Abstract 331: Mutation in the PPARG Ligand Binding Domain Impairs PPARG-Mediated Turnover of the p65 Subunit of NF-kB

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand activated transcription factor regulating metabolic and vascular function. PPARγ exerts anti-inflammatory actions, and recent data suggest this may be mediated by promoting the degradation of the p65 subunit of nuclear factor kappa B (NFκB). Transgenic mice expressing dominant negative PPARγ specifically in vascular smooth muscle or endothelium exhibited exacerbated atherosclerosis but the mechanism remains unknown. We hypothesized that PPARγ mutants promote inflammation because PPARγ-mediated p65 degradation is impaired. We tested this by co-transfection of HEK293T cells with vectors encoding p65, wildtype (WT) PPARγ, or various PPARγ mutants. The level of p65 protein expression was decreased by co-expression with WT-PPARγ (0.53±0.09 vs control, n=8). Whereas, the P467L PPARγ exhibited impaired degradation of p65 (1.0±0.06, n=10), the V290M (0.36±0.1), S273A (0.37±0.06), or K268R/K293R (0.41±0.03) mutations in PPARγ preserved p65 degradation. WT PPARγ was co-precipitated with p65 in co-transfected cells suggesting the mechanism of PPARγ-mediated p65 degradation involves a direct interaction between them. Consistent with this, the interaction between p65 and P467L PPARγ was severely impaired. To assess functional interactions between PPARγ and NFκB, we employed a model of interleukin-1β (IL-1β) mediated dysfunction in aortic rings. IL-1β dose-dependently induced NFκB activity as measured by increased phospho-p65 and decreased IκBα in aorta cultured for 2 hours with IL-1β. IL-1β dose-dependently reduced acetylcholine (Ach)-induced endothelial-dependent relaxation of aortic rings (80±12 vs 39±16, 20 pg/mL vs 11±3, 100 pg/mL, %). IL-1β-mediated loss of Ach vasodilation was reduced by the PPARγ agonist rosiglitazone (1 μM, 25 hr, n=3, p<0.05), or by transgenic over-expression of WT-PPARγ specifically in endothelium (n=6, p<0.05). We conclude that 1) p65 turnover may be regulated by PPARγ and that its mutation can result in impaired p65 degradation, 2) PPARγ activity can protect against vascular dysfunction associated with NFκB activation, and 3) loss of PPARγ-mediated p65 degradation may contribute to inflammation in hypertension and atherosclerosis.