HOMOLOGOUS UP-REGULATION OF KDR FLK-1 RECEPTOR EXPRESSION BY VASCULARENDOTHELIAL GROWTH-FACTOR IN-VITRO/

We investigated the possibility that vascular endothelial growth facto r (VEGF) treatment could regulate KDR/Flk-1 receptor expression in end othelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pM rhVEGF(165) 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 x 10(4)/cell versus 2.4 x 10(4)/ ce ll) without significantly affecting receptor binding affinity (K-d 76 pM versus 72 pM), Quantitative polymerase chain reaction analysis demo nstrated 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 bind ing also induced KDR up-regulation; in contrast, mutants with decrease d KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibitio n of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitog en-activated protein kinase activities all blocked VEGF-induced KDR up -regulation. Finally, co-incubation of nitric-oxide synthase inhibitor s with VEGF had no significant effect on KDR expression, but 100 mu M sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expres sion. 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 VEG F action in tumor and ischemia-induced angiogenesis.