Use of novel small molecule inhibitors to investigate the role of endogenous RIP2 kinase activity in modulating NOD2/RIP2-dependent cytokine signaling

RIP2 is a Ser/Thr protein kinase, which undergoes autophosphorylation and activates NF-κB and MAPKs following stimulation of the upstream NOD1/NOD2 receptors resulting in proinflammatory cytokine production. Previous studies using transient overexpression systems have suggested that RIP2 kinase activity mediates NOD1/2-dependent signaling by regulating cellular RIP2 protein levels. To interrogate whether this is also the case for endogenous RIP2 kinase, we used novel, potent and highly-selective small-molecule RIP2 kinase inhibitors to study MDP-stimulated signaling in both primary human monocytes and human colonic epithelial (HCT116) cells. We demonstrated that inhibition of RIP2 kinase activity blocks proinflammatory cytokine production in monocytes in the absence of detectable changes in total RIP2 protein levels. Using TNFα-primed HCT116 cells, we confirmed these observations and demonstrated a direct correlation between the inhibition of endogenous RIP2 kinase activity (measured as a decrease in pS176-RIPK2 levels) and a reduction in proinflammatory cytokine signaling in response to MDP-stimulation. Our data confirm the importance of endogenous RIP2 kinase activity in modulating proinflammatory cytokine release from both human monocytes and intestinal epithelial cells in response to bacterial ligands. Furthermore, our results suggest that RIP2 kinase inhibition may represent a novel therapeutic strategy for the treatment of intestinal inflammation.