Induction of c-fos and c-jun mRNA expression by basic fibroblast growth factor in cultured rat Müller cells

Abstract

Purpose: Exogenous basic fibroblast growth factor (bFGF) induces bFGF gene expression in cultured rat Müller cells. To elucidate the mechanism that links exogenous bFGF to transcriptional regulation of bFGF gene expression in these cells, the authors examined mRNA expression of the proto-oncogenes c-fos and c-jun in response to exogenous bFGF in cultured rat Müller cells.

Methods: Müller cells from 1- to 3-day-old Sprague-Dawley rats were isolated and cultured in essential modified Eagle's medium + 10% fetal calf serum. Cultured cells were identified by immunocytochemical analysis using antibodies against vimentin, carbonic anhydrase C, and glutamine synthetase. Cells of passages 1 through 4 were treated with bFGF (0.01, 0.1, 1, 10, and 100 ng/ml), either the protein kinase C (PKC) inhibitors H-7 (30 microM) and GF109203X (1 microM) or the PKC activator phorbol 12-myristate 13-acetate (PMA; 1, 10, 100, 500 nM), and either adenylate cyclase activator forskolin (5 microM) or adenylate cyclase inhibitor SQ22536 (100 microM). Northern blot analysis was performed to determine the mRNA expression of c-fos, c-jun, and bFGF.

Results: Addition of bFGF to culture medium induced c-fos and c-jun mRNA expression in a dose- and time-dependent manner. Induction of c-fos mRNA was observed as early as 10 minutes (9.6-fold) after exposure to bFGF at a dose of 10 ng/ml. It reached a maximum of 17.4-fold by 30 minutes. A rapid decline of c-fos mRNA level was observed after 45 minutes of bFGF treatment. The temporal pattern of c-jun gene expression was similar to that of c-fos, whereas a maximum induction of c-jun mRNA (8.2-fold) was seen after 45 minutes of treatment. Induction of c-fos and c-jun gene expression started at a bFGF concentration of 0.1 ng/ml. It reached peak levels of 15-fold for c-fos and 7.6-fold for c-jun mRNA at 10 ng/ml. A dose-dependent upregulation of c-fos and c-jun gene expression by the PKC activator PMA was also observed. A maximum induction was seen at 100 nM PMA. The induction of c-fos and c-jun gene expression by bFGF or by PMA was blocked by the PKC inhibitors H-7 (30 microM) or GF109203X (1 microM). SQ22536 (100 microM), an adenylate cyclase inhibitor, did not inhibit bFGF-induced c-fos and c-jun gene expression, whereas forskolin (5 microM), an adenylate cyclase activator, upregulated the expression.

Conclusions: These results indicate that exogenous bFGF induces c-fos and c-jun gene expression in cultured rat Müller cells through PKC activation. The proto-oncogenes c-fos and c-jun may play a role in the regulation of bFGF gene expression in response to exogenous bFGF in retinal Müller cells. These findings provide further insight into the roles of Müller cells and exogenous bFGF in protecting against photoreceptor degeneration.