iNOS (NOS2) at a glance

Abstract

Nitric oxide (NO) is an effector of the innate immune system. The innate immune system is a set of rapid host responses to pathogens. Cells of the innate immune system - macrophages, neutrophils and natural killer cells - use pattern recognition receptors to recognize molecular patterns associated with pathogens (Medzhitov, 2001). Activated macrophages then inhibit pathogen replication by releasing a variety of effector molecules, including NO.Resting immune cells lack the inducible NO synthase (iNOS or NOS2), the enzyme that synthesizes NO. However, a variety of extracellular stimuli can activate distinct signaling pathways that converge to initiate expression of NOS2. Cell wall components of bacteria and fungi can trigger the innate immune signaling cascade, leading to expression of NOS2. For example, lipopolysaccharide (LPS), a component of the wall of Gram-negative bacteria, can bind to LPS-binding protein (LBP), which delivers LPS to CD14, a high-affinity LPS receptor. Toll-like receptor 4 (TLR4) in conjunction with the small extracellular protein MD-2 interacts with the CD14-LPS complex, and then activates an intracellular signaling cascade via adaptors that include IRAK and MyD88, which in turn activate downstream molecules including TRAF6. The innate immune pathway then activates signaling pathways including the mitogen-activated protein kinase (MAPK) pathway and the nuclear factor κB (NF-κB) pathway. These pathways converge to activate NOS2 transcription.FIG1Cytokines released from infected host cells can also activate NO production, including tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Interferon γ (IFN-γ) produced by activated immune cells can activate NOS2 expression, and can synergistically activate NOS2 expression in combination with other agents such as LPS. IFN-γ interacts with the interferon receptor 1 (IFNR1) and IFNR2 complex, which activates kinases of the Jak family and signal transducers and activators of transcription (STAT) pathways (Darnell et al., 1994). The interferon signaling pathway can also activate NOS2 transcription.LPS-mediated activation of the innate immune pattern recognition receptors stimulates NOS2 mRNA transcription within 2-4 hours, and NOS2 translation within 6 hours. LPS activation of TLR4 leads to phosphorylation of inhibitor of NF-κB kinase (IKK), which phosphorylates the inhibitor of NF-κB (IκB), which releases the transcription factor NF-κB. NF-κB translocates from the cytoplasm to the nucleus, where it interacts with κB elements in the NOS2 5′ flanking region, triggering NOS2 transcription (Xie et al., 1994). IFN-γ triggers NOS2 transcription by activating the JAK/STAT pathway, leading to synthesis of the transcription factor interferon response factor 1 (IRF-1), which stimulates NOS2 transcription (Kamijo et al., 1994). IFN-γ also provides a synergistic boost to LPS induction of NOS2 transcription because IRF-1 interacts with NF-κB, altering the conformation of the NOS2 promoter (Saura et al., 1999a). Scaffolding proteins such as HMG-Y(I) and transcriptional activators such as CBP interact with IRF-1 and NF-κB, forming a multi-subunit complex that increases NOS2 transcription (Perrella et al., 1999). Other transcription factors, including Stat1α and hypoxia inducible factor-1 (HIF-1), can also regulate NOS2 expression.The 3′ untranslated region of NOS2 mRNA contains multiple AUUUA elements, which mediate stability of mRNAs of cytokines. The RNA-binding protein HuR interacts stabilizing NOS2 mRNA with the AUUUA elements of the NOS2 mRNA, (Rodriguez-Pascual et al., 2000). Lack of HuR leads to destabilization of NOS2 mRNA and decreased NOS2 expression.NOS2 does not synthesize NO until it forms homodimers. A set of proteins can inhibit NOS2 activity by preventing NOS2 homodimerization. For example, NOS-associated protein 110 kDa (NAP110) interacts with the N-terminus of NOS2, forming NOS2-NAP110 heterodimers, and blocking the formation of NOS2-NOS2 dimers (Ratovitski et al., 1999). Although NOS2 is ubiquitylated and phosphorylated, the significance of these post-translational modifications is unknown.Transforming growth factor β (TGF-β) inhibits NOS2 expression through transcriptional, post-transcriptional and post-translational mechanisms (Vodovotz et al., 1993). TGF-β inhibits NOS2 transcription in part by blocking expression of the scaffolding protein HMG-I(Y). A variety of other signaling molecules, including IL-4, IL-10, IL-13 and macrophage deactivation factor, inhibit NOS2 expression by unknown mechanisms.NOS2 contains a C-terminal reductase domain, which binds NADPH and transfers electrons from NADPH to FAD and then to FMN, and an N-terminal oxygenase domain, which contains a heme and binding sites for arginine, tetrahydrobiopterin and calmodulin. NOS2 utilizes oxygen and electrons from NADPH to oxidize the substrate L-arginine into the intermediate OH-L-arginine, which is then oxidized into NO and L-citrulline.In contrast to the other NO