Cathepsin L expression is up-regulated by hypoxia in human melanoma cells:: role of its 5′-untranslated region
Cathepsin L expression is up-regulated by hypoxia in human melanoma cells:: role of its 5′-untranslated region
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DOI：
10.1042/bj20071255
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发表时间：
2008-07-01
影响因子：
4.1
通讯作者：
Frade, Raymond
中科院分区：
文献类型：
--
作者：
Jean, Didier;Rousselet, Nathalie;Frade, Raymond
组织蛋白酶 L（一种半胱氨酸蛋白酶）的过度表达以及组织蛋白酶 L 原的分泌将人黑色素瘤细胞的表型转变为高度致瘤性和强转移性。这使我们鉴定出参与高度转移性人类黑色素瘤细胞中组织蛋白酶 L 表达调节的 DNA 调节序列。本研究的结果表明，在组织蛋白酶L基因下游的3'区域以及其启动子的GC/CCAAT位点的3'和5'侧翼区域中存在调控序列。此外，我们确定 5'-UTR（非翻译区）是组织蛋白酶 L 表达最重要的区域。该 5'-UTR 整合了一个替代启动子和参与转录后调节的序列。双顺反子报告载体和 RNA 的转染实验表明，组织蛋白酶 L 5'-UTR 包含功能性 IRES（内部核糖体进入位点）。该完整的 IRES 仅存在于三种剪接变体之一中，这三种剪接变体的 5'-UTR 有所不同。然后，我们分析了在缺氧条件下生长的人类黑色素瘤细胞系中组织蛋白酶 L 的表达。我们证明，在中度缺氧条件（1% O-2）下，组织蛋白酶 L 的细胞内表达上调。缺氧仅显着增加包含完整IRES的转录物的表达，但抑制启动子活性。这些结果表明 IRES 的存在使得组织蛋白酶 L mRNA 翻译在缺氧条件下有效。总而言之，我们的结果表明，体内肿瘤缺氧环境上调组织蛋白酶 L 的表达，从而促进肿瘤进展。
Overexpression of cathepsin L, a cysteine protease, and consequently procathepsin L secretion switch the phenotype of human melanoma cells to highly tumorigenic and strongly metastatic. This led us to identify the DNA regulatory sequences involved in the regulation of cathepsin L expression in highly metastatic human melanoma cells. The results of the present study demonstrated the presence of regulatory sequences in the 3' region downstream of the cathepsin L gene and in the 3'- and 5'-flanking regions of GC/CCAAT sites of its promoter. In addition, we established that the 5'-UTR (untranslated region) was the most important region for cathepsin L expression. This 5'-UTR integrated an alternative promoter and sequences involved in post-transcriptional regulation. Transfection experiments of bicistronic reporter vectors and RNAs demonstrated that the cathepsin L 5'-UTR contained a functional IRES (internal ribosome entry site). This complete IRES was present only in one of the three splice variants, which differed in their 5'-UTR. Then, we analysed cathepsin L expression in this human melanoma cell line grown under hypoxia. We demonstrated that under moderate hypoxic conditions (1 % O-2) intracellular expression of cathepsin L was up-regulated. Hypoxia significantly increased only the expression of the transcript which contains the complete IRES, but inhibited promoter activity. These results suggest that the presence of an IRES allowed cathepsin L mRNA translation to be efficient under hypoxic conditions. Altogether, our results indicated that in vivo a tumour hypoxic environment up-regulates cathepsin L expression which promotes tumour progression.