Angiotensin-converting Enzyme 2 Promoter Research Articles

Post-COVID-19 Pain Is Not Associated with DNA Methylation Levels of the ACE2 Promoter in COVID-19 Survivors Hospitalized Due to SARS-CoV-2 Infection.

One of theories explaining the development of long-lasting symptoms after an acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection include changes in the methylation pattern of the host. The current study aimed to investigate whether DNA methylation levels associated with the angiotensin-converting enzyme 2 (ACE2) promoter are different when comparing individuals previously hospitalized due to COVID-19 who then developed long-lasting post-COVID pain with those previously hospitalized due to COVID-19 who did not develop post-COVID-19 pain symptoms. Non-stimulated saliva samples were obtained from a cohort of 279 (mean age: 56.5, SD: 13.0 years old, 51.5% male) COVID-19 survivors who needed hospitalization. Clinical data were collected from hospital medical records. Participants were asked to disclose pain symptoms developed during the first three months after hospital admission due to COVID-19 and persisting at the time of the interview. Methylations of five CpG dinucleotides in the ACE2 promoter were quantified (as percentages). Participants were evaluated up to 17.8 (SD: 5.3) months after hospitalization. Thus, 39.1% of patients exhibited post-COVID-19 pain. Most patients (77.05%) in the cohort developed localized post-COVID-19 pain. Headache and pain in the lower extremity were experienced by 29.4% of the patients. Seven patients received a post-infection diagnosis of fibromyalgia based on the presence of widespread pain characteristics (11.6%) and other associated symptoms. No significant differences in methylation percentages at any CpG location of the ACE2 promoter were identified when comparing individuals with and without post-COVID-19 pain. The current study did not observe differences in methylation levels of the ACE2 promoter depending on the presence or absence of long-lasting post-COVID-19 pain symptoms in individuals who needed hospitalization due to COVID-19 during the first wave of the pandemic.

Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study.

Angiotensin-converting enzyme 2 (ACE2) is the receptor that enables SARS-CoV-2 to invade host cells. Previous studies have reported that reducing ACE2 expression may have an anti-SARS-CoV-2 effect. In this study, we constructed a pGL4.10-F2-ACE2 vector with double luciferase genes (firefly and Renilla luciferase) under the control of the ACE2 promoter and used it to screen compounds from Chinese traditional medicinal herbs (CTMHs) that can inhibit ACE2 transcription in human cells. We transfected HEK293T cells with pGL4.10-F2-ACE2 and treated them with CTMH compounds and then measured fluorescence to evaluate the indirect inhibition of ACE2 transcription. Out of 37 compounds tested, andrographolide demonstrated a dose-dependent inhibition of ACE2 transcription. We further confirmed by RT-qPCR and Western blot assays that andrographolide also reduced ACE2 expression in BEAS-2B cells in a dose-dependent manner. Moreover, pseudovirus infection assays in BEAS-2B cells demonstrated that andrographolide can inhibit SARS-CoV-2 infection in a dose-dependent manner. These results suggest that andrographolide has potential anti-SARS-CoV-2 activity and could be a candidate drug for COVID-19 prevention and treatment.

Exploring non-coding genetic variability in ACE2: Functional annotation and in vitro validation of regulatory variants

The surge in human whole-genome sequencing data has facilitated the study of non-coding region variations, yet understanding their biological significance remains a challenge. We used a computational workflow to assess the regulatory potential of non-coding variants, with a particular focus on the Angiotensin Converting Enzyme 2 (ACE2) gene. This gene is crucial in physiological processes and serves as the entry point for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 19 (COVID-19). In our analysis, using data from the gnomAD population database and functional annotation, we identified 17 significant Single Nucleotide Variants (SNVs) in ACE2, particularly in its enhancers, promoters, and 3′ untranslated regions (UTRs). We found preliminary evidence supporting the regulatory impact of some of these variants on ACE2 expression. Our detailed examination of two SNVs, rs147718775 and rs140394675, in the ACE2 promoter revealed that these co-occurring SNVs, when mutated, significantly enhance promoter activity, suggesting a possible increase in specific ACE2 isoform expression. This method proves effective in identifying and interpreting impactful non-coding variants, aiding in further studies and enhancing understanding of molecular bases of monogenic and complex traits.

