Cheng T, Cao W, Wen R, Steinberg R H, LaVail M M
Department of Physiology, University of California, San Francisco 94143-0730, USA.
Invest Ophthalmol Vis Sci. 1998 Mar;39(3):581-91.
To investigate the induction of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) gene expression by prostaglandin E2 (PGE2) in cultured rat Müller cells and to study the mechanism of the induction.
Müller cells were obtained from neonatal Sprague-Dawley rat retinas and cultured in essential modified Eagle's medium supplemented with 10% fetal calf serum for up to four passages. Cells were treated with PGE2, protein kinase A (PKA) inhibitors H-89 or SQ 22536, protein kinase C (PKC) inhibitors calphostin C or GF 109203X, PKC activator phorbol 12-myristate 13-acetate (PMA), or the PKA activator forskolin. Northern blot analysis was performed to determine the levels of VEGF and bFGF mRNA.
PGE2 induced VEGF and bFGF mRNA expression in a dose- and time-dependent manner. VEGF and bFGF mRNA reached peaks of 2- and 3.5-fold at 10 microM PGE2. No further increases were observed at 100 microM PGE2. When treated with 10 microM PGE2, the increases in VEGF and bFGF mRNA reached maximum by 2 hours, then slowly declined toward the control level within 24 hours of PGE2 treatment. The inductions of VEGF and bFGF mRNA expression by PGE2 were blocked by the specific PKA inhibitors H-89 (30 microM) or SQ 22536 (500 microM, 1000 microM). Forskolin (10 microM), a cyclic adenosine monophosphate activator, also stimulated VEGF and bFGF mRNA expression. However, the effects of forskolin and PGE2 on VEGF gene expression were not additive, whereas forskolin enhanced the effect of PGE2 on bFGF mRNA expression. The specific PKC inhibitors, GF 109203X (2 microM) and calphostin C (1 microM), did not inhibit PGE2-induced VEGF gene expression, whereas PGE2-induced bFGF expression was blocked by the PKC inhibitor GF 109203X. In addition, downregulation of PKC by PMA (0.8 microM) treatment did not block the induction of VEGF gene expression, whereas it did inhibit the induction of bFGF mRNA expression.
These results indicate that PGE2 stimulates VEGF and bFGF mRNA expression in cultured rat Müller cells. The induction of VEGF seems to occur through activation of the PKA pathway, whereas that of bFGF occurs through PKA and PKC activation. These findings raise the possibility that endogenous PGE2 stimulates VEGF and bFGF mRNA expression in Müller cells in vivo under conditions in which PGE2 production is increased, such as in injury.
研究前列腺素E2（PGE2）对培养的大鼠Müller细胞中血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）基因表达的诱导作用，并探讨其诱导机制。
从新生Sprague-Dawley大鼠视网膜获取Müller细胞，在添加10%胎牛血清的改良Eagle基本培养基中培养，传代至四代。细胞分别用PGE2、蛋白激酶A（PKA）抑制剂H-89或SQ 22536、蛋白激酶C（PKC）抑制剂calphostin C或GF 109203X、PKC激活剂佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）或PKA激活剂福斯高林处理。采用Northern印迹分析确定VEGF和bFGF mRNA水平。
PGE2以剂量和时间依赖性方式诱导VEGF和bFGF mRNA表达。在10 μM PGE2时，VEGF和bFGF mRNA分别达到2倍和3.5倍的峰值。在100 μM PGE2时未观察到进一步增加。用10 μM PGE2处理时，VEGF和bFGF mRNA在2小时达到最大值，然后在PGE2处理24小时内缓慢降至对照水平。PGE2对VEGF和bFGF mRNA表达的诱导被特异性PKA抑制剂H-89（30 μM）或SQ 22536（500 μM、1000 μM）阻断。环磷酸腺苷激活剂福斯高林（10 μM）也刺激VEGF和bFGF mRNA表达。然而，福斯高林和PGE2对VEGF基因表达的作用不是相加的，而福斯高林增强了PGE2对bFGF mRNA表达的作用。特异性PKC抑制剂GF 109203X（2 μM）和calphostin C（1 μM）不抑制PGE2诱导的VEGF基因表达，而PGE2诱导的bFGF表达被PKC抑制剂GF 109203X阻断。此外，PMA（0.8 μM）处理下调PKC不阻断VEGF基因表达的诱导，而抑制bFGF mRNA表达的诱导。
这些结果表明，PGE2刺激培养的大鼠Müller细胞中VEGF和bFGF mRNA表达。VEGF的诱导似乎通过PKA途径的激活发生，而bFGF的诱导通过PKA和PKC激活发生。这些发现增加了内源性PGE2在PGE2产生增加的情况下（如损伤时）刺激体内Müller细胞中VEGF和bFGF mRNA表达的可能性。
