Endothelial Cullin3 Mutation Causes Vascular Dysfunction, Arterial Stiffening, and Hypertension

Abstract

Cullin3 (Cul3) is a critical subunit of the Cul3‐Ring‐Ligase (CRL3) ubiquitin ligase complex that targets proteins for proteasomal degradation. Mutations in CULLIN3 gene (causing in‐frame deletion of exon 9, termed Cul3Δ9) cause hypertension in humans. Evidence suggests that the hypertension in these subjects is likely to be caused by a combination of renal tubular and vascular mechanisms. We have recently shown that smooth muscle expression of Cul3Δ9 causes vascular dysfunction and elevation of blood pressure (BP) via augmented RhoA/Rho‐kinase signaling, strongly supporting a vascular role of Cul3 in BP regulation. To test the importance of endothelial Cul3 in vivo, we bred the conditionally activatable Cul3Δ9 mice with Tek‐CREERT2 mice specifically expressing tamoxifen‐inducible Cre‐recombinase in the endothelium. The resultant mice (E‐Cul3Δ9) exhibited impaired endothelial‐dependent relaxation in carotid artery (maximal relaxation in response to 30 μM acetylcholine, 69% vs 84% in littermate control mice, n=5, p<0.05, two‐way ANOVA repeated measurements) and in the cerebral basilar artery (maximal relaxation in response to 100 μM acetylcholine, 41% vs 77% in littermate control mice, n=5, p<0.01). No difference in smooth muscle function was observed. Moreover, E‐Cul3Δ9 mice exhibited nocturnal hypertension as determined by radiotelemetry (night time peak systolic BP, 138±3 mmHg n=7 vs 122±4 mmHg n=5, p<0.01). However no difference was seen in daytime pressure. Preliminary data indicated that treatment with nitric oxide synthase (NOS) inhibitor Nω‐Nitro‐L‐arginine methyl ester markedly elevated nocturnal peak SBP in control mice (133±5 mmHg, n=3) but not in E‐Cul3Δ9 mice (143±3 mmHg, n=3), suggesting that NOS activity is low in E‐Cul3Δ9 mice. E‐Cul3Δ9 mice also exhibited arterial stiffening as indicated by elevated pulse wave velocity (3.7±0.3 m/s n=5 vs control mice 2.7±0.1 m/s n=7, p<0.01). To determine the molecular mechanism of endothelial dysfunction, primary aortic endothelial cells were isolated from mice carrying the inducible Cul3Δ9 construct and Cul3Δ9 expression was robustly induced by adenovirus carrying Cre recombinase gene in vitro. Expression of Cul3Δ9 resulted in marked decreases in wild type Cul3 protein, phosphorylated eNOS, and nitric oxide production. Because protein phosphatase 2A (PP2A) is a known Cul3 substrate which negatively regulates eNOS phosphorylation, we determined whether impaired eNOS activity was attributable to PP2A. Cul3Δ9‐induced impairment of eNOS activity was rescued by a selective PP2A inhibitor Okadaic Acid (4 nM), but not by a Protein Phosphatase 1 inhibitor Tautomycetin (4 nM). These data define a novel regulatory pathway involving Cullin‐3/PP2A/phospho‐eNOS in the endothelium. Selective endothelial expression of Cul3Δ9 partially phenocopies the hypertension observed in Cul3Δ9 patients, suggesting that mutations in Cullin‐3 cause human hypertension in part through a vascular mechanism characterized by endothelial dysfunction.Support or Funding InformationDr. Jing Wu is supported by an American Heart Association Postdoctoral Fellowship (17POST33660685). This project is funded by R01 HL125603 and R01 HL131689 to Dr. Curt Sigmund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.