Murphy B J, Andrews G K, Bittel D, Discher D J, McCue J, Green C J, Yanovsky M, Giaccia A, Sutherland R M, Laderoute K R, Webster K A
Pharmaceutical Discovery Division, SRI International, Menlo Park, California 94025, USA.
Cancer Res. 1999 Mar 15;59(6):1315-22.
Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and oxidants. They may also contribute to the regulation of cellular proliferation, apoptosis, and malignant progression. We reported previously that the human (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The present study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are transcriptionally activated by hypoxia through metal response elements (MREs) in their proximal promoter regions. These elements bind metal transcription factor-1 (MTF-1). Deletion and mutational analyses of the hMT-IIA promoter indicated that the hMRE-a element is essential for basal promoter activity and for induction by hypoxia, but that other elements contribute to the full transcriptional response. Functional studies of the mMT-I promoter demonstrated that at least two other MREs (mMRE-d and mMRE-c) are responsive to hypoxia. Multiple copies of either hMRE-a or mMRE-d conferred hypoxia responsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibroblasts with targeted deletions of both MTF-1 alleles (MTF-1(-/-); dko7 cells) were not induced by zinc and showed low responsiveness to hypoxia. A transiently transfected MT promoter was unresponsive to hypoxia or zinc in dko7 cells, but inductions were restored by cotransfecting a mouse MTF-1 expression vector. Electrophoretic mobility shift assays detected a specific protein-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Together, these results demonstrate that hypoxia activates MT gene expression through MREs and that this activation involves MTF-1.
金属硫蛋白（MTs）是一类应激诱导蛋白，具有多种生理功能，包括抵御金属毒性和氧化剂。它们还可能参与细胞增殖、凋亡及恶性进展的调控。我们之前报道过，人（h）MT-IIA亚型在暴露于低氧环境的癌细胞（A431、SiHa和HT29）中被诱导表达，低氧环境在实体瘤中较为常见。本研究表明，hMT-IIA和小鼠（m）MT-I的基因通过其近端启动子区域的金属反应元件（MREs）在转录水平上被缺氧激活。这些元件结合金属转录因子-1（MTF-1）。对hMT-IIA启动子的缺失和突变分析表明，hMRE-a元件对于基础启动子活性和缺氧诱导至关重要，但其他元件也有助于完整的转录反应。对mMT-I启动子的功能研究表明，至少另外两个MREs（mMRE-d和mMRE-c）对缺氧有反应。hMRE-a或mMRE-d的多个拷贝赋予最小MT启动子缺氧反应性。在两个MTF-1等位基因均有靶向缺失的成纤维细胞（MTF-1(-/-)；dko7细胞）中，小鼠MT-I基因转录本不受锌诱导，对缺氧的反应性也较低。在dko7细胞中，瞬时转染的MT启动子对缺氧或锌无反应，但通过共转染小鼠MTF-1表达载体可恢复诱导作用。电泳迁移率变动分析在缺氧细胞的核提取物中检测到一种含有MTF-1的特异性蛋白质-DNA复合物。总之，这些结果表明缺氧通过MREs激活MT基因表达，且这种激活涉及MTF-1。
