Lipid Rafts and Caveolin-1 Coordinate Interleukin-1β (IL-1β)-dependent Activation of NFκB by Controlling Endocytosis of Nox2 and IL-1β Receptor 1 from the Plasma Membrane

Abstract

Recent evidence suggests that signaling by the proinflammatory cytokine interleukin-1␤ (IL-1␤) is dependent on reactive oxygen species derived from NADPH oxidase. Redox signaling in response to IL-1␤ 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 IB kinase kinases) and ultimately for activation of the transcription factor NFB. 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-1␤ 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-1␤ signals caveolin-1-dependent endocytosis of both proteins into the redoxosome. We also show that inhibiting lipid raft-mediated endocytosis prevents NFB activation. Finally, we demonstrate that Vav1, a Rac1 guanine exchange factor and activator of Nox2, is recruited to lipid rafts following IL-1␤ stimulation and that it is required for NFB activation. Our results fill in an important mechanistic gap in the understanding of early IL-1R1 and Nox2 signaling events that control NFB activation, a redox-dependent process important in inflammation. IL-1␤2 is a potent proinflammatory cytokine that controls inflammation in response to a diverse collection of health problems, including ischemia/reperfusion injury, viral infection, bacterial infection, autoimmune diseases such as diabetes, allergies, trauma, and chemical exposure (1-8). A primary role for signaling by IL-1␤ in these inflammatory responses is the activation of NFB, a transcription factor that regulates a large number of immune molecules, apoptotic factors, anti-apoptotic factors, and other transcription factors (9). The importance of IL-1␤ and NFB in inflammation was highlighted by a clinical study on mortality in septic patients (10). A spectrum of cytokines and transcription factors was examined in this study, and two were identified as significant prognostic indictors of patient outcome. One prognostic indicator was NFB activity, with this transcription factor twice as active in non-survivors relative to survivors. The second prognostic indicator kinase was the ratio between IL-1␤ and its competitive antagonist IL-1ra, with survivors having a 50% higher IL-1ra/ IL-1␤ ratio than non-survivors.Because of the clinical importance of IL-1␤, elucidating the signaling events involved in IL-1␤-mediated NFB activation is of great significance. Among the early events that control IL-1␤ signaling is the induction of IL-1R1 dimerization following ligand binding (11,12). This event initiates binding of MyD88 to the TIR (Toll/IL-1R1) domains within the cytopl...

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