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Key Event Title
Dysregulation, ACE2 expression and activity
|Level of Biological Organization|
Key Event Components
|mRNA transcription||angiotensin-converting enzyme 2||increased|
|carboxypeptidase activity||angiotensin-converting enzyme 2||increased|
|protein localization to cell surface||angiotensin-converting enzyme 2||decreased|
|angiotensin-converting enzyme 2||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Viral spike protein interaction with ACE2 leads to microvascular disfunction||KeyEvent||Brendan Ferreri-Hanberry (send email)||Under development: Not open for comment. Do not cite|
|ACE2 dysregulation leads to gut dysbiosis||KeyEvent||Cataia Ives (send email)||Under development: Not open for comment. Do not cite||Under Development|
Key Event Description
The angiotensin-converting enzyme 2 (ACE2) is a membrane-anchored protein with wide tissue distribution https://www.proteinatlas.org/ENSG00000130234-ACE2. ACE2 has multiple functions, and is highly regulated at the transcriptional, post-transcriptional and post-translational levels. Modulation of the expression levels or functional activity of the ACE2 receptor is described in this KE.
ACE2 is bound to cell membranes, and in polarized cells it is exclusively targeted to the apical surface (Werner 2005; Ren 2006) [for more detailed description of the expression patterns of ACE2 see the applicability domain section].
Enzymatic function of ACE2 in interlinked bioactive peptide systems
ACE2 is mainly known and studied as a type I ectoenzyme i.e. a transmembrane protein with an extracellular amino-terminal domain harbouring a carboxypeptidase active site. ACE2 cleaves the carboxyterminal amino acid from a number of biologically active peptides (Figure 1 and 2), thus activating or deactivating them as agonists within the Renin Angiotensin System (RAS) (Santos et al., 2019) and within the Kinin–kallikrein system (KKS) (Kakoki & Oliver, 2009) in mammals.
The extracellular domain of ACE2 can itself be cleaved (shedding) in vitro and in vivo, releasing a soluble and catalytically active sACE2 (Guy et al., 2008 in human cardiac myofibroblasts; Peng Jia et al., 2009 in human lung epithelial cells and BALF; Werner et al., 2005 in canine epithelial polarised kidney MDCKII cells stabilly expressing ACE2). Constitutive shedding of ACE2 from the cellular membrane is mediated by another membrane-bound metalloprotease from the adamalysin family, ADAM17 (Iwata et al., 2009) also known as TACE, TNFalpha Converting Enzyme (Zunke 2017). Other proteins may also be involved in ACE2 protolithic modulation (Lambert et al, 2008).
The exact role of ACE2 shedding in modulating its function is not well understood, however, being catalytically active, the released sACE2 can, as the membrane-anchored full length ACE2, generate biologically active peptides which activate specific receptors in different cells/tissues/organs. Thus, the ACE2 function in the organism is mediated via its peptide products activating specific receptors on the same, (autocrine), nearby (paracrine) and potentially distant cells.
ACE2 is a homologue of angiotensin-converting enzyme (ACE), with whom it shares significant sequence similarity (Tipnis, 2000; Donoghue 2000), yet exhibits very distinct enzymatic activity. ACE2, as ACE, is a zink-metallopeptidase, however ACE2 is strictly a carboxypeptidase while ACE is an dipeptidase. Furthermore, the main substrate of ACE2 is the octopeptide Angiotensin II (Ang II), the enzymatic product of its homologue ACE. By cleaving a single amino acid from the C-terminus of Ang II, ACE2 generates a functionally different bioactive peptide Angiotensin 1-7 (Ang1-7) (Figure 1). ACE2 is key to the regulation of local and systemic Ang II levels.
Ultimately, the function of ACE2 at tissue level is mediated via the interaction of its main active product Ang1-7 with the Angiotensin II receptor 2 (AT2R), balanced by the activity of its homologue ACE and other peptidases in the RAS.
Figure1: Enzymatic activity of ACE2 compared to its homologue ACE and another protease relevant to the RAS (from Rice et al., 2004)
Figure 2: Simplified representation of the biological function of the enzymatic products of ACE2 activity in the KKS (adopted from Kakoki & Oliver, 2009).
