Intracellular signaling pathway of FGF-2-modulated corneal endothelial cell migration during wound healing in vitro

Abstract

After wounding, the corneal endothelium heals primarily by migration of adjacent cells into the denuded wound area. In this study, it has been attempted to identify elements of the intracellular signaling pathway activated through basic Fibroblast Growth Factor (FGF-2)- and Protein Kinase C (PKC)-modulated migration, using specific inhibitors and stimulators of second messengers in a cell culture model. Bovine corneal endothelial cells (BCEC) were grown to confluency and experiments performed with first passage cells under serum-free conditions. A central circular 'wound' was made with a specially designed trephine. In different experiments, cells were incubated with either FGF-2 (10 ng ml(-1)), pertussis toxin (PTX; 1-50 ng ml(-1)), phorbol 12-myristate 13-acetate (PMA; 50 ng ml(-1)), 2,4'-di-bromoacetophenone (DAP; 5 microM), 1-(5-iosquinolinesulphonyl)-2-methyl-piperazine dihydrochloride (H7; 10 microM), indomethacin (5 ng ml(-1)), nordihydroguaiaretic acid (NDGA; 10 ng ml(-1)), 2-(4-morpholinyl)-8-pheny-4H-1-benzopyran-4-one (LY294002; 10 microM) or different combinations of these agents. Unsupplemented cultures served as controls. Migration was quantitated by counting the cells inside the denuded area in one randomly chosen section from the wound edge 72 hr after wounding. Cell toxicity was determined with the trypan blue exclusion test. Results were statistically analysed by Student's t-test. FGF-2 and PMA (a protein kinase C activator) both stimulated migration of endothelial cells at 2.2- and 3.1-fold, respectively. The PLA(2) inhibitor DAP and the PKC inhibitor H7 both significantly reduced PMA-stimulated migration to control levels but had no effect (DAP) or even stimulated (H7) FGF-2-modulated migration. PTX did not affect FGF-2-stimulated migration. The phosphoinositol (3)-kinase inhibitor LY294002 significantly reduced FGF-2-mediated stimulation of endothelial migration similar to the rate of control cultures. LY294002 had no effect when applied together with PMA. The cyclooxygenase inhibitor indomethacin did not influence migration rates of the cells added either alone or in combination with PMA and FGF-2, respectively. The lipoxygenase inhibitor NDGA significantly reduced the number of migrating cells in cultures with no other supplements, or of those supplemented with either PMA or FGF-2. FGF-2-induced endothelial migration in vitro is not dependent on PKC/PLA(2) or pertussis-toxin sensitive G-protein pathways but rather requires activation of a phosphoinositol (3)-kinase-like enzyme and/or arachidonic acid release with subsequent liberation of lipoxygenase products. Independent of FGF-2, PKC is a major intracellular effector of corneal endothelial migration activity after wounding and stimulates migration via the PLA(2)-dependent generation of lipoxygenase metabolites.