cyclin-d1 and 11-12-epoxy-5-8-14-eicosatrienoic-acid

Cytochrome P450 (CYP) epoxygenase products, such as 11,12-epoxyeicosatrienoic acid (EET), stimulate endothelial cell proliferation. We set out to identify the signal transduction cascade linking EET generation to enhanced proliferation and angiogenesis. In human endothelial cells overexpressing CYP 2C9, cell number was increased compared with control cells and was inhibited by the CYP 2C9 inhibitor, sulfaphenazole. CYP 2C9 overexpression was associated with the activation of Akt and an increase in cyclin D1 expression, effects that were abolished by the epidermal growth factor (EGF) receptor inhibitor, AG1478, which also prevented the CYP 2C9-induced increase in cell proliferation. Stimulation of EGF receptor overexpressing cells with 11,12-EET or transfection of these cells with CYP 2C9 enhanced the tyrosine phosphorylation of the EGF receptor. Endothelial tube formation in a fibrin gel was significantly enhanced (6-fold) in CYP 2C9 overexpressing cells and was comparable with the tube formation induced by EGF. In the chick chorioallantoic membrane, 11,12-EET stimulated vessel formation (3.5-fold) and induced vessel convergence, an effect that was abolished by cotreatment with either an EGF receptor-neutralizing antibody or AG1478. These results indicate that CYP 2C9-derived EETs stimulate angiogenesis by a mechanism involving the activation of the EGF receptor.

Topics: 8,11,14-Eicosatrienoic Acid; Allantois; Animals; Aryl Hydrocarbon Hydroxylases; Cell Division; Cell Line; Chick Embryo; Chorion; Cyclin D1; Cytochrome P-450 CYP2C9; Endothelium, Vascular; Enzyme Inhibitors; ErbB Receptors; Humans; Neovascularization, Physiologic; Phosphorylation; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor Cross-Talk; Tyrphostins

Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) are important modulators of endothelial cell homeostasis. We investigated the signaling pathway linking the activation of CYP 2C9 to enhanced endothelial cell proliferation. Overexpression of CYP 2C9 in cultured human endothelial cells markedly increased proliferation. This effect was paralleled by an up-regulation of the G(1) phase regulatory protein, cyclin D1. The specific CYP 2C9 inhibitor, sulfaphenazole, prevented both the enhanced cell proliferation and up-regulation of cyclin D1. CYP 2C9 overexpression also decreased the activity of the c-Jun N-terminal kinase (JNK). Coexpression of wild type JNK with CYP 2C9 attenuated the CYP 2C9-induced increase in cyclin D1 expression and abolished the CYP 2C9-induced proliferation response. In contrast, cotransfecting dominant negative JNK with CYP 2C9 restored the CYP 2C9-mediated up-regulation of cyclin D1 and proliferation. The inactivation of JNK is linked to its dephosphorylation by dual specificity mitogen-activated protein (MAP) kinase phosphatases (MKPs). Overexpression of CYP 2C9 significantly increased the expression of MKP-1, as did incubation with 11,12-EET. These data demonstrate that the mitogenic effect of CYP 2C9 is due to the generation of EETs, which promote the MKP-1-mediated dephosphorylation and inactivation of JNK, effects ultimately culminating in the expression of cyclin D1 and endothelial cell proliferation.

Topics: 8,11,14-Eicosatrienoic Acid; Aryl Hydrocarbon Hydroxylases; Cell Cycle Proteins; Cell Division; Cells, Cultured; Culture Media, Serum-Free; Cyclin D1; Cytochrome P-450 Enzyme System; Dual Specificity Phosphatase 1; Endothelium, Vascular; Enzyme Induction; Gene Expression Regulation; Humans; Hydrogen Peroxide; Immediate-Early Proteins; JNK Mitogen-Activated Protein Kinases; Kinetics; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Steroid 16-alpha-Hydroxylase; Steroid Hydroxylases; Sulfaphenazole; Time Factors; Transfection; Umbilical Veins