Basic fibroblast growth factor induces expression of VEGF receptor KDR through a protein kinase C and p44/p42 mitogen-activated protein kinase-dependent pathway

Vascular endothelial growth factor (VEGF) and basic fibroblast growth facto r (bFGF) are angiogenic molecules whose combined mitogenic activity is pote ntly synergistic. However, the molecular mechanism underlying this synergy is incompletely understood. We examined whether VEGF and bFGF affect expres sion of each other or alter expression of the VEGF receptor KDR in retinal capillary endothelial cells. In addition, we investigated the intracellular signaling mechanisms involved in this response. VEGF-induced [H-3]thymidin e uptake was tightly correlated with KDR mRNA and protein concentrations, s uggesting that increased KDR expression might account for VEGF's synergisti c activity in the presence of bFGF. bFGF (10 ng/ml) induced KDR mRNA expres sion within 4 h and attained a 4.0-fold increase after 24 h. KDR protein ex pression was increased 7.5-fold after 48 h. VEGF (=50 ng/ml) did not alter bFGF, VEGF, or KDR mRNA expression under serum-deprived conditions. In cont rast, VEGF increased KDR mRNA expression 87% under growth conditions and 2. 9-fold under serum-deprived conditions in the presence of bFGF. The protein kinase C (PKC) agonist phorbol myristate acetate (PMA) induced KDR mRNA ex pression 5.1-fold at 100 nmol/l. bFGF increased p44/p42 mitogen-activated p rotein kinase (MAPK) phosphorylation within 5 min, reaching a maximum withi n 15 min and remaining significantly elevated for >6 h. bFGF-induced MAPK p hosphorylation and KDR mRNA expression were almost completely inhibited by 5 mu mol/l GFX a non-isoform-selective PKC inhibitor. MAPK inhibitor PD9805 9 reduced KDR mRNA expression 72% at concentrations that inhibited bFGF-ind uced MAPK phosphorylation 100%, suggesting that pathways in addition to MAP K might also be involved. Inhibitors of the beta isoform of PKC (LY333531), protein kinase A (PKA) (H89), and phosphotidylinositol (PI) 3 kinase (wort mannin) had no significant effect. These data suggest that bFGF stimulates KDR expression through a PKC and p44/p42 MAPK-dependent pathway not primari ly involving the beta isoform of PKC, PKA, or PI-3 kinase. Since bFGF induc es VEGF expression and since increased KDR expression potentiates VEGF acti on, resulting in additional KDR expression and marked mitogenic activity, t hese data provide a novel mechanistic explanation for the angiogenic synerg y between VEGF and bFGF.