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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Maddala
Rupalatha
R
Department of Ophthalmology, Duke University Medical Center, Durham, NC, USA.
Reddy
Venkat N
VN
Epstein
David L
DL
Rao
Vasantha
V
D000199:Actins / Q000378:metabolism*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D006133:Growth Substances / Q000494:pharmacology*; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects* / Q000201:enzymology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D015398:Signal Transduction; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000199:Actins; D004791:Enzyme Inhibitors; D006133:Growth Substances; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins