Abstract 59: Loss of Cullin-3 in Smooth Muscle Increases RhoA, Vascular Contraction and Arterial Pressure
Mutations in Cullin-3 result in early-onset hypertension in humans through an undefined mechanism. Cullin-3 RING E3 ubiquitin ligase complex mediates proteasomal degradation of RhoA, a key mediator of vascular smooth muscle tone. We reported that smooth muscle-specific expression of dominant negative PPARγ (S-P467L) in transgenic mice causes hypertension and aortic dysfunction via increased RhoA/Rho-kinase signaling. Dominant negative PPARγ caused decreased Cullin-3 protein (0.31±0.04-fold of Non-Transgenic (NT); P<0.05) and decreased ratio of active Nedd8-Cullin-3 / Cullin-3 (0.06±0.04 vs 0.51±0.18 NT; P<0.05), which correlated with increased protein levels of Cullin-3 substrates RhoA (2.6±0.2-fold of NT; P<0.05) and Cyclin E (2.0±0.1-fold of NT; P<0.05) in S-P467L medial aorta. We tested the hypothesis that Cullin-3 controls arterial pressure by regulating vascular function. siRNA-mediated knockdown of Cullin-3 (72 hr), confirmed by Western blot, increased RhoA (2.5±0.1-fold of NC1 negative control siRNA; P<0.05) and Cyclin E (1.9±0.2-fold of NC1; P<0.05) protein in primary rat aortic smooth muscle cells. Inhibition of cullin-RING ligase activity using the Nedd8-activating enzyme inhibitor, MLN4924 (1μM, 16 hr), also increased RhoA (4.3±1.0-fold of DMSO vehicle). Treatment of aortic rings from control mice with MLN4924 (1μM in DMEM/F12, 16 hr) resulted in enhanced agonist-mediated contraction in response to endothelin 1 (30nM: 0.35±0.05g vs 0.19±0.02g DMSO vehicle; P<0.05), serotonin (3μM: 1.2±0.07g vs 0.9±0.08g; P<0.05), and phenylephrine (3μM: 1.1±0.05g vs 0.7±0.05g, P<0.05) that was Rho-kinase-dependent, despite decreased contraction to KCl (100mM: 0.7±0.03g vs 1.0±0.03g; P<0.05). Finally, administration of MLN4924 (30mg/kg TID, 2 days) to control mice in vivo increased mean arterial pressure during the light phase (121±4 vs. 108±3mmHg baseline, P<0.05; dark phase: 122±4 vs. 123±4mmHg baseline, NS) as measured by radiotelemetry. Our results demonstrate that interference with PPARγ in smooth muscle causes vascular dysfunction via impaired Cullin-3-mediated regulation of RhoA/Rho-kinase signaling and provide a mechanistic link between mutations in Cullin-3 and hypertension.
- © 2012 by American Heart Association, Inc.