Glycogen synthase kinase-3 regulates cytoskeleton and translocation of Rac1 in long cellular extensions of human keratinocytes.
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.
Koivisto
Leeni
L
Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, Vancouver, BC, Canada.
Häkkinen
Lari
L
Matsumoto
Kazue
K
McCulloch
Christopher A
CA
Yamada
Kenneth M
KM
Larjava
Hannu
H
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*
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