Figure 3

Induction of TRAF3 and phosphorylation of ASK-1 and MKK4 are essential in JNK-activation and CD40-mediated apoptosis. (a) Retrovirus transduction was used to establish the EJ derivative line EJ-TRAF3-KD, which stably expresses an anti-TRAF3 shRNA, and its isogenic control shRNA-expressing cell line EJ-Con. Immunoblotting experiments demonstrate that ligation of CD40 in the control cells caused induction of TRAF3 expression (3 h), but this is significantly attenuated in EJ-TRAF3-KD cells. Further Western blotting experiments show that in EJ-Con, CD40 ligation triggers the phosphorylation of ASK-1 (3 h) as well as MKK4 (6 h) and JNK (6 h). No visible changes in MKK7 phosphorylation (6 h) were observed. These changes are followed by the induction of Bak and Bax (24 h). By contrast, TRAF3 knockdown abrogates the activation of ASK-1, subsequent phosphorylation of MKK4 and JNK, as well as induction of Bak and Bax. CK18 detection was used for comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) to ensure equal loading. Time points are indicated on the right hand side for clarity. Results are representative of three independent experiments. Of note, in all immunoblotting experiments, expression of cytokeratin proteins (CK8 or CK18) was employed to confirm equal loading rather than total unphosphorylated protein (as explained in the Methods). (b) Retrovirus transduction was used to establish EJ cell line derivatives EJ-ASK1-KD, EJ-MKK4-KD and EJ-MKK7-KD that stably express anti-ASK-1, anti-MKK4 and anti-MKK7 shRNAs, respectively. Immunoblotting experiments in these cell lines demonstrated that CD40-mediated phosphorylation of ASK-1 (3 h) and MKK4 (6 h) is blocked by the respective shRNAs when compared to their isogenic control cells (EJ-Con – as shown in (a) above as well as in Supplementary Figure 8), while basal phospho-MMK7 expression (6 h) is reduced. CK18 detection was used to assess equal loading. Results are representative of at least two independent experiments. (c) Western blots for the detection of phosphorylated JNK following CD40 ligation (6 h) in EJ-ASK1-KD, EJ-MKK4-KD and EJ-MKK7-KD demonstrate that shRNA-mediated knockdown of ASK-1 and MKK4, but not MKK7, significantly attenuates CD40-mediated p-JNK induction observed in control cells (EJ-Con in Figure 2a above). CK18 detection was used to assess equal loading. Results are representative of two independent experiments. (d) CD40-mediated apoptosis is abrogated by TRAF3, MKK4, ASK-1 knockdown as indicated by the blockade of caspase-3/7 activation in EJ-TRAF3-KD, EJ-ASK1-KD and EJ-MKK4-KD cells, in comparison to EJ-Con. By contrast, MKK7 knockdown had no effect on apoptosis. Bars represent mean fold caspase activity ±s.d. (n=5). Results are representative of three independent experiments. (e) Immunoblotting experiments demonstrate that ligation of CD40 in HCT116 cells triggers upregulation of TRAF3 (3 h), as well as phosphorylation of MKK4 (6 h) and JNK (6 h), which is followed by the induction of Bax (12 h). CK8 detection was used for comparison of controls (‘–’) versus mCD40L-treated cells (‘+’) to confirm equal loading. Results are representative of at least two independent experiments. (f) Retrovirus transduction was used to establish the HCT116 derivative lines HCT116-TRAF3-KD and HCT116-Bax-KD, which stably express anti-TRAF3 and anti-Bax shRNA, respectively, and their isogenic control shRNA-expressing cell line HCT116-Con. CD40-mediated apoptosis is attenuated by TRAF3 and Bax knockdown as indicated by the blockade of caspase-3/7 activation in HCT116-TRAF3-KD and HCT116-Bax-KD cells, in comparison to HCT116-Con. Bars represent mean fold caspase activity ±s.d. (n=5).