Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor

A common feature of most isolated cell systems is low or undetectable level s of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKc14, that expressed an active regi o- and enantioselective arachidonic acid (AA) epoxygenase, Site-specific mu tagenesis was used to convert bacterial P450 BM-3 into an active regio- and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeico satrienoic acid (EET) production (241.82 ng/10(8) cells, >97% of total EETs ), whereas no detectable EETs were seen in cells transfected with vector al one. In F87V BM-3 cells, AA stimulated [H-3]thymidine incorporation and inc reased cell proliferation, which was blocked by the tyrosine kinase inhibit or, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wort mannin and LY294002, and by the mitogen-activated protein kinase kinase inh ibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cyt ochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increas ed EET production in F87V BM-3 cells, which was completely abolished by pre treatment with either 17-ODYA or the phospholipase A(2) (PLA(2)) inhibitor, quinacrine, Compared with vector-transfected cells, F87 BM-3 transfected c ells demonstrated marked increases in both the extent and sensitivity of DN A synthesis in response to EGF. These changes occurred in the absence of si gnificant differences in EGF receptor expression. As seen with exogenous AA , EGF increased ERK tyrosine phosphorylation to a significantly greater ext ent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in th ese control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhi bitors reduced ERK tyrosine phosphorylation to levels indistinguishable fro m that seen in cells transfected with vector alone. These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis and provide compelling evidence for the PL2-AA-EET pathway as an important intracellular-signaling pathway in cells expressing high le vels of cytochrome P450 epoxygenase.