Dominant Signals Leading to Inhibitor κB Protein Degradation Mediate CD40 Ligand Rescue of WEHI 231 Immature B Cells from Receptor-Mediated Apoptosis

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Recently, we demonstrated maintenance of nuclear factor (NF)-κB/Rel factors plays a major role in B cell survival. Treatment of WEHI 231 immature B cells with an Ab against the surface IgM protein (anti-IgM) induces apoptosis that can be rescued by engagement of CD40 receptor. The dramatic decrease in high basal levels of NF-κB/Rel activity induced by anti-IgM treatment led to cell death. CD40 ligand (CD40L) treatment prevented the drop in NF-κB/Rel factor binding by inducing a sustained decrease in inhibitor (I) κB-α and transient decrease in IκB-β protein levels. In this study, we have investigated the regulation of these NF-κB/Rel-inhibitory proteins. In exponentially growing WEHI 231 cells, the IκB-α and IκB-β proteins decayed with an approximate t1/2 of 38 and 76 min, respectively, which was blocked effectively upon addition of the proteasome-specific inhibitor (benzylcarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO). Anti-IgM treatment stabilized IκB-α and IκB-β proteins. CD40L treatment resulted in a dramatic decrease in t1/2 (<5 min) for both IκB molecules, which was inhibited by addition of Z-LLF-CHO. CD40L treatment also caused a delayed increase in IκB-β mRNA levels, most likely contributing to the observed recovery of IκB-β levels. Microinjection of IκB-α-glutathione S-transferase fusion protein into nuclei of WEHI 231 cells ablated protection by CD40L from receptor-mediated killing. Furthermore, CD40L rescued apoptosis induced upon microinjection of a vector expressing wild-type IκB-α, but not a 32A/36A mutant form of IκB-α, unable to be phosphorylated and hence degraded. Thus, control of turnover of IκB proteins by CD40L plays a major role in maintenance of NF-κB/Rel and resultant rescue of WEHI 231 cells from apoptosis.