Inhibitory effect of PGE2 on EGF-induced MAP kinase activity and rabbit corneal epithelial proliferation.
OBJECTIVE
To determine in rabbit corneal epithelial cells in culture whether epidermal growth factor (EGF)-induced increases in prostaglandin (PG) E2 production inhibit both the extracellular signal-regulated kinase 2 (Erk-2), a mitogen-activated protein kinase (MAPK), cascade activation, and the mitogenic response to this growth factor.


METHODS
Serum starvation for 24 to 36 hours was used to synchronize cultures of SV40-transformed rabbit corneal epithelial (RCE) cells. The effects of exogenous PGE2, inhibition of PGE2 synthesis, and modulation of protein kinase A (PKA) activity on EGF-induced Erk-2 activation were assessed by immunoprecipitation, kinase assays, and Western blot analysis. PGE2 synthesis was measured by using enzyme-linked immunosorbent assay. [3H]-Thymidine incorporation was used to measure RCE cell proliferation rates.


RESULTS
EGF (5 ng/ml) significantly increased PGE2 production in a time-dependent manner up to 94%+/-8% after 3 hours. EGF-induced PGE2 production was suppressed by AACOCF3, a phospholipase A2 (cPLA2) inhibitor. EGF-induced Erk-2 activation reached a maximal level at 15 minutes, followed by a decline toward the control level after 3 hours. In the presence of either PGE2 (50 microg/ml) or 8-CPT-cAMP (100 microM), the EGF-induced Erk-2 activation was lessened. PKA was activated by applications of EGF or PGE2 and suppressed by AACOCF3. On the other hand, either inhibition of PGE2 production with AACOCF3 or H-89, a PKA inhibitor, enhanced EGF-induced Erk-2 activity. Raf-1 activity was stimulated by EGF to maximal activity at 5 minutes and returned toward its control level after 60 minutes. As with the dependence of Erk-2 activity on PKA activity, in the presence of H-89, the EGF-induced Raf-1 activation was significantly enhanced. DNA synthesis was increased 59%+/-5% (n = 4) after EGF stimulation, indicating a mitogenic effect of EGF in RCE cells. Inhibition of cPLA2 activity with AACOCF3 increased DNA synthesis in RCE cells by another 64% relative to the effect of EGF alone. In contrast, with either PGE2 or 8-CPT-cAMP present the mitogenic response to EGF was totally suppressed.


CONCLUSIONS
EGF-induced increases in PGE2 production dampened the mitogenic response to this growth factor. This suppression appears to be a consequence of PGE2-elicited increases in PKA activity, which leads to inhibition of EGF-induced activation of MAPK cascades at the level of Raf-1 and further affects downstream events including Erk-2. These results indicate that the mitogenic response to EGF in vivo in the proliferating basal cell layer may be dependent on the level of its PKA activity.
Kang
S S
SS
Department of Biological Sciences, SUNY College of Optometry, New York, New York, USA.
Li
T
T
Xu
D
D
Reinach
P S
PS
Lu
L
L
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D015232:Dinoprostone / Q000096:biosynthesis / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D007546:Isoquinolines / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011817:Rabbits; D013449:Sulfonamides; D013997:Time Factors
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; C081565:arachidonyltrifluoromethane; D004815:Epidermal Growth Factor; D004247:DNA; D000242:Cyclic AMP; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D015232:Dinoprostone; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide