CD40 regulation of nuclear factor-kappa B/Rel and c-myc expression and their involvement in apoptosis of murine B cells

Engagement of surface immunoglobulin M (sIgM) on WEHI 231 murine B lymphoma cells leads to abortive activation and apoptosis, suggesting that this cell line may represent a model for tolerance. Loss of viability in these cells is preceded by an early increase in c-myc RNA levels followed by a decline below control values, implicating c-myc in the control of apoptosis. Co-stimulation with CD40 ligand (CD40L) rescues WEH1 231 cells from anti-sIgM induced apoptosis. Therefore, the effects of CD40L on c-myc expression were measured. Treatment of these cells with the combination cD40L and anti-sIgM led to induction and maintenance of elevated levels of c-myc RNA and protein. Since transcriptional regulation of c-myc is mediated through two NF-{dollar}kappa{dollar}B sites in WEHI 231, the effects of CD40L on DNA binding by this family of transcription factors were evaluated. CD40L induced and sustained the levels of NF-{dollar}kappa{dollar}B binding to both of these sites, paralleling the changes in c-myc RNA levels. Elevated levels of Nf-{dollar}kappa{dollar}B were partially achieved through a sustained decrease in the steady state amount of NF-{dollar}kappa{dollar}B/Rel specific inhibitory protein, I{dollar}kappa{dollar}B-{dollar}alpha{dollar}, and a transient decrease in I{dollar}kappa{dollar}B-{dollar}beta{dollar}. To determine the mechanism of decrease of the two inhibitors, mRNA levels were first examined. CD40L treatment caused an early increase in I{dollar}kappa{dollar}B-{dollar}alpha{dollar} mRNA levels, presumably in response to the rapid, elevated NF-{dollar}kappa{dollar}B activity and a slower increase in I{dollar}kappa{dollar}B-{dollar}beta{dollar} mRNA levels. Thus the drop in I{dollar}kappa{dollar}B expression did not appear due to a transcriptional control mechanism. In exponentially growing WEHI 231 cells, the I{dollar}kappa{dollar}B-{dollar}alpha{dollar} and I{dollar}kappa{dollar}B-{dollar}beta{dollar} proteins decayed with half-lives of approximately 38 and 76 min., respectively. CD40L treatment caused a sudden, dramatic decrease in half-life for both I{dollar}kappa{dollar}B molecules (t 1/2 {dollar}<{dollar} 5 min.). Addition of the proteosome-specific inhibitor, Z-LLF-CHO, effectively blocked the degradation of both I{dollar}kappa{dollar}B-{dollar}alpha{dollar} and I{dollar}kappa{dollar}B-{dollar}beta{dollar} in exponentially growing WEHI 231 cells and following CD40L treatment. These findings indicate engagement of CD40 promotes I{dollar}kappa{dollar}B protein turnover, leading to enhanced NF-{dollar}kappa{dollar}B/Rel and c-Myc expression and ultimately, cell survival.