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

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-Δ^12,14-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.