非经典性NF-KB活性与TRAF3在霍奇金淋巴瘤细胞中的作用机制

摘要

目的 探讨非经典性NF-κB信号通路及肿瘤坏死因子受体相关分子(TRAF)3在B细胞源性霍奇金淋巴瘤(HL)细胞中的作用,并试图对非经典性NF-κB信号通路在HL中异常活化作出合理解释.方法 采用凝胶迁移电泳实验测定L428和KM-H2 HL细胞的细胞核中NF-κB的活性,并用TransAMTM ELISA方法定量分析L428细胞的细胞核中各κB DNA-蛋白质复合物的活性成分.采用Western blot法检测细胞质中各NF-κB家族成员以及TRAF3蛋白的表达情况;瞬时转染TRAF3表达载体检测其对NF-κB表达的影响.结果 L428和KM-H2细胞中NF-κB活性持续性存在.经典性以及非经典性NF-κB活性在L428细胞的细胞核中均呈持续性高表达.L428和KM-H2细胞中出现p52蛋白积聚和RelB表达增加.另外,L428细胞和KM-H2细胞中TRAF3的表达均非常弱,而过表达TRAF3后抑制了p100蛋白质加工和p52蛋白积聚.结论 B细胞源性HL细胞中经典性和非经典性NF-κB活性呈持续性活化;TRAF3的低表达很可能是非经典性NF-κB活化的原因之一.%Objective To investigate the function of unclassical NF-κB signaling pathway and TNF associated-factor(TRAF)-3 in Hodgkin's lymphoma(HL)cells,and explore a reasonable explanation for alternative NF-κB signaling pathway activation in HL cells.Methotis The intranuclear NF-κB activity in L428 and KM-H2 cells Was examined by electrophoretie mobility shift assay(EMSA).The NF-κB DNA complex in L428 cell nuclear extracts Was further quantified by an ELISA-based NF-κB family transcription factor activity assay.The expression of other NF-κB family members in the cytoplasm,and the TRAF3 expression were detected by Western blot analysis.The effects of TRAF3 on the unclassical NF-κB signaling pathway in L428 cells were studied by transient expression of TRAF3.Results The NF-κB signaling pathway activety persistently remained in L428 and KM-H2 cells.Both classical and unclassical NF-κB activity in L428 cells was highly expressed.There were enhaneed p52 protein accumulation and RelB expression and a very weak expression of TRAF3 in both L428 and KM-H2 cells.Transient transfeetion of TRAF3-exprssion vector increased the TRAF expression and blocked the p100 processing and p52 protein accumulation in both cells.Conclusion The classical as well as the unclassical NF-κB activities characterized by p100 processing and D52-RelB nuclear localization are persistently present in HL cells.Lack of the TRAF3 expression might be one of the reasons for the aberrent expression of the unclassical NF-κB activity.