Early growth response gene-1 regulates hypoxia-induced expression of tissue factor in glioblastoma multiforme through hypoxia-inducible factor-1-independent mechanisms.

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

• Authors: Yuan Rong, Fang Hu, Ruopan Huang, Nigel Mackman, Jonathan M Horowitz, Randy L Jensen, Donald L Durden, Erwin G Van Meir, Daniel J Brat
• Journal: Cancer research (Cancer Res) Vol. 66 Issue 14 Pg. 7067-74 (Jul 15 2006) ISSN: 0008-5472 [Print] United States
• PMID: 16849552 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• EGR1 protein, human
• Early Growth Response Protein 1
• Hypoxia-Inducible Factor 1
• NF-kappa B
• Proto-Oncogene Proteins c-jun
• Sp1 Transcription Factor
• Transcription Factor AP-1
• Vascular Endothelial Growth Factor A
• Thromboplastin

Topics

• Cell Hypoxia (physiology)
• Cell Line, Tumor
• Early Growth Response Protein 1 (biosynthesis, genetics)
• Glioblastoma (genetics, metabolism)
• Humans
• Hypoxia-Inducible Factor 1 (biosynthesis, genetics)
• NF-kappa B (biosynthesis, genetics)
• Promoter Regions, Genetic
• Proto-Oncogene Proteins c-jun (biosynthesis, genetics)
• Sp1 Transcription Factor (biosynthesis, genetics)
• Thromboplastin (biosynthesis, genetics)
• Transcription Factor AP-1 (biosynthesis, genetics)
• Transfection
• Up-Regulation
• Vascular Endothelial Growth Factor A (genetics)