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 gamma (PPAR gamma) in macrophages, The transcription activity of a cloned 8'-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 PPAR gamma ligands, 15-deoxy-(12,14)-prostaglandin J(2) (PGJ(2)), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPAR gamma also significantly suppressed transcription, and further addition of PGJ(2) or TRO augmented the suppression. Deletion analysis showed that the element responsible for the PPAR gamma effect is located in a region containing the nuclear factor E2 (NF-EB)/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-EB-related factor 2). Moreover, the complex was decreased with PGJ(2), TRO, or in vitro translated PPAR gamma. The transcription suppression by PPAR gamma was confirmed using this truncated NRF2-binding element (-98/-88) by the reporter gene assay. Finally, a direct interaction between PPAR gamma 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 PPAR gamma via a protein-protein interaction with NRF2 in macrophages.