Abstract 3608: High Mobility Group A1 Protein - A New Regulator for Peroxisome Proliferator-Activated Receptor Gamma Transcriptional Repression in Human Aortic Smooth Muscle Cells

Abstract

The study aimed to identify new nuclear cofactors for PPARgamma (peroxisome proliferator-activated receptor gamma)-dependent gene transcription in human aortic smooth muscle cells (HASMC) in order to develop new PPARgamma-ligands with improved clinical safety in the absence of deleterious cardiovascular side effects. Using an Oligo GEArray® Human Nuclear Receptors and Coregulators Microarray for gene expression profiling, we identified the transcriptional regulator and chromatin modifying High Mobility Group (HMG) A1 protein highly expressed in unstimulated HASMC. PPARgamma-dependent gene regulation was studied by analysis of PMA-induced MMP-9 (matrix metalloproteinase 9) expression ± pioglitazone (pio 10μM). PMA (50ng/ml) stimulated MMP-9 mRNA expression by 46.3±22.3-fold (p<0.05 vs. vehicle) which was markedly blocked by pio (10μM: 17.4±4.8-fold vs. PMA alone p<0.05). Pio also blocked PMA-induced MMP-9 promoter activity by 45% in transactivation assays in HEK293 using a pGL3-MMP-9 2.2 kb construct. To evaluate the role of HMGA1, gene silencing experiments with siRNA for HMGA1 were performed (91 % in HASMC and 80.2% in HEK293 reduction of HMGA1 protein expression). HMGA1 siRNA completely abolished PPARgamma-mediated MMP9-mRNA repression (control siRNA: pio-mediated MMP-9 regulation vs. PMA alone: −66.8 % in HASMC and −59.3% in HEK293 p<0.01; HMGA1 siRNA: pio-mediated MMP-9 regulation vs. PMA alone: +10.7 % in HASMC and +14.7% in HEK293 vs. PMA alone; p=n.s.). Knockdown of HMGA1 expression reverse trans-repression of MMP9 by PPARgamma in HASMCs. By using ChIP assay we could demonstrate that pio-induced PPARgamma activation leads to a potent recruitment of PPARgamma (3.0 fold vs.1.15 fold PMA alone) and HMGA1 complexes (1.24 fold vs. 0.0 fold PMA alone) to the MMP9 promoter in HASMC. In consonance with reduced promoter activity, RNA-Polymerase II was removed from the MMP9 promoter by pio (0.08 fold vs 1.04 fold PMA alone). In conclusion, HMGA1 is required for PPARgamma-mediated repression of MMP-9 gene transcription. Ligand-induced HMGA1-PPARgamma interactions might be an important determinant for ligand-specific anti-atherosclerotic actions.