Cullin-3 (CUL3) mutations (CUL3Δ9) were previously identified in hypertensive patients with pseudohypoaldosteronism type-II (PHAII), but the mechanism causing hypertension and whether this is driven by renal tubular or extratubular mechanisms remains unknown. We report that selective expression of CUL3Δ9 in smooth muscle acts by interfering with expression and function of endogenous CUL3, resulting in impaired turnover of the CUL3 substrate RhoA, increased RhoA activity, and augmented RhoA/Rho kinase signaling. This caused vascular dysfunction and increased arterial pressure under baseline conditions and a marked increase in arterial pressure, collagen deposition, and vascular stiffness in response to a subpressor dose of angiotensin II, which did not cause hypertension in control mice. Inhibition of total cullin activity increased the level of CUL3 substrates cyclin E and RhoA, and expression of CUL3Δ9 decreased the level of the active form of endogenous CUL3 in human aortic smooth muscle cells. These data indicate that selective expression of the Cul3Δ9 mutation in vascular smooth muscle phenocopies the hypertension observed in Cul3Δ9 human subjects and suggest that mutations in CUL3 cause human hypertension in part through a mechanism involving smooth muscle dysfunction initiated by a loss of CUL3-mediated degradation of RhoA.
Larry N. Agbor, Stella-Rita C. Ibeawuchi, Chunyan Hu, Jing Wu, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund
(A) Vasorelaxation in response to acetylcholine in aortas from nontransgenic (NT) and S-CUL3Δ9 mice (n = 14–16). 3- to 4-mm aortic sections were equilibrated at 0.5 g for 45 minutes in an organ bath, preconstricted with PGF2α to 45%–50% of maximal response to U46619, and the dose-response to ACh was assessed. *P < 0.05, S-CUL3Δ9 vs. NT by 2-way repeated-measure ANOVA. (B) Expression of the indicated proteins in aortas from NT and S-CUL3Δ9 mice. (C) Quantification of neddylated CUL3WT, unneddylated CUL3WT, and RhoA in aortas from NT and S-CUL3Δ9 mice. n = 4–9/genotype. *P < 0.05, S-CUL3Δ9 vs. NT by 2-tailed t test. (D) Levels of active RhoA-GTP from aortic rings from NT and S-CUL3Δ9 mice using a pulldown assay. Aortas were equilibrated to 0.5 g tension, with or without serotonin (5-HT) (1 μmol/l, 5 minutes) as indicated. The level of RhoA-GTP was quantified. n = 5–6/genotype/treatment. *P < 0.05, 5-HT vs. baseline; #P < 0.05, 5-HT S-CUL3Δ9 vs. 5-HT NT by 1-way ANOVA. This is a representative of 3 independent experiments. (E) Levels of active RhoA-GTP from aortic rings from C57BL/6 mice using a pulldown assay. Aortic rings were incubated with the RhoA activator CN03 or saline (5 μg/ml, 4 hours). The level of RhoA-GTP was quantified (n = 5–7/treatment). Error bars represent mean ± SEM. *P < 0.05, CN03 vs. saline by Student’s t test. This is a representative of 2 independent experiments. NS, nonspecific bands; SMC, smooth muscle cell; GDP, guanosine diphosphate; GTPγs, guanosine triphosphate γ S.