RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity
Masashi Mukohda, Shi Fang, Jing Wu, Larry N. Agbor, Anand R. Nair, Stella-Rita C. Ibeawuchi, Chunyan Hu, Xuebo Liu, Ko-Ting Lu, Deng-Fu Guo, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund
Masashi Mukohda, Shi Fang, Jing Wu, Larry N. Agbor, Anand R. Nair, Stella-Rita C. Ibeawuchi, Chunyan Hu, Xuebo Liu, Ko-Ting Lu, Deng-Fu Guo, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund
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Research Article Vascular biology

RhoBTB1 protects against hypertension and arterial stiffness by restraining phosphodiesterase 5 activity

Abstract

Mice selectively expressing a PPARγ dominant-negative mutation in vascular smooth muscle exhibit RhoBTB1 deficiency and hypertension. Our rationale was to use genetic complementation to uncover the mechanism of action of RhoBTB1 in vascular smooth muscle. Inducible smooth muscle–specific restoration of RhoBTB1 fully corrected hypertension and arterial stiffness by improving vasodilator function. Notably, the cardiovascular protection occurred despite the preservation of increased agonist-mediated contraction and RhoA and Rho kinase activity, suggesting that RhoBTB1 selectively controls vasodilation. RhoBTB1 augmented the cyclic 3′,5′-monophosphate (cGMP) response to NO by restraining the activity of phosphodiesterase 5 (PDE5) through its action as a substrate adaptor delivering PDE5 to the Cullin-3 E3 ring ubiquitin ligase complex for ubiquitination, thereby inhibiting PDE5. Angiotensin II infusion also caused RhoBTB1 deficiency and hypertension, which were prevented by smooth muscle–specific RhoBTB1 restoration. We conclude that RhoBTB1 protected against hypertension, vascular smooth muscle dysfunction, and arterial stiffness in at least 2 models of hypertension.

Authors

Masashi Mukohda, Shi Fang, Jing Wu, Larry N. Agbor, Anand R. Nair, Stella-Rita C. Ibeawuchi, Chunyan Hu, Xuebo Liu, Ko-Ting Lu, Deng-Fu Guo, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund

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Figure 1

Inducible RhoBTB1 experimental model.

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Inducible RhoBTB1 experimental model. (A) Schematic of the inducible Rho...
(A) Schematic of the inducible RhoBTB1 transgene construct illustrating removal of the loxP-STOP-loxP with consequent expression of RhoBTB1 and tdTomato in response to Cre recombinase. (B) Western blot detecting RhoBTB1 or Myc-tagged RhoBTB1, tdTomato, and GAPDH in HEK293 cells transfected with Myc-RhoBTB1 or RhoBTB1IND, with or without a Cre recombinase expression vector. Actual size markers transferred from the blots are shown. (C) Schematic of breeding to generate triple-transgenic mice expressing dominant-negative PPARγ (S-P467L) in vascular smooth muscle along with inducible smooth muscle–specific expression of RhoBTB1 (S-RhoBTB1). (D) Relative mRNA expression of RhoBTB1, tdTomato, and human PPARγ (hPPARγ) was determined by qPCR in aorta from mice of the indicated strains 3–4 weeks after injection of Tx. Data were normalized to the average control value, set to 1.0. All data represent the mean ± SEM. *P < 0.05 versus control; #P < 0.05, S-P467L versus S-P467L/S-RhoBTB1 mice; 1-way ANOVA. (E) Western blot of total aortic protein from the indicated mouse strains (treated with Tx) probed for PPARγ, tdTomato, and GAPDH. Actual size markers transferred from the blots are shown. Shown are 3 representative blots from 7 total samples analyzed for each genotype. (F) Immunostaining of aorta from control and S-P467L/S-RhoBTB1 mice. Red indicates tdTomato and green vWF, a marker of endothelium. DAPI staining (blue) labels nuclei. Scale bars: 100 μm (left panels) and approximately 15 μm (right panels).