RESUMO
A specific splice variant of the CD44 cell- surface protein family, CD44v6, has been shown to act as a coreceptor for the receptor tyrosine kinase c-Met on epithelial cells. Here we show that also on endothelial cells (ECs), the activity of c-Met is dependent on CD44v6. Furthermore, another receptor tyrosine kinase, VEGFR-2, is also regulated by CD44v6. The CD44v6 ectodomain and a small peptide mimicking a specific extracellular motif of CD44v6 or a CD44v6-specific antibody prevent CD44v6-mediated receptor activation. This indicates that the extracellular part of CD44v6 is required for interaction with c-Met or VEGFR-2. In the cytoplasm, signaling by activated c-Met and VEGFR-2 requires association of the CD44 carboxy-terminus with ezrin that couples CD44v6 to the cytoskeleton. CD44v6 controls EC migration, sprouting, and tubule formation induced by hepatocyte growth factor (HGF) or VEGF-A. In vivo the development of blood vessels from grafted EC spheroids and angiogenesis in tumors is impaired by CD44v6 blocking reagents, suggesting that the coreceptor function of CD44v6 for c-Met and VEGFR-2 is a promising target to block angiogenesis in pathologic conditions.
Assuntos
Receptores de Hialuronatos/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/imunologia , Imunoprecipitação , Camundongos , Camundongos SCID , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
CD44 isoforms act as coreceptors for the receptor tyrosine kinases c-Met and VEGFR-2. However, Cd44 knockout mice do not show overt phenotypes, in contrast to Met and Vegfr-2 knockout mice. We hypothesized that CD44 is being compensated for by another factor in Cd44 null mice. Using RNAi technology and blocking experiments with antibodies, peptides, and purified ectodomains, as well as overexpression studies, we identified intercellular adhesion molecule-1 (ICAM-1) as a new coreceptor for c-Met in CD44-negative tumor cells and in primary hepatocytes obtained from Cd44 null mice. Most strikingly, after partial hepatectomy, CD44v6-specific antibodies inhibited liver cell proliferation and c-Met activation in wild-type mice, whereas ICAM-1-specific antibodies interfered with liver cell proliferation and c-Met activation in Cd44 knockout mice. These data show that ICAM-1 compensates for CD44v6 as a coreceptor for c-Met in Cd44 null mice. Compensation of proteins by members of the same family has been widely proposed to explain the lack of phenotype of several knockout mice. Our experiments demonstrate the functional substitution of a protein by a heterologous one in a knockout mouse.