, and migration, all linked to a loosening of cell-cell adhesion structures. The transcription factor Slug is required for this process, and EGF treatment of human keratinocytes induced activating phosphorylation of Erk5 that coincides with slug transcription. Accordingly, ectopic activation of Erk5 led to increased Slug mRNA levels and faster wound healing, whereas keratinocyte migration was totally blocked by Erk5 pathway inhibition. Expression of a shRNA specific for Erk5 strongly diminished Erk5 levels in keratinocytes and significantly decreased their motility response to EGF, along with induction of Slug expression. These Erk5-deprived keratinocytes showed an altered, more compact morphology, along with disruption of desmosome organization. Accordingly, they displayed an altered ability to form cell aggregates. These results implicate a novel EGFR/Erk5/Slug pathway in the control of cytoskeleton organization and cell motility in keratinocytes treated with EGF.

Published
2008
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5. Cellular localization of apelin and its receptor in the anterior pituitary: evidence for a direct stimulatory action of apelin on ACTH release

Author

Marie-Thérèse Bluet-Pajot, Rodrigo Alvear-Perez, Philippe Zizzari, Catherine Llorens-Cortes, Annabelle Réaux-Le Goazigo, Jacques Epelbaum, Neuropeptides centraux et régulations hydrique et cardiovasculaire, Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Labex MemoLife, Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Neurobiologie de la Croissance et de la Senescence, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre interdisciplinaire de recherche en biologie (CIRB), École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Labex MemoLife, Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Collège de France ( CdF ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), and Llorens Cortes, Catherine

Subjects
Male, Pituitary gland, Endocrinology, Diabetes and Metabolism, MESH: Amino Acid Sequence, MESH: Receptors, G-Protein-Coupled, MESH: Base Sequence, MESH : Tissue Distribution, Receptors, G-Protein-Coupled, Rats, Sprague-Dawley, 0302 clinical medicine, MESH: Cricetulus, MESH: Pituitary Gland, MESH: Ligands, MESH : CHO Cells, Tissue Distribution, MESH: Animals, MESH: Peptide Fragments, MESH : Ligands, 0303 health sciences, MESH : Amino Acid Sequence, MESH : Cyclic AMP, MESH: Pituitary Gland, Anterior, MESH : Cricetinae, MESH : Protein Binding, [ SDV.MHEP.EM ] Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, [SDV.MHEP.EM]Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Apelin, MESH : Adrenocorticotropic Hormone, Intercellular Signaling Peptides and Proteins, MESH : Carrier Proteins, medicine.medical_specialty, endocrine system, MESH : Cell Membrane, MESH : Cricetulus, MESH: Carrier Proteins, Adrenocorticotropic hormone, 03 medical and health sciences, Adrenocorticotropic Hormone, Anterior pituitary, Pituitary Gland, Anterior, MESH: CHO Cells, Physiology (medical), MESH: Protein Binding, MESH: Tissue Distribution, MESH: Adrenocorticotropic Hormone, MESH: Humans, MESH: Molecular Sequence Data, MESH : Humans, Peptide Fragments, Endocrinology, Carrier Proteins, 030217 neurology & neurosurgery, MESH : Molecular Sequence Data, Physiology, MESH: Corticotrophs, MESH: Cricetinae, MESH : Models, Biological, MESH: Rats, Sprague-Dawley, Rats, Inbred WKY, MESH : Pituitary Gland, Receptor, Corticotrophs, Cellular localization, MESH: Cyclic AMP, [SDV.MHEP.EM] Life Sciences [q-bio]/Human health and pathology/Endocrinology and metabolism, Apelin Receptors, MESH : Rats, MESH : Forskolin, medicine.anatomical_structure, MESH: Pituitary Hormones, Anterior, MESH: Calcium, MESH : Receptors, G-Protein-Coupled, MESH : Transfection, MESH: Forskolin, hormones, hormone substitutes, and hormone antagonists, MESH: Rats, Inbred WKY, Endocrine gland, MESH: Rats, MESH : Male, MESH : Rats, Inbred WKY, Biology, Models, Biological, MESH : Pituitary Hormones, Anterior, MESH : Pituitary Gland, Anterior, Pituitary Hormones, Anterior, Internal medicine, medicine, Animals, MESH : Calcium, 030304 developmental biology, Apelin receptor, MESH: Transfection, MESH : Peptide Fragments, MESH: Models, Biological, MESH : Corticotrophs, MESH : Rats, Sprague-Dawley, MESH: Male, Rats, MESH : Peptide Hormones, MESH: Peptide Hormones, MESH : Base Sequence, MESH : Animals, MESH: Cell Membrane
Abstract

