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Author Notes:

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@emory.edu

Acknowledgments We thank Dr. John Svaren for providing JDM590 and JDM499 plasmids.

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Research Funding:

Grant support: USPHS, NIH awards NS-42934, CA-109382, NS053727 (D.J. Brat), CA-86335, and CA-87830 (E.G. VanMeir), Georgia Cancer Coalition (D.L. Durden), Musella Foundation (E.G. VanMeir), and Pediatric Brain Tumor Foundation of the United States (E.G. VanMeir).

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

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Journal Title:

Cancer Research

Volume:

Volume 66, Number 14

Publisher:

, Pages 7067-7074

Type of Work:

Article | Post-print: After Peer Review

Abstract:

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-κB (NF-κB), 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-κB-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-1α on tissue factor expression, we used glioma cells stably transfected with a HIF-1α siRNA expression vector and found that HIF-1α 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.

Copyright information:

©2006 American Association for Cancer Research. References

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