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The angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system, which is involved in the regulation of blood pressure. Alterations in ACE expression or activity are associated with various pathological phenotypes, particularly cardiovascular diseases. In human endothelial cells, ACE was shown to be negatively regulated by tumor necrosis factor (TNF) α. To examine, whether or not, epigenetic factors were involved in ACE expression regulation, methylated DNA immunoprecipitation and RNA interference experiments directed against regulators of DNA methylation homeostasis i.e., DNA methyltransferases (DNMTs) and ten-eleven translocation methylcytosine dioxygenases (TETs), were performed. TNFα stimulation enhanced DNA methylation in two distinct regions within the ACE promoter via a mechanism linked to DNMT3a and DNMT3b, but not to DNMT1. At the same time, TET1 protein expression was downregulated. In addition, DNA methylation decreased the binding affinity of the transcription factor MYC associated factor X to the ACE promoter. In conclusion, DNA methylation determines the TNFα-dependent regulation of ACE gene transcription and thus protein expression in human endothelial cells., Competing Interests: The authors have declared that no competing interests exist.

In addition to being a peptidase, the angiotensin-converting enzyme (ACE) can be phosphorylated and involved in signal transduction. We evaluated the role of ACE in granulocyte-colony-stimulating factor (G-CSF)-induced hematopoietic progenitor cell (HPC) mobilization and detected a significant increase in mice-lacking ACE. Transplantation experiments revealed that the loss of ACE in the HPC microenvironment rather than in the HPCs increased mobilization. Indeed, although ACE was expressed by a small population of bone-marrow cells, it was more strongly expressed by endosteal bone. Interestingly, there was a physical association of ACE with the G-CSF receptor (CD114), and G-CSF elicited ACE phosphorylation on Ser1270 in vivo and in vitro. A transgenic mouse expressing a non-phosphorylatable ACE (ACE S/A ) mutant demonstrated increased G-CSF-induced HPC mobilization and decreased G-CSF-induced phosphorylation of STAT3 and STAT5. These results indicate that ACE expression/phosphorylation in the bone-marrow niche interface negatively regulates G-CSF-induced signaling and HPC mobilization.

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 focusing 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 mellitus in mice was studied as a pathophysiological model of altered AMPK activity. Diabetes mellitus 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: 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 mellitus.

Human monocytes/macrophages express the angiotensin-converting enzyme (ACE) but nothing is known about its role under physiological conditions. As adipose tissue contains resident macrophages that have been implicated in the generation of insulin resistance in expanding fat mass, we determined whether adipocytes release factors that affect ACE expression and function in monocytes. Incubation of human monocyte-derived macrophages with conditioned medium from freshly isolated human adipocytes (BMI = 25.4 ± 0.96) resulted in a 4-fold increase in ACE expression. The effect was insensitive to denaturation and different proteases but abolished after lipid extraction. mRNA levels of the major histocompatibility complex class II protein increased in parallel with ACE, whereas the expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6, and cyclooxygenase-2 decreased. As a consequence of the reduction in MCP-1, monocyte recruitment was also attenuated. Moreover, adipocyte-conditioned medium prevented the interferon (IFN)-γ induced formation of TNF-α, IL-6, and MCP-1, all markers of classically-activated (M1 type) macrophages. The decrease in cytokine expression in adipocyte-conditioned medium-treated macrophages was sensitive to ACE silencing by small interfering RNA (siRNA). Accordingly, ACE overexpression in THP-1 cells mimicked the effect of adipocyte-conditioned medium. In both cell types, ACE inhibition failed to affect the changes induced by adipocyte conditioned-medium treatment and ACE overexpression. Thus, the modulation of macrophage polarization by ACE appears to be mediated independently of enzyme activity, probably via intracellular signaling. Interestingly, human macrophage ACE expression was also upregulated by IL-4 and IL-13, which promote the "alternative" activation of macrophages and decreased by LPS and IFN-γ. Mechanistically, adipocyte-conditioned medium stimulated the phosphorylation of the AMP-activated protein kinase and AMPK inhibition prevented the increase in ACE expression. Moreover, ACE expression was reduced in spleen derived-monocytes from AMPKα1(-/-) mice versus their wild-type littermates. These data indicate that mature adipocytes modulate the expression profile of macrophages by releasing lipid mediators that increase ACE expression via AMPK. This prevents the pro-inflammatory cytokine production by macrophages.

