CD40L and TNF both activate the classical NF-κB pathway, which is not required for the CD40L induced alternative pathway in endothelial cells.

CD40L and TNF signal through engagement of their respective receptors, which are both members of the TNF receptor family. They use partially common signaling molecules leading, among others, to activation of the NF-κB pathway. However, whereas TNF activates the classical, CD40L has been reported to activate the alternative NF-κB pathway, leading to the anticipation that differences in the pattern of inflammatory gene expression would occur. Here, we have compared the gene expression repertoire of CD40L (CD154) and TNF stimulated HUVEC and report that unexpectedly, apart from a stronger response to TNF, no major qualitative differences could be observed. This applies for the period of up to 6 h, a time where the alternative pathway has already been activated. Analysis of the early events after receptor engagement revealed that both TNF and CD40L activate the classical NF-κB pathway, and confirm activation of the alternative by the latter. Furthermore, using genetic and pharmacological inhibition of the classical pathway we show that activation of the alternative occurs independently of the former. This reveals novel insights into NF-κB signaling by CD40L and TNF in endothelial cells.