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Early Growth Response Gene-1 Regulates Hypoxia-Induced Expression of Tissue Factor in Glioblastoma Multiforme through Hypoxia-Inducible Factor-1–Independent Mechanisms

Departments of ¹ Pathology and Laboratory Medicine, ² Psychiatry and Behavioral Science, ³ Obstetrics and Gynecology, ⁴ Pediatrics, ⁵ Hematology/Oncology, and ⁶ Neurosurgery, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia; ⁷ Department of Immunology, The Scripps Research Institute, La Jolla, California; ⁸ Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina; and ⁹ Department of Neurosurgery, University of Utah, Salt Lake City, Utah

Requests for reprints: Daniel J. Brat, Department of Pathology and Laboratory Medicine, Emory University Hospital, H-176, 1364 Clifton Road Northeast, Atlanta, GA 30322. Phone: 404-712-1266; Fax: 404-727-3133; E-mail: dbrat{at}emory.edu.

Hypoxia strongly up-regulates tissue factor and promotes plasma clotting by glioblastoma multiforme, but transcriptional mechanisms remain undefined. Here, we investigated the potential roles of early growth response gene-1 (Egr-1), Sp1, nuclear factor-B (NF-B), activator protein-1 (AP-1), and hypoxia-inducible factor-1 (HIF-1) in the hypoxic regulation of tissue factor by glioblastoma multiforme cells in vitro. Hypoxia (1% O₂) strongly induced Egr-1 mRNA within 1 hour and led to nuclear localization of Egr-1 protein. Using luciferase reporter plasmids in glioma cells, we found that hypoxia dramatically increased luciferase activity in cells with constructs containing Egr-1-binding sites but not in cells with constructs containing AP-1- or NF-B-binding sites. Electrophoretic mobility shift assays revealed hypoxia-induced Egr-1, but not Sp1, binding to oligonucleotides containing the Egr-1/Sp1 motif of tissue factor gene promoter. Using an expression vector containing the minimal tissue factor promoter (–111 to +14 bp) and small interfering RNA (siRNA) directed at Egr-1 and Sp1 mRNAs, we found that Egr-1 was required for maximal hypoxic induction of promoter activity. Forced overexpression of Egr-1 but not Sp1 by cDNA transfection caused up-regulation of tissue factor in glioma cells under normoxia (21% O₂), whereas siRNA directed at Egr-1 strongly attenuated hypoxia-induced tissue factor expression. To examine the effects of HIF-1 on tissue factor expression, we used glioma cells stably transfected with a HIF-1 siRNA expression vector and found that HIF-1 mRNA silencing did not affect tissue factor expression under hypoxia. We conclude that hypoxic up-regulation of tissue factor in glioblastoma multiforme cells depends largely on Egr-1 and is independent of HIF-1. (Cancer Res 2006; 66(14): 7067-74)

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