Fibroblast growth factor 2 uses PLC-gamma1 for cell proliferation and PI3-kinase for alteration of cell shape and cell proliferation in corneal endothelial cells.

PURPOSE

Fibroblast growth factor 2 (FGF-2) induces endothelial-mesenchymal modulation in corneal endothelial cells, including stimulation of cell proliferation and cell shape change and induction of fibrillar collagen. In the present study, we investigated whether FGF-2 uses distinct signaling pathways for individual biological activities.

METHODS

Specific metabolic inhibitors were used to block cell proliferation, while reversion of cellular morphology (modulated with FGF-2) was determined using specific antibodies and inhibitors. Immunocytochemical analysis was performed to identify any changes observed in the cytoskeleton in relation to cell shape. Association of cytoskeleton molecules with phosphatidylinositol 3-kinase was determined using co-precipitation. Cell proliferation was assayed using a colorimetric method for determining the number of viable cells.

RESULTS

The fibroblastic morphology induced by FGF-2 reverted to a polygonal shape in cells treated with anti-FGF-2 antibody, anti-phosphatidylinositol 3-kinase antibody, LY294002, and genistein, while anti-phospholipase C gamma1 antibody did not to reverse the modulated cell morphology. Cell proliferation mediated by FGF-2 was blocked by metabolic inhibitors (genistein, LY294002 and wortmannin); genistein inhibited FGF-mediated cell proliferation in a dose-response manner and had a maximum inhibition of 80% at 100 microM, while inhibitors of phosphatidylinositol 3-kinase had less inhibitory effect than did genistein. When cytoskeleton proteins were examined, the characteristic punctated staining profiles of vinculin observed in normal cells were maintained in fibroblastic corneal endothelial cells treated with FGF-2. The inhibitors that cause reversion of cell shape also demonstrated the punctated staining potential. Likewise, the staining profiles of alpha-actinin and smooth muscle alpha-actin were not altered, regardless of cell shape. Filamentous actin and alpha-actinin were co-localized to the cytoskeleton and phosphatidylinositol 3-kinase was associated with the cytoskeleton, regardless of cell shape.

CONCLUSIONS

These findings indicate that FGF-2 uses distinct and/or dual signaling pathways for individual biological activities.