ASC directs NF-kappaB activation by regulating receptor interacting protein-2 (RIP2) caspase-1 interactions

Receptor interacting protein-2 (RIP2) is a Caspase recruitment domain (CARD)-containing kinase that interacts with Caspase-1 and plays an important role in NF-kappaB activation. Apoptosis-associated speck-like protein containing a CARD (ASC) is a PYRIN and CARD-containing molecule, important in the induction of Apoptosis and Caspase-1 activation. Although RIP2 has also been linked to Caspase-1 activation, RIP2 knockout Animals fail to show a defect in caspase-1-mediated processing of proIL-1beta to its active form. Therefore, RIP2 function in binding to Caspase-1 remains poorly understood. We hypothesized that Caspase-1 may serve as a scaffolding molecule that promotes RIP2 interaction with IkappaB kinase-gamma thus inducing NF-kappaB activation. We further hypothesized that ASC, which also interacts with Caspase-1 via its CARD, may interfere with the Caspase-1 RIP2 interaction. In HEK293 cells, ASC induced prominent activation of Caspase-1 and proIL-1beta processing. RIP2 transient transfection induced transcription of an NF-kappaB reporter gene. This RIP2-induced NF-kappaB activity and Caspase-1 binding was inhibited in a dose-dependent fashion by ASC. Consistent with a role for Caspase-1 as a scaffold for RIP2, Caspase-1 knockout macrophages were suppressed in their ability to activate NF-kappaB, and septic Caspase-1 knockout Animals produced less IL-6, a functional marker of NF-kappaB activity. Lastly, THP-1 cells treated with small interfering RNA for ASC decreased their Caspase-1 activity while enhancing their NF-kappaB signal. These data suggest that ASC may direct Caspase-1 away from RIP2-mediated NF-kappaB activation, toward caspase-1-mediated processing of proIL-1beta by interfering with the RIP2 Caspase-1 interaction.