Although most studies have focused on the role of ACE2 in angiotensin metabolism in the RAS, the enzyme has broad substrate specificity and it also hydrolyses a number of other biologically active peptides including des-Arg9-bradykinin (DABK), apelin-13, neurotensin(1–11), dynorphin A-1–13), β-casomorphin-(1–7), and ghrelin (Vickers 2002, Humming, 2007.
ACE2 cleavage of DABK to bradykinin 1-7 (Figure 2) has been demonstrated in chemico (Donoghue 2000, Vickers 2002), with human polarised primary lung cells in vitro and in mice broncho-alveolar lavage (BALF) (Sodhi 2018). Deactivation of DBAK, a preferencial bradikynin receptor 1 (B1K) agonist (Coulson et al., 2019), is an important regulatory function of ACE2 in the KKS (Figure 2).
1.2 ACE2 chaperone function for transporters of amino acid transfer (B0AT1)
Somewhat less known is the RAS independent function of ACE2 in the gut, where it regulates intestinal amino acid homeostasis, expression of antimicrobial peptides, and the gut microbiome (Camargo et al., 2020). ACE2 was identified as an important regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis in mice (Hashimoto et al., 2012). The mechanism by which ACE2 regulates amino acid transport in the intestine involves interaction with the broad neutral (0) amino acid transporter 1 (B0AT1) (Slc6a19) which mediates the uptake of neutral dietary amino acids, such as tryptophan and glutamine, into intestinal cells in a sodium-dependent manner (Camargo et al, 2009). A crystal structure study revealed a complex dimer of ACE2/ B0AT1 heterodimers (Yan et al., 2020), previously suggested by immunoprecipitation of intestinal membrane proteins in mice (Fairweather et al., 2012). Immunofluorescence showed co-localization of B0AT1 with ACE2 at the luminal surface of human small intestine (Vuille-dit-Bille et al., 2015). ACE2 seems to be necessary not only for the amino acid transfer by B0AT1, but also for its membrane expression (Camargo et al., 2008; Hashimoto et al., 2012).
REGULATION OF ACE2 LEVELS and ACTIVITY
ACE2 is regulated at the transcriptional, post-transcriptional and post-translational level, the final potentially differing in the different organisational contexts: cell membrane versus tissue (plasma and/or interstitial). In addition, all of these regulatory processes may be differentially modulated in different tissues.
Age, sex and species specific differences in aspects of the regulation, and also tissue specific regulation, have been reported (reviewed in Saponaro 2020).
Loss of function of ACE2 in vivo in ACE2 knockout (KO) mice has been associated with elevated levels of Ang II in heart, kidney and plasma as well as histological and functional perturbations in the lungs and in the cardiovascular (Crackower et al, Nature 2002) and renal (Oudit et al, 2010) systems, mostly in the presence of a particular stress factor, in some cases potentiated by aging (reviewed in Humming 2007).
Furthermore, Ace2 KO mice exhibited reduced serum levels of tryptophan, together with downregulated expression of small intestinal antimicrobial peptides and altered gut microbiota, which was re-established by tryptophan supplementation (Hashimoto et a., 2012).
At transcriptional level
Overall, the transcriptional regulatory elements of the ace2 gene are not well characterised.
Ace2, human but not mouse, was identified as an Interferon Stimulated Gene (ISG) in airway epithelial cells (Ziegler, 2020), indicating species specific regulation and its importance for human viral infections mediated via ACE2 (e.g. SARS-COV2). Influenza virus infection also induced ACE2 mRNA synthesis in human lung tissue (Ziegler, 2020).
In vitro in normal kidney tubular epithelial cell line (HK-2) ACE2 mRNA is down-regulated following Ang II treatment (Koka 2008). The exact transcriptional regulatory mechanism is not clear, but the observed ACE2 mRNA up-regulation in this system appears to be mediated by the activation of the ERK1/2 and p38 MAP kinase pathway and dependent on the activation of AT1R receptor by AngII, as demonstrated by specific AT1R, MAP kinase and ERK1/2 MAP kinase inhibitors (Koka et al., 2008). Regulation of ACE2 expression mediated by AT1R activation is an important endogenous regulatory mechanism for ACE2 activity within the RAS system (ref…..).
