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) Western blot and quantification of the phosphorylated form of myosin phosphatase target subunit 1 (MYPT1) from nontransgenic (NT) and S-CUL3Δ9 aortas (n = 3–4/genotype). *P < 0.05, S-CUL3Δ9 vs. NT by Student’s t test. (B and C) Effect of the Rho-kinase inhibitor, Y-27632 (1 μmol/l, 30 minutes), on acetylcholine-mediated relaxation in aortas (B, n = 4–6) and basilar arteries (C, n = 5) from S-CUL3Δ9 mice compared with NT mice. Error bars represent the mean ± SEM. *P < 0.05. S-CUL39 vs. all other curves by 2-way repeated-measure ANOVA. (D) Effect of the Rho-kinase inhibitor, Y-27632 (1 μmol/l, 30 minutes), on acetylcholine-mediated relaxation in basilar arteries treated with CN03. n = 3/genotype or treatment. Error bars represent the mean ± SEM. *P < 0.05, CN03 vs. all other curves by 2-way repeated-measure ANOVA.