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.
Authors
Larry N. Agbor, Stella-Rita C. Ibeawuchi, Chunyan Hu, Jing Wu, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund
(A) Schematic of construct used to generate inducible cell-specific CUL3Δ9 transgenic mice. CUL3Δ9 cDNA was generated by overlap splice extension PCR and cloned into a vector, which we previously described (26, 30). (B) Aortic smooth muscle cells (SMC) were isolated from CUL3Δ9 transgenic mice and infected with adenovirus expressing either Cre recombinase (AdCRE) or GFP (AdGFP). The first two control lanes are HEK293 cells transfected with either empty vector (V) or CUL3Δ9 construct (Δ9) to provide a marker for the position of the CUL3Δ9 protein band. A low and high exposure of the same blot are shown to show the decrease in neddylated CUL3WT (top) and the expression of CUL3Δ9 (bottom). This is a representative of 4 independent experiments. (C) Quantification of neddylated CUL3WT (Nedd-CUL3WT) (n = 8). Error bars represent the mean ± SEM. *P < 0.001 by 2-tailed t test. (D) PCR analysis of genomic DNA providing evidence of Cre-mediated excision in smooth muscle–enriched tissues from vehicle- or tamoxifen-treated S-CUL3Δ9 mice. The positions of the transgene and recombined transgene after excision of the lox-STOP-lox (LSL excised) sequence are indicated. (E) Aortic sections from tamoxifen-treated nontransgenic (NT) and S-CUL3Δ9 mice were immunostained for the endothelial marker CD31 (green), stained with DAPI (blue), and were visualized for intrinsic fluorescence of tdTomato reporter (red). Original magnification, ×10; scale bar: 200 μm (top). Original magnification, ×60; scale bar: 10 μm (bottom). NS, nonspecific band; V, vehicle; NT, nontransgenic mice.