11,12-Epoxyeicosatrienoic Acid-induced Inhibition of FOXO Factors Promotes Endothelial Proliferation by Down-Regulating p27

Abstract

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 p27Kip1 in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induced a time-and dose-dependent decrease in p27Kip1 protein expression, whereas p21 Cip1 was not significantly affected. This effect on p27Kip1 protein was associated with decreased mRNA levels as well as p27Kip1 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 p27Kip1 , whereas transfection of a constitutive active Akt decreased p27Kip1 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 p27Kip1 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 p27Kip1 .The epoxyeicosatrienoic acids (EETs) 1 are biologically active eicosanoids generated by cytochrome P450 epoxygenases (1, 2). Epoxygenases of the CYP2B, -2C, and -2J subfamilies are expressed in vascular endothelial cells and metabolize arachidonic acid into a series of regio-and stereo-selective EETs (5,8,11,and 14, that are potent vasodilators and have been identified as endothelium-derived hyperpolarizing factors (3-5).Recent reports have shown that EETs exert multiple effects on the vascular wall that are not directly related to changes in membrane potential or vascular tone. Indeed, EETs seem to act as second messengers in numerous signaling pathways (6, 7) and are involved in the regulation of inflammation, migration, apoptosis, hypoxia-reoxygenation injury, and platelet aggregation (8 -13). Moreover, we and others have recently been able to demonstrate that EETs stimulate endothelial cell proliferation and elicit an angiogenic response (14 -16).Progression through the mammalian cell cycle requires the activation of cyclin-dependent kinases (CDKs) through association with regulatory subunits (cyclins) that phosphorylate the retinoblastoma ...

Characterization of 5,6- and 8,9-Epoxyeicosatrienoic Acids (5,6- and 8,9-EET) as Potent in Vivo Angiogenic Lipids

“…In agreement with our data, treatment of human endothelial cells with 11,12-EET leads to transient Akt activation (38), and this event seems to be related to the ability of this eicosanoid to induce EGF receptor phosphorylation (10). Moreover, treatment of bovine artery endothelial cells with 14,15-EET activates ERK1/2, which then stimulates the phosphorylation and activation of endothelial nitric-oxide synthase (39).…”

“…Our results suggest that these eicosanoids are capable of stimulating the same cell function by activating quite different cellular pathways. In this regard, it has been shown that the ability of 11,12-EET to induce endothelial cell proliferation results from PI3K/Akt-mediated down-regulation of the cyclin-dependent kinase inhibitor p27 kip1 (38). Moreover, studies with CYP2C9-transfected endothelial cells suggested that the mitogenic activity of 11,12-EET, one of the EETs formed by the enzyme (18), results from activation of the EGF receptor and subsequent PI3K/Akt-mediated activation (10).…”

AKT Pathway is Activated by 1,25-Dihydroxyvitamin D3 and Participates in its Anti-Apoptotic Effect and Cell Cycle Control in Differentiating HL60 Cells

“…2B), an event that would be expected to decrease the p27Kip1 expression. 40,41 Indeed, there is also the question why the PI3K/AKT pathway, generally considered to be mitogenic, 42,43 is activated by 1,25D. We have experimentally confirmed that the downstream, pro-mitogenic targets of PI3K/AKT, GSK3β and p70S6K, are phosphorylated in concert with AKT (Fig.…”

Molecular Determinants of Cardiac KATP Channel Activation by Epoxyeicosatrienoic Acids

“…EETs are known to regulate vital physiological functions including vasoreactivity (23,24), inflammation (25), and cell proliferation (26,27). EETs have been considered to be candidates of endothelium-derived hyperpolarizing factors in human coronary vessel bed (23,28).…”