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(Received for publication, February 13, 1996, and in revised form, July 5, 1996)
From the 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
Volume 271, Number 45,
Issue of November 8, 1996
pp. 28220-28228
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
in Human Glioma Cells
POSSIBLE ROLES OF SP-1
,
,
,
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
(TNF-), and interleukin 1 most
potently enhanced VEGF mRNA levels of a glioma cell line, U251.
Incubation of the glioma cells with bFGF or TNF- 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-. Gel
mobility shift assays demonstrated that only SP-1 binding activity was
increased 1 h after exposure to bFGF or TNF-, 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- but not that by hypoxia.
Western blot analysis also demonstrated an increase in cellular amounts
of VEGF by TNF- 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- 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--dependent activation. These
data indicated that the expression of the VEGF gene enhanced by bFGF or
TNF- 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.
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