Various bioactive metabolites of arachidonic acid have been implicated in the mitogenic response of cultured cells to stimulation by epidermal growth factor (EGF). Among the earliest events in the proliferative response to EGF is the transient increase in the expression of proto- oncogenes which regulate the expression of genes required for cell cycle progression and DNA synthesis. Our laboratory has demonstrated that neonatal rat lens epithelium has a high 12(S)-hydroxyeicosatetraenoic acid 12(S)-HETE synthetic capacity, which decreases as epithelial cell proliferation decreases with age. Recent work has demonstrated that inhibitors of 12(S)-HETE synthesis decreased the EGF-stimulated proto-oncogene expression and reduced DNA synthesis in cultured rat lens epithelial cells. Exogenous 12(S)-HETE, but not 5(S)-HETE or 15(S)-HETE reversed the inhibitor's effect and restored proto-oncogene expression and DNA synthesis. These results suggest that endogenously synthesized l2(S)-HETE mediates the EGF response on proto-oncogene expression and DNA synthesis in rat lens epithelial cells. The proposed studies will gain an understanding of the molecular mechanism(s) involving the modulation of c-myc, c-fos and c-jun protooncogene expression and DNA synthesis by 12(S)-HETE in the lens epithelium. Proto-oncogene transcriptional regulation will be assessed using Northern blot and nuclear run-on assays to determine the impact of 12(S)-HETE or 12-lipoxygenase inhibitors. Inhibitors of 12(S)-HETE synthesis and other hydroxy-fatty acids will bee tested with other lens- active growth factors, such as fibroblast growth factor (FGF) to determine if synthesis of 12(S)-HETE modulates their DNA synthesis and proto-oncogene expression. These studies will advance our understanding of what intracellular signals regulate normal lens growth and address how lipid mediators and growth factors interact in the proliferative response.