Inhibitors of the angiotensin-converting enzyme (ACE) decrease angiotensin II production and activate an intracellular signaling cascade that affects gene expression in endothelial cells. Because ACE inhibitors have been reported to delay the onset of type 2 diabetes, we determined ACE signaling-modulated gene expression in endothelial cells and adipocytes. Using differential gene expression analysis, several genes were identified that were 3-fold up- or down-regulated by ramiprilat in cells expressing wild-type ACE versus cells expressing a signaling-dead ACE mutant. One up-regulated gene was the cellular retinol-binding protein 1 (CRBP1). In adipocytes, the overexpression of CRBP1 enhanced (4- to 5-fold) the activity of promoters containing response elements for retinol-dependent nuclear receptors [retinoic acid receptor (RAR) and retinoid X receptor (RXR)] or peroxisome proliferator-activated receptors (PPAR). CRBP1 overexpression also enhanced the promoter activity (by 470 +/- 40%) and expression/release of the anti-inflammatory and antiatherogenic adipokine adiponectin (cellular adiponectin by 196 +/- 24%, soluble adiponectin by 228 +/- 74%). Significantly increased adiponectin secretion was also observed after ACE inhibitor treatment of human preadipocytes, an effect prevented by small interfering RNA against CRBP1. Furthermore, in ob/ob mice, ramipril markedly potentiated both the basal (approximately 2-fold) and rosiglitazonestimulated circulating levels of adiponectin. In patients with coronary artery disease or type 2 diabetes, ACE inhibition also significantly increased plasma adiponectin levels (1.6- or 2.1-fold, respectively). In summary, ACE inhibitors affect adipocyte homeostasis via CRBP1 through the activation of RAR/RXR-PPAR signaling and up-regulation of adiponectin. The latter may contribute to the beneficial effects of ACE inhibitors on the development of type 2 diabetes in patients with an activated renin-angiotensin system.

The binding of angiotensin-converting enzyme (ACE) inhibitors to ACE initiates a signaling cascade that involves the phosphorylation of the enzyme on Ser1270 as well as activation of the c-Jun NH2-terminal kinase (JNK) and leads to alterations in gene expression. To clarify how ACE inhibitors activate this pathway, we determined their effect on the ability of the enzyme to dimerize and the role of ACE dimerization in the initiation of the ACE signaling cascade. In endothelial cells, ACE was detected as a monomer as well as a dimer in native gel electrophoresis and dimerization/oligomerization was confirmed using the split-ubiquitin assay in yeast. ACE inhibitors elicited a rapid, concentration-dependent increase in the dimer/monomer ratio that correlated with that of the ACE inhibitorinduced phosphorylation of ACE. Cell treatment with galactose and glucose to prevent the putative lectin-mediated self-association of ACE or with specific antibodies shielding the N terminus of ACE failed to affect either the basal or the ACE inhibitor-induced dimerization of the enzyme. In ACE-expressing Chinese hamster ovary cells, ACE inhibitors elicited ACE dimerization and phosphorylation as well as the activation of JNK with similar kinetics to those observed in endothelial cells. However, these effects were prevented by the mutation of the essential Zn2+-complexing histidines in the C-terminal active site of the enzyme. Mutation of the N-terminal active site of ACE was without effect. Together, our data suggest that ACE inhibitors can initiate the ACE signaling pathway by inducing ACE dimerization, most probably via the C-terminal active site of the enzyme.

