ACE2 Ubiquitination is Differentially Regulated by Sex Hormones in Hypertension

Abstract

Angiotensin Converting Enzyme 2 (ACE2) is a compensatory enzyme converting Angiotensin (Ang)-II to Ang-(1-7). Previously, we reported that ACE2 is expressed on neurons in the brain and the lack of ACE2 on the cell surface, partly resulting from Ang-II-mediated internalization, ubiquitination, and lysosomal degradation, contributes to neurogenic hypertension. In the current study, we aimed to identify ACE2 ubiquitination partners and investigate their regulation by sex hormones in hypertension. C57BI/6J mice (3-month-old, n=10/group) were divided into 4 groups: control males (CM), castrated males (CX), control females (CF) and ovariectomized females (OVX). Following castration or ovariectomy, mice were implanted with blood pressure (BP) transducers (telemetry) and upon recovery, baseline BP was recorded for 24 hours. Mice were then implanted subcutaneously with an osmotic pump containing Ang-II (490 ng/Kg/min/4 weeks) or saline. BP was recorded weekly, for 4 weeks while baroreflex (sequence method) and autonomic (pharmacological method) functions were assessed before and after Ang-II. Hypothalamus was isolated from all groups and subjected to LCMS analysis for identification of ubiquitination hits followed by validation using capillary western. Ang-II infusion induced a significant mean BP increase in all groups (CM: 133 ±0.6; CX: 141 ±0.6; CF: 134 ±0.7; OVX: 130 ±0.3 mmHg; n=6: one-way ANOVA, P< 0.001). Proteomics analysis pointed to an Ang-II-mediated increase in UBR1 and NEDD4-L E3 ligases, while BRCC3, a deubiquitinase, appeared downregulated. Capillary western analysis confirmed that Ang-II-mediated hypertension was associated with a 4-fold increase in UBR1 expression in the hypothalamus of males but not in females (P<0.001) and castration blunted this effect by 50% while ovariectomy had no effect. Similarly, NEDD4-L expression in the hypothalamus was upregulated by 4-fold and completely prevented by castration (P<0.001). NEDD4-L expression was unchanged in females with or without estrogens. On the other hand, males infused with Ang-II showed no change in BRCC3 expression while hypertensive females exhibited a dramatic reduction (8-fold) of this deubiquitinase (P<0.001). Baseline expression of BRCC3 was reduced by 50% (P<0.001) following ovariectomy and further reduced by Ang-II. To establish a link between UBR1, NEDD4-L and ACE2, endothelial cells were treated with UBR1 and NEDD4-L siRNA, resulting in a significant increase of ACE2 levels by 5-fold compared to control cells (P<0.001). Our data show that Ang-II-mediated hypertension leads to UBR1 and NEDD4-L upregulation in males, a process mediated by testosterone, while BRCC3 expression depends on estrogens and is downregulated by Ang-II in females. UBR1 and NEDD4-L are involved in ACE2 downregulation. ACE2 expression in hypertension is differentially regulated by E3 ligases and deubiquitinases that are themselves dependent on sex hormones. Inhibition of UBR1 and NEED4L could be a new therapeutic approach to preserve ACE2 compensatory activity in hypertension.