TRAF6在B淋巴瘤细胞系NF-κB和AP-1信号传导通路中的作用

Role of TRAF6 in NF-κB and AP-1 signaling transduction pathway in human B lymphoma cell line

目的利用人B淋巴瘤细胞系模型探讨TRAF6在调控NF-κB和AP-1信号系统中的作用。方法将融合有黄色荧光素(YFP)的功能缺失型TRAF6(DN-TRAF6)质粒和小干扰RNA-TRAF6质粒转染至人类B淋巴细胞系,过夜培养后经流式细胞仪或G418筛选阳性转染细胞,通过Western blot、ELISA等方法研究TRAF6对NF-κB通路中IκBα磷酸化和转录因子(P65,P50和c-Rel)向细胞核内转移以及AP-1通路中ERK、JNK、P38磷酸化和转录因子(C-FOS,C-JUN,ATF和CREB)向细胞核内转移等的影响。结果B细胞过度表达DN-TRAF6或转染小干扰RNA-TRAF6均可抑制IκBα和JNK的磷酸化水平以及P65、P50、c-Rel和c-Fos、C-JUN的核内转录。结论TRAF6可选择性地作用于人B淋巴细胞的NF-κB和AP-1信号传导系统中部分激酶,在上述两条信号系统活化中起重要作用。

Objective To investigate the role of TRAF6 in NF-κB and AP-1 signaling pathway in human B cell line. Methods Human Ramos B cells were transfected with plasmids expressing YFP fusion dominant-negative TRAF6 （DN-TRAF6）, or transfected with shRNA-TRAF6 plasmid. After incubation overnight, cells were either sorted with flowcytometry or screened by Gall 8. Activation of NF-κB and AP-1 pathway, including phosphorylation of IκBα, ERK, JNK and P38, as well as nuclear translocation of NF-κB subunits （ P65, PSO and c-Rel） and AP-1 subunits（C-FOS, C-JUN, CREB and ATF） were detected by Western blot and ELISA. Results In ceils which overexpress DN-TRAF6 or endogenous TRAF6 expression were knocked-down by shRNA, the phosphorylation of IκBα, as well as phosphorylation of JNK were inhibited. Furthermore, nuclear translocation of NF-κB subunits P65, P50, c-Rel, and AP-1 subunits C-FOS and C-JUN were also inhibited in B cells through overexpression of DN-TRAF6. Conclusion TRAF6 selectively activates some kinases in CD40 mediated NF-κB and AP-1 signaling pathway, and plays an important role in their activation.