RESUMO
Urokinase-type plasminogen activator (uPA) additionally elicits a whole array of pro-angiogenic responses, such as differentiation, proliferation, and migration. In this study, we demonstrate that in endothelial cells uPA also protects against apoptosis by transcriptional up-regulation and partially by mRNA stabilization of inhibitor of apoptosis proteins, most prominently the X-linked inhibitor of apoptosis protein (XIAP). The antiapoptotic activity of uPA was dependent on its protease activity, the presence of uPA receptor (uPAR) and low-density lipoprotein receptor-related protein (LRP), but independent of the phosphatidylinositol 3 (PI3) kinase pathway, whereas vascular endothelial growth factor (VEGF)-induced antiapoptosis was PI3 kinase dependent. uPA-induced cell survival involved phosphorylation of p21-activated kinase 1 (Pak1) and the IkappaB kinase alpha that leads to nuclear factor kappaB (NF-kappaB) p52 activation. Indeed, blocking NF-kappaB activation by using specific NF-kappaB inhibitors abolished uPA-induced cell survival as it blocked uPA-induced XIAP up-regulation. Furthermore, down-regulating XIAP expression by small interfering RNA (siRNA) significantly reduced uPA-dependent endothelial cell survival. This mechanism is also important for VEGF-induced antiapoptosis because VEGF-dependent up-regulation of XIAP was found defective in uPA(-/-) endothelial cells. This led us to conclude that uPA is part of a novel NF-kappaB-dependent cell survival pathway.
Assuntos
Apoptose/fisiologia , Endotélio Vascular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Quinase I-kappa B/metabolismo , Immunoblotting , Técnicas Imunoenzimáticas , Imunoprecipitação , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Quinases Ativadas por p21/metabolismoRESUMO
In this study we have investigated the role of a specific corepressor of EGR-1, NAB2, to down-regulate vascular endothelial growth factor (VEGF)-induced gene expression in endothelial cells and to inhibit angiogenesis. Firstly, we show a reciprocal regulation of EGR-1 and NAB2 following VEGF treatment. During the initial phase EGR-1 is rapidly induced and NAB2 levels are down-regulated. This is followed by a reduction of EGR-1 and a concomitant increase of NAB2. Secondly, using the tissue factor gene as a readout for VEGF-induced and EGR-1-regulated gene expression we demonstrate that NAB2 can completely block VEGF-induced tissue factor reporter gene activity. Thirdly, by adenovirus-mediated expression we show that NAB2 inhibits up-regulation of tissue factor, VEGF receptor-1, and urokinase plasminogen activator mRNAs even when a combination of VEGF and bFGF is used for induction. In addition, NAB2 overexpression significantly reduced tubule and sprout formation in two different in vitro angiogenesis assays and largely prevented the invasion of cells and formation of vessel-like structures in the murine Matrigel model. These data suggest that NAB2 regulation represents a mechanism to guarantee transient EGR-1 activity following exposure of endothelial cells to VEGF and that NAB2 overexpression could be used to inhibit signals involved in the early phase of angiogenesis.
