title: Role of guanine nucleotide exchange factor-H1 in complement-mediated RhoA activation in glomerular epithelial cells.
abstract: Visceral glomerular epithelial cells (GEC), also known as podocytes, are vital for the structural and functional integrity of the glomerulus. The actin cytoskeleton plays a central role in maintaining GEC morphology. In a rat model of experimental membranous nephropathy (passive Heymann nephritis (PHN)), complement C5b-9-induced proteinuria was associated with the activation of the actin regulator small GTPase, RhoA. The mechanisms of RhoA activation, however, remained unknown. In this study, we explored the role of the epithelial guanine nucleotide exchange factor, GEF-H1, in complement-induced RhoA activation. Using affinity precipitation to monitor GEF activity, we found that GEF-H1 was activated in glomeruli isolated from rats with PHN. Complement C5b-9 also induced parallel activation of GEF-H1 and RhoA in cultured GEC. In GEC in which GEF-H1 was knocked down, both basal and complement-induced RhoA activity was reduced. On the other hand, GEF-H1 knockdown augmented complement-mediated cytolysis, suggesting a role for GEF-H1 and RhoA in protecting GEC from cell death. The MEK1/2 inhibitor, U0126, and mutation of the ERK-dependent phosphorylation site (T678A) prevented complement-induced GEF-H1 activation, indicating a role for the ERK pathway. Further, complement induced GEF-H1 and microtubule accumulation in the perinuclear region. However, both the perinuclear accumulation and the activation of GEF-H1 were independent of microtubules and myosin-mediated contractility, as shown using drugs that interfere with microtubule dynamics and myosin II activity. In summary, we have identified complement-induced ERK-dependent GEF-H1 activation as the upstream mechanism of RhoA stimulation, and this pathway has a protective role against cell death.
authors.affiliation: From the Department of Medicine, McGill University Health Centre, Montreal, Quebec H3A 2B4, Canada and.
authors.forename: Flaviana
authors.initials: F
authors.lastname: Mouawad
authors.forename: Lamine
authors.initials: L
authors.lastname: Aoudjit
authors.forename: Ruihua
authors.initials: R
authors.lastname: Jiang
authors.forename: Katalin
authors.initials: K
authors.lastname: Szaszi
authors.forename: Tomoko
authors.initials: T
authors.lastname: Takano
mesh_terms: D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D015938:Complement Membrane Attack Complex / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000235:genetics / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D009218:Myosins / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D002070:Butadienes; D015938:Complement Membrane Attack Complex; D009570:Nitriles; D064067:Rho Guanine Nucleotide Exchange Factors; C113580:U 0126; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D009218:Myosins; D020742:rhoA GTP-Binding Protein
keywords: Actin; ERK; Guanine Nucleotide Exchange Factor (GEF); Podocytes; Rhoa