Induction of basic fibroblast growth factor mRNA by basic fibroblast growth factor in Müller cells.
OBJECTIVE
To investigate the induction of basic fibroblast growth factor (bFGF) gene expression in cultured rat Müller cells by bFGF and to study the mechanism of induction.


METHODS
Müller cells from 1- to 3-day-old Sprague-Dawley rats were isolated and cultured with Dulbecco's modified Eagle's medium with 10% fetal calf serum. Cultured cells were identified by immunocytochemistry using antibodies against vimentin, carbonic anhydrase II, and glutamine synthetase. Cells of passages 1 through 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 cylase activator, forskolin; or the adenylate cyclase inhibitor, SQ22536. Northern blot analysis was performed to determine the mRNA expression of bFGF, ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF).


RESULTS
Addition of bFGF to culture medium induced bFGF gene expression in a dose- and time-dependent manner. Induction of bFCF mRNA started at a bFGF concentration of 0.1 ng/ml. The bFGF mRNA level was elevated by 2-fold at 1 ng/ml of bFGF, 2.8-fold at 5 ng/ml, and reached a peak 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) after 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 gene expression was blocked by PKC inhibitors H-7 (30 microM). The PKC activator PMA (0.1 microM) also upregulated bFGF gene expression, but the effects of bFGF and PMA were not additive. An adenylate cyclase inhibitor, SQ22536 (100 microM), did not inhibit bFGF-induced bFGF gene expression. Although forskolin (5 microM), an adenylate cyclase activator, also upregulated the level of bFGF mRNA, the effects of forskolin and bFGF were additive. In addition, no inhibitory effect on bFGF-induced expression of bFGF mRNA was found using H-89 (1 microM). Exogenous bFGF did not alter the mRNA levels of CNTF and BDNF.


CONCLUSIONS
These results indicate that bFGF induces bFGF gene expression in cultured rat Müller cells through PKC activation. The authors' findings raise the possibility that Müller cells in vivo also respond to available bFGF (for example, that released from the endogenous reservoirs in the case of injury) or to exogenous bFGF by producing more bFGF, which could in turn promote photoreceptor survival.
Cao
W
W
Department of Physiology, University of California, San Francisco 94143-0730, USA.
Wen
R
R
Li
F
F
Cheng
T
T
Steinberg
R H
RH
D000818:Animals; D015152:Blotting, Northern; D002256:Carbonic Anhydrases / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016222:Fibroblast Growth Factor 2 / Q000235:genetics* / Q000494:pharmacology*; D015870:Gene Expression; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D007150:Immunohistochemistry; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D014746:Vimentin / Q000378:metabolism
D012333:RNA, Messenger; D014746:Vimentin; D016222:Fibroblast Growth Factor 2; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002256:Carbonic Anhydrases; D005974:Glutamate-Ammonia Ligase