Abstract 065: Expression of a Dominant Negative Cullin 3 Mutant (Cul3[[Unable to Display Character: ∆]]9) Impairs Endogenous Cullin 3 Activity and Causes Impaired Vasorelaxation of Aorta

Abstract

Patients with dominant loss-of-function mutations in Cullin 3 (Cul3) resulting in deletion of exon 9 (Cul3[[Unable to Display Character: ∆]]9) exhibit severe early onset hypertension correlated with reduced degradation of Cul3 substrates. We have shown that knockout of Cul3 induced by clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 in HEK293T cells resulted in accumulation of the Cul3 substrate RhoA. To gain insight into the mechanism of attenuated Cul3[[Unable to Display Character: ∆]]9-mediated protein degradation, we utilized CRISPR-Cas9 genome editing to generate Cul3[[Unable to Display Character: ∆]]9-expressing cell lines. We hypothesized that expression of the Cul3[[Unable to Display Character: ∆]]9 protein will impair endogenous Cul3 wildtype (WT) function and accumulate Cul3 substrates. CRISPR guide RNAs targeting endogenous Cul3 were cloned into a plasmid expressing humanized Cas9. HEK293T cells were transfected and single cell clones selected. Sequencing revealed that genome editing induced a 548 bp deletion spanning exon 9 (Chromosome 2:224503433-224503979). Sequencing of the mRNA transcript also revealed that exon 9 was deleted, and Cul3[[Unable to Display Character: ∆]]9 mRNA and protein was expressed. Although RhoA protein levels were normal, Cul3 substrates WNK4 and PLK1 were significantly increased in Cul3[[Unable to Display Character: ∆]]9-expressing cells (WNK4: 1.5±0.12 Cul3[[Unable to Display Character: ∆]]9 vs 1.0±0.02 WT; PLK1: 1.4±0.06 Cul3[[Unable to Display Character: ∆]]9 vs 1.0±0.03 WT, P<0.001). Interestingly, Cul3[[Unable to Display Character: ∆]]9-expressing cells exhibited impaired Cul3 ubiquitin ligase activity compared to WT. We also generated a novel transgenic mouse model inducibly expressing Cul3[[Unable to Display Character: ∆]]9 protein specifically in smooth muscle (termed S-Cul3[[Unable to Display Character: ∆]]9) and assessed vascular responses in the aorta using a wire myograph. Aorta from S-Cul3[[Unable to Display Character: ∆]]9 transgenic mice exhibited impaired ACh relaxation compared to WT (at 30 μM: 55±2% sCul3[[Unable to Display Character: ∆]]9 vs 71±7% WT, p<0.0001). In contrast, vasodilation to the nitric oxide donor, nitroprusside (SNP) was normal, as was constriction to endothelin-1. There was a significant increase in RhoA protein expression in aorta of S-Cul3[[Unable to Display Character: ∆]]9 transgenic mice compared to WT (1.6±0.2 sCul3[[Unable to Display Character: ∆]]9 vs 1.0±0.1 WT, P<0.05). These findings suggest a mechanism whereby Cul3[[Unable to Display Character: ∆]]9 protein may interfere with endogenous Cul3 and impair the degradation of Cul3 substrates such as RhoA or WNK4, contributing, at least in part-, to hypertension.