Induction of Vascular Endothelial Growth Factor by Tumor Necrosis Factor α in Human Glioma Cells

doi:10.1074/jbc.271.45.28220

Induction of Vascular Endothelial Growth Factor by Tumor Necrosis Factor α in Human Glioma Cells

POSSIBLE ROLES OF SP-1*

From the ^‡ Department of Biochemistry, Kyushu University School of Medicine, Maidashi, Fukuoka 812-82, Japan,
^¶ Department of Gene Expression, National Biotechnology Research Center (GBF), 318124 Braunschweig, Germany, and
^§ Department of Molecular Biology, University of Occupational and Environmental Health, Kita-Kyushu 807, Japan

^¶ To whom correspondence should be addressed. Tel.: 81-92-641-1151 (ext. 3351); Fax: 81-92-632-4198.

Abstract

The expression of vascular endothelial growth factor (VEGF) has been implicated in brain tumor angiogenesis, and the promoter region for the VEGF gene contains several SP-1 and AP-1 (c-Fos and c-Jun) binding motifs. Among eight human glioma cell lines, cellular mRNA levels of transcription factors SP-1 and AP-1 (c-Fos and c-Jun) were found to be closely correlated with those of VEGF. VEGF expression appears to be highly susceptible to hypoxia or exogenous cytokines and growth factors. Of various cytokines and growth factors, basic fibroblast growth factor (bFGF), tumor necrosis factor α (TNF-α), and interleukin 1 most potently enhanced VEGF mRNA levels of a glioma cell line, U251. Incubation of the glioma cells with bFGF or TNF-α increased both VEGF and SP-1 mRNA at 30 min and c-Fos mRNA at 1-3 h, over 5-fold. Nuclear run-on assays showed an apparent increase of the transcription of the VEGF gene as well as the SP-1 gene by bFGF or TNF-α. Gel mobility shift assays demonstrated that only SP-1 binding activity was increased 1 h after exposure to bFGF or TNF-α, and also that AP-1, but not SP-1, activity was significantly activated by hypoxia. Mithramycin, an inhibitor of SP-1, at 1-10 nM inhibited activation of the VEGF gene by bFGF or TNF-α but not that by hypoxia. Western blot analysis also demonstrated an increase in cellular amounts of VEGF by TNF-α and a decrease by co-administration with mithramycin. The promoter activity of the VEGF gene, which contains five SP-1 binding sites and one AP-1 binding site but not hypoxia regulatory elements, was enhanced by bFGF or TNF-α but not by hypoxia. The chloramphenicol acetyltransferase assay with VEGF promoter deletion constructs demonstrated that four clusterized SP-1 binding sites in the proximal promoter were essential for the basal transcription and the TNF-α-dependent activation. These data indicated that the expression of the VEGF gene enhanced by bFGF or TNF-α appeared to be mediated in part through the transcription factor SP-1, suggesting a different mechanism from that for hypoxia-induced activation of the VEGF gene.

Footnotes

↵* This study was supported by a grant-in-aid for cancer research and the International Scientific Research Program, Joint Research, from the Ministry of Education, Science and Culture of Japan, the Fukuoka 21th Century Medical Fund and Fukuoka Anticancer Research Fund (to M. O., K. K., and M. K.), the Kimura Memorial Cardiovascular Fund (to M. O.), and the Yasuda Memorial Medical Fund (to M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

bFGF

basic fibroblast growth factor

VEGF

vascular endothelial growth factor

TGF

transforming growth factor

EGF

epidermal growth factor

PDGF

platelet-derived growth factor

IL

interleukin

TNF

tumor necrosis factor

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

FBS

fetal bovine serum

PBS

phosphate buffered saline

DMEM

Dulbecco's modified Eagle's medium

NF

nuclear factor

LDL

low density lipoprotein

CAT

chloramphenicol acetyltransferase.
↵² M. Ryuto and M. Kuwano, unpublished observation.
- Received February 13, 1996.
- Revision received July 5, 1996.