RESUMEN
The endothelial cell-specific chemotaxis receptor (ECSCR) is a cell-surface protein selectively expressed by endothelial cells (ECs), with roles in EC migration, apoptosis and proliferation. Our previous study (Verma, A., Bhattacharya, R., Remadevi, I., Li, K., Pramanik, K., Samant, G. V., Horswill, M., Chun, C. Z., Zhao, B., Wang, E., Miao, R. Q., Mukhopadhyay, D., Ramchandran, R., and Wilkinson, G. A. (2010) Blood 115, 4614-4622) showed that loss of ECSCR in primary ECs reduced tyrosine phosphorylation of vascular endothelial growth factor (VEGF) receptor 2/kinase insert domain receptor (KDR) but not VEGF receptor 1/FLT1. Here, we show that ECSCR biochemically associates with KDR but not FLT1 and that the predicted ECSCR cytoplasmic and transmembrane regions can each confer association with KDR. Stimulation with VEGF165 rapidly and transiently increases ECSCR-KDR complex formation, a process blocked by the KDR tyrosine kinase inhibitor compound SU5416 or inhibitors of endosomal acidification. Triple labeling experiments show VEGF-stimulated KDR(+)/ECSCR(+) intracellular co-localization. Silencing of ECSCR disrupts VEGF-induced KDR activation and AKT and ERK phosphorylation and impairs VEGF-stimulated KDR degradation. In zebrafish, ecscr interacts with kdrl during intersomitic vessel sprouting. Human placenta and infantile hemangioma samples highly express ECSCR protein, suggesting a role for ECSCR-KDR interaction in these tissues.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de la Membrana/metabolismo , Proteolisis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Reguladoras de la Apoptosis , Línea Celular , Endosomas/genética , Endosomas/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Hemangioma/genética , Hemangioma/metabolismo , Hemangioma/patología , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Indoles/farmacología , Masculino , Proteínas de la Membrana/genética , Placenta/metabolismo , Placenta/patología , Embarazo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt , Pirroles/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Pez CebraRESUMEN
Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from -1170 bp to -1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain- and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.