Nox proteins in signal transduction

Abstract

The NADPH oxidase (Nox) family of superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2))-producing proteins has emerged as an important source of reactive oxygen species (ROS) in signal transduction. ROS produced by Nox proteins Nox1-5 and Duox1/2 are now recognized to play essential roles in the physiology of the brain, the immune system, the vasculature, and the digestive tract as well as in hormone synthesis. Nox-derived ROS have been implicated in regulation of cytoskeletal remodeling, gene expression, proliferation, differentiation, migration, and cell death. These processes are tightly controlled and reversible. In this review, we will discuss recent literature on Nox protein tissue distribution, subcellular localization, activation, and the resulting signal transduction mechanisms.

Fig 1. Nox family members and their regulatory subunits

Although no 3-dimensional crystallization of Nox proteins has been performed, they are believed to contain six transmembrane domains based on hydrophobicity analysis (seven for Duox1/2). Oxidase activity occurs when NADPH binds to Nox on the cytosolic side, where it transfers electrons to FAD and the heme centers (not shown) and finally to oxygen on the outer membrane surface, resulting in O₂^•− formation. In Nox1-4, the transmembrane subunit p22phox associates with active and inactive Nox. It is believed to have between two and four transmembrane segments. Nox1 is believed to primarily interact with the cytosolic subunits NoxO1, NoxA1 and GTP-Rac upon activation; however p47phox and p67phox can replace NoxO1 and NoxA1, respectively. Nox2 activation involves association with GTP-Rac, p47phox, p67phox and p40phox. Nox3 activation is less well defined, but is believed to primarily involve GTP-Rac, p47phox and NoxA1 in the inner ear. Nox4 is constitutively active when associating with the cytosolic p22phox subunit. Nox5 and Duox1/2 activation involves Ca2⁺ binding to EF-hand domains in the cytosol. Duox1/2 require the association of DuoxA1/2, respectively, for localization to the plasma membrane.

Fig 2. Nox1 signal transduction pathways

Nox1 is localized to cavaolae in the plasma membrane and endosomes. TNF-α stimulates TNF receptor-1 (TNFR1), resulting in the recruitment of TRADD, RIP1, Rac and Nox1 to the receptor. The complex produces ROS that activate JNK to initiate necrosis. Other activators of Nox1 include Ang II, thrombin, and PDGF. H₂O₂ produced by Nox1 activation initiates hypertrophy by activating p38 MAPK, which associates with MAPKAPK2 and Akt. Nox1 also activates SSH1L, which activates cofilin by dephosphorylation to promote cell migration. In a parallel pathway, Nox1-derived ROS increase cSrc phosphorylation, which activates PDK1, followed by PAK1. Nox1 also stimulates growth by activating Ras and ERK1/2, which activate the transcription factor Ets-1 by phosphorylation. Ets-1 upregulates Cyclin D, promoting passage through the cell cycle.

Fig 3. Nox2 signal transduction pathways

Nox2 is localized to endosome and phagosome membranes. In necrosis, TNF-α activates TNFR1, which recruits TRADD to the receptor. TRAF2 then binds to TRADD in a Nox2-derived ROS dependent manner and activates IKK, leading to NFκB activation and necrosis. Nox2 in endosomes is also activated by thrombin, VEGF and angiopoietin-1. Nox2-derived ROS promote angiogenesis by activating VE-cadherin, Akt and cSrc. Nox2 acts in host defense in phagosomes by producing O₂^•−, which is dismutated to H₂O₂. The reaction of H₂O₂ with Cl⁻ is catalyzed by MPO to form HOCl, which is bacteriocidal.

Fig 4. Nox4 signal transduction pathways

Nox4 is constitutively active, but activity and/or expression can be increased by insulin binding to the InsR, Ang II activating the AT1R and TGF-β1 binding to TGF-βR. Nox4 is involved in the inhibition of insulin signaling by inhibiting the phosphatase PTP1B, which prolongs the phosphorylation of the insulin receptor. Nox4 promotes migration by activating MMP2. Nox4 promotes cell differentiation by multiple mechanisms. Nox4 derived-ROS activate p38 MAP kinase, which phosphorylates and activates MEF2C to promote differentiation. In addition, H₂O₂ produced by Nox4 activates MKP-1, which inhibits the activation of ERK1/2. Since ERK1/2 normally promotes growth, its inhibition may allow for differentiation to occur. Nox4 also promotes growth and survival by several pathways. Nox4 derived ROS inhibit LMW-PTP, which prolongs the phosphorylation of JAK2 to promote growth. Nox4 also promotes phosphorylation of pRb and elF4E to promote growth and hypertrophy.

Fig 5. Nox5 signal transduction pathways

Nox5 is activated by PKC, IP₃ and Ca²⁺ produced by PDGFR activation and cytokine receptor activation (IL-4/IL-4R). Nox5-derived ROS can increase inflammatory gene expression by activating NFκB and through positive feedback of the IL-4R by inhibiting PTP1B. H₂O₂ activates ROS production by Nox5 via an association between Nox5 and c-Abl. Nox5-derived ROS activate growth and proliferation by phosphorylating JAK2, which phosphorylates STAT3 to promote proliferation.