Repurposing UDCA, an FXR Inhibitor, to Prevent SARS-Cov-2 Infection

Brevini T, Maes M, Webb GJ, et al. FXR Inhibition May Protect From SARS-CoV-2 Infection by Reducing ACE2. Nature 2022 Dec 5. https://doi.org/10.1038/s41586-022-05594-0. Viruses need to access the host’s machinery to replicate and propagate, and do so by binding to their respective receptors on host cells. Angiotensin-converting enzyme 2 (ACE2) was identified early in the pandemic as the entryway for the severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Since then, scientists have been exploring ways to prevent SARS-Cov-2 infection by targeting ACE2. In a study published in Nature, Brevini et al identified farnesoid X receptor (FXR) as a regulator of ACE2 expression. FXR is a ubiquitous bile acid sensing protein. Upon its activation by chenodeoxycholic acid, ACE2 increased in biliary, airway, and intestinal organoids. FXR directly bound the ACE2 promoter and activated its transcription. Furthermore, suppressing FXR signaling with ursodeoxycholic acid (UDCA) or Z-guggulsterone–reduced ACE2 expression. To test its relevance to coronavirus disease 2019 (COVID-19) in vitro, organoids were stimulated with chenodeoxycholic acid and infected with SARS-Cov-2 in the presence or absence of UDCA or Z-guggulsterone. Reduced viral infection was measured when drugs were present. In vivo, UDCA treatment decreased ACE2 expression in tissues of mice and hamsters. Furthermore, the Syrian Hamster model showed replication and transmission of SARS-Cov-2 in all untreated animals, whereas only 3 of 9 UDCA-treated animals were infected, and they presented a milder disease course. Interestingly, the authors took several approaches to validate their findings in the human context. They first used donated organs that were unfit for transplantation. They performed ex situ normothermic perfusion on a pair of lungs, whereby 1 lung received UDCA in the perfusate and the other served as a matched control. UDCA treatment decreased ACE2 expression in lung cells and ACE2 activity in the perfusate. Importantly, UDCA treatment decreased infection when lungs were exposed to SARS-Cov-2. Similar observations were seen with liver grafts. Additionally, they determined that UDCA reduced ACE2 expression in the nasal epithelium of healthy volunteers who received UDCA. They also retrospectively found an inverse correlation between UDCA and serum ACE2 levels in a serum proteomics dataset of a patient cohort. Finally, the authors interrogated 2 COVID-19 databases, one composed of patients with chronic liver disease and the other of vaccinated liver transplant recipients. In both scenarios, patients receiving UDCA had better COVID-19 outcomes compared with matched controls. Brevini et al were clever in their approaches. They repurposed donated organs found unfit for transplantation, giving them a new benefit for preclinical drug testing with potentially better predictive power. More important, they repurposed an approved and off-patent drug, UDCA, to prevent SARS-Cov-2 infection, a novel approach against viral infection that is directed toward the host rather than the virus. Because ACE2 is a receptor for many viruses, UDCA may have broader use and is less susceptible to immune escape by viruses. Although the experimental data shown were strong, it remains to be seen how UDCA performs in clinical trials involving larger and controlled cohorts of patients. Nonetheless, the repurposing of a cost-effective and readily available drug to prevent viral infection alongside vaccination is exciting and promising.

Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells.

