Differential Effects of TRAFs in the Activation of NF-KappB in Hodgkin’s Lymphoma Cells.

CD30, a member of the TNF receptor (TNFR) superfamily, is a lymphocyte-specific receptor that is originally recognized as a surface molecule overexpressed on Hodgkin’s lymphoma cells. Engagement of CD30 with its ligand CD30L activates the nuclear factor kB (NF-kB), which are mediated by interactions with TNFR-associated factor (TRAFs), and supports proliferation of Hodgkin’s cells. However, the role of individual TRAFs in the CD30 signaling pathway in Hodgkin’s cells has not been fully addressed yet. In this study, we found that except TRAF3, all other TRAFs were expressed consistently in B cell-derived Hodgkin’s cell lines (L428 and KM-H2) either in mRNA or protein level. In L428 but not KM-H2 cells, the classical (p50-RelA) and alternative (p52-RelB) NF-kB activity were constitutively activated measured by Western Blotting and EMSA. To better understand the CD30 signaling properties in Hodgkin’s lymphoma cells, we silenced the expression of TRAFs individually by means of RNAi. Successful downregulation of TRAF1 protein expression led to the apoptosis of L428 cells, which was companied by the reduction of both classical and alternative NF-kB activity. Furthermore, the expression of targeting genes of NF-kB, such as A20, c-Flip, ICAM-1, and Cyclin D1 was also decreased. Using siRNA targeting TRAF2 expression, the classical NF-kB activity was reduced while the alternative NF-kB activity moderately induced especially upon CD30L treatment, which was companied by the induction of p100 processing and RelB nuclear localization. The survival rate was decreased when TRAF2 was knockdown. TRAF5 knockdown manifested similar results as that of TRAF2. These observations were only took place in L428 cells but not KM-H2 cells. In addition, the phosphorylation of the extracellular signal-regulated kinases (ERK) 1 and 2, and subsequent activation of JunB in Hodgkin’s cells were staying unchanged in individual TRAFs knockdown experiment. Taken together, the study here further investigates the features of CD30-TRAFs-NF-kB signaling pathway in Hodgkin’s lymphoma cells. At least TRAF1, 2, and 5 are involved in CD30 signaling pathway and protecting Hodgkin’s lymphoma cells from undergoing apoptosis. The interaction between TRAFs is necessary to facilitate the signaling and proper activation of classical NF-kB, which is responsible for the antiapoptotic effect of Hodgkin’s cells. Among TRAF1, 2, and 5, the redundant function is unlikely. Furthermore, the sequential activation of ERK and JunB is unlikely dependent on TRAFs-NF-kB pathway in Hodgkin’s lymphoma cells. Thus, these data reveal that the classical NF-kB activity downstream of CD30 signaling is TRAFs-dependent in Hodgkin’s lymphoma cells, which is regulated by the relative level of individual TRAF in a cell-specific manner.