Homologous Up-regulation of KDR/Flk-1 Receptor Expression by Vascular Endothelial Growth Factor in Vitro

doi:10.1074/jbc.273.45.29979

Homologous Up-regulation of KDR/Flk-1 Receptor Expression by Vascular Endothelial Growth Factor *in Vitro**

From the Departments of ^‡Pharmacokinetics and Metabolism and ^§Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080

Abstract

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pmrhVEGF₁₆₅ for 0–7 days. Western blot analysis showed a 3–5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2–3-fold increase in high affinity receptor number (5.0 × 10⁴/cellversus 2.4 × 10⁴/cell) without significantly affecting receptor binding affinity (K _d 76 pm versus 72 pm). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 μm sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.

Footnotes

↵* 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.
↵¶ To whom correspondence should be addressed: Dept. of Pharmacokinetics and Metabolism, Genentech, Inc., MS #70, 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-3269; Fax: 650-225-6452; E-mail: zioncheck.tom{at}gene.com.
Abbreviations:

VEGF

vascular endothelial growth factor

PLC-γ

phospholipase C-γ

PKC

protein kinase C

MAPK

mitogen-activated protein kinase

ACE

adrenal cortex capillary endothelial

l-NAME

l-N ^G-nitroarginine methyl ester

NNA

Nω-nitro-l-arginine

SNP

sodium nitroprusside

RT

reverse transcriptase

PCR

polymerase chain reaction

FGF

fibroblast growth factor

HGF

hepatocyte growth factor

pRL-CMV

Renilla luciferase cytomegalovirus.
- Received June 1, 1998.
- Revision received August 17, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.