Angiotensin-converting enzyme 2 (ACE2) is the receptor of COVID-19 pathogen SARS-CoV-2, but the transcription factors (TFs) that regulate the expression of the gene encoding ACE2 (ACE2) have not been systematically dissected. In this study we evaluated TFs that control ACE2 expression, and screened for small molecule compounds that could modulate ACE2 expression to block SARS-CoV-2 from entry into lung epithelial cells. By searching the online datasets we found that 24 TFs might be ACE2 regulators with signal transducer and activator of transcription 3 (Stat3) as the most significant one. In human normal lung tissues, the expression of ACE2 was positively correlated with phosphorylated Stat3 (p-Stat3). We demonstrated that Stat3 bound ACE2 promoter, and controlled its expression in 16HBE cells stimulated with interleukin 6 (IL-6). To screen for medicinal compounds that could modulate ACE2 expression, we conducted luciferase assay using HLF cells transfected with ACE2 promoter-luciferase constructs. Among the 64 compounds tested, 6-O-angeloylplenolin (6-OAP), a sesquiterpene lactone in Chinese medicinal herb Centipeda minima (CM), represented the most potent ACE2 repressor. 6-OAP (2.5 µM) inhibited the interaction between Stat3 protein and ACE2 promoter, thus suppressed ACE2 transcription. 6-OAP (1.25–5 µM) and its parental medicinal herb CM (0.125%–0.5%) dose-dependently downregulated ACE2 in 16HBE and Beas-2B cells; similar results were observed in the lung tissues of mice following administration of 6-OAP or CM for one month. In addition, 6-OAP/CM dose-dependently reduced IL-6 production and downregulated chemokines including CXCL13 and CX3CL1 in 16HBE cells. Moreover, we found that 6-OAP/CM inhibited the entry of SARS-CoV-2 S protein pseudovirus into target cells. These results suggest that 6-OAP/CM are ACE2 inhibitors that may potentially protect lung epithelial cells from SARS-CoV-2 infection.

The accessible promoter-mediated supplementary effect of host factors provides new insight into the tropism of SARS-CoV-2

In the past year, the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in the worldwide coronavirus disease 2019 (COVID-19) pandemic. Yet our understanding of the SARS-CoV-2 tropism mechanism is still insufficient. In this study, we examined the chromatin accessibility at the promoters of host factor genes (ACE2, TMPRSS2, NRP1, BSG, CTSL, and FURIN) in 14 tissue types, 23 tumor types, and 189 cell lines. We showed that the promoters of ACE2 and TMPRSS2 were accessible in a tissue- and cell-specific pattern, which is accordant with previous clinical research on SARS-CoV-2 tropism. We were able to further verify that type I interferon (IFN) could induce angiotensin-converting enzyme 2 (ACE2) expression in Caco-2 cells by enhancing the binding of HNF1A, the transcription factor of ACE2, to ACE2 promoter without changing chromatin accessibility. We then performed transcription factor (TF)-gene interactions network and pathway analyses and discovered that the TFs regulating host factor genes are enriched in pathways associated with viral infection. Finally, we established a novel model that suggests that open chromatin at the promoter mediates the host factors’ supplementary effect and ensures SARS-CoV-2 entry. Our work uncovers the relationship between epigenetic regulation and SARS-CoV-2 tropism and provides clues for further investigation of COVID-19 pathogenesis.

Prenatal dexamethasone exposure induced pancreatic β-cell dysfunction and glucose intolerance of male offspring rats: Role of the epigenetic repression of ACE2

The prevalence of diabetes in children and adolescents has been rising gradually, which is relevant to adverse environment during development, especially prepartum. We aimed to explore the effects of prenatal dexamethasone exposure (PDE) on β-cell function and glucose homeostasis in juvenile offspring rats. Pregnant Wistar rats were subcutaneously administered with dexamethasone [0.1, 0.2, 0.4mg/(kg.d)] from gestational day 9 to 20. PDE impaired glucose tolerance in the male offspring rather than the females. In male offspring, PDE impaired the development and function of β-cells, accompanied with lower H3K9ac, H3K14ac and H3K27ac levels in the promoter region of angiotensin-converting enzyme 2 (ACE2) as well as suppressed ACE2 expression. Meanwhile, PDE increased expression of glucocorticoid receptor (GR) and histone deacetylase 3 (HDAC3) in fetal pancreas. Dexamethasone also inhibited ACE2 expression and insulin production in vitro. Recombinant expression of ACE2 restored insulin production inhibited by dexamethasone. In addition, dexamethasone activated GR and HDAC3, increased protein interaction of GR with HDAC3, and promoted the binding of GR-HDAC3 complex to ACE2 promoter region. Both RU486 and TSA abolished dexamethasone-induced decline of histone acetylation and ACE2 expression. In summary, suppression of ACE2 is involved in PDE induced β-cell dysfunction and glucose intolerance in juvenile male offspring rats.

