Growth factor induced activation of Rho and Rac GTPases and actin cytoskeletal reorganization in human lens epithelial cells

• Published in: Molecular vision Vol. 9; pp. 329 - 336
• Main Authors: Maddala, Rupalatha, Reddy, Venkat N, Epstein, David L, Rao, Vasantha
• Format: Journal Article
• Language: English
• Published: United States 17.07.2003
• Subjects: Actins - metabolism; Cell Line, Transformed; Cells, Cultured; Cytoskeleton - metabolism; Enzyme Activation; Enzyme Inhibitors - pharmacology; Epithelial Cells - drug effects; Epithelial Cells - enzymology; Fluorescent Antibody Technique, Indirect; Growth Substances - pharmacology; Humans; Lens, Crystalline - drug effects; Lens, Crystalline - enzymology; Microscopy, Confocal; Microscopy, Fluorescence; rac GTP-Binding Proteins - antagonists & inhibitors; rac GTP-Binding Proteins - metabolism; rho GTP-Binding Proteins - antagonists & inhibitors; rho GTP-Binding Proteins - metabolism; Signal Transduction
• ISSN: 1090-0535

To determine the involvement of the Rho GTPases-mediated signaling pathway in growth factor-stimulated actomyosin cytoskeletal organization and focal adhesion formation in lens epithelial cells. Serum starved human lens epithelial cells (SRA01/04) were treated with different growth factors including epidermal growth factor (EGF), basic-fibroblast growth factor (b-FGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGF-beta), insulin-like growth factor 1 (IGF-1), lysophosphatidic acid (LPA), and thrombin. Growth factor stimulated activation of Rho and Rac GTPases were evaluated by GTP-loading pull-down assays. Changes in actin cytoskeletal organization and focal adhesions were determined by fluorescence staining using FITC-phalloidin and anti-vinculin antibody/rhodamine-conjugated secondary antibody, respectively. Fluorescence images were recorded using either confocal or fluorescence microscopy. Rho GTPase activity was significantly augmented in human lens epithelial cells treated with EGF, b-FGF, TGF-beta, IGF-1, and LPA. Rac GTPase activation, in contrast, was significantly enhanced in response to only EGF or b-FGF. Serum starved human lens epithelial cells exhibited a strong increase in cortical actin stress fibers and integrin-mediated focal adhesions in response to b-FGF, PDGF, TGF-beta, thrombin, and LPA. While EGF induced a striking increase in membrane ruffling and a marginal increase on focal adhesion formation, IGF-1 had no effect on either. Pretreatment of lens epithelial cells with C3-exoenzyme (an irreversible inhibitor of Rho-GTPase), lovastatin (an isoprenylation inhibitor), or the Rho kinase inhibitor Y-27632 abolished the ability of the different growth factors to elicit actin stress fiber and focal adhesion formation. EGF induced membrane ruffling, however, was not suppressed by Y-27632 and C3-exoenzyme. These results demonstrate that different growth factors induce actin cytoskeleton reorganization and formation of cell-ECM interactions in lens epithelial cells and this response of growth factors appears to be mediated, at least in part, through the Rho/Rho kinase-mediated signaling pathway. The ability of growth factors to trigger activation of Rho and Rac GTPases along with actomyosin cytoskeletal reorganization and formation of focal adhesions might well play a crucial role in lens epithelial cell proliferation, migration, elongation and survival.