Cytochrome P450 epoxygenase metabolism of arachidonic acid inhibits apoptosis

The ubiquitous cytochrome P450 hemoproteins play important functional roles in the metabolism and detoxification of foreign chemicals. However, other than established roles in cholesterol catabolism and steroid hormone biosyn thesis, their cellular and/or organ physiological functions remain to be fu lly characterized. Here we show that the cytochrome P450 epoxygenase arachi donic acid metabolite 14,15-epxyeicosatrienoic acid (14,15-EET) inhibits ap optosis induced by serum withdrawal, H2O2, etoposide, or excess free arachi donic acid (AA), as determined by DNA laddering, Hoechst staining, and fluo rescein isothiocyanate-labeled annexin V binding. In the stable transfectan ts (BM3 cells) expressing a mutant bacterial P450 AA epoxygenase, F87V BM3, which was genetically engineered to metabolize arachidonic acid only to 14 ,15-EET, AA did not induce apoptosis and protected against agonist-induced apoptosis. Ceramide assays demonstrated increased AA-induced ceramide produ ction within 1 h and elevated ceramide levels for up to 48 h, the longest t ime tested, in empty-vector-transfected cells (Vector cells) but not in BM3 cells. Inhibition of cytochrome P450 activity by 17-octadecynoic acid rest ored AA-induced ceramide production in BM3 cells. Exogenous C2-ceramide mar kedly increased apoptosis in quiescent Vector cells as well as BM3 cells, a nd apoptosis was prevented by pretreatment of Vector cells with exogenous 1 4,15-EET and by pretreatment of BM3 cells with AA. The ceramide synthase in hibitor famonisin BI did not affect AA-induced ceramide production and apop tosis; in contrast, these effects of AA were blocked by the neutral sphingo myelinase inhibitor scyphostatin. The pancaspase inhibitor Z-VAD-fmk had no effect on AA-induced ceramide generation but abolished AA-induced apoptosi s. The antiapoptotic effects of 14,15-EET were blocked by two mechanistical ly and structurally distinct phosphatidylinositol-3 (PI-3) kinase inhibitor s, wortmannin and LY294002, but not by the specific mitogen-activated prote in kinase kinase inhibitor PD98059. Immunoprecipitation followed by an in v itro kinase assay revealed activation of Akt kinase within 10 min after 14, 15-EET addition, which was completely abolished by either wortmannin or LY2 94002 pretreatment. In summary, the present studies demonstrated that 14,15 -EET inhibits apoptosis by activation of a PI-3 kinase-Akt signaling pathwa y. Furthermore, cytochrome P450 epoxygenase promotes cell survival both by production of 14,15-EET and by metabolism of unesterified AA, thereby preve nting activation of the neutral sphingomyelinase pathway and proapoptotic c eramide formation.