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

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 six hours, 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-kappaB 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- kappaB signaling by CD40L and TNF in endothelial cells Human Umbilical Vein Endothelial Cells (HUVEC) in passage 4 were seeded in 6 well plates, 80.000 cells/cm2. After reaching post-confluency, the cells were stimulated with 50ng/ml sMCD40L (Enzo Life Sciences) or 10ng/ml TNF (R&D) for 0, 0.5, 1, 2.5, and 6 hours. The experiment was done in triplicates. Total RNA was isolated using the Qiagen RNA Plus Universal mini Kit. Samples were then pooled, and processed for analysis using the Affymetrix PrimeView Human Gene Expression Arrays according to the manufacturer's instructions