Possible Therapeutic Interventions in COVID-19 Induced ARDS by Cotinine as an ACE-2 Promoter and AT-1R Blocker.

In these challenging times of the pandemic, as coronavirus disease 2019 (COVID-19) has taken over the planet, its complications such as acute respiratory distress syndrome (ARDS) have the potential to wipe out a large portion of our population. Whereas a serious lack of ventilators, vaccine being months away makes the condition even worse. That's why promising drug therapy is required. One of them is suggested in this article. It is the angiotensin-converting enzyme-2 (ACE-2) to which the COVID-19 virus binds and upon downregulation of which the pulmonary permeability increases and results in the filling of alveoli by proteinaceous fluids, which finally results in ARDS. ARDS can be assisted by angiotensin-II type-1 receptor (AT-1R) blocker and ACE-2 upregulator. AT-1R blocker will prevent vasoconstriction, the pro-inflammatory effect seen otherwise upon its activation. ACE-2 upregulation will ensure less formation of angiotensin II, vasodilatory effects due to the formation of angiotensin (1-7), increased breakdown of bradykinin at lung level. Overall, decreased vasoconstriction of vessels supplying lungs and decreased vasodilation of lung tissues will ensure decreased pulmonary permeability and eventually relieve ARDS. It should also be considered that all components of the renin-angiotensin-aldosterone system (RAAS) are located in the lung tissues. A drug with the least plasma protein binding is required to ensure its distribution across these lung tissues. Cotinine appears to be a promising candidate for COVID-19- induced ARDS. It acts across the board and acts as both an AT-1R blocker, and ACE-2 upregulator. It also has a weak plasma protein binding that helps to spread through the lung tissues. In this review, we summarized that cotinine, along with COVID-19 virus replication blocker anti-virals, may prove to be a promising therapy for the treatment of COVID-19 induced ARDS.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

ACE2 correlated with immune infiltration serves as a prognostic biomarker in endometrial carcinoma and renal papillary cell carcinoma: implication for COVID-19.

Angiotensin-converting enzyme 2 (ACE2) is a member of the renin-angiotension system, however, the correlation between ACE2 and prognosis in UCEC (Uterine Corpus Endometrial Carcinoma) and KIRP (Kidney Renal Papillary Cell Carcinoma) is not clear. We analyzed the expression levels of ACE2 in the Oncomine and TIMER databases, the correlation between ACE2 and overall survival in the PrognoScan, GEPIA and Kaplan-Meier plotter databases. The correlation between ACE2 and immune infiltration level and the type markers of immune cells was investigated in TIMER database. A prognosis analysis based on the expression levels of ACE2 was further performed in related immune cells subgroup. The ACE2 promoter methylation profile was tested in the UALCAN database. In addition, we used GSE30589 and GSE52920 databases to elucidate the changes of ACE2 expression in vivo and in vitro after SARS-CoV infection. ACE2 was elevated in UCEC and KIRP, and high ACE2 had a favorable prognosis. The expression of ACE2 was positively correlated with the level of immune infiltration of macrophage in KIRP, B cell, CD4+T cell, neutrophil and dendritic cell immune infiltration levels in UCEC. ACE2 was significantly positively correlated with the type markers of B cells and neutrophils, macrophages in UCEC, while ACE2 in KIRP was positively correlated with the type markers of macrophages. High ACE2 expression level had a favorable prognosis in different enriched immune cells subgroups in UCEC and KIRP. And the promoter methylation levels of ACE2 in UCEC and KIRP were significantly reduced. What’s more, we found that the expression of ACE2 decreased in vivo and in vitro after SARS-CoV infection. In conclusion, ACE2 expression increased significantly in UCEC and KIRP, elevated ACE2 was positively correlated with immune infiltration and prognosis. Moreover, tumor tissues may be more susceptible to SARS-CoV-2 infection in COVID-19 patients with UCEC and KIRP, which may worsen the prognosis.

CCAAT/enhancer-binding protein β overexpression alleviates myocardial remodelling by regulating angiotensin-converting enzyme-2 expression in diabetes.

