Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor gamma in macrophages via an interaction withNRF2

We have studied the transcription regulation of the rat thromboxane synthas e (TXS) gene by peroxisome proliferator-activated receptor gamma (PPAR gamm a) in macrophages, The transcription activity of a cloned 8'-flanking regio n (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)-prostaglan din J(2) (PGJ(2)), and the thiazolidinedione troglitazone (TRO) in a dose-d ependent manner. Overexpression of PPAR gamma also significantly suppressed transcription, and further addition of PGJ(2) or TRO augmented the suppres sion. Deletion analysis showed that the element responsible for the PPAR ga mma 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 TX S gene. By electrophoretic mobility shift assay using the NF-E2/AP-1 site a nd nuclear extracts from macrophages, we observed a specific protein-DNA co mplex formation, which was inhibited by a specific antibody against the tra nscription factor NRF2 (NF-EB-related factor 2). Moreover, the complex was decreased with PGJ(2), TRO, or in vitro translated PPAR gamma. The transcri ption suppression by PPAR gamma was confirmed using this truncated NRF2-bin ding element (-98/-88) by the reporter gene assay. Finally, a direct intera ction between PPAR gamma and NRF2 was confirmed by glutathione S-transferas e pull-down assay. In conclusion, the NRF2-binding site (-98/-88) is the ma jor promoter of 5'FL-TXS which can be suppressed by activated PPAR gamma vi a a protein-protein interaction with NRF2 in macrophages.