Vitamin D Receptor (VDR) may also emerge as an ACE2 transcriptional regulator/repressor (Saponaro 2020 and Glinsky 2020, unreviewed pre-print). VDR has already been implicated in the transcriptional repression of Renin, at least in vitro (Yuan 2007).
17b-estradiol (E2) has also been indicated in the transcriptional regulation of ace2 in a tissue specific manner (recently reviewed by Saponaro 2020). E2 down-regulated ace2 transcription particularly in kidney and differentiated airway epithelial cells. However, in human atrial tissue, E2 appeared to up-regulate ACE2 mRNA and protein. This change was associated with decreased levels of ACE homologue protein. The exact mechanism for this regulation remains to be elucidated as it may represent a significant modulating factor in the differential sex susceptibility to ACE2 dysregulation under varied stress conditions (e.g SARS-COV infection).
Epigenetic transcriptional regulation of ace2 has also been indicated (recently reviewed by Saponaro 2020. Transcription of ace2 is repressed by histone methylation and stimulated by NAD+-dependent deacetylase SIRT1 during cellular energy stress. Interestingly, in children ACE2 is normally hypermethylated and poorly expressed in the lung and in other organs (Saponaro 2020, ref therein).
Gut microbiota have also been implicated in the transcriptional regulation of ACE2 expression in the gut (Yang et al., 2020) but also in the lung (Koester et al., 2021). Whether this is directly or indirectly occurring via microbial metabolites remains to be elucidated, but the study by Koester ar e al, 2020 observed variability in intestinal Ace2 expression in gnotobiotic mice colonized with different microbiota, partially attributable to differences in microbiome-encoded proteases and peptidases.
At post-transcriptional level
Generally, modulation of ACE2 mRNA and protein levels appear to follow consistent pattern. However, it has been demonstrated that under certain conditions and in some tissues, mRNA and protein levels appear to follow a different pattern, suggesting important role of post-transcriptional or post-translational (see next section) regulation of ACE2 expression and function.
For example, hypertension in humans has been associated with different modulation of mRNA and protein levels in the heart tissue (Koka 2008). Specifically, heart tissue from patients with hypertension showed decreased levels of ACE2 mRNA while protein levels were comparable to normal tissue. In contrast, ACE mRNA and protein levels appeared consistently up-regulated in hart tissue of hypertensive patients (Koka 2008). In the same study, in the kidney tissue from patients with hypertensive nephropathy, both, ACE2 mRNA and protein levels, appeared consistently down-regulated compared to normal kidney tissue (Koka 2008). Significant suppression of ACE2 mRNA and protein expression was also observed in vitro in normal kidney tubular epithelial cell line (HK-2) treated with AngII (linked to hypertension in vivo) in a dose and time dependent manner Koka 2008). AngII treatment in vitro with myocardia- derived cells was not examined in this study and the discrepancy of mRNA and protein level modulation in the hypertensive human heart tissue biopsies was attributed by the authors to limitations of the detecting methods (Koka 2008).
Clear discrepancy in the modulation of mRNA versus protein level has been observed in vivo in mice in myocardial tissue (Patel 2014). Namely, up-regulation of mRNA synthesis was associated with down-regulation of ACE2 protein levels following 1 or 2 week treatment by exogenous circulating AngII (Patel 2014). In this study, down-regulation of ACE2 protein levels was alleviated by AT1R blockage/inhibitors, while mRNA up-regulation was not dependent on AT1R signalling. This strongly suggests involvement of post-transcriptional mechanism step(s) mediated by AngII/AT1R fot the regulation of ACE2 protein/function, at least in myocardial tissue under certain stress conditions.
Modulation of ACE2 protein and activity levels by AngII is clinically relevant phenomenon and AngII activity blockers (ACE inhibitors and AT1R blockers) are used to move the balance of the RAS from the ACE/AngII/AT1R axis towards the protective ACE2/Ang1-7/MasR axis. This is particularly relevant in the lung where ACE/ACE2 activity ratio is high (Roca-Ho, 2017-mice, human and other ref……?..).
