Prostaglandin E2 induces vascular endothelial growth factor and basic fibroblast growth factor mRNA expression in cultured rat Müller cells.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Cheng
T
T
Department of Physiology, University of California, San Francisco 94143-0730, USA.
Cao
W
W
Wen
R
R
Steinberg
R H
RH
LaVail
M M
MM
D000818:Animals; D000831:Animals, Newborn; D015152:Blotting, Northern; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000187:drug effects* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D008222:Lymphokines; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D016222:Fibroblast Growth Factor 2; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate