title: 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.affiliation: Department of Pathology and Laboratory Medicine, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Rong
authors.forename: Fang
authors.initials: F
authors.lastname: Hu
authors.forename: Ruopan
authors.initials: R
authors.lastname: Huang
authors.forename: Nigel
authors.initials: N
authors.lastname: Mackman
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Horowitz
authors.forename: Randy L
authors.initials: RL
authors.lastname: Jensen
authors.forename: Donald L
authors.initials: DL
authors.lastname: Durden
authors.forename: Erwin G
authors.initials: EG
authors.lastname: Van Meir
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Brat
mesh_terms: D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D051766:Early Growth Response Protein 1 / Q000096:biosynthesis / Q000235:genetics*; D005909:Glioblastoma / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000096:biosynthesis / Q000235:genetics*; D016328:NF-kappa B / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000096:biosynthesis / Q000235:genetics; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D018808:Transcription Factor AP-1 / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
chemical_list: C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D051793:Hypoxia-Inducible Factor 1; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D042461:Vascular Endothelial Growth Factor A; D013925:Thromboplastin