RESUMO
In this study, we examine the effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the phosphorylation status of specific phosphotyrosine residues on the vascular endothelial cell growth factor receptor-2 (VEGFR-2) cytoplasmic tail and examine the effects on associated downstream signaling pathways. To focus on metalloproteinase-independent mechanisms, we used the TIMP-2 analog known as Ala+TIMP-2 that is deficient in matrix metalloproteinase-inhibitory activity. Our experiments are designed to compare the effects of VEGF-A stimulation with or without Ala+TIMP-2 pretreatment, as well as basal responses in human microvascular endothelial cells. Our results show that Ala+TIMP-2 selectively alters the phosphorylation pattern of VEGFR-2 after VEGF-A stimulation and disrupts the downstream activation of PLC-gamma, Ca(+2) flux, Akt, and eNOS, as well as decreasing cGMP levels. Moreover, we observed an Ala+TIMP-2-induced reduction in cGMP levels typically elevated by exogenous NO donors implicating Ala+TIMP-2 in the direct activation of an isobutylmethylxanthine (IBMX)-sensitive cGMP phosphodiesterase activity. TIMP-2 suppression of endothelial mitogenesis and angiogenesis involves at least two mechanisms, one mediated by protein tyrosine phosphatase inhibition of VEGFR-2 activation as well as downstream signaling and a second mechanism involving direct activation of an IBMX-sensitive phosphodiesterase activity.
Assuntos
Células Endoteliais/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , 1-Metil-3-Isobutilxantina , Cálcio/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , GMP Cíclico/metabolismo , Ativação Enzimática , Matriz Extracelular/metabolismo , Humanos , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
We have previously reported that tissue inhibitor of metalloproteinases-2 (TIMP-2), an endogenous inhibitor of matrix metalloproteinase, modulates angiogenic responses through the MMP inhibition-independent activity. In this study, we investigate the molecular mechanisms of TIMP-2-mediated growth inhibition in response to fibroblast growth factor-2 (FGF-2). Pre-treatment with a protein tyrosine phosphatase inhibitor orthovanadate or expression of a dominant negative Shp-1 mutant fails to induce TIMP-2 inactivation of FGF-2 signaling pathways in human microvascular endothelial cells. We also show that TIMP-2 inhibition of FGF-2-induced p42/44(MAPK) activation and cell proliferation is associated with TIMP-2 binding to integrin alpha3beta1 on endothelial cell surfaces, as demonstrated by use of anti-integrin alpha3 or beta1 blocking antibodies, or disruption of integrin alpha3 expression by siRNA. Collectively, our results indicate that TIMP-2 inhibits FGF-2 signaling pathways through association with integrin alpha3beta1 and Shp-1-dependent inhibition of p42/44(MAPK) signaling, which in turn, results in suppression of FGF-2-stimulated endothelial cell mitogenesis.
Assuntos
Células Endoteliais/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Humanos , Integrina alfa3beta1/antagonistas & inibidores , Integrina alfa3beta1/genética , Integrina alfa3beta1/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteinases da Matriz/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Fisiológica , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , RNA Interferente Pequeno/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Proteínas Recombinantes/farmacologia , Transdução de Sinais/fisiologia , Inibidor Tecidual de Metaloproteinase-2/fisiologiaRESUMO
Tissue inhibitors of metalloproteinases (TIMPs) suppress matrix metalloproteinase (MMP) activity critical for extracellular matrix turnover associated with both physiologic and pathologic tissue remodeling. We demonstrate here that TIMP-2 abrogates angiogenic factor-induced endothelial cell proliferation in vitro and angiogenesis in vivo independent of MMP inhibition. These effects require alpha 3 beta 1 integrin-mediated binding of TIMP-2 to endothelial cells. Further, TIMP-2 induces a decrease in total protein tyrosine phosphatase (PTP) activity associated with beta1 integrin subunits as well as dissociation of the phosphatase SHP-1 from beta1. TIMP-2 treatment also results in a concomitant increase in PTP activity associated with tyrosine kinase receptors FGFR-1 and KDR. Our findings establish an unexpected, MMP-independent mechanism for TIMP-2 inhibition of endothelial cell proliferation in vitro and reveal an important component of the antiangiogenic effect of TIMP2 in vivo.