Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor γ in macrophages via an interaction with NRF2

Abstract

We have studied the transcription regulation of the rat thromboxane synthase (TXS) gene by peroxisome proliferator-activated receptor γ (PPARγ) in macrophages. The transcription activity of a cloned 5'-flanking region (1.6 kilobases) of the rat TXS gene (5'FL-TXS) was examined by luciferase reporter gene assay. TXS mRNA expression and the transcription activity of 5'FL-TXS were inhibited by PPARγ ligands, 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPARy also significantly suppressed transcription, and further addition of PGJ2 or TRO augmerited the suppression. Deletion analysis showed that the element responsible for the PPARγ effect is located in a region containing the nuclear factor E2 (NF-E2)/AP-1 site (-98/-88), which was indicated to be the major promoter of the TXS gene. By electrophoretic mobility shift assay using the NF-E2/AP-1 site and nuclear extracts from macrophages, we observed a specific protein-DNA complex formation, which was inhibited by a specific antibody against the transcription factor NRF2 (NF-E2-related factor 2). Moreover, the complex was decreased with PGJ2, TRO, or in vitro translated PPARγ. The transcription suppression by PPARy was confirmed using this truncated NRF2-binding element (-98/-88) by the reporter gene assay. Finally, a direct interaction between PPARγ and NRF2 was confirmed by glutathione S-transferase pull-down assay. In conclusion, the NRF2-binding site (-98/-88) is the major promoter of 5'FL-TXS which can be suppressed by activated PPARγ via a protein-protein interaction with NRF2 in macrophages.