title: Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/β Signaling Node Leading to TNF Secretion.
abstract: Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow-derived mast cells from wild-type and Fyn-deficient mice. Fyn(-/-) cells showed higher LPS-induced secretion of preformed and de novo-synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn(-/-) cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn(-/-) cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/β, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation-associated protein (AHNAK, desmoyokin). LPS-induced PKCα/β activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn(-/-) MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/β necessary for the secretion of TNF by VAMP3(+) carriers.
authors.affiliation: Departamento de Farmacobiología, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Sede Sur, Tlalpan, CP 14330 Mexico City, Mexico;
authors.forename: Alejandro
authors.identifier: 0000-0003-0343-1685
authors.initials: A
authors.lastname: Martín-Ávila
authors.affiliation: Departamento de Farmacobiología, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Sede Sur, Tlalpan, CP 14330 Mexico City, Mexico;
authors.forename: Jaciel
authors.identifier: 0000-0003-4205-0949
authors.initials: J
authors.lastname: Medina-Tamayo
authors.affiliation: Departamento de Farmacobiología, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Sede Sur, Tlalpan, CP 14330 Mexico City, Mexico;
authors.forename: Alfredo
authors.identifier: 0000-0003-1195-1808
authors.initials: A
authors.lastname: Ibarra-Sánchez
authors.affiliation: Departamento de Biología Celular y del Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, CP 04510 Mexico City, Mexico;
authors.forename: Genaro
authors.identifier: 0000-0002-2319-8772
authors.initials: G
authors.lastname: Vázquez-Victorio
authors.affiliation: Departamento de Farmacobiología, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Sede Sur, Tlalpan, CP 14330 Mexico City, Mexico;
authors.forename: Jorge Iván
authors.identifier: 0000-0002-3278-2348
authors.initials: JI
authors.lastname: Castillo-Arellano
authors.affiliation: Departamento de Farmacobiología, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Sede Sur, Tlalpan, CP 14330 Mexico City, Mexico;
authors.forename: Alma Cristal
authors.initials: AC
authors.lastname: Hernández-Mondragón
authors.affiliation: National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892-1820; and.
authors.forename: Juan
authors.initials: J
authors.lastname: Rivera
authors.affiliation: INSERM UMRS1149, Faculté de Médecine, Université Paris-Diderot, Site X, Bichat, Paris 75018, France.
authors.forename: Iris K
authors.identifier: 0000-0002-0741-9248
authors.initials: IK
authors.lastname: Madera-Salcedo
authors.affiliation: INSERM UMRS1149, Faculté de Médecine, Université Paris-Diderot, Site X, Bichat, Paris 75018, France.
authors.forename: Ulrich
authors.identifier: 0000-0002-2661-2983
authors.initials: U
authors.lastname: Blank
authors.affiliation: Departamento de Biología Celular y del Desarrollo, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, CP 04510 Mexico City, Mexico;
authors.forename: Marina
authors.identifier: 0000-0002-3972-0983
authors.initials: M
authors.lastname: Macías-Silva
authors.affiliation: Departamento de Farmacobiología, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Sede Sur, Tlalpan, CP 14330 Mexico City, Mexico; cgonzal@cinvestav.mx.
authors.forename: Claudia
authors.identifier: 0000-0001-7332-3829
authors.initials: C
authors.lastname: González-Espinosa
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation; D008070:Lipopolysaccharides / Q000276:immunology; D008407:Mast Cells / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D050766:Qb-SNARE Proteins / Q000378:metabolism; D050767:Qc-SNARE Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051040:Vesicle-Associated Membrane Protein 3 / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C107017:Snap23 protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D051040:Vesicle-Associated Membrane Protein 3; C494953:vesicle-associated membrane protein 3, mouse; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; C496630:Prkca protein, mouse; D051571:Protein Kinase C-alpha; D054648:Protein Phosphatase 2