Nedd4-2 up-regulation is associated with ACE2 ubiquitination in hypertension

Abstract

Aims: Angiotensin-converting enzyme 2 (ACE2) is a critical component of the compensatory renin-angiotensin system that is down-regulated during the development of hypertension, possibly via ubiquitination. However, little is known about the mechanisms involved in ACE2 ubiquitination in neurogenic hypertension. This study aimed at identifying ACE2 ubiquitination partners, establishing causal relationships and clinical relevance, and testing a gene therapy strategy to mitigate ACE2 ubiquitination in neurogenic hypertension.

Methods and results: Bioinformatics and proteomics were combined to identify E3 ubiquitin ligases associated with ACE2 ubiquitination in chronically hypertensive mice. In vitro gain/loss of function experiments assessed ACE2 expression and activity to validate the interaction between ACE2 and the identified E3 ligase. Mutation experiments were further used to generate a ubiquitination-resistant ACE2 mutant (ACE2-5R). Optogenetics, blood pressure telemetry, pharmacological blockade of GABAA receptors in mice expressing ACE2-5R in the bed nucleus of the stria terminalis (BNST), and capillary western analysis were used to assess the role of ACE2 ubiquitination in neurogenic hypertension. Ubiquitination was first validated as leading to ACE2 down-regulation, and Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) was identified as a E3 ligase up-regulated in hypertension and promoting ACE2 ubiquitination. Mutation of lysine residues in the C-terminal of ACE2 was associated with increased activity and resistance to angiotensin (Ang)-II-mediated degradation. Mice transfected with ACE2-5R in the BNST exhibited enhanced GABAergic input to the paraventricular nucleus (PVN) and a reduction in hypertension. ACE2-5R expression was associated with reduced Nedd4-2 levels in the BNST.

Conclusion: Our data identify Nedd4-2 as the first E3 ubiquitin ligase involved in ACE2 ubiquitination in Ang-II-mediated hypertension. We demonstrate the pivotal role of ACE2 on GABAergic neurons in the maintenance of an inhibitory tone to the PVN and the regulation of pre-sympathetic activity. These findings provide a new working model where Nedd4-2 could contribute to ACE2 ubiquitination, leading to the development of neurogenic hypertension and highlighting potential novel therapeutic strategies.

Keywords: Autonomic regulation; Blood pressure; GABA; Sex differences; Sympathetic drive.

Figure 1

Ubiquitination reduces ACE2 expression and activity. (A) Representative blot showing the levels of ubiquitination of ACE2 in control HEK293T cells before and after Ang-II treatment. The first two lanes are from cells transfected with GFP-tagged ACE2, AT₁R, HA-tagged ubiquitin, and pcDNA3.1, and the last two lanes are from cells transfected for 48 h, serum-starved for 24 h, and subsequently treated with (left) Ang-II (100 nM for 4 h) or Ang-II (100 nM) and losartan (1 μM) for 4 h. GFP was then immunoprecipitated from the cells, and immunoblotting was performed against HA, as described in the Methods section. (B) ACE2 activity in HEK293T cells transfected with pcDNA3.1 or HA-tagged ubiquitin (in duplicate from three separate transfections, n = 6). (C) ACE2 activity (n = 6) and (D) expression (n = 3) normalized to β-actin in HEK293T cells transfected with pcDNA3.1 and treated with PYR-41 (50 μM for 2 h), a cell-permeable inhibitor of E1 ligases, PR-619 (20 µM, 6 h), a broad-spectrum reversible inhibitor of cysteine-reactive deubiquitinases, or Ang-II (100 nM). (E) ACE2 expression in Neuro2A cells treated with the same drugs (n = 5–6). Two-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 2

Predicted ACE2-interacting Nedd4-2 is up-regulated in hypertension. (A) Bioinformatic analysis (UbiBrowser v1.0) showing E3 ubiquitin ligases predicted to interact with ACE2 based on E3 recognition motifs, network loops, and enriched gene ontology pairs. (B) Identification of four potential Nedd4-2 (also known as NEDD4L) recognition motifs (green) in the amino acid sequence of ACE2. Close-by lysine residues (K) are highlighted as potential ubiquitination sites. (C) Changes in E3 ubiquitin ligase abundance identified by proteomic analysis from the hypothalamus of normotensive and Ang-II-infused mice from both sexes. Representative pictures and capillary western analysis of Nedd4-2 (130 kDa) and ACE2 (120 kDa) expression in the mouse (n = 6/group) brain (D), heart (E), kidney (F), and cardiac left ventricles (n = 6/group) from African American donors (G) normalized to GAPDH (∼37 kDa). Two-way ANOVA followed by Tukey’s test for multiple comparisons. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 3

