RESUMO
Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.
Assuntos
Inibidores do Crescimento/metabolismo , Substâncias de Crescimento/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Caveolina 1 , Caveolinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Clonagem Molecular , Endotélio/citologia , Endotélio/efeitos dos fármacos , Endotélio/enzimologia , Endotélio/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Inibidores do Crescimento/química , Inibidores do Crescimento/genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ácido Palmítico/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação , Alinhamento de SequênciaRESUMO
Although the function of vascular endothelial growth factor in the induction of tumor angiogenesis is well understood, the role of a second group of angiogenic factors, the fibroblast growth factors (FGFs), remains elusive. We used a recombinant adenovirus expressing soluble FGF receptor (AdsFGFR) to interfere with FGF function in tumor angiogenesis. AdsFGFR repressed endothelial cell proliferation in vitro and inhibited tumor angiogenesis in an ex vivo bioassay, in which endothelial cells were cocultured with angiogenic tumor biopsies in a collagen gel. Moreover, AdsFGFR repressed tumor angiogenesis and hence tumor growth in vivo in allograft transplantation experiments. Whereas adenoviral expression of a soluble form of VEGF receptor 1 (AdsFlt) predominantly affected the initiation of tumor angiogenesis, soluble FGF receptor (sFGFR) appeared to impair the maintenance of tumor angiogenesis. The combination of sFGFR and soluble Flt exhibited a synergistic effect in the repression of tumor growth. Finally, i.v. injection of AdsFGFR resulted in a dramatic repression of tumor growth in a transgenic mouse model of pancreatic beta cell carcinogenesis. Similar to control infections with AdsFlt, tumor-associated vessel density was decreased, indicating that the expression of sFGFR impaired tumor angiogenesis. These data indicate that FGFs play a critical role in tumor angiogenesis.
