Abstract
We investigated the molecular mechanism of cyclic GMP-induced down-regulation of soluble guanylyl cyclase expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an allosteric activator of this enzyme, decreased the expression of soluble guanylyl cyclase alpha(1) subunit mRNA and protein. This effect was blocked by the enzyme inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase alpha(1) mRNA-degrading activity was increased in protein extracts from YC-1-exposed aorta and was attenuated by pretreatment with actinomycin D and NS2028. Gelshift and supershift analyses using an adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region of the alpha(1) mRNA and a monoclonal antibody directed against the mRNA-stabilizing protein HuR revealed HuR mRNA binding activity in aortic extracts, which was absent in extracts from YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this decrease was prevented by NS2028. Similarly, down-regulation of HuR by RNA interference in cultured rat aortic smooth muscle cells decreased alpha(1) mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha(1) mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha(1) mRNA and lowering alpha(1) subunit expression as a negative feedback response.
Highlights
The hemoprotein soluble guanylyl cyclase1 is the predominant intracellular nitric oxide (NO) receptor in vascular smooth muscle cells [1]
Influence of YC-1 on the Expression of soluble guanylyl cyclase (sGC)␣1 mRNA and Protein in Rat Aorta—To assess the effect of increased cGMP formation on the sGC␣1 subunit expression in rat aorta, freshly isolated endothelium-denuded aortic rings from Wistar Kyoto rats were kept under organ culture conditions, either in the absence or presence of the sGC activator molecule YC-1 (10 M), and the specific sGC inhibitor NS2028 (10 M)
According to densitometric analysis of the reverse transcriptase-polymerase chain reaction (RT-PCR) product (Fig. 1A) and the immunoreactive protein (␣1 ϭ 82 kDa, Fig. 1B) the abundance of sGC␣1 subunit mRNA and protein was markedly lower in YC-1-exposed aorta compared with controls
Summary
The hemoprotein soluble guanylyl cyclase (sGC) is the predominant intracellular nitric oxide (NO) receptor in vascular smooth muscle cells [1]. An up-regulation of sGC expression was found in aortic tissue from nitroglycerin-tolerant rats [5] and from rats suffering from chronic heart failure [6], despite diminished vasodilator responses to NO This apparently discrepant finding indicates that altered sGC expression does not necessarily translate into predictable changes in cGMP-dependent functional responses, but that other mechanisms, such as altered NO bio-availability, may overrun the influence of altered sGC expression. There is evidence that expression of sGC is controlled by second messenger cyclic nucleotides via a post-transcriptional mechanism: in various cells cyclic AMP-eliciting agonists decrease the expression of sGC mRNA and protein [7, 8] by a destabilization of the sGC mRNA This effect is mimicked by activation of the cGMP signaling pathway, e.g. application of NO donors, stimulation of particulate guanyl cyclase by atrial natriuretic factor, and stimulation of cGMP-dependent protein kinase by the stable cGMP-analogue 8-chlorophenylthio-cGMP [9]. We observed that the elav family protein HuR [10] stabilizes the sGC␣1 mRNA by binding to AU-rich elements (ARE) in its 3Ј-untranslated region (UTR), and that an increase in intracellular cGMP strongly decreases HuR expression and sGC␣1 mRNA binding activity, leading to accelerated mRNA degradation
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call