Retinoic acid decreases nitric oxide production in endothelial cells: A role of phosphorylation of endothelial nitric oxide synthase at Ser ¹¹⁷⁹

The effects of retinoic acid (RA) on nitric oxide (NO) production are controversial. Furthermore, it has never been studied whether these effects are mediated by direct modulation of phosphorylation of endothelial nitric oxide synthase (eNOS). Using bovine aortic endothelial cells, we found that all-trans RA (atRA) dose- and time-dependently decreased NO production without alteration in eNOS expression. This decrease was accompanied by reduction in eNOS-Ser ¹¹⁷⁹ phosphorylation. However, atRA did not alter the phosphorylation of eNOS-Ser ¹¹⁶ or eNOS-Thr ⁴⁹⁷. Concurrently, atRA also decreased the expressions of vascular endothelial growth factor (VEGF) and its receptor KDR/Flk-1, and Akt phosphorylation. Co-treatment with troglitazone, an activator of VEGF expression, reversed the atRA-induced reductions in eNOS-Ser ¹¹⁷⁹ phosphorylation and NO production, with concomitant restoration in VEGF expression. Direct treatment with VEGF also reversed these inhibitory effects, suggesting an important role for VEGF. Nonetheless, the RARα antagonist Ro 41-5253 did not block all the inhibitory effects of atRA, indicating that these inhibitory effects are not mediated by the RA response element (RARE). Thus, atRA decreases eNOS-Ser ¹¹⁷⁹ phosphorylation through a mechanism that depends on VEGF-KDR/Flk-1-mediated Akt phosphorylation but is independent of RARE, leading to reduction in NO production.