The AMP-Activated Protein Kinase Regulates Endothelial Cell Angiotensin-Converting Enzyme Expression via p53 and the Post-Transcriptional Regulation of microRNA-143/145

Subject codes: [128] ACE/Angiotension receptors [95] Endolthelium/vascular type/nitric oxide [138] Cell signaling/ signal transduction ABSTRACT Rationale: High angiotensin-converting enzyme (ACE)-levels are associated with cardiovascular disease, but little is known about the regulation of its expression. Objective: To assess the molecular mechanisms regulating endothelial ACE expression focussing on the role of the AMP-activated protein kinase (AMPK) and miR-143/145. Methods and Results: Shear stress decreased ACE expression in cultured endothelial cells, an effect prevented by downregulating AMPKα2 but not AMPKα1. AMPKα2-/-mice expressed higher ACE levels than wild-type littermates resulting in impaired hindlimb-vasodilatation to the ACE substrate, bradykinin. The latter response was also evident in animals lacking the AMPKα2 subunit only in endothelial cells. In cultured endothelial cells miR-143/145 levels were increased by shear stress in an AMPKα2-dependent manner and miR-143/145 overexpression decreased ACE expression. The effect of shear stress was unrelated to an increase in miR-143/145 promoter activity and transcription but could be attributed to post-transcriptional regulation of precursor-miR-143/145 by AMPKα2. The AMPK substrate, p53, can enhance the post-transcriptional processing of several microRNAs, including miR-143/145. We found that shear stress elicited the AMPKα2-dependent phosphorylation of p53 (on Ser15) and that p53 downregulation prevented the shear stress-induced decrease in ACE expression. Streptozotocin-induced diabetes in mice was studied as a pathophysiological model of altered AMPK activity. Diabetes increased tissue phosphorylation of the AMPK substrates, p53 and acetyl-coenzyme A carboxylase, changes that correlated with increased miR-143/145 levels and decreased ACE-expression. Conclusions: The AMPKα2 suppresses endothelial ACE expression via the phosphorylation of p53 and upregulation of miR-143/145. Post-transcriptional regulation of miR-143/145 may contribute to the vascular complications associated with diabetes. Nonstandard Abbreviations: AMPK AMP-activated protein kinase ACE angiotensin converting enzyme Ang II Angiotensin II eNOS endothelial nitric oxide synthase KLF2 Krüppel-like factor 2 miRNA microRNA NO nitric oxide INTRODUCTION Angiotensin II (Ang II) has been implicated in the pathobiology of atherosclerosis and the arterial response to injury and restenosis via mechanisms that include vascular hypertrophy, extracellular matrix production, and cytokine induction. Although the angiotensin converting enzyme (ACE) is only one of several enzymes that can generate Ang II, 1 the enzyme has been allocated a central role in cardiovascular disease development mainly on the basis of the positive effects observed in response to ACE inhibitor therapy and experimental work involving protein downregulation. 2 Certainly, ACE levels seem to