Apelin is a bioactive peptide recently identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. The presence of apelin-immunoreactive nerve fibers, together with the detection of apelin receptor mRNA in the parvocellular part of the paraventricular nucleus and the stimulatory action of apelin on corticotropin-releasing hormone release, indicate that apelin modulates adrenocorticotropin (ACTH) release via an indirect action on the hypothalamus. However, a direct action of apelin in the anterior pituitary cannot be excluded. Here, we provided evidence for the existence of an apelinergic system within the adult male rat pituitary gland. Double immunofluorescence staining indicated that apelin is highly coexpressed in the anterior pituitary, mainly in corticotrophs (96.5 ± 0.3%) and to a much lower extent in somatotropes (3.2 ± 0.2%). Using in situ hybridization combined with immunohistochemistry, a high expression of apelin receptor mRNA was also found in corticotrophs, suggesting a local interaction between apelin and ACTH. In an ex vivo perifusion system of anterior pituitaries, apelin 17 (K17F, 10−6M) significantly increased basal ACTH release by 41%, whereas apelin 10 (R10F, 10−6M), an inactive apelin fragment, was ineffective. In addition, K17F but not R10F induced a dose-dependent increase in K+-evoked ACTH release, with maximal increase being observed for a 10−6M concentration. Taken together, these data outline the potential role of apelin as an autocrine/paracrine-acting peptide on ACTH release and provide morphological and neuroendocrine basis for further studies that explore the physiological role of apelin in the regulation of anterior pituitary functions.

Published
2007
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6. Missense Mutations in SLC26A8, Encoding a Sperm-Specific Activator of CFTR, Are Associated with Human Asthenozoospermia

Author

Thierry Bienvenu, Pierre F. Ray, Nathalie Da Silva, Mathilde Jollivet, Aminata Touré, Pierre Tufféry, Baptiste Rode, Denise Escalier, Gérard Gacon, Emmanuel Dulioust, Frédéric Becq, Jean-Philippe Wolf, Caroline Norez, Thassadite Dirami, Natacha Gaitch, Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de physiologie et biologie cellulaires (IPBC), Université de Poitiers-Centre National de la Recherche Scientifique (CNRS), Physiopathologie des maladies génétiques d'expression pédiatrique, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biochimie et Génétique Moléculaire, Université Paris Descartes - Paris 5 (UPD5), Molécules Thérapeutiques in silico (MTI), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service d'Histologie-Embryologie, Biologie de la Reproduction (CECOS Paris Cochin), Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), AGeing and IMagery (AGIM), Centre National de la Recherche Scientifique (CNRS)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Joseph Fourier - Grenoble 1 (UJF)-Université Pierre Mendès France - Grenoble 2 (UPMF), Université Joseph Fourier - Grenoble 1 (UJF), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Morillon, Christelle, Université Pierre Mendès France - Grenoble 2 (UPMF)-Université Joseph Fourier - Grenoble 1 (UJF)-École Pratique des Hautes Études (EPHE), Institut Cochin ( UM3 (UMR 8104 / U1016) ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de physiologie et biologie cellulaires ( IPBC ), Université de Poitiers-Centre National de la Recherche Scientifique ( CNRS ), Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université Pierre et Marie Curie - Paris 6 ( UPMC ), Université Paris Descartes - Paris 5 ( UPD5 ), Molécules Thérapeutique in silico, Recherche de molécules à visée thérapeutique par approches In Silico ( MTI ), Université Paris Diderot - Paris 7 ( UPD7 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Service d'Histologie-Embryologie, Biologie de la Reproduction ( CECOS ), Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Cochin [AP-HP], AGeing and IMagery ( AGIM ), Université Pierre Mendès France - Grenoble 2 ( UPMF ) -Université Joseph Fourier - Grenoble 1 ( UJF ) -École pratique des hautes études ( EPHE ) -Centre National de la Recherche Scientifique ( CNRS ), and Université Joseph Fourier - Grenoble 1 ( UJF )

