Tumor Necrosis Factor-α Regulates Expression of Vascular Endothelial Growth Factor Receptor-2 and of Its Co-receptor Neuropilin-1 in Human Vascular Endothelial Cells

doi:10.1074/jbc.273.34.22128

Tumor Necrosis Factor-α Regulates Expression of Vascular Endothelial Growth Factor Receptor-2 and of Its Co-receptor Neuropilin-1 in Human Vascular Endothelial Cells*

From the Vascular Biology Laboratory, Department of Genetics, Biology and Biochemistry, Medical School, University of Torino, Torino, 10126 Italy, the ^§Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080, the ^¶Department of Vascular and Connective Tissue Research Gaubius, Laboratory TNO-PG, 2333 CK Leiden, The Netherlands,^**Departments of Urology and Surgery, Children’s Hospital, Harvard Medical School, and the ^‡Departments of Surgery and Pathology, Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115

Abstract

Tumor necrosis factor-α (TNF-α) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-α does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-α up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-α increased the cellular amounts of VEGFR-2 protein and tripled the high affinity¹²⁵I-VEGF-A₁₆₅ capacity without affecting theK _d of ligand-receptor interaction. As a consequence, TNF-α enhanced the migration and the wound healing triggered by VEGF-A₁₆₅. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-α is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-α-mediated pathological settings.

Footnotes

↵* This study was supported by grants from the European Community (Biomed-2 Project: BMHL-CT96–0669), the Italian Association for Cancer Research, Istituto Superiore Ri Sanità (X AIDS Project, Multiple-Sclerosis Project, Program on Tumor Therapy), the Dutch Cancer Society (TNOP 97–1511), National Institutes of Health (CA 37392 and 45548), Centro Nazionale Ricerche (P. F. Biotecnologie), and Ministero Dell’ Universita e Della Ricerca Scientifica Technologica (60%).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.
↵‡ Supported by a grant from the Italian Foundation for Cancer Research.
↵‖ To whom correspondence should be addressed: Dept. of Genetics, Biology and Biochemistry, Via Santena 5bis., 10126 Torino, Italy. Tel.: 39-11-6706684; Fax: 39-11-6635663; E-mail:Bussol{at}molinette.unito.it.
↵2 H. Gerber and J. Park, personal communication.
Abbreviations:

TNF-α

tumor necrosis factor-α

BSA

bovine serum albumin

FCS

fetal calf serum

FGF

fibroblast growth factor

PBS

phosphate-buffered saline

VEGF

vascular endothelial growth factor

VEGFR

VEGF receptor

kb

kilobase pair(s)

MOPS

3-(N-morpholino)propanesulfonic acid

TES

N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid

PIPES

piperazine-N,N′-bis(2-ethanesulfonic acid)

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
- Received March 12, 1998.
- Revision received June 14, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.