Nitric oxide production and regulation of endothelial nitric-oxide synthase phosphorylation by prolonged treatment with troglitazone: Evidence for involvement of peroxisome proliferator-activated receptor (PPAR) γ-dependent and PPARγ-independent signaling pathways

Recently, peroxisome proliferator-activated receptor γ (PPARγ) ligands have been reported to increase endothelial NO, but the signaling mechanisms involved are unknown. Using troglitazone, a PPARγ ligand known as an antidiabetic compound, we investigated the molecular mechanism of its effect on NO production in bovine aortic endothelial cells. Troglitazone increased endothelial NO production in a dose- and time-dependent manner with no alteration in endothelial nitric-oxide synthase (eNOS) expression. The maximal increase (∼3.1-fold) was achieved with 20 μM troglitazone treatment for 12 h, and this increase was accompanied by increases in the expression of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, and in Akt phosphorylation. Analysis with antibodies specific for each phosphorylated site demonstrated that troglitazone (20 μM treatment for 12 h) significantly increased both the phosphorylation of Ser¹¹⁷⁹ of eNOS (eNOS-Ser¹¹⁷⁹) and the dephosphorylation of eNOS-Ser ¹¹⁶ but did not alter eNOS-Thr⁴⁹⁷ phosphorylation. Treatment with anti-VEGF antibody to scavenge the increased VEGF induced by troglitazone partially inhibited troglitazone-stimulated NO production. This was accompanied by the attenuation of troglitazone-stimulated increases in the phosphorylation of Akt and eNOS-Ser¹¹⁷⁹ with no alteration in eNOS-Ser¹¹⁶ dephosphorylation. We also found that bisphenol A diglycidyl ether, a PPARγ antagonist, partially inhibited troglitazone-stimulated NO production with a concomitant reduction in VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser¹¹⁷⁹ phosphorylation but with no alteration in eNOS-Ser¹¹⁶ dephosphorylation induced by troglitazone. Taken together, our results demonstrate that prolonged treatment with troglitazone increases endothelial NO production by at least two independent signaling pathways: PPARγ-dependent, VEGF-KDR/Flk-1-Akt-mediated eNOS-Ser¹¹⁷⁹ phosphorylation and PPARγ-independent, eNOS-Ser¹¹⁶ dephosphorylation.