Induction of RIP-2 kinase by proinflammatory cytokines is mediated via NF-κB signaling pathways and involves a novel feed-forward regulatory mechanism

The transcription factor NF-κB (nuclear factor κB) is a central mediator of inflammatory and apoptotic signaling in the cell. The protein kinase RIP-2 is a member of the CARD protein family (caspase activation and recruitment domain, also known as CARD3, Ripk2, CARDIAK, RICK, and CCK), and has been shown to be an activator of NF-κB. In this study, it was demonstrated by transcriptional profiling and protein expression analysis that the inflammatory cytokines TNF-α, IL-1β, and IFN-γ induced RIP-2 transcription and translation in endothelial cells. Two mechanistically distinct inhibitors of NF-κB signaling, sulfasalazine (NF-κB inhibitor) and WY-14643 [PPARα (peroxisome proroliferator-activated receptor α) agonist] that interfere with the transcription factor RELA (p65), suppressed TNF-α induced RIP-2 gene expression, which indicated that NF-κB signaling was involved in the cytokine-induced transcriptional activation of RIP-2 gene expression. Consistent with these observations, multiple NF-κB response elements were found in the upstream regions of the human and mouse RIP-2 genes. NF-κB-mediated regulation of RIP-2 gene and protein expression suggests an additional step in the regulation of NF-κB function as RIP-2 has been shown to positively modulate NF-κB by binding IKKγ (IκB kinase γ), a component of the IKK complex. These findings support a positive feed-forward mechanism of NF-κB regulation that involves NF-κB-dependent induction of RIP-2 transcription and a subsequent increase in RIP-2 protein levels in response to inflammatory cytokines. Elevated RIP-2 protein levels are then available to promote NF-κB function via interaction with IKKγ. RIP-2 is the first reported NF-κB-dependent protein kinase that positively regulates NF-κB activity.