11,12-Epoxyeicosatrienoic Acid-induced Inhibition of FOXO Factors Promotes Endothelial Proliferation by Down-Regulating p27[sup Kip1].

Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. In this study, we investigated the involvement of the forkhead box, class O (FOXO) family of transcription factors and their downstream target p27[sup Kip1] in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12EET induced a time- and dose-dependent decrease in p27[sup Kip1] protein expression, whereas p21[sup Cip1] was not significantly affected. This effect on p27[sup Kip1] protein was associated with decreased mRNA levels as well as p27[sup Kip1] promoter activity, 11,12-EET also stimulated the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominantnegative or an "Akt-resistant"/constitutively active FOXO3a mutant reversed the 11,12-EET-induced downregulation of p27[sup Kip1], whereas transfection of a constitutive active Akt decreased p27[sup Kip1] expression independently of the presence or absence of 11,12-EET. To determine whether these effects are involved in EETinduced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicited endothelial cell proliferation and this effect was inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. Reducing FOXO expression using RNA interference, on the other hand, attenuated p27[sup Kip1] expression and stimulated endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt.dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27[sup Kip1]. [ABSTRACT FROM AUTHOR]