The up-regulation of ACE2 mRNA observed in mouse myocardial tissue by Patel et al., 2014 appears contradictory to the finding of ACE2 mRNA down-regulation observed in the heart of hypertensive patients observed in the study of Koka et al., 2008 (if the latter result is accepted despite potential method limitations). However, it should be noted that the base level of ACE2 and also the relative ACE2/ACE ratio in the case of chronic hypertensive patients, many of whom have been on AT1R inhibitor treatment (Koka 2008), and in healthy mice treated with AngII for relatively short time (Patel 2014), may be different leading to response to the stressor (hypertension and AngII) over time. Consistent with this, distinct ACE/ACE2 activity ratios have been demonstrated in different organs of normal, non-obese diabetic (NOD) and insulin treated NOD mice, which varied additionally over the time course after the onset of diabetes (Roca-Ho et al., 2014).
Finally, species specific regulatory differences may be involved that would warrant further examination. But, overall, the studies discussed above illustrate the complex regulatory mechanisms of ACE2 mRNA, protein and activity levels in different tissues and under different stress conditions for the RAS system.
Rapid and transient up-regulation of ACE2 mRNA followed by down-regulation of ACE2 protein levels has been reported in the lung as a result of LPS induced acute inflammation in mice (Sodhi 2018). In this case the increase of ACE2 mRNA appears to be a rapid and transient compensatory effect to ACE2 protein/activity down-regulation mediated by NFk- B signalling in response to acute inflammation. Inhibiting NF- B signaling by Bay11-7082 restored ACE2 activity, again demonstrating post transcriptional or translational regulation of ACE2 in the lungs. In addition, this study examined the effect of ACE2 dysregulation on the KKS and demonstrated that attenuation of ACE2 activity under conditions of LPS induced inflammation leads to impaired DABK inactivation and enhanced BKB1R signalling (Sodhi 2018).
The underlying mechanisms of post-transcriptional mRNA regulation remain to be elucidated further. Some evidence on involvement of miRNA in the process [to be competed]
At post-translational level – enzymatic activity including shedding
Complexity of analysing the regulation of ACE2 function is emphasized even further when enzymatic activity is considered, including its spatial distribution between cell/tissue versus interstitium/plasma, mediated by shedding.
The exact role of ACE2 shedding is not well understood, but proteolytic ectodomain shedding of membrane proteins is a fundamental post-translational regulatory mechanism of the activity/function of a wide variety of proteins, including growth factors, cytokines, receptors and cell adhesion proteins (Lichtenthaler et al., 2018).
sACE2 activity is increased in patients with heart failure (HF) and correlates with disease severity (Epelman 2008).
In mice in vivo, shedding of ACE2 by TACE was induced by sub-chronic (2 weeks) exogenous AngII treatment (mimicking HF), leading to decreased ACE2 protein level and increased ACE2 mRNA in myocardial tissue with concurrent elevated sACE2 activity (Patel et al., 2014).
How It Is Measured or Detected
- using fluorescently labeled peptide substrates (Rice, 2004; Sodhi, 2008; Roca-Ho, 2017 1; Lu and Sun, 2020; Xiao 2017)
- measuring catalytic products (direct) or markers of activation of receptors for the products (indirect) of ACE2 activity (e.g Ferrario 2005)
- mRNA by RT-PCR (Sodhi 2018; Roca-Ho, 2017) or scRNA seq (e.g. Qi. et al 2020)
- protein in tissue extracts/preparations by immunoprecipitation or Western blotting (Koka 2008)
- protein in live tissues or cultured cells by immunostaining (Humming 2007; Fraga-Silva et al., 2011; Ren 2006; Warner 2005)
high throughput and quantitative measurement of protein by quantitative proteomic analysis (Park 2020; Stegbauer 2020)
Domain of Applicability
ACE2 is expressed in a wide variety of tissues affecting their function.
ACE2 was initially identified in human lymphoma cDNA library (Tipnis, 2000), and from a human cDNA library of ventricular cells with heart failure (Donoghue 2000). Expression of ACE2 has later been also identified in the heart, kidney, and testis (Donoghue et al. (2000b). However, subsequent studies have shown a much broader distribution, including the upper airways, lungs, gut, and liver (reviewed recently by Saponaro, 2020).
