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Inhibitory Effect of PGE₂ on EGF-Induced MAP Kinase Activity and Rabbit Corneal Epithelial Proliferation

¹ From the Department of Biological Sciences, SUNY College of Optometry, New York, New York; and the ² Department of Physiology and Biophysics, Wright State University, School of Medicine, Dayton, Ohio.

PURPOSE. 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 µg/ml) or 8-CPT–cAMP (100 µM), 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.

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