Diabetic cardiomyopathy, a major cardiac complication, contributes to heart remodelling and heart failure. Our previous study discovered that CCAAT/enhancer‐binding protein β (C/EBPβ), a transcription factor that belongs to a family of basic leucine zipper transcription factors, interacts with the angiotensin‐converting enzyme 2 (ACE2) promoter sequence in other disease models. Here, we aimed to determine the role of C/EBPβ in diabetes and whether ACE2 expression is regulated by C/EBPβ. A type 1 diabetic mouse model was generated by an intraperitoneal injection of streptozotocin. Diabetic mice were injected with a lentivirus expressing either C/EBPβ or sh‐C/EBPβ or treated with valsartan after 12 weeks to observe the effects of C/EBPβ. In vitro, cardiac fibroblasts and cardiomyocytes were treated with high glucose (HG) to investigate the anti‐fibrosis, anti‐apoptosis and regulatory mechanisms of C/EBPβ. C/EBPβ expression was down‐regulated in diabetic mice and HG‐induced cardiac neonatal cells. C/EBPβ overexpression significantly attenuated collagen deposition and cardiomyocyte apoptosis by up‐regulating ACE2 expression. The molecular mechanism involved the binding of C/EBPβ to the ACE2 promoter sequence. Although valsartan, a classic angiotensin receptor blocker, relieved diabetic complications, the up‐regulation of ACE2 expression by C/EBPβ overexpression may exert greater beneficial effects on patients with diabetic cardiomyopathy.

Epigenetic regulation of angiotensin-converting enzyme 2 (ACE2) by SIRT1 under conditions of cell energy stress

ACE2 (angiotensin-converting enzyme 2) counterbalances the actions of ACE (angiotensin-converting enzyme) by metabolizing its catalytic product, the vasoactive and fibrogenic peptide AngII (angiotensin II), into Ang-(1-7) [angiotensin-(1-7)]. Enhanced ACE2 expression may be protective in diabetes, cardiovascular disease and cancer. However, relatively little is known about the specific physiological factors regulating ACE2 expression. In the present paper, we show, by Western blotting and qPCR (quantitative real-time PCR), that ACE2 expression is increased under conditions of cell stress, including hypoxic conditions, IL (interleukin)-1β treatment and treatment with the AMP mimic AICAR (5-amino-4-imidazolecarboxamide riboside). The NAD+-dependent deacetylase SIRT1 (silent information regulator T1) was found to be up-regulated after AICAR treatment but, conversely, was down-regulated after IL-1β treatment. ChIP analysis demonstrated that SIRT1 bound to the ACE2 promoter and that binding was increased after AICAR treatment, but decreased after IL-1β treatment. Inhibition of SIRT1 activity ablated the AICAR-induced increase in ACE2. In conclusion, we have established that the expression of the ACE2 transcript is controlled by the activity of SIRT1 under conditions of energy stress.

Apelin is a positive regulator of ACE2 in failing hearts

Angiotensin converting enzyme 2 (ACE2) is a negative regulator of the renin-angiotensin system (RAS), catalyzing the conversion of Angiotensin II to Angiotensin 1-7. Apelin is a second catalytic substrate for ACE2 and functions as an inotropic and cardioprotective peptide. While an antagonistic relationship between the RAS and apelin has been proposed, such functional interplay remains elusive. Here we found that ACE2 was downregulated in apelin-deficient mice. Pharmacological or genetic inhibition of angiotensin II type 1 receptor (AT1R) rescued the impaired contractility and hypertrophy of apelin mutant mice, which was accompanied by restored ACE2 levels. Importantly, treatment with angiotensin 1-7 rescued hypertrophy and heart dysfunctions of apelin-knockout mice. Moreover, apelin, via activation of its receptor, APJ, increased ACE2 promoter activity in vitro and upregulated ACE2 expression in failing hearts in vivo. Apelin treatment also increased cardiac contractility and ACE2 levels in AT1R-deficient mice. These data demonstrate that ACE2 couples the RAS to the apelin system, adding a conceptual framework for the apelin-ACE2-angiotensin 1-7 axis as a therapeutic target for cardiovascular diseases.