ACE2 is ubiquitinated by Nedd4-2. (A) Impact of WT Nedd4-2 and its catalytically inactive mutant (C/S) transfection on ACE2 activity in HEK293T cells exposed to Ang-II (n = 5). (B) Modulation of ACE2 cellular levels by Nedd4-2 in HEK293T cells (n = 4). (C) Subcellular localization of ACE2 and Nedd4-2 in HAEC in the absence (control) or presence of Ang-II (100 nM for 4 h). (D) Representative pictures and capillary western analysis of Nedd4-2 and ACE2 expression in HAEC treated with Ang-II in the presence or absence of Nedd4-2 siRNA. Two-way ANOVA followed by Tukey’s test for multiple comparisons, except for panel (B) (one-way ANOVA followed by Bonferroni’s test). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Scale bars are 10 µm.

Figure 4

Ubiquitination-resistant ACE2. (A) Amino acid sequence of the native ACE2 C-terminus and the six mutants (Δ) where lysine residues were replaced by arginine. The distal predicted Nedd4-2 recognition motif is highlighted in green. (B) ACE2 activity for WT and hACE2 mutants in the absence and presence of Ang-II (100 nM, 4 h). (C) Representative western electrophoresis and quantified data showing the impact of Ang-II (100 nM, 4 h) on total cellular levels of ACE2 (n = 6) in HEK293T cells transfected with WT ACE2 or ACE2-5R. All cells were transfected with AT₁R plasmids. (D) Schematic of the hACE2-5R (Mutant 6) adeno-associated viral vector. Two-way ANOVA. Statistical significance: *P<0.05 and **P < 0.01 vs. WT.

Figure 5

ACE2 is expressed on GABAergic neurons. (A) Schematic of the injection protocol and representative expression of AAV-ChR2-eYFP in the dorsal (dBNST) and ventral (vBNST) bed nucleus of the stria terminalis. Scale bar: 200 µm. (B) Immunohistochemistry pictures showing the co-localization of GABAergic neuron-targeted channelrhodopsin (ChR2) expression (green) with ACE2 (red). Photostimulations of ChR2 (10 Hz, 2 min, n = 7) with blue LED on GABAergic cell bodies in the BNST (C) and neuronal projections in the PVN (D) lead to an immediate reduction in systolic BP and HR, while green LED is ineffective. Inserts show magnifications (×2) of boxed regions. Scale bars: 100 µm. (E) Changes in systolic BP and HR initiated by photoactivation of ChR2 in the BNST are prevented by GABA_A receptor blockade by bicuculline (1 mM) in the PVN. Abbreviations: 3V, third ventricle; AC, anterior commissure.

Figure 6

Ubiquitination-resistant ACE2-5R blunts neurogenic hypertension. Twenty-four-hour recording of BP and HR traces (A) and average data at 4 weeks (B) show that BNST expression of hACE2-5R blunted the development of Ang-II-mediated hypertension in mice during the active phase (grey box; n = 5–6/group). The resting and active phases are indicated by the sun and moon symbols, respectively. Cumulative traces and the area under the curve (C) show that PVN blockade of GABA_A receptors with bicuculline (1 mM) mediates an enhanced systolic pressure response in Ang-II-infused mice expressing ACE2-5R in the BNST (n = 4–6/group). Arrows indicate bicuculline bilateral injections. Capillary western analysis of Nedd4-2 (D) (top band), ACE2 (E), AT₁R (F), Mas1R (G), and MDM2 (H) expression in the BNST normalized to total proteins (n = 7–10/group). Two-way ANOVA followed by Tukey’s test for multiple comparisons. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 7

Working model. Schematic of the role of ACE2 ubiquitination in neurogenic hypertension. ACE2 is expressed on GABAergic neurons within the BNST where elevated Ang-II levels lead to Nedd4-2 up-regulation and ACE2 ubiquitination. Ubiquitinated ACE2 is internalized and degraded, preventing the conversion of Ang-II to Ang-(1-7). This is associated with a reduction in the GABAergic inhibitory input to pre-sympathetic neurons in the PVN, contributing to the development of neurogenic hypertension. BNST-targeted expression of a ubiquitination-resistant ACE2-5R mutant preserved ACE2 expression on the cell surface, leading to Ang-II conversion to Ang-(1-7), a reduction of Nedd4-2 expression, enhanced GABAergic inhibitory tone to the PVN, a and reduction in neurogenic hypertension.