Abstract 001: Mutation in the PPARγ Ligand Binding Domain Impairs the Anti-inflammatory Action of PPARγ

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) has been proposed to antagonize the activities of nuclear factor kappa B (NFκB) to regulate inflammation. Transgenic mice expressing dominant negative (DN) PPARγ specifically in vascular smooth muscle cells (SMC) exhibited exacerbated atherosclerosis but the mechanism remains unknown. We hypothesized that DN PPARγ promotes NFκB-induced inflammation in SMC. To test this, we cultured mesenteric SMCs from transgenic mice that would conditionally express wild-type (WT) or DN PPARγ (P467L) in response to adenovirus expressing Cre-recombinase (AdCRE). PPARγ expression remained silent in control SMC infected with AdGFP. TNF-α (0.05 ng/ml, 6 hr) induced NFκB target gene (MCP-1, iNOS and MMP9) expression to a greater extent in P467L-Cre compared to P467L-GFP. For example, MMP9 expression was induced 6.3±0.2-fold in P467L-Cre vs 3.2±0.5-fold in P467L-GFP (p<0.01). The NFκB subunit, p65, mRNA level was not altered in these cells. There was no induction of the PPARγ target aP2 in P467L-Cre, but it was induced 6.5±1.7-fold in WT-SMC infected with AdCRE (WT-Cre). The ability of TNF-α to induce NFκB target gene expression was blunted or abrogated in WT-Cre cells, and their expression was significantly reduced in TNF-α-treated WT-Cre compared to WT-GFP (MMP9: 0.7±1.2 vs 6.0±0.3, p<0.01). To examine mechanisms in vivo, we crossbred transgenic mice expressing WT PPARγ specifically in SMC (S-WT) with mice expressing luciferase under control of a NFκB-responsive promoter. TNF-α (500 ng/ml, 24 hr)-induced NFκB activity was decreased in aorta and carotid artery from S-WT mice compared to control mice (aorta: 4.7±1.1 v s 6.7±0.7, p<0.05, carotid artery: 4.0±0.6 vs 8.7±1.2, p<0.01). Finally, to assess the mechanism preventing anti-inflammatory activity by DN PPARγ, we assessed its interaction with p65 protein when co-expressed in HEK293T cells. WT PPARγ co-precipitated with p65, but the interaction between p65 and P467L PPARγ was severely impaired (n=6). Other mutants (R165T, V290M) could bind to p65, suggesting that loss of the ability is specific to P467L PPARγ. We conclude that SMC-PPARγ has anti-inflammatory effects mediated through inhibition of NFκB activity, which is abolished by P467L mutation.