RhoBTB1, a Novel PPARγ Target Gene Regulates Vascular Function

Peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand activated transcription factor known to regulate fatty acid metabolism. Synthetic agonists of PPARγ, thiazolidinediones have been shown to be cardioprotective and lower blood pressure independent of glycemic control. In contrast, loss of PPARγ function in patients carrying DN PPARγ mutations (V290M or P467L) causes insulin resistance and severe early onset hypertension. We previously showed that PPARγ affects blood pressure through its activities in vascular smooth muscle cells (SMCs), since transgenic mice expressing a dominant‐negative PPARγ mutation (P467L) specifically in SMC (S‐P467L) are hypertensive and exhibit severe aortic dysfunction. These mice also display reduced expression of a novel PPARγ target gene, RhoBTB1. We hypothesized that RhoBTB1 may play a role in the PPARγ‐mediated regulation of vascular function that is disrupted in S‐P467L mice. To test this, we generated transgenic mice with tamoxifen‐inducible, SMC‐specific expression of RhoBTB1 (S‐RhoBTB1) by crossing CAG‐RhoBTB1 transgenic mice with SMC‐CreERT2 mice. These mice were then crossed with S‐P467L to produce triple transgenic (S‐P467L x S‐RhoBTB1) mice, predicted to maintain RhoBTB1 expression during P467L‐induced PPARγ dysfunction in SMC. Following tamoxifen injection (75 mg/kg, IP, for 5 days), RhoBTB1 mRNA expression in aorta was increased from the reduced level observed in S‐P467L mice, and was restored to the level of NT mice. The mRNA of a reporter co‐expressed with RhoBTB1, tdTomato, was also increased in aorta from S‐P467L x S‐RhoBTB1 mice. Thoracic aorta from S‐P467L mice showed impaired acetylcholine (ACh)‐induced endothelial‐dependent relaxation (maximum relaxation; 43.3+4.4 %, n=6). Notably, tamoxifen itself (in the absence of the RhoBTB1 transgene) did not improve the impaired relaxation in response to ACh or SNP in aorta or basilar artery from S‐P467L mice. We also confirmed that expression of the tdTomato reporter did not have any effects on vascular reactivity in aorta utilizing SMC‐CreERT2 mice bred with ROSA transgenic mice with Cre‐inducible expression of tdTomato (n=4). However, overexpression of RhoBTB1 in SMC of S‐P467L (S‐P467L x S‐RhoBTB1) restored the ACh response to the same level as NT mice (maximum relaxation; 74.2+1.1 %, n=6). A similar improvement was observed in basilar artery (19.9+6.7 vs 48.1+12.3 %, p<0.05, n=6). Aorta from S‐P467L mice also displayed a severely impaired vasodilator response to sodium nitroprusside (SNP) that was also normalized in aorta from tamoxifen‐treated S‐P467L x S‐RhoBTB1 mice (p<0.01, n=6). Interestingly, contraction induced by ET‐1, but not KCl, was enhanced in S‐P467L aorta, and was not improved by restoring RhoBTB1 expression (n=6). This suggests there may be some selectivity in the function of RhoBTB1 whereby it promotes vasodilation but does not affect vasoconstriction. We conclude that loss of RhoBTB1 function explains the impaired vasodilation observed in response to interference with PPARγ in smooth muscle. Moreover, these study define RhoBTB1 as a novel PPARγ target gene that plays an important role in facilitating vasodilatation. Support or Funding Information This work was supported by National Institutes of Health Grants HL048058, HL062984 and HL08307 (to Curt D. Sigmund).