H ₂ O ₂ Regulation of Vascular Function Through sGC mRNA Stabilization by HuR

Abstract

Objective-Hydrogen peroxide (H 2 O 2 ) is an important mediator in the vasculature, but its role in the regulation of soluble guanylate cyclase (sGC) activity and expression is not completely understood. The aim of this study was to test the effect of H 2 O 2 on sGC expression and function and to explore the molecular mechanism involved. Methods and Results-H 2 O 2 increased sGC␤1 protein steady-state levels in rat aorta and aortic smooth muscle cells (RASMCs) in a time-and dose-dependent manner, and this effect was blocked by catalase. sGC␣2 expression increased along with ␤1 subunit, whereas ␣1 subunit remained unchanged. Vascular relaxation to an NO donor (sodium nitroprusside) was enhanced by H 2 O 2 , and it was prevented by ODQ (sGC inhibitor). cGMP production in both freshly isolated vessels and RASMCs exposed to H 2 O 2 was greatly increased after sodium nitroprusside treatment. The H 2 O 2 -dependent sGC␤1 upregulation was attributable to sGC␤1 mRNA stabilization, conditioned by the translocation of the mRNA-binding protein HuR from the nucleus to the cytosol, and the increased mRNA binding of HuR to the sGC␤1 3Ј untranslated region. HuR silencing reversed the effects of H 2 O 2 on sGC␤1 levels and cGMP synthesis. has been proposed to be an endothelium-derived hyperpolarizing factor in peripheral arteries. 12,13 H 2 O 2 may also dilate arteries by alternative mechanisms, such as the stimulation of the production of prostanoids 14 or the activation of soluble guanylate cyclase (sGC). 15 Moreover, under some circumstances, H 2 O 2 may increase the vascular content of endothelial nitric oxide synthase and to stimulate its activity, with subsequently increased synthesis of nitric oxide (NO). 16 -18 Most of cellular effects of NO depend on the interaction with its main receptor, sGC, and ROS could interact with this receptor, thus modifying the cellular response to NO. In fact, several studies have shown that cGMP levels increase during ischemia, a pathological condition characterized by increased ROS synthesis. 19,20 In addition, sGC can be modulated by ROS, modifying its activity after brief exposition to oxidants, [21][22][23] and recent work suggests that sGC expression and activity may be attenuated by ROS in rat aortic smooth muscle cells (RASMCs) 24 and ovine pulmonary artery smooth muscle cells. 25 Moreover, Courtois et al 26 proposed that a superoxide aniondependent sGC downregulation may be involved in the genesis of the hypertension induced by chronic lead administration. Conclusion-OurThe present study was devoted to analyzing the ability of ROS, particularly H 2 O 2 , to modulate the cellular content of MethodsDetails regarding materials, animal studies, RASMC isolation, and immunohistochemical studies; measurement of cGMP, RNA isolation, and RNA interference; RNA-electrophoretic mobility shift assay (RNA-EMSA); and statistics are provided in an expanded Methods section in the online Data Supplement, available at http://atvb.ahajournals.org. Vascular ReactivityThe preparation of the aortic ri...

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