Yaekashiwa Masahiro, Wang Lee-Ho
Division of Hematology, Department of Internal Medicine, University of Texas-Houston, Houston, Texas 77030, USA.
DNA Cell Biol. 2003 Aug;22(8):479-87. doi: 10.1089/10445490360708883.
Thromboxane A(2) synthase (TXAS) converts prostaglandin H(2) to thromboxane A(2), a potent inducer of vaso-constriction and platelet aggregation. TXAS expression level is cell type preferential; high in hematopoietic cells and low in nonhematopoietic cells. We previously showed that p45 NF-E2 activated the TXAS promoter in hematopoietic cells via binding to the nucleotides -86/-77 from the transcriptional start site [Yaekashiwa and Wang (2002) J. Biol. Chem. 277, 22497-22508]. We reported here that, by transient transfection analysis, this region was also critical for TXAS trans-activation in the A549 and WI-38 lung cells. Mutation of the NF-E2 site greatly reduced TXAS promoter activity in these two types of cells. Using stably transfected A549 cells, we showed that an NF-E2 mutation retained only 0.25% of the wild-type promoter activity. Ecotopic expression of NF-E2 related factors showed that Nrf2, but not Nrf1, Nrf3, or Bach1, activated TXAS promoter in a dose-dependent manner. Furthermore, chromatin immunoprecipitation assay using the stably transfected A549 cells demonstrated that Nrf2 bound the TXAS NF-E2 site in vivo. TXAS gene thus utilizes the same cis-acting element but different trans-acting factors to confer cell-preferential expression. We also showed that forced expression of p300 upregulated TXAS gene in a dose-dependent manner. Mutation of NF-E2 site, but not TATA or initiator site, abolished the p300-mediated activation of TXAS gene.
血栓素A(2)合酶（TXAS）将前列腺素H(2)转化为血栓素A(2)，后者是血管收缩和血小板聚集的强效诱导剂。TXAS的表达水平具有细胞类型偏好性；在造血细胞中高表达，而在非造血细胞中低表达。我们之前表明，p45 NF-E2通过与转录起始位点上游-86/-77核苷酸结合，在造血细胞中激活TXAS启动子[矢柏泽和王（2002年）《生物化学杂志》277, 22497 - 22508]。我们在此报告，通过瞬时转染分析，该区域对于A549和WI - 38肺细胞中TXAS的反式激活也至关重要。NF-E2位点的突变极大地降低了这两种细胞类型中TXAS启动子的活性。使用稳定转染的A549细胞，我们发现NF-E2突变仅保留了野生型启动子活性的0.25%。NF-E2相关因子的异位表达表明，Nrf2而非Nrf1、Nrf3或Bach1以剂量依赖方式激活TXAS启动子。此外，使用稳定转染的A549细胞进行的染色质免疫沉淀分析表明，Nrf2在体内与TXAS的NF-E2位点结合。因此，TXAS基因利用相同的顺式作用元件但不同的反式作用因子来赋予细胞偏好性表达。我们还表明，p300的强制表达以剂量依赖方式上调TXAS基因。NF-E2位点的突变而非TATA或起始位点的突变消除了p300介导的TXAS基因激活。
