[CANCER RESEARCH 59, 1315–1322, March 15, 1999] Activation of Metallothionein Gene Expression by Hypoxia Involves Metal Response Elements and Metal Transcription Factor-1 1 Brian J. Murphy, 2 Glen K. Andrews, Doug Bittel, Daryl J. Discher, Jesica McCue, Christopher J. Green, Marianna Yanovsky, Amato Giaccia, Robert M. Sutherland, Keith R. Laderoute, and Keith A. Webster Pharmaceutical Discovery Division, SRI International, Menlo Park, California 94025 [B. J. M., C. J. G., M. Y., K. R. L., K. A. W.]; Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160 [G. K. A., D. B.]; Department of Molecular and Cellular Pharmacology, University of Miami, Florida 33136 [D. J. D., J. M., K. A. W.]; Department of Radiation Biology, Stanford University, California 94305 [A. G.]; and Varian Biosynergy, Inc., Palo Alto, California 94304 [R. M. S.] ABSTRACT 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 previ- ously 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). Dele- tion 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 respon- siveness 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. INTRODUCTION MTs 3 are ubiquitous, low molecular weight proteins characterized by high cysteine content and high affinities for metals such as zinc and cadmium (reviewed in Ref. 1). Both the constitutive and stress- inducible expression of MT appear to be dependent on activation of MTF-1, a member of the Cys 2 His 2 family of zinc finger transcription factors (2, 3). MTs have well-established roles in metal homeostasis and in the detoxification of heavy metals. Moreover, they also confer protection against reactive oxygen intermediates, electrophilic anti- neoplastic agents, various mutagens, ionizing radiation, and nitric oxide (4, 5). Other studies indicate roles for MTs in the regulation of cellular proliferation and apoptosis (6 –9), perhaps through an inter- action of MT with nuclear factor-B-DNA complexes (10). These properties of MTs reflect their potential importance for malignant progression; high expression of MTs correlates with poor prognosis and progressive disease in a number of human tumors (11, 12). In this context, MTF-1 may be important for tumorigenesis not only through MT expression but also through its regulation of other genes. For example, MTF-1 activity is necessary for the expression of -glu- tamylcysteine synthetase (13), the rate-limiting enzyme for the syn- thesis of GSH (14). GSH is a major contributor to cellular defenses against environmental alkylating agents and oxidants, including reac- tive oxygen species generated by ionizing radiation (14). Thus, MTF-1 may be critical for modulating gene expression associated with malignant phenotypes such as resistance to therapy. The mammalian MT family consists of two ubiquitous isoforms (MT-I and MT-II) and two tissue-specific isoforms (MT-III and MT-IV). Although only one isoform of mammalian MT-II has been identified (MT-IIA), at least seven unique functional isoforms have been described for the hMT-I gene (15). The expression and regulation of some of the hMT genes (e.g., MT-IA, MT-IB, MT-1G, and MT-IIA) have been described (15, 16). MT-IIA is the predominant human MT isoform and is expressed in most cultured human cells (15). In contrast, MT-I is the predominant MT isoform in the mouse. The mammalian MT-I and MT-II genes are transcriptionally regulated by metal ions, such as zinc and cadmium, and by a wide variety of other stimuli. These latter agonists include bacterial endotoxin (lipopolysac- charides), phorbol esters, xenobiotics, and oxidative stress (3). The hMT-IIA gene is also induced by hypoxia, growth factors, cytokines, UV radiation, glucocorticoids, X-irradiation, and some specific DNA- damaging agents (4, 15, 17). Although the regulatory mechanisms of these nonmetallic inducers are not well understood, some appear to involve redox stresses (3). Both MT-I and MT-II promoters contain multiple copies of specific cis-acting elements that cooperate to direct metal inducibility (MREs; 1). The MRE-associated transcription factor (MTF-1) that binds to MREs and activates MT transcription has been cloned from mouse and human cells (18, 19). The mechanism by which metals activate transcription through MTF-1 has not been well established, although it appears to involve interactions at the zinc finger domain of MTF-1 (20). We have observed that oxidative stress activates the mMT-1 gene through MTF-1 binding to the MREs in the proximal promoter (21, 22). In addition to the MREs, proximal mMT-1 promoters contain Sp1 binding sites, USF binding sites, AREs, glucocorticoid-responsive elements, and consensus TATA box sequences (23, 24). The hMT-IIA promoter is also complex and contains a glucocorticoid-responsive element, Sp1 and AP1 binding sites, three putative AP2 sites, four metal response elements (MRE-a through MRE-d), and a TATA box (15). We reported previously that low pO 2 levels, similar to those meas- ured in tumors 1 mm in diameter (25), significantly induced hMT- IIA transcript and protein expression in a variety of carcinoma cell lines (17). In this study, we present the following major observations: Received 9/11/98; accepted 1/14/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This study was supported by NIH Grants CA57692 (to B. J. M.), ES05704 (to G. K. A.), CA67166, Project 5 (to K. R. L.), and HL44578 (to K. A. W.) and by a grant from the Cigarette and Tobacco Surtax of the State of California through the Tobacco- Related Disease Research Program of the University of California, 1RT-402 (to K. A. W.). D. B. is supported by NIH postdoctoral fellowship NRSA: F32 ES05753. 2 To whom requests for reprints should be addressed, at Pharmaceutical Discovery, SRI International, Room LA257, 333 Ravenswood Avenue, Menlo Park, CA 94025. E-mail: bmurphy@unix.sri.com. 3 The abbreviations used are: MT, metallothionein; MTF, metal transcription factor; hMT, human MT; mMT, mouse MT; MRE, metal response element; GSH, glutathione; ARE, antioxidant response element; USF, upstream stimulatory element; MEF, mouse embryo fibroblast; Luc, luciferase; HIF, hypoxia-inducible factor; HRE, hypoxia response element; CMV, cytomegalovirus. 1315 Research. on December 5, 2021. © 1999 American Association for Cancer cancerres.aacrjournals.org Downloaded from