Subjects
Male, MESH : Immunohistochemistry, [SDV]Life Sciences [q-bio], MESH: Cricetinae, Cystic Fibrosis Transmembrane Conductance Regulator, Antiporters, MESH : Antiporters, 0302 clinical medicine, MESH: Cricetulus, Cricetinae, MESH : CHO Cells, Missense mutation, Genetics(clinical), MESH: Animals, MESH : Immunoblotting, Genetics (clinical), Sperm motility, Genetics, MESH: Cystic Fibrosis Transmembrane Conductance Regulator, 0303 health sciences, 030219 obstetrics & reproductive medicine, MESH: Immunoblotting, Chinese hamster ovary cell, MESH: Spermatozoa, MESH : Cricetinae, MESH: Genetic Predisposition to Disease, MESH: Anion Transport Proteins, MESH: Sperm Motility, Immunohistochemistry, Spermatozoa, Cystic fibrosis transmembrane conductance regulator, Transport protein, [SDV] Life Sciences [q-bio], Sulfate Transporters, Asthenozoospermia, MESH: Sperm Capacitation, Sperm Motility, MESH : Asthenozoospermia, MESH : Sperm Capacitation, MESH : Male, Urology, MESH : Cricetulus, Immunoblotting, Anion Transport Proteins, Mutation, Missense, CHO Cells, Biology, 03 medical and health sciences, Cricetulus, MESH: CHO Cells, Capacitation, Report, MESH : Spermatozoa, medicine, Animals, Humans, Genetic Predisposition to Disease, 030304 developmental biology, MESH: Mutation, Missense, MESH: Humans, [ SDV ] Life Sciences [q-bio], business.industry, Activator (genetics), MESH : Cystic Fibrosis Transmembrane Conductance Regulator, MESH : Humans, MESH: Immunohistochemistry, medicine.disease, Sperm, Molecular biology, MESH: Male, MESH: Asthenozoospermia, MESH : Sperm Motility, MESH : Anion Transport Proteins, biology.protein, MESH : Genetic Predisposition to Disease, MESH: Antiporters, MESH : Animals, business, Sperm Capacitation, MESH : Mutation, Missense
Abstract

International audience; The cystic fibrosis transmembrane conductance regulator (CFTR) is present in mature sperm and is required for sperm motility and capacitation. Both these processes are controlled by ions fluxes and are essential for fertilization. We have shown that SLC26A8, a sperm-specific member of the SLC26 family of anion exchangers, associates with the CFTR channel and strongly stimulates its activity. This suggests that the two proteins cooperate to regulate the anion fluxes required for correct sperm motility and capacitation. Here, we report on three heterozygous SLC26A8 missense mutations identified in a cohort of 146 men presenting with asthenozoospermia: c.260G>A (p.Arg87Gln), c.2434G>A (p.Glu812Lys), and c.2860C>T (p.Arg954Cys). These mutations were not present in 121 controls matched for ethnicity, and statistical analysis on a control population of 8,600 individuals (from dbSNP and 1000 Genomes) showed them to be associated with asthenozoospermia with a power > 95%. By cotransfecting Chinese hamster ovary (CHO)-K1 cells with SLC26A8 variants and CFTR, we showed that the physical interaction between the two proteins was partly conserved but that the capacity to activate CFTR-dependent anion transport was completely abolished for all mutants. Biochemical studies revealed the presence of much smaller amounts of protein for all variants, but these amounts were restored to wild-type levels upon treatment with the proteasome inhibitor MG132. Immunocytochemistry also showed the amounts of SLC26A8 in sperm to be abnormally small in individuals carrying the mutations. These mutations might therefore impair formation of the SLC26A8-CFTR complex, principally by affecting SLC26A8 stability, consistent with an impairment of CFTR-dependent sperm-activation events in affected individuals.

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