TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.

Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear.

Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector.

In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells.

Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells.

No relevant conflicts of interest to declare.