Role of endothelial cell extracellular signal-regulated kinase(1/2) in urokinase-type plasminogen activator upregulation and in vitro angiogenesis byfibroblast growth factor-2

Downstream signaling triggered by the binding of fibroblast growth factor-2 (FGF2) to its tyrosine-kinase receptors involves the activation of mitogen -activated protein kinase kinase (MEK) with consequent phosphorylation of e xtracellular signal-regulated kinases (ERKs), Here we demonstrate that FGF2 induces ERK1/2 activation in bovine aortic endothelial (BAE) cells and tha t the continuous presence of the growth factor is required for sustained ER K1/2 phosphorylation, This is prevented by the MEK inhibitors PD 098059 and U0126, which also inhibit FGF(2)-mediated upregulation of urokinase-type p lasminogen activator (uPA) and in vitro formation of capillary-like structu res in three-dimensional type I collagen gel, Various FGF2 mutants originated by deletion or substitution of basic amino acid residues in the amino terminus or in the carboxyl terminus of FGF2 ret ained the capacity to induce a long-lasting activation of ERK1/2 in BAE cel ls, Among them, K(128)Q/R(129)Q-FGF2 was also able to stimulate uPA product ion and morphogenesis whereas R(129)Q/K(134)Q-FGF2 caused uPA upregulation only In contrast, K(27,30)Q/R(31)Q-FGF2, K(128)Q/K(138)Q-FGF2 and R(115,129 )QM(119,128)Q-FGF2 exerted a significant uPA-inducing and morphogenic activ ity in an ERK1/2-dependent manner only in the presence of heparin, Furtherm ore, no uPA upregulation and morphogenesis was observed in BAE cells treate d with the deletion mutant Delta(27-32)-FGF2 even in the presence of solubl e heparin, Thus, mutational analysis of FGF2 dissociates the capacity of th e growth factor to induce a persistent activation of ERK1/2 from its abilit y to stimulate uPA upregulation and/or in vitro angiogenesis. In conclusion, the data indicate that ERK1/2 phosphorylation is a key step in the signal transduction pathway switched on by FGF2 in endothelial cells , Nevertheless, a sustained ERK1/2 activation is not sufficient to trigger uPA upregulation and morphogenesis. FGF2 mutants may represent useful tools to dissect the signal transduction pathway(s) mediating the complex respon se elicited by an angiogenic stimulus in endothelial cells.