RESUMO
Receptor-interacting protein 2 (RIP2) is a member of the receptor interacting protein (RIP) family and plays an important role in the innate and adaptive immune responses. Overexpression of RIP2 mediates divergent signaling pathways including NF-κB activation and cell death. To further investigate the biological activity of RIP2 in vitro, a large amount of purified protein is required. For this purpose, the full length of RIP2 was cloned from human Ramos (human Burkitt lymphoma) tumor cells and inserted in a prokaryotic expression vector pET22b, and then the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. The expression of RIP2 was induced with IPTG. SDS-PAGE analysis showed that recombinant human RIP2 (rhRIP2) was mainly expressed as soluble fraction in the supernatant of the cell lysate. The recombinant protein was subsequently purified by His Trap FF crude to a purity of 90 %. MTT assay of the purified rhRIP2 showed its functional diversity in different cell lines, a specific inhibitory effect on MCF7 cells, but a promotion on the proliferation of Ramos cells. Furthermore, we identified that rhRIP2 could suppress activation of canonical NF-κB in MCF7 cells and activate non-canonical NF-κB signaling in Ramos cells, these data suggested that RIP2 participates in different signaling pathways contributing to its specific effects in vitro. Our results provided new clues to further explore the regulation mechanisms of RIP2 in tumorigenesis.
Assuntos
Expressão Gênica , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Plasmídeos , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/isolamento & purificação , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/farmacologia , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacosRESUMO
Receptorinteracting protein 2 (RIP2) has recently been reported to be involved in tumor infiltration and cancer metastasis. However, the function of RIP2 in human astrocytoma remains unclear. In the present study, we showed that the expressions of RIP2 and BclxL were positively correlated with the malignant grade in 28 cases of astrocytoma of various grades and 6 cases of normal human tissues. In addition, increased activity of the NFκB and p38 signaling pathways in astrocytoma tissue was observed. Cytological experiments indicated that RIP2 promoted human glioblastoma cell proliferation by inducing expression of BclxL, and knockdown of endogenous RIP2 promoted cell apoptosis. Mechanistically, knockdown of RIP2 suppressed downstream events including the canonical and alternative NFκB pathway as well as the mitogenactivated protein kinase (p38) pathway. In addition, the present study also demonstrated that tumor necrosis factor receptorassociated factor 3 (TRAF3), as a novel RIP2 binding partner, was downregulated in glioma tissues and functionally was a negative regulator involved in RIP2induced glioma cell growth. Taken together, the present study established a negative link between RIP2 and TRAF3 proteins and identifies a new pathway for regulating astrocytoma progression.
Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Fator 3 Associado a Receptor de TNF/genética , Proteína bcl-X/genética , Adulto , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Gradação de Tumores , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Proteína bcl-X/metabolismoRESUMO
Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a highly versatile immune regulator that positively controls type I interferon production, but negatively regulates the activation of mitogen-activated protein kinase and alternative nuclear factor-κB signaling. The precise function of TRAF3 in different signaling pathways remains unclear. Thus, in a yeast two-hybrid assay, TRAF3 was used as the bait to screen a human spleen cDNA library for TRAF3 interactors that may potentially mediate TRAF3-regulated functions. Receptor-interacting protein 2 (RIP2) was identified as a TRAF3 binding partner. The interaction between TRAF3 and RIP2 was further confirmed by mammalian two-hybrid, co-immunoprecipitation and GST pull-down assays, and this interaction was also verified by immunoprecipitation of endogenous proteins in Ramos cells, a human B lymphoma cell line. RIP2 is an activator of NF-κB. We therefore examined the effect of TRAF3 in RIP2-induced NF-κB activation. The result showed that TRAF3 could inhibit RIP2-induced NF-κB activation. Given the high expression of RIP2 in the B lymphoma cell line and endogenous interaction between TRAF3 and RIP2 in Ramos cells, the role of RIP2 was further studied. The result demonstrated that RIP2 knockdown was capable of increasing the expression of TRAF3 and suppressing the activation of alternative NF-кB pathway in Ramos cells. These findings suggest that functional interactions between RIP2 and TRAF3 may provide some clues to the mechanisms of TRAF3-involvement in both positive and negative regulatory functions.