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

Yuan Rong, Fang Hu, Ruo Pan Huang, Nigel Mackman, Jonathan M. Horowitz, Randy L. Jensen, Donald L. Durden, Erwin G. Van Meir, Daniel J. Brat

Research output: Contribution to journalArticle

75 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)7067-7074
Number of pages8
JournalCancer Research
Volume66
Issue number14
DOIs
StatePublished - Jul 15 2006

Fingerprint

Hypoxia-Inducible Factor 1
Thromboplastin
Glioblastoma
Growth
Genes
Glioma
Small Interfering RNA
Up-Regulation
Transcription Factor AP-1
Luciferases
Messenger RNA
Hypoxia
Binding Sites
Cell Hypoxia
Electrophoretic Mobility Shift Assay
Oligonucleotides
Transfection
Plasmids
Complementary DNA

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Early growth response gene-1 regulates hypoxia-induced expression of tissue factor in glioblastoma multiforme through hypoxia-inducible factor-1-independent mechanisms. / Rong, Yuan; Hu, Fang; Huang, Ruo Pan; Mackman, Nigel; Horowitz, Jonathan M.; Jensen, Randy L.; Durden, Donald L.; Van Meir, Erwin G.; Brat, Daniel J.

In: Cancer Research, Vol. 66, No. 14, 15.07.2006, p. 7067-7074.

Research output: Contribution to journalArticle

Rong, Yuan ; Hu, Fang ; Huang, Ruo Pan ; Mackman, Nigel ; Horowitz, Jonathan M. ; Jensen, Randy L. ; Durden, Donald L. ; Van Meir, Erwin G. ; Brat, Daniel J. / Early growth response gene-1 regulates hypoxia-induced expression of tissue factor in glioblastoma multiforme through hypoxia-inducible factor-1-independent mechanisms. In: Cancer Research. 2006 ; Vol. 66, No. 14. pp. 7067-7074.
@article{e98a20bf070f41a789079dd1a234d058,
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-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.",
author = "Yuan Rong and Fang Hu and Huang, {Ruo Pan} and Nigel Mackman and Horowitz, {Jonathan M.} and Jensen, {Randy L.} and Durden, {Donald L.} and {Van Meir}, {Erwin G.} and Brat, {Daniel J.}",
year = "2006",
month = "7",
day = "15",
doi = "10.1158/0008-5472.CAN-06-0346",
language = "English (US)",
volume = "66",
pages = "7067--7074",
journal = "Cancer Research",
issn = "0008-5472",
publisher = "American Association for Cancer Research Inc.",
number = "14",

}

TY - JOUR

T1 - Early growth response gene-1 regulates hypoxia-induced expression of tissue factor in glioblastoma multiforme through hypoxia-inducible factor-1-independent mechanisms

AU - Rong, Yuan

AU - Hu, Fang

AU - Huang, Ruo Pan

AU - Mackman, Nigel

AU - Horowitz, Jonathan M.

AU - Jensen, Randy L.

AU - Durden, Donald L.

AU - Van Meir, Erwin G.

AU - Brat, Daniel J.

PY - 2006/7/15

Y1 - 2006/7/15

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33746925159&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33746925159&partnerID=8YFLogxK

U2 - 10.1158/0008-5472.CAN-06-0346

DO - 10.1158/0008-5472.CAN-06-0346

M3 - Article

C2 - 16849552

AN - SCOPUS:33746925159

VL - 66

SP - 7067

EP - 7074

JO - Cancer Research

JF - Cancer Research

SN - 0008-5472

IS - 14

ER -