Abstract
Recent evidence suggests that signaling by the proinflammatory cytokine interleukin-1beta (IL-1beta) is dependent on reactive oxygen species derived from NADPH oxidase. Redox signaling in response to IL-1beta is known to require endocytosis of its cognate receptor (IL-1R1) following ligand binding and the formation of redox-active signaling endosomes that contain Nox2 (also called redoxosomes). The consequent generation of reactive oxygen species by redoxosomes is responsible for the downstream recruitment of IL-1R1 effectors (IRAK, TRAF6, and IkappaB kinase kinases) and ultimately for activation of the transcription factor NFkappaB. Despite this knowledge of the signaling events that occur downstream of redoxosome formation, an understanding of the mechanisms that coordinate the genesis of redoxosomes following IL-1beta stimulation has been lacking. Here, we demonstrate that lipid rafts play an important role in this process. We show that Nox2 and IL-1R1 localize to plasma membrane lipid rafts in the unstimulated state and that IL-1beta signals caveolin-1-dependent endocytosis of both proteins into the redoxosome. We also show that inhibiting lipid raft-mediated endocytosis prevents NFkappaB activation. Finally, we demonstrate that Vav1, a Rac1 guanine exchange factor and activator of Nox2, is recruited to lipid rafts following IL-1beta stimulation and that it is required for NFkappaB activation. Our results fill in an important mechanistic gap in the understanding of early IL-1R1 and Nox2 signaling events that control NFkappaB activation, a redox-dependent process important in inflammation.