Nox2 and Rac1 Regulate H2O2-Dependent Recruitment of TRAF6 to Endosomal Interleukin-1 Receptor Complexes

Receptor stimulation at the plasma membrane has been implicated in the production of cellular reactive oxygen species (ROS) by a significant number of ligands, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide, angiotensin II, platelet-derived growth factor (PDGF), insulin, epidermal growth factor, transforming growth factor β1, and interleukin-1β (IL-1β) (40). Early studies demonstrated that enhanced cellular clearance of H2O2 by catalase abrogated PDGF and epidermal growth factor receptor signals, implicating H2O2 as a critical redox-signaling intermediate (3, 45). It is currently believed that H2O2 functions as a signaling intermediate by inhibiting cellular phosphatases at cysteines in the catalytic site (40) and by altering protein structure by oxidation of reactive thiols (15). For example, a recent study has implicated mitochondrial superoxide as a source of H2O2 responsible for the oxidative inactivation of JNK phosphatases important in TNF-mediated apoptosis (22). Similarly, peroxiredoxin II has been shown to act as a negative regulator of PDGF signaling by controlling the activity of protein tyrosine phosphatases important in PDGF receptor inactivation (7). In addition, cytosolic peroxiredoxin II attenuates the activation of JNK and p38 but potentiates ERK activation following TNF-α treatment (24). The major ROS-generating systems in cells include the mitochondria and seven known NADPH oxidase catalytic subunits (Nox1, Nox2gp91phox, Nox3, Nox4, Nox5, Duox1, and Duox2) (26). NADPH oxidases generate superoxide (·O2−) by transferring an electron from NADPH to molecular oxygen. The most widely characterized NADPH oxidases include the phagocytic gp91phox (Nox2), which has also been found to be expressed in a variety of other nonphagocytic cell types (36, 47, 50). The active form of the phagocytic NADPH oxidase is composed of a multisubunit membrane complex. Subunit recruitment of p67phox, p47phox, p40phox, p22phox, and Rac1/2, a small GTPase, plays an important role in activating ·O2− production for this complex (23, 26). Given the obvious potential toxicity of ROS, cells have also developed mechanisms to control both the production and clearance of ROS. Clearance of ROS is carefully regulated by a number of enzymes that dismutate ·O2− to H2O2 (SOD-1, -2, and -3) or degrade H2O2 (glutathione peroxidases, catalase, and peroxiredoxins) (13, 41). As mentioned above, peroxiredoxin II is recruited to the PDGF receptor and regulates its phosphorylation status. In this context, peroxiredoxin II controls the availability of H2O2 which, in turn, regulates the activity of protein tyrosine phosphatases (7). This mechanism is an example of ROS-mediated regulation of signal transduction by the clearance of H2O2. On the contrary, cellular mechanisms that direct the production of Nox-derived superoxide to selectively influence certain receptor signaling pathways remain poorly understood. A link between Nox protein activation and cellular signaling is only recently becoming recognized. For example, expression of a dominant negative Nox4 attenuates insulin-stimulated H2O2 production and downstream phosphatase signaling involved in adipocyte insulin receptor activation (32). TNF-α and IL-1β, two major proinflammatory cytokines well recognized for their abilities to activate NF-κB (16, 42), have also been hypothesized to utilize Nox-dependent H2O2 production in their activation cascades. Supportive evidence includes the demonstration that inhibition of Rac1 or p47phox abrogates TNF-α- and IL-1-stimulated H2O2 production and NF-κB activation (10, 14, 17, 21, 28). Similarly, enhanced clearance of H2O2 by GPx1 expression abrogates NF-κB activation (25, 29). However, the dependence of NF-κB activation on ROS still remains highly controversial (19). To this end, it is presently unclear how ligand binding to membrane receptors coordinates the production of ROS through NADPH oxidase(s) and downstream receptor signals that influence NF-κB activation. IL-1β is a potent proinflammatory cytokine that plays an important role in many disease processes. IL-1β exerts its pleiotropic effects by binding to the IL-1 receptor (IL-1R1) on the plasma membrane and initiating the formation of an intracellular receptor-associated protein complex responsible for transducing receptor signals (35). Formation of an active IL-1R1 signaling complex involves the early binding of several accessory factors to the receptor, including IL-1RacP (20), MyD88 (34, 49), and Tollip (5). These factors then act in a context-dependent fashion to mediate the recruitment of IL-1 receptor-associated kinases (IRAK) (30, 34). Once associated with the receptor complex, IRAK subsequently leads to the recruitment of TRAF6, a member of the TNF receptor-associated factor (TRAF) family of adaptor proteins (38). TRAF6 is required for the activation and the recruitment of kinases such as TAK1 and NIK to the IL-1R1 complex (35, 38, 48). In the context of NF-κB activation, the IL-1R1 complex transmits signals through TAK1- and/or NIK-mediated phosphorylation of the IκB kinase (IKK) complex (16). Once the IKK complex is phosphorylated, it in turn phosphorylates IκBα/β, and NF-κB is mobilized to the nucleus. In the present study, we have used the redox dependence of IL-1β-mediated NF-κB activation to study how Nox-derived ROS coordinate receptor signals required for efficient NF-κB activation. To this end, we have demonstrated that following IL-1β stimulation, MyD88-dependent endocytosis of IL-1R1 was required for the redox-dependent activation of NF-κB by the endosomal compartment. In the absence of endocytosis, the IL-1 receptor complex formation failed to incorporate TRAF6, and NF-κB activation was significantly abrogated. Similarly, clearance of ROS from within the endosomal compartment significantly retarded both IKK and NF-κB activation following IL-1β stimulation. ROS production by the endosomal compartment was facilitated by Rac1-dependent internalization of Nox2 with IL-1R1 and directed the H2O2-dependent recruitment of TRAF6 to ligand-activated IL-1R1/MyD88 complexes in endomembranes. Through this process, the generation of ·O2− by endosomal Nox2, and its conversion to H2O2, facilitated the redox-dependent formation of an IL-1R1 complex capable of activating the IKK complex and NF-κB. This mechanism helps clarify how cells can partition Nox-derived ROS to selectively influence receptor activation from the plasma membrane.