title: Glycogen synthase kinase-3 regulates cytoskeleton and translocation of Rac1 in long cellular extensions of human keratinocytes.
abstract: Wound keratinocytes form long cellular extensions that facilitate their migration from the wound edge into provisional matrix. We have previously shown that similar extensions can be induced by a long-term exposure to EGF or rapidly by staurosporine in cultured cells. This morphological change depends on the activity of glycogen synthase kinase-3 (GSK-3). Here, we have characterized the cytoskeletal changes involved in formation of these extended lamellipodia (E-lam) in human HaCaT keratinocytes. E-lams contained actin filaments, stable microtubules and keratin intermediate filaments. E-lam formation was prevented by cytochalasin D, colchicine and low concentrations of taxol and nocodazole, suggesting that actin and microtubule organization and dynamics are essential for E-lam formation. Staurosporine induced recruitment of filamentous actin (F-actin), cortactin, filamin, Arp2/3 complex, Rac1 GTPase and phospholipase C-gamma1 (PLC-gamma1) to lamellipodia. Treatment of cells with the GSK-3 inhibitors SB-415286 and LiCl(2) inhibited E-lam formation and prevented the accumulation of Rac1 and Arp2/3 complex at lamellipodia. The formation of E-lams was dependent on fibronectin-binding integrins and normally regulated Rac1, and expression of either dominant-negative or constitutively active forms of Rac1 prevented E-lam formation. Overexpression of either RhoA or Cdc42 GTPases suppressed E-lam formation. We conclude that extended lamellipodia formation in keratinocytes requires actin and tubulin assembly at the leading edge, and this process is regulated by Rac1 downstream of GSK-3.
authors.affiliation: Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Leeni
authors.initials: L
authors.lastname: Koivisto
authors.forename: Lari
authors.initials: L
authors.lastname: Häkkinen
authors.forename: Kazue
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Christopher A
authors.initials: CA
authors.lastname: McCulloch
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Yamada
authors.forename: Hannu
authors.initials: H
authors.lastname: Larjava
mesh_terms: D000627:Aminophenols / Q000494:pharmacology; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D018021:Lithium Chloride / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D015739:Nocodazole / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D011554:Pseudopodia / Q000737:chemistry / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: C417520:3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione; D000627:Aminophenols; D004791:Enzyme Inhibitors; D008301:Maleimides; D019384:Nucleic Acid Synthesis Inhibitors; D015638:Cytochalasin D; D038362:Glycogen Synthase Kinase 3; D020830:rac1 GTP-Binding Protein; D018021:Lithium Chloride; D019311:Staurosporine; D017239:Paclitaxel; D015739:Nocodazole; D003078:Colchicine