TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-kappaB pathway by TRAF-binding TNFRs

Abstract

TNF family members and their receptors contribute to increased gene expression for inflammatory processes and intracellular cascades leading to programmed cell death, both via activation of NF-kappaB. TNF receptor (TNFR)-associated factors (TRAFs) are cytoplasmic adaptor proteins binding to various receptors of the TNFR family. In an attempt to delineate the role of individual TRAFs, we compared NF-kappaB activation by CD40(wt) and CD40 mutants with different TRAF recruitment patterns. Recognized only recently, NF-kappaB signaling occurs at least via two different pathways. Each pathway results in nuclear translocation of two different Reldimers, the canonical p50/RelA and the noncanonical p52/RelB. Here, we show that via TRAF6, CD40 mediates only the activation of the canonical NF-kappaB pathway. Via TRAF2/5, CD40 activates both the canonical and the noncanonical NF-kappaB pathways. We observed that TRAF3 specifically blocked the NF-kappaB activation via TRAF2/5. This inhibitory effect of TRAF3 depends on the presence of an intact zinc finger domain. Paradoxically, suppression of TRAF2/5-mediated NF-kappaB activation by TRAF3 resulted in enhanced transcriptional activity of TRAF6-mediated canonical NF-kappaB emanating from CD40. We also observed that 12 TNFR family members (p75TNFR, LTbetaR, RANK, HVEM, CD40, CD30, CD27, 4-1BB, GITR, BCMA, OX40, and TACI) are each capable of activating the alternative NF-kappaB pathway and conclude that TRAF3 serves as a negative regulator of this pathway for all tested receptors.

Fig. 1.

NF-κB activation and TRAF recruitment by CD40 and CD40 mutants. (A) Schematic presentation of the TRAF interaction sites in the CD40-signaling domain. Shown are the interactions sites of TRAF6 and TRAF2/3/5 as well as the mutations relevant in this study. (B) CD40-mediated activation of NF-κB ceases only when the TRAF6, the TRAF2/3/5, and two further QE motifs in the CD40 C terminus are mutated or deleted (correlation with TRAF recruitment). All assays were done as described in Materials and Methods. Induction of NF-κB activity was determined after 3 h of stimulation with CD40L⁺ cells. The results were normalized on the basis of receptor expression. TRAF recruitment is expressed as the percentage of the CD40_wt control (arbitrarily assumed to be 100%).

Fig. 2.

TRAF3 inhibits NF-κB signaling of CD40 mutants with TRAF6 recruitment defects (dependence on zinc finger motifs). CD40_wt or CD40 mutants were transfected into 293T cells together with TRAF3, ΔTRAF3_89–567, or ΔTRAF3_324–567 expression plasmids and a NF-κB reporter plasmid. Eighteen hours later the cells were stimulated for 3 h with CD40L⁺ cells, then lysed and Luciferase activity was determined. All results were normalized on the basis of CD40 expression. Shown are the average values of duplicate determinations of one representative experiment of four.

Fig. 3.

TRAF3 inhibits TRAF2/5- but not TRAF6-mediated NF-κB activation. Subconfluent 293T cells were transfected with CD40 or CD40 mutant expression plasmids together with TRAF3_wt (A), ΔTRAF3_324–567 (B), or a control plasmid (A and B). The cells were harvested 26 h after transfection, and nuclear extracts were prepared as described in ref. . Five micrograms of protein of each extract was analyzed by Western blot. The p100 degradation was analyzed in the cytoplasmic fraction of the same experiment. CD40 or CD40_mut expression was also determined in the cytoplasmic fractions and is shown as the percentage of the control experiment with CD40_wt. Shown is one representative result of four experiments.

Fig. 4.

Targeting of TRAF2, TRAF5, or NIK expression with siRNA reduces CD40-mediated activation of the noncanonical NF-κB pathway. Subconfluent 293T cells were transfected in a two-step procedure with the indicated siRNA constructs and with CD40 or the indicated CD40 mutants. Nuclear extracts were prepared and analyzed as described in Fig. 3.

Fig. 5.

TRAF3 inhibits noncanonical NF-κB activation by TRAF-binding TNFRSF members. (A) TRAF2, -3, and -5 recruitment to TNFRSF members. TRAF recruitment to TNFRSF members was determined by coexpression of the indicated receptor and the TRAF of interest in 293T cells. TRAF-binding was quantified by ELISA as described. TRAF-binding to CD40 was used as a reference. The receptor expression in each individual experiment was determined and used to normalize the recruitment data. Shown is the data and standard deviation of three independent experiments. (B) TRAF3 inhibits the activation of noncanonical NF-κB through TNFRSF members. Shown is the nuclear translocation of p52 in response to the indicated TNFRSF members in 293T cells expressing TRAF3 at low versus high levels. Transfections were done as described above. Receptor expression was adjusted in pilot experiments to intermediate p52 signal intensity. The amount of receptor-expression plasmid used in the experiment and the resulting receptor expression are shown. Receptor expression was determined in the cytoplasmic fractions of the nuclear extracts used for the detection of nuclear p52.