INDUCTION OF BASIC FIBROBLAST GROWTH-FACTOR MESSENGER-RNA BY BASIC FIBROBLAST GROWTH-FACTOR IN MULLER CELLS

Purpose. To investigate the induction of basic fibroblast growth facto r (bFGF) gene expression in cultured rat Mailer cells by bFGF and to s tudy the mechanism of induction. Methods. Muller cells from 1- to 3-da y-old Sprague-Dawley rats were isolated and cultured with Dulbecco's m odified Eagle's medium with 10% fetal calf serum. Cultured cells were identified by immunocytochemistry using antibodies against vimentin, c arbonic anhydrase II, and glutamine synthetase. Cells of passages 1 th rough 4 were treated with bFGF,the protein kinase C (PKC) inhibitor, H -7; calphostin C, or the PKC activator, PMA; and protein kinase A (PKA ) inhibitor, H-89; as well as the adenylate cyclase activator, forskol in; or the adenylate cyclase inhibitor, SQ22536. Northern blot analysi s was performed to determine the mRNA expression of bFGF, ciliary neur otrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF). R esults. Addition of bFGF to culture medium induced bFGF gene expressio n in a dose- and time-dependent manner. Induction of bFGF mRNA started at a bFGF concentration of 0.1 ng/ml. The bFGF mRNA level was elevate d by 2-fold at 1 ng/ml of bFGF, 2.8-fold at 5 ng/ml, and reached a pea k of 4-fold at 10 ng/ml and 3.7-fold at 50 ng/ml. At 10 ng/ml of bFGF, induction of bFGF mRNA was observed as early as 2 hours (2-fold) afte r treatment. The bFGF mRNA level continued to increase to 3.7-fold by 4 hours, and reached a maximum of 4.4-fold by 8 hours. A slow decline of the bFGF mRNA level was observed after 8 hours of bFGF treatment (3 .5-fold by 12 hours, and 3-fold by 24 hours). This induction of bFGF g ene expression was blocked by PKC inhibitors H-7 (30 mu M) and calphos tin C (1 mu M). The PKC activator PMA (0.1 mu M) also upregulated bFGF gene expression, but the effects of bFGF and PMA were not additive. A n adenylate cyclase inhibitor, SQ22536 (100 mu M), did not inhibit bFG F-induced bFGF gene expression. Although forskolin (5 mu M), an adenyl ate cyclase activator, also upregulated the level of bFGF mRNA, the ef fects of forskolin and bFGF were additive. In addition, no inhibitory effect on bFGF-induced expression of bFGF mRNA was found using H-89 (1 mu M). Exogenous bFGF did not alter the mRNA levels of CNTF and BDNF. Conclusions. These results indicate that bFGF induces bFGF gene expre ssion in cultured rat Mailer cells through PKC activation. The authors ' findings raise the possibility that Mailer cells in vivo also respon d to available bFGF (for example, that released from the endogenous re servoirs in the case of injury) or to exogenous bFGF by producing more bFGF, which could in turn promote photoreceptor survival.