RESUMO
We have previously shown that a molecular interaction between insulin-like growth factor binding protein 2 (IGFBP2) and integrin alpha5 is necessary for the enhancement of cell migration in IGFBP2-overexpressing gliomas. In the present study, we examined the mechanism through which the IGFBP2/integrin alpha5 interaction mediates enhanced glioma cell migration. Although both ERK and JNK MAP kinases were activated, JNK was specifically involved in IGFBP2-mediated migration as shown by inhibitor analysis of IGFBP2-overexpressing cells. Because gliomas are solid tumors that require contact with a surface (e.g., other cells, extracellular matrix) for migration, we used the extracellular matrix (ECM) protein fibronectin, which is the sole ligand of the alpha5beta1 integrin receptor, to show that integrin alpha5 is an important mediator of JNK activation. In addition, we found the IGFBP2/integrin alpha5 pathway to be activated in a significantly shorter interval in cells seeded onto fibronectin-coated surfaces compared to cells seeded onto plastic alone. The activation of JNK was downstream of the IGFBP2/integrin alpha5 interaction, as shown by alpha5 knockdown experiments using IGFBP2-overexpressing cells. Based on these data we propose that the interaction between IGFBP2 and integrin alpha5 accelerates cell adhesion, and this, in turn, enhances JNK-mediated glioma cell migration.
Assuntos
Neoplasias Encefálicas/patologia , Movimento Celular/genética , Glioma/patologia , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Integrina alfaV/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , RNA Interferente Pequeno/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioma/genética , Glioma/metabolismo , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Integrina alfaV/genética , Integrina alfaV/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transfecção , Células Tumorais CultivadasRESUMO
In the study we report here, we tested the hypothesis that insulin-like growth factor-binding protein 2 (IGFBP2) promotes cell mobility through its interaction with integrin alpha5. Our previous microarray studies showed that IGFBP2 activates the expression of integrin alpha5. In addition, IGFBP2 has an Arg-Gly-Asp (RGD) domain, which is a known integrin binding motif. We first confirmed our microarray results by showing that the expression of integrin alpha5 is indeed up-regulated at the protein level in IGFBP2-overexpressing SNB19 glioma cells. Using co-immunoprecipitation, we confirmed that IGFBP2 does interact with integrin alpha5. To confirm that IGFBP2 interacts directly with integrin alpha5 through the RGD domain, we created an RGD --> RGE mutant (D306E) IGFBP2 and stably overexpressed the mutant IGFBP2 in the same cell line. Co-immunoprecipitation then showed that D306E-IGFBP2 had no detectable binding with integrin alpha5. We further observed that IGFBP2-overexpressing cells have extensive cell surface lamellipodia, whereas D306E-IGFBP2-overexpressing cells show abundant cell surface focal adhesions. Consistent with this, phenotype analysis then showed that IGFBP2-overexpressing cells have elevated migration rates compared with vector control; in contrast, the migration rates of the D306E-IGFBP2-overexpressing cells were not elevated and were comparable with that of vector control. Decreased expression of integrin alpha5 by small interference RNA in IGFBP2-overexpressing cells also reduced cell mobility. Therefore, we have concluded that one mechanism by which IGFBP2 activates IGFBP2-induced cell mobility is through its interaction with integrin alpha5 and this interaction is specifically mediated through the RGD domain on IGFBP2.