Tissue and sub-cellular distribution of ACE2 (protein and mRNA)
Protein expression patterns
Immunostaining methods show that ACE2 is chiefly bound to cell membranes, predominantly in the smooth muscle cells and in the endothelium of the vasculature, while negligible levels can be detected in the circulation. In blood cells, it has been observed in platelets and macrophages, but not in B and T lymphocytes (Hamming et al., 2004; Fraga-Silva et al., 2011).
ACE2 shows differential sub-cellular distribution which can be significant for its basal/constitutive and modulated function. It is mainly detectable at the cell-surface with little intracellular localization, and the protein does not readily internalize (Warner et al., 2005). In polarized cells, ACE2 is exclusively targeted to the apical surface in vivo in kidney (Warner 2005) and in vitro in polarised cells derived from the colon, lung and kidney (Ren 2006). This is in contrast to its sequence homologue and functional “balancer” ACE, which distributes equally between apical and basolateral surfaces (Warner 2005).
In the small intestines, ACE2 is highly expressed on enterocytes and via its local RAS function participates in the regulation of the intestinal glucose transport. Intestinal ACE2 generates locally Ang 1-7 from luminal Ang II. Ang II was shown to inhibit SGLT1-dependent intestinal glucose uptake in a dose-dependent manner in vitro and in vivo in rats (Wong et al, 2007; 2009) and in human biopsies via AT1R activation (Casselbrant et al., 2015).
mRNA expression patterns
More recently, Qi. et al 2020 analysed 13 human tissues by scRA sequencing and report that ACE2 mRNA was mainly expressed in the ileum enterocytes, kidney proximal tubules and lung AT2 cells. ACE2 mRNA was also detected but to a lesser extent in the colon enterocytes, esophagus and keratinocytes and minimally in the cholangiocytes (biliary cells of the liver).
Evidence for Perturbation by Stressor
Despite its significant role in the regulation/balancing of the RAS and KKS systems, the ACE2 enzyme has found its particular fame only since the outbreak of the SARS-COV2 pandemic in 2020 (ref…) because ACE2 turned out to act also as the receptor for the SARS-COV2, enabling virus entry into the cells, much like its cousin SARS-COV (Glowacka, 2010, ………….).
Interaction of the S proteins of SARS-COV1 and 2 with ACE2 on the cell membrane leads to:
- Down-regulation of its level and activity on the cellular membrane ( ref )
- Up-regulation of the level of sACE2 and enzymatic activity in the supernatant of cultured cells (……..), enhanced enzymatic activity in vitro (Lu and Sun, 2020) and in vivo in COVID-19 patients ( )
- Up-regulation of ACE2 mRNA level in in BALF from infected patients (Garvin 2020, others?).
A number of reviews and hypothesis research papers have been able to link both up-regulation and down-regulation of ACE2 (at mRNA or protein/activity) to the observed perturbations in the RAS and KKS systems that ultimately drive organ damage in SARS and in COVID-19 (Garvin 2020, Nicplau, 2000, van de Veerdonk 2020, Jiqi Wang 2020) This is not surprising given the complexity and interconnectedness of these systems (Fig. 2).
It is possible that both mechanisms (up and down-regulation) may play a role in a tissue/organ specific and time dependent manner.
Gastrointestinal (GI) symptoms (anorexia, diarrhoea, vomiting, and abdominal pain) are commonly observed in patients with COVID-19 (Bourgonje et all, 2020 and references therein). Given the tissue distribution of ACE2 and its high expression in the GI tract luminal side, SARS-CoV-2 could directly invade the gastrointestinal epithelium via ACE2. Consistent with this, viral RNA in faeces could be detected in patients even after viral RNA in the respiratory tract became negative and infectious virus could be isolated from the stool [120, 121]. However, there are also conflicting studies that could not find evidence for the presence of infectious virus in RNA-positive stool samples .
The influenza virus is another biological stressor that appears to modulate ACE2 activity, but without binding to ACE2. Influenza infection also appears to up-regulate ACE2 mRNA in human nasal epithelia (Ziegler, 2020).
in epithelial kidney cells (Liu 2014, …..) and in mouse models of the disease where ACE2 modulation is associated with the severity of the virus induced disease ( Yang 2014, Zou 2014,…….).
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