title: Protein disulfide isomerase is required for platelet-derived growth factor-induced vascular smooth muscle cell migration, Nox1 NADPH oxidase expression, and RhoGTPase activation.
abstract: Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.
authors: Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, Brazil 05403-000.
authors: Luciana A
authors: LA
authors: Pescatore
authors: Diego
authors: D
authors: Bonatto
authors: Fábio L
authors: FL
authors: Forti
authors: Amine
authors: A
authors: Sadok
authors: Hervé
authors: H
authors: Kovacic
authors: Francisco R M
authors: FR
authors: Laurindo
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D020868:Gene Silencing; D006801:Humans; D009124:Muscle Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000235:genetics; D010982:Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D009124:Muscle Proteins; D010982:Platelet-Derived Growth Factor; C107953:RAC1 protein, human; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; C066737:RHOA protein, human; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C498910:NOX4 protein, human; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D019704:Protein Disulfide-Isomerases