Regulation of thioredoxin gene expression by vitamin A in human airway epithelial cells.
Regulation of thioredoxin gene expression by vitamin A in human airway epithelial cells.
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维生素 A 对人气道上皮细胞中硫氧还蛋白基因表达的调节。
DOI：
10.1165/ajrcmb.26.5.4276
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发表时间：
2002
影响因子：
6.4
通讯作者：
Wu,Reen
中科院分区：
文献类型：
--
作者：
Chang,Wen-Hsing;Reddy,SekharP-M;Di,Yuan-PuPeter;Yoneda,Ken;Harper,Richart;Wu,Reen
人硫氧还蛋白（Trx）是一种12 kD的蛋白质，已知参与细胞生长和细胞损伤修复所必需的各种还原/氧化反应。我们以前证明，基于核运行的分析，视黄酸（RA）刺激呼吸道上皮细胞在转录水平上的Trx基因表达。人类Trx基因5 ′侧翼区的核苷酸测序显示，在-28处存在TATA盒，在-426、-453、-507和-626 nt处存在四个RA反应元件（RARE）样半位点。瞬时转染试验与Trx启动子报告基因，氯霉素乙酰转移酶（CAT），表现出剂量依赖性的参与，这四个RARE样的半位点在RA增强启动子活性。当将这4个RARE样半位点（-357至-671 nt）侧翼的DNA片段导入tk-CAT 2载体的异源启动子时，观察到基础和RA刺激的CAT活性。定点诱变方法证明了RARE-I和RARE-II在-426和-453 nt处分别起着重要作用，RARE-III在-507 nt处在基础和RA刺激的CAT活性中起着辅助作用。Bothin vivoandin vitrogenomic footprinting实验进一步证明了这些“推定的”RARE-I/II/III半位点中的特异性蛋白质-DNA相互作用。凝胶电泳迁移率变动分析表明，这些罕见的半网站与从RA处理的文化获得的核提取物的具体相互作用。抗RAR- α 抗体超移位实验进一步证实了RARE-I/II位点与RAR- α核受体的相互作用。这些结果表明，一个经典的RARE/RAR的相互作用参与RA刺激的Trx基因在人气道上皮细胞的表达。
Human thioredoxin (Trx) is a 12-kD protein known to be involved in various reduction/oxidation reactions essential for cell growth and cellular injury repair. We previously demonstrated, based on nuclear run-on assay, that retinoic acid (RA) stimulated Trx gene expression in airway epithelial cells at the transcriptional level. Nucleotide sequencing of the 5 ′ -flanking region of the human Trx gene revealed the presence of a TATA box at –28 and four RA response element (RARE)-like half sites at –426, − 453, − 507, and –626 nt. Transient transfection assays with a Trx promoter-reporter gene, chloramphenicol acetyltransferase (CAT), demonstrated a dose-dependent involvement of these four RARE-like half sites in RA-enhanced promoter activity. When the DNA fragment that flanks these four RARE-like half sites from − 357 to − 671 nt was introduced into a heterologous promoter of the tk-CAT2 vector, both basal and RA-stimulated CAT activities were observed. A site-directed mutagenesis approach demonstrated an essential role for RARE-I and RARE-II at − 426 and − 453 nt, respectively, and an auxiliary role for RARE-III at − 507 nt in both basal and RA-stimulated CAT activities. Bothin vivoandin vitrogenomic footprinting experiments further demonstrated specific protein–DNA interactions in these “putative” RARE-I/II/III half sites. Gel electrophoretic mobility shift assays demonstrated specific interactions of these RARE-like half sites with the nuclear extracts obtained from RA-treated cultures. The anti–RAR- α antibody super-shift experiment further confirmed the interactions of RARE-I/II sites with RAR- α nuclear receptor. These results suggest a classic RARE/RAR interaction involved in RA-stimulated Trx gene expression in human airway epithelium.