Activation of metallothionein gene expression by hypoxia involves metal response elements and metal transcription factor-1

Metallothioneins (MTs) are a family of stress-induced proteins with diverse physiological functions, including protection against metal toxicity and o xidants, They may also contribute to the regulation of cellular proliferati on, apoptosis, and malignant progression. We reported previously that the h uman (h)MT-IIA isoform is induced in carcinoma cells (A431, SiHa, and HT29) exposed to low oxygen, conditions commonly found in solid tumors. The pres ent study demonstrates that the genes for hMT-IIA and mouse (m)MT-I are tra nscriptionally 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 fu ll transcriptional response. Functional studies of the mMT-I promoter demon strated 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 respo nsiveness to a minimal MT promoter. Mouse MT-I gene transcripts in fibrobla sts with targeted deletions of both MTF-1 alleles (MTF-1(-/-); dko7 cells) were not induced by zinc and showed low responsiveness to hypoxia. A transi ently transfected MT promoter was unresponsive to hypoxia or zinc in dko7 c ells, but inductions were restored by cotransfecting a mouse MTF-1 expressi on vector. Electrophoretic mobility shift assays detected a specific protei n-DNA complex containing MTF-1 in nuclear extracts from hypoxic cells. Toge ther, these results demonstrate that hypoxia activates MT gene expression t hrough MREs and that this activation involves MTF-1.