Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor gamma 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 (PPARgamma) 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 PPARgamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPARgamma also significantly suppressed transcription, and further addition of PGJ(2) or TRO augmented the suppression. Deletion analysis showed that the element responsible for the PPARgamma 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 PGJ(2), TRO, or in vitro translated PPARgamma. The transcription suppression by PPARgamma was confirmed using this truncated NRF2-binding element (-98/-88) by the reporter gene assay. Finally, a direct interaction between PPARgamma 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 PPARgamma via a protein-protein interaction with NRF2 in macrophages.
Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan.
Y
Y
Ikeda
A
A
Sugawara
Y
Y
Taniyama
A
A
Uruno
K
K
Igarashi
S
S
Arima
S
S
Ito
K
K
Takeuchi
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002839:Chromans / Q000494:pharmacology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology*; D007004:Hypoglycemic Agents / Q000494:pharmacology; D016350:Leucine Zippers; D008264:Macrophages / Q000201:enzymology / Q000502:physiology*; D008969:Molecular Sequence Data; D051267:NF-E2-Related Factor 2; D015230:Prostaglandin D2 / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D045162:Thiazolidinediones; D013930:Thromboxane-A Synthase / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000187:drug effects / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D000077288:Troglitazone
D002839:Chromans; D004268:DNA-Binding Proteins; D007004:Hypoglycemic Agents; D051267:NF-E2-Related Factor 2; C495637:Nfe2l2 protein, rat; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D013844:Thiazoles; D045162:Thiazolidinediones; D015534:Trans-Activators; D014157:Transcription Factors; C037112:9-deoxy-delta-9-prostaglandin D2; D013930:Thromboxane-A Synthase; D000077288:Troglitazone; D015230:Prostaglandin D2