Both v-Ha-Ras and v-Raf Stimulate Expression of the Vascular Endothelial Growth Factor in NIH 3T3 Cells

doi:10.1074/jbc.270.43.25915

Both v-Ha-Ras and v-Raf Stimulate Expression of the Vascular Endothelial Growth Factor in NIH 3T3 Cells (*)

From the Institute for Molecular Medicine, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg, Germany

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Abstract

Stimulation of NIH 3T3 cells with platelet-derived growth factor (PDGF)-BB and 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances vascular endothelial growth factor (VEGF) gene expression. To address the question of whether Ras and Raf are involved in the induction of VEGF gene expression by PDGF and TPA, we examined the effects of both factors on NIH 3T3 cells stably transfected with v-Ha-ras or v-raf. In serum-starved NIH 3T3 cells, only low levels of mRNA expression can be detected, whereas both ras and raf transformed cell lines express enhanced levels of a 4.3-kilobase VEGF transcript. Stimulation with PDGF or TPA resulted in increased VEGF mRNA in all cell lines, with highest levels found in the transformed cells. Immunofluorescence studies confirmed that the elevated VEGF mRNA expression correlated with enhanced protein levels. Positive immunofluorescence signals could be detected in v-Ha-ras or v-raf transformed cell lines but not in unstimulated NIH 3T3 cells. VEGF from conditioned medium of v-raf transformed NIH 3T3 cells was partially purified by chromatography on heparin-Sepharose. Biological activity of this VEGF protein was demonstrated by competition with binding of recombinant I-VEGF to human umbilical vein endothelial cells and by its ability to stimulate proliferation of these cells.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

VEGF

vascular endothelial growth factor

PDGF

platelet-derived growth factor

TPA

12-O-tetradecanoylphorbol-13-acetate

PKC

protein kinase C

HUVE

human umbilical vein endothelial.
↵² S. Grugel and U. R. Rapp, unpublished data.
↵³ J. Lyons, S. M. Storm, and U. R. Rapp, unpublished data.
- Received December 14, 1994.
- Revision received June 7, 1995.