The phosphorylation of the short C-terminal cytoplasmic domain of the somatic angiotensin-converting enzyme (ACE) is involved in the regulation of enzyme shedding. We determined whether the phosphorylation of the cytoplasmic domain of ACE (ACEct) on Ser1270 regulates the cleavage/secretion of the enzyme by affecting its association with other proteins. ACE was associated with beta-actin and the nonmuscle myosin heavy chain IIA (MYH9) in endothelial cells, as determined by coimmunoprecipitation experiments as well as an ACEct affinity column. The ACE-associated MYH9 immunoprecipitated from (32)P-labeled endothelial cells was basally phosphorylated and cell stimulation with ACE inhibitors, or with bradykinin, increased the phosphorylation of MYH9. Casein kinase 2 (CK2) but not protein kinase C phosphorylated MYH9 in vitro, CK2 coprecipitated with MYH9 from endothelial cells and the phosphorylation of MYH9 in intact cells paralleled the phosphorylation of ACE on Ser1270 by CK2. The CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole attenuated the phosphorylation of ACE and MYH9, disrupted their association, and enhanced the cleavage/secretion of ACE from the plasma membrane. Cytochalasin D decreased the interaction between ACE and MYH9 and stimulated ACE shedding. Although MYH9 was still able to associate with residual amounts of a nonphosphorylatable S1270A ACE mutant, no ACE inhibitor-induced increase in MYH9 phosphorylation could be detected in S1270A-expressing cells. These data indicate that the interaction of ACE with MYH9 determines ACE shedding and is modulated by phosphorylation processes. Furthermore, because ACE inhibitors affect the phosphorylation of MYH9, the phosphorylation of this class II myosin might contribute to the phenomenon of ACE signaling in endothelial cells.

Purpose of Review: The renin-angiotensin system is not what it was, or for that matter not necessarily where we thought it should be. For example, there is a novel angiotensin I-metabolizing enzyme that generates angiotensin 1-7 rather than angiotensin II. Moreover, we are slowly realizing the importance of local rather than circulating angiotensin II., Recent Findings: Rather than concentrating on the systemic renin-angiotensin system, recent work has concentrated on elucidating the consequences of increasing angiotensin II production within specific organs, such as the heart and vasculature, as well as in the pancreas and in adipose tissue. Inhibition of angiotensin II production either using angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers not only reverses remodelling but also increases tissue insulin sensitivity. Targeting the renin-angiotensin system clinically delays the onset of type 2 diabetes, but the mechanisms involved are not clearly understood. Moreover, at least one other angiotensin-converting enzyme homologue (ACE2) plays a significant role in the regulation of heart and kidney function, and as it generates angiotensin 1-7 from angiotensin I, it is proposed to counteract the detrimental effects associated with the activation of the classic renin-angiotensin system., Summary: There is a need to re-evaluate the role(s) played by the molecular components of the "extended" local renin-angiotensin system and their role in vascular disease and type 2 diabetes.

Inhibition of the angiotensin-converting enzyme (ACE) protects against the progression of several cardiovascular diseases. Recent evidence suggests that some of the beneficial effects of ACE inhibitors can be attributed to the activation of a distinct ACE signaling cascade rather than to the changes in angiotensin II and bradykinin levels. Moreover, at least one other ACE homolog (ACE2) plays a significant role in the regulation of heart and kidney function.

