Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells -: Roles of the Oct-1 element in the 5′-flanking region and the AP-2 element in the untranslated exon 1
Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells -: Roles of the Oct-1 element in the 5′-flanking region and the AP-2 element in the untranslated exon 1
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DOI：
10.1074/jbc.m007688200
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发表时间：
2000-12-22
影响因子：
4.8
通讯作者：
Urade, Y
中科院分区：
文献类型：
--
作者：
Fujimori, K;Kanaoka, T;Urade, Y
人造血前列腺素D合酶（H-PGDS）基因在人巨核细胞中高表达，其中佛波酯诱导其表达。我们表征了人巨核细胞Dami细胞中人H-PGDS基因的5'侧翼区和非翻译外显子1（-1044至+290）的启动子活性，使用荧光素酶报告基因的瞬时表达分析表明，5'侧翼区和非翻译外显子1足以在Dami细胞中有效表达H-PGDS基因，但在单核U937细胞中则不然。与整个区域相比，5'侧翼区域中 Oct-1 元件的删除和定点突变使启动子活性降低了约 30%（从 -1044 到 +290）。电泳迁移率变动分析表明 Oct-1 特异性结合到启动子区域。有趣的是，即使只有非翻译外显子 1（+1 至 +290）也显示出与 -1044 至 +290 整个区域的 60% 的启动子活性相似。非翻译外显子 1 内 AP-2 元件的定点诱变消除了基础启动子活性及其佛波酯介导的上调。在 AP-2 缺陷的 HepG2 细胞中，H-PGDS 启动子活性通过与 AP-2 α 共表达而增强。这些发现表明，5'侧翼区域中的 Oct-1 元件充当正顺式作用元件，并且非翻译外显子 1 中的 AP-2 元件对于巨核 Dami 细胞中基础和佛波酯介导的人 H-PGDS 基因表达的上调至关重要。
The human hematopoietic prostaglandin D synthase (H-PGDS) gene is highly expressed in human megakaryoblastic cells, in which phorbol ester induces its expression. We characterized the promoter activity of the 5'-flanking region and the untranslated exon 1 (-1044 to +290) of the human H-PGDS gene in human megakaryoblastic Dami cells, Transient expression analysis using the luciferase reporter gene revealed that the 5'-flanking region and the untranslated exon 1 were sufficient for efficient expression of the H-PGDS gene in Dami cells, but not in monocytic U937 cells. Deletion and site-directed mutagenesis of the Oct-1 element in the 5'-flanking region decreased the promoter activity by similar to 30% compared with that of the entire region from -1044 to +290. An electrophoretic mobility shift assay demonstrated that Oct-1 specifically bound to the promoter region. Interestingly, even only untranslated exon 1 (+1 to +290) showed similar to 60% of the promoter activity of the entire region from -1044 to +290. Site-directed mutagenesis of the AP-2 element within the untranslated exon 1 abolished the basal promoter activity as well as its phorbol ester-mediated up-regulation. In AP-2-deficient HepG2 cells, the H-PGDS promoter activity was enhanced by coexpression with AP-2 alpha. These findings indicate that the Oct-1 element in the 5'-flanking region acts as a positive cis-acting element and that the AP-2 element in the untranslated exon 1 is crucial for both basal and phorbol ester-mediated up-regulation of human H-PGDS gene expression in megakaryoblastic Dami cells.