Document Type

Dissertation

Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Pharmacology

First Advisor

Curt D. Sigmund

Abstract

Hypertension and type II diabetes are key components of metabolic syndrome affecting one third of US population. Insulin-sensitizing thiazolidinediones (TZDs) are high affinity synthetic ligands for Peroxisome Proliferator-Activated Receptor gamma (PPARG), a nuclear receptor and ligand-activated transcription factor. Clinical data show that TZDs are cardioprotective and lower blood pressure even with increased water and salt retention, suggesting a direct role for PPARG in blood pressure regulation. Human subjects with PPARG mutations exhibit severe early onset hypertension, insulin resistance and type II diabetes. These PPARG mutations, V290M and P467L, affect the ligand-binding domain of PPARG and have dominant negative effect on transcriptional activity. However, the mechanism by which PPARG regulates blood pressure remains elusive. A common feature of hypertension is increased RhoA activity and transgenic mice expressing dominant negative PPARG in vascular smooth muscle cells (S-P467L) exhibit hypertension and severe aortic dysfunction dependent on over-activation of the RhoA/ROCK signaling.

Previously published data from our group report that smooth muscle-specific interference of PPARG impairs Cullin-3 E3 ubiquitin ligase-mediated regulation of RhoA and identify Cullin-3 as a novel regulator of vascular function. Patients with de novo mutations that lead to deletion of 57 amino acids encoded by exon 9 in Cullin-3 have early onset hypertension, but the mechanistic basis for this effect is lacking. We show that Cul3 mutation resulted in reduced RhoA ubiquitination and degradation. Reduced Cullin-3 activity increases the RhoA pool that can be activated to a GTP bound state by cellular RhoGEFs in response to contractile agonists promoting hypertension.

We have identified a novel PPARG target gene-RhoBTB1, a component of the Cullin-3 RING E3 ubiquitin ligase (CRL3) complex. SiRNA-mediated knockdown of RhoBTB1 significantly decreased Cul3 protein levels resulting in a modest increase in RhoA protein. To understand the fundamental mechanisms by which vascular PPARG regulate vascular function, we generated a transgenic mice with smooth muscle-specific overexpression of RhoBTB1 (S-RhoBTB1) and crossed with the S-P467L mice. We show that replacement of RhoBTB1 in the vascular smooth muscle complements the defects observed in S-P467L due to PPARG interference. This study will advance our understanding on how PPARG regulates blood pressure so that new therapies that maximize the beneficial effects of PPARG can be developed.

Public Abstract

Thiazolidinediones (TZDs) are insulin sensitizers prescribed to type II diabetes patients to address the problem of insulin resistance. TZDs are synthetic agonist of PPARγ and have been shown to reduce blood pressure in diabetic patients, strongly suggesting that PPARγ may play a direct role in regulating blood pressure. In addition, persons with certain genetic mutations in PPARγ have early onset hypertension as well as diabetes and insulin resistance. Mouse models expressing PPARγ mutations are also hypertensive. My research is aimed at understanding how TZDs lower blood pressure and the mechanisms involved. The activation of PPARγ results in expression of several genes. We have identified a new PPARγ target gene called RhoBTB1. RhoBTB1 directly binds to Cullin-3, which regulates turnover of several proteins. We believe PPARγ regulate blood pressure through RhoBTB1 and Cullin-3. Recently, mutations in Cullin-3 that cause human hypertension were identified. We found that RhoBTB1 gene and protein expressions are significantly decreased in mouse models expressing PPARγ mutations. This also correlated with decreased Cullin-3 expression. In addition, reintroduction of RhoBTB1 into mice with the PPARγ mutations reestablishes normal vascular function. Although TZDs lower blood pressure in diabetic patients, recent clinical studies reported serious concerns of TZDs particularly in some patients with heart failure, so there is an urgent need to understand the mechanism and direct actions of PPARγ in cardiovascular physiology so that more specific drugs might be developed that maximize the beneficial effects of PPARγ activation but eliminate the detrimental side effects.

Keywords

publicabstract

Pages

xii, 110

Bibliography

99-110

Copyright

Copyright 2015 Stella-Rita C Ibeawuchi

Included in

Pharmacology Commons

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