Abstract 5679: The Critical Role of p90 Ribosomal S6 Kinase (p90RSK) in Endothelial eNOS Expression is Mediated by ERK5 Transactivation via Its SUMOylation and Phosphorylation
Emerging evidence shows the reduction of vascular reactivity in diabetic patients. We have reported that advanced glycated end products and reactive oxygen species inhibit laminar steady flow (s-flow) mediated-eNOS expression in endothelial cells (EC) via ERK5-SUMOylation, which subsequently inhibits ERK5 and KLF2 transactivation. However, it remains unknown how ERK5-SUMOylation is regulated in EC. In this study we investigated the involvement of redox sensitive kinase p90RSK on ERK5 activation and eNOS induction. Inhibition of ERK5 transactivation by H2O2 was reversed by dominant negative p90RSK (21±7%-increase compared with H2O2 treated cells, p<0.05), indicating that this inhibition is p90RSK-dependent. Transduction of an adenovirus vector expressing DN-p90RSK (Ad-DN-p90RSK) abolished H2O2-mediated inhibition of s-flow-mediated eNOS expression. Ad-DN-p90RSK significantly inhibited H2O2-mediated ERK5-SUMOylation, suggesting that p90RSK inhibits ERK5 by increasing ERK5-SUMOylation. To elucidate how p90RSK increases ERK5-SUMOylation, we investigated the effect of de-SUMOylation enzyme SENP2, which contains potential p90RSK phosphorylation sites. Depletion of SENP2 by SENP2 siRNA increased ERK5-SUMOylation. Over-expression of SENP2 inhibited ERK5-SUMOylation in a dose-dependent manner, suggesting that ERK5 is a substrate for SENP2. An in vitro kinase assay revealed that active p90RSK phosphorylated SENP2, suggesting SENP2 as a novel substrate of p90RSK. In addition, H2O2 induced SENP2 nuclear export, which was inhibited by Ad-DN-p90RSK. These data suggest that p90RSK inactivates SENP2 by phosphorylating it, thus increasing ERK5-SUMOylation and decreasing ERK5/KLF2 transactivation and eNOS expression. En face confocal microscopy of normal mouse aorta showed p90RSK localization in the perinuclear region in EC. Interestingly, while p-p90RSK exhibited nuclear localization in s-flow areas, p-p90RSK localized in the cytoplasm of EC in disturbed flow (d-flow) areas which are prone to atherosclerosis. ApoE−/− mice revealed increased p90RSK activation in d-flow areas and early atherosclerotic lesions, also supporting the patho-physiological role of the p90RSK-SENP2 axis on EC dysfunction.
This research has received full or partial funding support from the American Heart Association, National Center.