Inhibitory effect of PGE(2) on EGF-induced MAP kinase activity and rabbit corneal epithelial proliferation

PURPOSE. TO determine in rabbit corneal epithelial cells in culture whether epidermal growth factor (EGF)-induced increases in prostaglandin (PG) E-2 production inhibit both the extracellular signal-regulated kinase 2 (Erk-2) , a mitogen-activated protein kinase (MAPK), cascade activation, and the mi togenic response to this growth factor. METHODS. Serum starvation for 24 to 36 hours was used to synchronize cultur es of SV40-transformed rabbit corneal epithelial (RCE) cells. The effects o f exogenous PGE(2), inhibition of PGE(2) synthesis, and modulation of prote in kinase A (PKA) activity on EGF-induced Erk-2 activation were assessed by immunoprecipitation, kinase assays, and Western blot analysis. PGE(2) synt hesis was measured by using enzyme-linked immunosorbent assay. [H-3]-Thymid ine incorporation was used to measure RCE cell proliferation rates. RESULTS. EGF (5 ng/ml) significantly increased PGE(2) production in a time- dependent manner up to 94% +/- 8% after 3 hours. EGF-induced PGE(2) product ion was suppressed by AACOCF3, a phospholipase A2 (cPLA2) inhibitor. EGF-in duced 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 PGE(2) (50 mu g/ml) or 8-CPT-cAMP (100 mu M), the EGF-induced Erk-2 activat ion was lessened. PKA was activated by applications of EGF or PGE(2) and su ppressed by AACOCF3. On the other hand, either inhibition of PGE(2) product ion with AACOCF3 or H-89, a PKA inhibitor, enhanced EGF-induced Erk-2 activ ity. 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 depende nce of Erk-2 activity on PKA. activity, in the presence of H-89, the EGF-in duced Raf-1 activation was significantly enhanced. DNA synthesis was increa sed 59% +/- 5% (n = 4) after EGF stimulation, indicating a mitogenic effect of EGF in RCE cells. Inhibition of cPLA2 activity with AACOCF3 increased D NA synthesis in RCE cells by another 64% relative to the effect of EGF alon e. In contrast, with either PGE(2) or 8-CPT-cAMP present the mitogenic resp onse to EGF was totally suppressed. CONCLUSIONS. EGF-induced increases in PGE(2) production dampened the mitoge nic response to this growth factor. This suppression appears to be a conseq uence of PGE(2)-elicited increases in PKA activity, which leads to inhibiti on of EGF-induced activation of MAPK cascades at the level of Raf-1 and fur ther affects downstream events including Erk-2. These results indicate that the mitogenic response to EGF in vivo in the proliferating basal cell laye r may be dependent on the level of its PKA activity.