Cao W, Li F, Steinberg R H, LaVail M M
Department of Physiology, University of California, San Francisco, USA.
Invest Ophthalmol Vis Sci. 1998 Mar;39(3):565-73.
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.
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.
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.
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.
外源性碱性成纤维细胞生长因子（bFGF）可诱导培养的大鼠Müller细胞中bFGF基因表达。为阐明外源性bFGF与这些细胞中bFGF基因表达转录调控之间的联系机制，作者检测了原癌基因c-fos和c-jun在培养的大鼠Müller细胞中对外源性bFGF的mRNA表达情况。
从1至3日龄的Sprague-Dawley大鼠分离出Müller细胞，并在改良的Eagle基本培养基+10%胎牛血清中培养。使用抗波形蛋白、碳酸酐酶C和谷氨酰胺合成酶的抗体，通过免疫细胞化学分析鉴定培养的细胞。对第1至4代细胞用bFGF（0.01、0.1、1、10和100 ng/ml）、蛋白激酶C（PKC）抑制剂H-7（30 μM）和GF109203X（1 μM）或PKC激活剂佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA；1、10、100、500 nM），以及腺苷酸环化酶激活剂福斯可林（5 μM）或腺苷酸环化酶抑制剂SQ22536（100 μM）进行处理。进行Northern印迹分析以确定c-fos、c-jun和bFGF的mRNA表达情况。
向培养基中添加bFGF以剂量和时间依赖性方式诱导c-fos和c-jun mRNA表达。在以10 ng/ml剂量暴露于bFGF后10分钟（9.6倍）即可观察到c-fos mRNA诱导。到30分钟时达到最大值17.4倍。在bFGF处理45分钟后观察到c-fos mRNA水平迅速下降。c-jun基因表达的时间模式与c-fos相似，而在处理45分钟后观察到c-jun mRNA的最大诱导（8.2倍）。c-fos和c-jun基因表达的诱导在bFGF浓度为0.1 ng/ml时开始。在10 ng/ml时，c-fos mRNA达到峰值水平15倍，c-jun mRNA达到7.6倍。PKC激活剂PMA也观察到对c-fos和c-jun基因表达的剂量依赖性上调。在100 nM PMA时观察到最大诱导。bFGF或PMA对c-fos和c-jun基因表达的诱导被PKC抑制剂H-7（30 μM）或GF109203X（1 μM）阻断。腺苷酸环化酶抑制剂SQ22536（100 μM）不抑制bFGF诱导的c-fos和c-jun基因表达，而腺苷酸环化酶激活剂福斯可林（5 μM）上调其表达。
这些结果表明，外源性bFGF通过PKC激活在培养的大鼠Müller细胞中诱导c-fos和c-jun基因表达。原癌基因c-fos和c-jun可能在视网膜Müller细胞中对外源性bFGF的bFGF基因表达调控中发挥作用。这些发现为Müller细胞和外源性bFGF在预防光感受器变性中的作用提供了进一步的见解。
