Bifunctional Property of PPAR Gamma1 for KDR Gene Expression. Interaction of PPAR Gamma1 with Sp1 and Sp3

Abstract

Abstract: : Purpose: Recent evidences in our laboratory revealed that peroxisome proliferator-activated receptorγ (PPARγ) ligands inhibited intraocular angiogenesis. However, the precise mechanisms are still unclear. We investigated the role of PPARγ ligands and PPARγ itself in the expressional regulation of VEGF receptor 2 (KDR), which plays a critical role in the vasculogenic and angiogenic processes including diabetic retinopathy. Methods and Results:We demonstrated the potent and novel inhibitory activity of PPAR γ ligands (15-deoxy Δ12, 14-prostaglandin J2 (15d-PGJ2) or insulin-sensitizing thiazolidinedione pioglitazone (Pio)) on KDR gene and protein expression in bovine retinal capillary endothelial cells (BRECs) by Northern bloting and western blotting. We demonstrated the inhibitory activity of 15d-PGJ2 on KDR gene expression through the suppression of both DNA-Sp1and –Sp3 binding by electrophoretic mobility shift assay (EMSA). PPAR γ1 was also shown to physically interact with Sp1 and Sp3, and PGJ2 dose-dependently suppressed Sp1- and Sp3-PPARγ interaction by glutathione S-transferase (GST) pull down assay. Moreover, the results of transactivation and EMSA indicated that PPAR γ1 protein increased KDR promoter activity by enhansement of the Sp1-KDR promoter binding without its ligands. We also demonstrated that the ligand binding domain (LBD), but not DNA binding domain (DBD), of PPAR γ1 enhanced the Sp1-KDR promoter binding by EMSA. Conclusions: The interaction between PPARγ1, especially LBD of PPARγ1, and Sp1/Sp3 appears to regulate binding between Sp family and KDR promoter region. PPARγ1 ligands bind to LDB of PPARγ1 and suppressed Sp family-KDR promoter binding, resulting in the inhibition of KDR gene expression in BRECs. Thus, PPAR γ1 seems to have bifunctional property for KDR gene regulation.

Keywords: diabetic retinopathy • signal transduction • gene/expression