The transcription factor HNF1α induces expression of angiotensin-converting enzyme 2 (ACE2) in pancreatic islets from evolutionarily conserved promoter motifs

Pancreatic angiotensin-converting enzyme 2 (ACE2) has previously been shown to be critical for maintaining glycemia and β-cell function. Efforts to maintain or increase ACE2 expression in pancreatic β-cells might therefore have therapeutic potential for treating diabetes. In our study, we investigated the transcriptional role of hepatocyte nuclear factor 1α (HNF1α) and hepatocyte nuclear factor 1β (HNF1β) in induction of ACE2 expression in insulin-secreting cells. A deficient allele of HNF1α or HNF1β causes maturity-onset diabetes of the young (MODY) types 3 and 5, respectively, in humans. We found that ACE2 is primarily transcribed from the proximal part of the ACE2 promoter in the pancreas. In the proximal part of the human ACE2 promoter, we further identified three functional HNF1 binding sites, as they have binding affinity for HNF1α and HNF1β and are required for induction of promoter activity by HNF1β in insulinoma cells. These three sites are well-conserved among mammalian species. Both HNF1α and HNF1β induce expression of ACE2 mRNA and lead to elevated levels of ACE2 protein and ACE2 enzymatic activity in insulinoma cells. Furthermore, HNF1α dose-dependently increases ACE2 expression in primary pancreatic islet cells. We conclude that HNF1α can induce the expression of ACE2 in pancreatic islet cells via evolutionarily conserved HNF1 binding sites in the ACE2 promoter. Potential therapeutics aimed at counteracting functional HNF1α depletion in diabetes and MODY3 will thus have ACE2 induction in pancreatic islets as a likely beneficial effect.

Endothelin-1 Downregulates Angiotensin-Converting Enzyme-2 Expression in Human Bronchial Epithelial Cells

Background/Aims: Both endothelin-1 (ET-1) and the renin-angiotensin system (RAS) are implicated in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). In the present study, we explored the interaction between ET-1 and the RAS by examining the effect of ET-1 on angiotensin-converting enzyme-2 (ACE2) expression and activity in human bronchial epithelial cells (HBEpCs). Methods: HBEpCs were treated with ET-1 (1, 10, 20, 40 or 50 nmol/l) for 6, 12, 18, 24 or 30 h with or without the transcription inhibitor actinomycin D, endothelin A (ETA) receptor blocker BQ123, endothelin B receptor blocker BQ788, or different kinase inhibitors. Results: ET-1 decreased the ACE2 mRNA level in a dose- and time-dependent manner within 24 h, which led to dose-dependent downregulation of the ACE2 promoter activity, protein level and the cell membrane ACE2 activity. Actinomycin D (1 mg/ml), BQ123 (1 μmol/l), and the p38 mitogen-activated protein kinase (MAPK) siRNA and inhibitor PD169316 (25 μmol/l) completely abolished the effect of ET-1 on ACE2 expression in HBEpCs. Conclusion: ET-1 downregulates ACE2 expression and activity at the transcription level in HBEpCs via the ETA receptor by a p38 MAPK-dependent mechanism. This is the first evidence of crosstalk between the ET-1/ETA axis and the RAS in regard to the pathogenesis and progression of COPD.

Hepatocyte nuclear factors 1α and 1β (HNF1α and HNF1β) are powerful inducers of the enzymatic activity of angiotensin‐converting enzyme 2 (ACE2) in insulin‐secreting cells

