RhoBTB1, a Novel PPARγ Target Gene Regulates Vascular Function
Authors: Mukohda Masashi , Ibeawuchi Stella‐Rita C , Hu Chunyan , Davis Debbie R , Guo Deng Fu , Rahmouni Kamal , Quelle Frederick W , Sigmund Curt D 
Published: November 2023

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand activated transcription factor regulating metabolic and vascular function. We previously reported that mice (S‐DN) expressing dominant‐negative PPARγ in smooth muscle cells (SMC) are hypertensive and exhibit impaired vascular relaxation and enhanced contraction due to increased RhoA/Rho kinase (ROCK) activity, and display reduced expression of a novel PPARγ target gene, RhoBTB1. We hypothesized that RhoBTB1 may play a protective role in vascular function that is disrupted in S‐DN mice. We generated transgenic mice (termed R+) with tamoxifen‐inducible, Cre‐dependent expression of RhoBTB1 in SMC. R+ mice were crossed with S‐DN to produce mice (S‐DN/R+) in which tamoxifen‐treatment (75 mg/kg, ip, 5 days) restored the deficiency in RhoBTB1 expression observed in the aorta of S‐DN mice to normal. Thoracic aorta and basilar artery from S‐DN showed impaired acetylcholine (ACh)‐induced endothelial‐dependent relaxation (p<0.01, n=7–9), which was completely reversed by rescue of RhoBTB1 expression in SMC (p<0.01, n=9). On the contrary, S‐DN mice exhibited enhanced contraction to serotonin (5‐HT) and endothelin‐1 (ET1) in aorta (p<0.01, n=6–9), but this was not reversed by replacement of RhoBTB1 in SMC (n=6–9). Importantly, rescue of RhoBTB1 expression reversed the hypertensive phenotype observed in S‐DN mice (p<0.01, n=8–10). To identify the mechanism of RhoBTB1, RhoA/ROCK activity was assessed. A ROCK inhibitor, Y‐27632, completely improved the impaired ACh relaxation and enhanced contraction induced by 5‐HT or ET‐1 in S‐DN aorta (p<0.05, n=4–6), and also reversed the enhanced contraction in S‐DN/R+ (p<0.05, n=4–6). Consistent with this, increase ROCK‐dependent phosphorylation of myosin phosphatase targeting protein (MYPT) was observed in S‐DN and this was preserved S‐DN/R+ aorta, indicating that restoration of RhoBTB1 did not affect increased RhoA/ROCK activity in S‐DN. Since the NOS inhibitor, L‐NAME, abolished ACh relaxation in S‐DN/R+ and phosphorylation and expression of eNOS were not difference in S‐DN/R+ aorta compared to NT and S‐DN, we next examined the SMC‐dependent relaxation pathway. Aorta from S‐DN mice displayed severely decreased sodium nitroprusside (SNP)‐ and soluble guanylate cyclase (sGC) stimulator, BAY 41‐2722‐induced endothelial‐independent relaxation with a right‐shifted dose‐response (p<0.01, n=7–9), which was reversed in tamoxifen‐treated S‐DN/R+ mice (p<0.01, n=9). Finally, we found that relaxation induced by a cGMP analog, 8‐Bromo‐cGMP, was impaired in S‐DN aorta (p<0.05, n=6), which was reversed in S‐DN/R+ mice (p<0.05, n=7). cGMP‐dependent protein kinase (PKG) 1 expression was not different between genotypes, suggesting RhoBTB1 may affect downstream targets of PKG. We conclude that loss of RhoBTB1 function explains the impaired vasodilation and hypertension observed in response to interference with PPARγ in smooth muscle. Moreover, these studies define RhoBTB1 as a novel PPARγ target gene that plays an important role in selectively facilitating vasodilatation.

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Grants from the NIH and AHA

Support from the Roy J, Carver Trust