Angiotensin-converting enzyme (ACE) inhibitors elicit outside-in signaling via ACE in endothelial cells. This involves the CK2-mediated phosphorylation of ACE on Ser1270 and the activation of the c-Jun N-terminal kinase (JNK)/c-Jun pathway, resulting in an enhanced endothelial ACE expression. Because cyclooxygenase-2 (COX-2) expression is reported to be increased in subjects treated with ACE inhibitors, we determined the role of ACE signaling in this phenomenon and the transcription factors involved. In lungs from mice treated with the ACE inhibitor ramipril for 5 days, COX-2 expression was increased. A similar (1.5- to 2-fold) increase in COX-2 protein was detected in primary cultures of human endothelial cells treated with ramiprilat. In an endothelial cell line stably expressing human somatic ACE, ramiprilat increased COX-2 promoter activity, an effect not observed in ACE-deficient cells or cells expressing a nonphosphorylatable ACE mutant (S1270A). The ramiprilat-induced, ACE-dependent increase in COX-2 expression and promoter activity (both 1.5- to 2-fold greater than control) was prevented by the inhibition of JNK. Ramiprilat significantly enhanced the DNA binding activity of activator protein-1 in cells expressing ACE but not S1270A ACE. Activator protein-1 decoy oligonucleotides prevented the ACE inhibitor-induced increase in COX-2 promoter activity and protein expression. As a consequence of the ramiprilat-induced increase in COX-2 expression, prostacyclin and prostaglandin E2, but not thromboxane A2, production was increased and was inhibited by the COX-2 inhibitor celecoxib. These results indicate that ACE signaling may underlie the increase in COX-2 and prostacyclin levels in patients treated with ACE inhibitors.

Not all of the cardiovascular effects of angiotensin-converting enzyme (ACE) inhibitors can be attributed to changes in angiotensin II and bradykinin levels. Because the cytoplasmic tail of ACE is phosphorylated, we determined whether ACE inhibitors affect the phosphorylation of ACE and whether ACE possesses the characteristics of a signal transduction molecule. The ACE inhibitors ramiprilat and perindoprilat, and the substrate bradykinin (but not angiotensin I), enhanced the activity of ACE-associated CK2 and the phosphorylation of ACE Ser1270 in cultured endothelial cells. Mitogen-activated protein kinase kinase 7 and c-Jun N-terminal kinase (JNK) coprecipitated with ACE, and stimulation of endothelial cells with ACE inhibitors increased the activity of ACE-associated JNK and elicited the accumulation of phosphorylated c-Jun in the nucleus. Ramiprilat was however unable to activate JNK or to stimulate the nuclear accumulation of c-Jun in endothelial cells expressing a S1270A ACE mutant or in ACE-deficient cells. Because the ACE inhibitor-induced increase in ACE expression has been linked to the formation of c-Jun homodimers, we investigated whether ACE signaling via JNK contributes to this response in vitro and in vivo. Prolonged ramiprilat treatment increased ACE expression in primary cultures of human endothelial cells and in vivo (mouse lung), a response that was prevented by pretreatment with the JNK inhibitor SP600125. Thus, ACE is involved in outside-in signaling in endothelial cells and "ACE signaling" may be an important cellular mechanism contributing to the beneficial effects of ACE inhibitors.

Soluble angiotensin-converting enzyme (ACE) is derived from the membrane-bound form by proteolytic cleavage of its C-terminal domain. Because intracellular events might be involved in the regulation of the cleavage process, we determined whether the cytoplasmic tail of ACE is phosphorylated and whether this process regulates secretion. Immunoprecipitation of ACE (180 kDa) from (32)P-labeled endothelial cells revealed that ACE is phosphorylated. Phosphorylation was not observed in endothelial cells overexpressing a mutant form of ACE (ACEDeltaS, all five cytoplasmic serine residues replaced by alanine). CK2 coprecipitated with ACE from endothelial cells, and CK2 phosphorylated both ACE and a peptide corresponding to the cytoplasmic tail. Mutation of serine(1270) within the CK2 consensus sequence almost abolished ACE phosphorylation. In ACE-overexpressing endothelial cells, ACE was mostly localized to the plasma membrane. However, no ACE was detected in the plasma membrane of ACEDeltaS-overexpressing cells, although a precursor ACE (170 kDa) was prominent in the endoplasmic reticulum and the cell supernatant contained substantial amounts of the soluble protein (175 kDa). A correlation between ACE-phosphorylation and secretion was confirmed in endothelial cells treated with the CK2-inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which time-dependently decreased the phosphorylation of ACE and increased its shedding. These results indicate that the CK2-mediated phosphorylation of ACE regulates its retention in the plasma membrane and may determine plasma ACE levels.