Maturity onset diabetes of the young (MODY) types 3 and 5 are caused by deficiency of hepatocyte nuclear factor 1α (HNF1α) and hepatocyte nuclear factor 1β (HNF1β) which bind to similar DNA motifs. Our laboratory has previously shown that: 1) pancreatic angiotensin‐converting enzyme 2 (ACE2) is reduced in diabetes, 2) pancreatic ACE2 gene therapy improves glucose control in diabetic mice, and 3) HNF1β induces expression of ACE2 mRNA in insulinoma cells. However, discordance between ACE2 expression at the mRNA and enzyme activity level is often reported. Furthermore, HNF1α is more prevalent and thought to be more important than HNF1β in differentiated beta cells. Hypothesis: Both HNF1α and HNF1β lead to increased ACE2 mRNA expression as well as elevated ACE2 enzymatic activity in insulin‐secreting cells. Transfection of rat 832/13 insulinoma cells with expression plasmids for HNF1α and HNF1β led to marked (25‐ and 95‐fold, respectively, p < 0.001) induction of ACE2 mRNA and a strong increase (12‐ and 41‐fold, p < 0.001) in ACE2 enzymatic activity. Furthermore, HNF1α and HNF1β induced expression from both of the two known ACE2 promoter regions in mouse βTC‐3 insulinoma cells. The data demonstrate a link between HNF1α/HNF1β, ACE2 activity, and glucose homeostasis. These observations could lead to new therapeutic strategies for the treatment of MODY and type 2 diabetes. Support: NIH/NIDDK (DK084466).

The PPAR‐γ agonist Rosiglitazone increases angiotensin‐converting enzyme 2 (ACE2) promoter activity in neurons

Angiotensin converting enzyme 2 (ACE2) converts angiotensin‐(Ang)‐II to the vasodilator peptide Ang‐(1–7). Our laboratory has previously observed that ACE2 expression and activity in brain regions involved in the central regulation of blood pressure (BP) and volume homeostasis (e.g. SFO, PVN, and RVLM) are reduced in mice with elevated brain Ang‐II levels. Other investigators have shown that Rosiglitazone, a peroxisome proliferator‐activator receptor gamma (PPAR‐γ) agonist, increases ACE2 mRNA in adipocytes. Here we assessed the potential of PPAR‐γ to activate the ACE2 gene promoter. A reporter plasmid containing the human ACE2 proximal promoter sequence was constructed, and luciferase activity was measured after transfection of mouse Neuro‐2a cells in the presence or absence of Rosiglitazone (1 μM) and Ang‐II (100 nM) for 24 hours. Human ACE2 promoter activity increased by 2‐fold (P<0.05) with Rosiglitazone treatment independently of Ang‐II treatment, suggesting that PPAR‐γ may bind the proximal promoter and induce expression of hACE2. On the other hand, our data suggest that Ang‐II regulation of the hACE2 gene expression is not dependent on its proximal promoter. Altogether, we show that PPAR‐γ positively regulates the hACE2 promoter activity, supporting a beneficial role in modulating Ang‐II levels in neurogenic hypertension and in the regulation of volume homeostasis. (Funding: HL093178)

Stimulation of angiotensin‐converting enzyme 2 promoter activity by hepatocyte nuclear factor 1β (HNF1β) in insulinoma cells

We previously showed that Angiotensin-converting enzyme 2 (ACE2) gene therapy increases insulin secretion (Bindom et al, 2010). Moreover, others reported that exogenous expression of hepatocyte nuclear factor 1β (HNF1β) activates the ACE2 promoter in HEK293 cells that have no endogenous HNF1β. However, physical interaction with the ACE2 promoter was not shown and the relevance to diabetes is unclear. Our objective was to determine whether HNF1β directly binds to the ACE2 gene promoter and whether HNF1β overexpression stimulates promoter activity in insulinoma cells. Chromatin immunoprecipitation assays showed binding of HNF1β to the ACE2 gene promoter region in HEK293T cells transfected with an HNF1β expression plasmid. Human and mouse ACE2 promoter regions containing 2 putative HNF1 binding sites were cloned into the luciferase vector pGL3-Basic. The ACE2 promoter sequences were active in driving the expression of luciferase in insulinoma cell lines βTC-3 and 832/13. Furthermore, overexpression of HNF1β markedly induced expression of luciferase (> 20-fold in βTC-3 cells and > 100-fold in 832/13 cells) and ACE2 mRNA (> 10-fold in 832/13 cells). Our data show that HNF1β binds and stimulates ACE2 promoter activity in insulinoma cells. These findings bring new evidence for a role of ACE2 in type 2 diabetes by providing a possible new mechanism leading to insulin secretion. Support: NIH/NIDDK (DK084466).