Identification of a cis-acting element that is responsible for cadmium-mediated induction of the human heme oxygenase gene.
Identification of a cis-acting element that is responsible for cadmium-mediated induction of the human heme oxygenase gene.
复制标题
鉴定负责镉介导的人血红素加氧酶基因诱导的顺式作用元件。
DOI：
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发表时间：
1994
影响因子：
4.8
通讯作者：
Shigeki Shibahara
中科院分区：
文献类型：
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作者：
K. Takeda;S. Ishizawa;Masaaki Sato;T. Yoshida;Shigeki Shibahara
血红素加氧酶是血红素催化过程中的一种重要酶，可被镉等多种环境因素诱导。镉处理后HeLa细胞血红素氧合酶的活性和mRNA水平显著升高。作为研究这种诱导的分子机制的第一步，我们在包括HeLa在内的四种人类细胞系中进行了瞬时表达测定，以分析镉介导的融合基因的诱导，所述融合基因含有萤火虫荧光素酶基因作为人类血红素加氧酶基因启动子下的报告基因。通过测定转染细胞中表达的荧光素酶活性，我们发现在血红素加氧酶基因的转录起始位点上游约4.5和4个酶对之间的区域，该区域赋予镉介导的对融合基因的诱导。然后，该区域在HeLa细胞中进行了进一步的功能分析，这使我们能够将镉响应元件定位到20个碱基对。凝胶迁移率变动分析表明，这20个碱基对的元素是由HeLa细胞的核蛋白（S）的特异性结合，但其结合活性不变的镉处理。使用合成的镉反应元件含有各种碱基的变化，我们已经确定了一个10个碱基对的序列，TGCTAGATTT，所需的镉介导的诱导和体外蛋白结合。因此，我们认为这种结合蛋白参与了镉介导的血红素加氧酶基因的激活。顺便提及，AP-1结合位点的共有序列TGAGTCA存在于该镉响应元件的下游。然而，我们提供的证据表明，AP-1是不直接参与镉介导的人类血红素加氧酶基因的诱导。
Heme oxygenase is an essential enzyme in heme catabolism and is inducible by various environmental derangements such as cadmium. The activity and mRNA levels of heme oxygenase were remarkably increased in HeLa cells by the treatment with cadmium. As a first step in studying the molecular mechanisms of this induction, we performed transient expression assays in four human cell lines including HeLa to analyze the cadmium-mediated inducibility of the fusion genes, containing the firefly luciferase gene as a reporter under the human heme oxygenase gene promoter. By determining the luciferase activity expressed in the transfected cells, we found the region between about 4.5 and 4 kilobase pairs upstream from the transcriptional initiation site of the heme oxygenase gene which confers cadmium-mediated inducibility on the fusion gene. The region was then subjected to further functional analysis in HeLa cells, which allowed us to localize the cadmium-responsive element to 20 base pairs. Gel mobility shift assays demonstrated that this 20-base pair element is specifically bound by nuclear protein(s) of HeLa cells, the binding activities of which were however unchanged by the treatment with cadmium. Using the synthetic cadmium-responsive elements containing various base changes, we have identified a 10-base pair sequence, TGCTAGATTT, required for the cadmium-mediated inducibility and in vitro protein binding. We thus suggest that this binding protein(s) is involved in the cadmium-mediated activation of the heme oxygenase gene. Incidentally, the consensus sequence of AP-1 binding site, TGAGTCA, is present downstream of this cadmium-responsive element. However, we provide evidence that AP-1 is not directly involved in the cadmium-mediated induction of the human heme oxygenase gene.