Previously, we reported that aphidicolin acutely increases nitric oxide (NO) production in bovine aortic endothelial cells (BAECs) by increasing phosphorylation of endothelial nitric oxide synthase at serine 1179 (p-eNOS-Ser1179) and decreasing phosphorylation at serine 116 (p-eNOS-Ser116) without altering eNOS protein expression. Here, we demonstrate that prolonged treatment with aphidicolin (over 24 h) also increased NO production in BAECs. Unlike the acute effects of aphidicolin, however, prolonged treatment increased expression of eNOS protein and dimerization with elevated level of tetrahydrobiopterin, an essential cofactor for eNOS dimerization. The NOS inhibitor, Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME), completely reduced aphidicolin-induced NO production, despite no change of eNOS protein. Prolonged treatment with aphidicolin increased mRNA expression of eNOS. A promoter assay using 5′-serially deleted eNOS gene promoters (−1600 to +22; −962 to +22; −873 to +22; −428 to +22) revealed that the Tax-responsive element (TRE) site, a cAMP response element (CRE)-like site, located at -962 to -873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Ectopic expression of a dominant-negative inhibitor of CRE binding protein (CREB), A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. We also found that aphidicolin increased CREB activity, as evidenced by increased level of p-CREB-Ser133. Co-treatment with LY294002, a phosphoinositide 3-kinase inhibitor, but not H-89, an inhibitor of protein kinase A, decreased the aphidicolin-stimulated increase in p-CREB-Ser133 levels, eNOS expression, and NO production. Furthermore, ectopic expression of a dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin also increased phosphorylation of ataxia telangiectasia mutated kinase (ATM) at Ser1981 (p-ATM-Ser1981) and the knockdown of ATM using small interfering RNA (siRNA) attenuated all stimulatory effects of aphidicolin on p-Akt-Ser473, p-CREB-Ser133, eNOS expression, and NO production. Lastly, aphidicolin significantly decreased BAEC viability, which was further decreased by co-treatment with the NO scavenger, 2-phenyl4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide or the NOS inhibitor, L-NAME. Similarly, the knockdown of eNOS using siRNA further aggravated EC damage, suggesting a protective role for NO in EC death caused by aphidicolin. Taken together, our results suggest that aphidicolin increases NO production in BAECs by increasing eNOS expression via an ATM/Akt/CREB signaling cascade, which contributes to the attenuation of aphidicolin-induced EC death.
18 - 21 May 2019
European Society of Endocrinology