Homeostatic Modulation of Cell Surface KDR and Flt1 Expression and Expression of the Vascular Endothelial Cell Growth Factor (VEGF) Receptor mRNAs by VEGF

Dongfang Wang,David B Donner,Robert S Warren

doi:10.1074/jbc.m001847200

Abstract

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.

Highlights

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer
The cells were subjected to acidic washes to promote ligand dissociation, rendering cell surface VEGF receptors that had not been internalized available for interaction with subsequently applied 125I-VEGF
The unique importance of this angiogenic factor is illustrated by the observation that the loss of even a single allele for VEGF is lethal [45, 46]

Summary

Homeostatic Modulation of VEGF Receptors by VEGF

Aortic endothelial cells and NIH 3T3 cells overexpressing KDR/ Flk or Flt, bovine aortic endothelial cells, immortalized endothelial cells, and HUVEC have begun to identify the signaling proteins downstream of KDR/Flk or Flt1 [25, 33,34,35,36,37,38,39,40,41]. We demonstrate that VEGF or tumor-conditioned medium down-regulates cell surface expression of KDR and Flt on HUVEC. Such down-regulation diminishes the ability of VEGF to stimulate MAPK (ERK1 and ERK2) and p38 MAPK activities. Whereas VEGF or tumorconditioned medium down-regulated cell surface receptor expression, expression of the mRNAs for KDR and Flt was increased by VEGF or tumor-conditioned medium. The demonstration that loss of cell surface VEGF receptor expression and responsiveness to VEGF occur coincidentally with up-regulation of the VEGF receptor mRNAs indicates the operation of a homeostatic mechanism that replenishes cell surface receptor expression and restores HUVEC responsiveness once exogenous levels of VEGF are diminished

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION