Nitric Oxide Decreases the Expression of Endothelin-Converting Enzyme-1 Through mRNA Destabilization

Abstract

Objective-Endothelial function depends on the equilibrium in the synthesis of vasoactive endothelial factors. It is well known that endothelin and nitric oxide (NO) exhibit reciprocal regulation. We assessed the ability of NO to regulate endothelin-converting enzyme-1 (ECE-1) expression in vascular endothelial cells. Methods and Results-Bovine aortic endothelial cells were incubated with 2 different NO donors as well as with a cyclic-GMP analog, dibutyryl-cGMP (dB-cGMP). ECE-1 protein content and mRNA expression were evaluated by Western blot and Northern blot, respectively, promoter activity by transfection experiments, ECE-1 activity by ELISA, and cGMP production by radioimmunoassay. Both NO donors decreased ECE-1 protein content, mRNA expression, and ECE-1 activity. ODQ, an inhibitor of soluble guanylyl cyclase, blocked those effects. NO donors raised cGMP levels, and dB-cGMP mimicked their effects on ECE-1 expression, which were blocked by KT5823, a nonspecific PKG inhibitor. The changes on ECE-1 expression were due to a destabilization on 3Ј-untranslated region (3Ј-UTR) of this mRNA, because the activity of a luciferase reporter construct containing the 3Ј-UTR of the ECE-1 gene was reduced by dB-cGMP in a PKG-dependent manner. The biological relevance of this regulation was confirmed in bovine aortic endothelial cells coincubated with macrophages in the presence of lipopolysaccharide, in eNOS-deficient mice, and in Wistar rats treated with NO donors. In every case, an inverse relationship was observed between NO and ECE-1 protein content. Conclusion-Our

Endothelin-Converting Enzyme-1 (ECE-1) Is Post-Transcriptionally Regulated by Alternative Polyadenylation

“…The data presented here suggest that this phenomenon is purely a function of transcript length rather than the result of elimination of specific sequences; replacement of the deleted regions with the reverse complement restored the repression of gene expression. This is at odds with the recent reports in rats, in which specific regulatory regions in the 3′UTR were identified [10], [11], and suggests that different mechanisms may exist in different cell types and/or species. Work is ongoing to identify the precise molecular mechanisms underlying the 3′UTR-induced suppression of ECE-1 expression, but our findings suggest this may be a novel mechanism responsible for ECE-1 over-expression in cancer cells and may therefore represent a novel therapeutic target.…”

“…In the course of this study it was reported that nitric oxide (NO) causes a decrease in ECE-1 expression, via destabilization of the transcript via the 3′UTR. NO acts through a cGMP-dependent mechanism and a region of the 3′UTR containing regulatory elements involved in this mechanism was identified [10]. A study of hepatic wound healing detected upregulation of ECE protein expression and identified two 3′UTR-binding proteins which cause stabilization of the transcript [11].…”

“…Previous studies have provided evidence that ECE-1 may be subject to post-transcriptional regulation in hepatic stellate cells and vascular endothelial cells [10], [11]. Post-transcriptional regulation, including the processing, export, localisation, turnover and translation of messenger RNAs (mRNAs), is increasingly recognised as a major means of altering gene expression levels [12].…”

Matrix Metalloproteinase Enhances Big-Endothelin-1 Constriction in Mesenteric Vessels of Pregnant Rats With Reduced Uterine Blood Flow

“…Although this hypothesis could explain the relative lack of contribution of MMPs to the conversion of bET-1 to ET-1 in L-NAME-treated vessels, it fails to account for the relative increase in overall constriction in the wake of NOS inhibition. NO, however, has also been shown to inhibit ECE-1 expression; 23 thus, it is possible that in the presence of intact NO, ECE is largely inhibited and the additional bET-1 is processed in RUPP animals by MMPs, which are largely absent in Sham animals. In the presence of L-NAME, the loss of NO-mediated inhibition of ECE may provide a more dominant synthesis pathway for bET-1, with a resultant lowered contribution by MMPs.…”

Pharmacological hypothesis: Nitric oxide‐induced inhibition of ADAM‐17 activity as well as vesicle release can in turn prevent the production of soluble endothelin‐converting enzyme

“…(Raoch et al. ) Their opposing effects on the vasculature have led to numerous studies examining the mechanisms behind their reciprocal regulation (Kolb‐Bachofen et al. ; Bourque et al.…”