* 163730

NITRIC OXIDE SYNTHASE 2; NOS2


Alternative titles; symbols

NOS2A
NOS2A, INDUCIBLE, HEPATOCYTE
NITRIC OXIDE SYNTHASE, INDUCIBLE; INOS
NITRIC OXIDE SYNTHASE, MACROPHAGE


HGNC Approved Gene Symbol: NOS2

Cytogenetic location: 17q11.2     Genomic coordinates (GRCh38): 17:27,756,766-27,800,529 (from NCBI)


Gene-Phenotype Relationships
Location Phenotype Phenotype
MIM number
Inheritance Phenotype
mapping key
17q11.2 {Malaria, resistance to} 611162 3

TEXT

Description

Nitric oxide (NO) is a messenger molecule with diverse functions throughout the body. In the brain and peripheral nervous system, NO displays many properties of a neurotransmitter; it is implicated in neurotoxicity associated with stroke and neurodegenerative diseases, neural regulation of smooth muscle, including peristalsis, and penile erection. NO is also responsible for endothelium-derived relaxing factor (EDRF) activity regulating blood pressure. In macrophages, NO mediates tumoricidal and bactericidal actions, as indicated by the fact that inhibitors of NO synthase (NOS) block these effects. Neuronal NOS (163731) and macrophage NOS are distinct isoforms (Lowenstein et al., 1992). Both the neuronal and the macrophage forms are unusual among oxidative enzymes in requiring several electron donors: FAD, FMN, NADPH, and tetrahydrobiopterin.


Cloning and Expression

Lowenstein et al. (1992) cloned cDNA for the macrophage form of NOS and expressed the enzyme in human kidney cells. Macrophage enzyme showed 50% sequence identity to the neuronal enzyme. Like the neuronal form, NOS2 has recognition sites for FAD, FMN, and NADPH, and also has a consensus calmodulin binding site. Macrophage NOS mRNA was found to be strikingly inducible; it was absent in quiescent macrophages or spleen but prominent 2 to 6 hours after endotoxin treatment.

Nitric oxide is synthesized from L-arginine by the action of nitric oxide synthase(s), generating citrulline as a coproduct. The NO synthases, P450-type heme proteins, are all NADPH-, FMN-, and tetrahydrobiopterin-dependent. In endothelial cells and neuronal tissues, NO synthase activity is constitutively expressed and has a requirement for Ca(2+) and calmodulin. In contrast, NO synthase is inducible in macrophages and some other tissues. The inducible NO synthase in these cells does not require exogenous Ca(2+) or calmodulin for activity, and induction is inhibited by glucocorticoids. NO synthase is inducible in articular chondrocytes. Interleukin-1 beta (IL1B; 147720) induces the enzyme in human chondrocytes where induction is Ca(2+)-independent and marginally affected by glucocorticoids. Charles et al. (1993) isolated a cDNA clone that encoded a protein of 1,153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. The deduced amino acid sequence of the human chondrocyte inducible NO synthase showed 51% identity and 68% similarity with the endothelial NO synthase (163729) and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species. The induction of NO synthase in human cells had previously been shown only for hepatocytes (Nussler et al., 1992). Geller et al. (1993) cloned cDNA for an inducible NO synthase from human hepatocytes. The sequence differed from the human chondrocyte sequence at 7 amino acid positions.

Using a bovine NOS II-related cDNA to screen 2 human genomic libraries, Bloch et al. (1995) isolated clones containing 3 independent genes. One clone encoded the previously identified gene previously called NOS2 and called by them NOS2A. The 2 other genes specified amino acids homologous, but not identical, to those specified by the NOS2A gene; these were designated NOS2B (600719) and NOS2C (600720).


Gene Structure

Using both murine macrophage and human hepatocyte inducible NOS cDNAs as probes, Chartrain et al. (1994) isolated overlapping cosmid clones for the NOS2 gene from a human genomic library. The gene was estimated to be 37 kb long and consisted of 26 exons and 25 introns. They mapped the transcriptional initiation site 30 basepairs downstream of a TATA sequence. Xu et al. (1996) concluded that in fact the NOS2 open reading frame is encoded by 27 exons, with translation initiation and termination in exon 2 and exon 27, respectively.


Mapping

By Southern analysis of somatic cell hybrid lines, Marsden et al. (1994) mapped the NOS2 gene to chromosome 17; they refined the assignment to 17q11.2-q12 by a fluorescence in situ hybridization. Jenkins et al. (1994), who referred to the inducible NO synthase of mouse macrophages as Nos-1, found that the gene maps to mouse chromosome 11 in a region homologous to human chromosome 17q. They predicted that the human gene might lie in the region 17q11.2. Mehrabian et al. (1994) likewise mapped the mouse homolog to chromosome 11, using RFLVs in linkage analysis in an interspecific backcross. Chartrain et al. (1994) used polymerase chain reaction analysis of a human/rodent genomic DNA somatic cell hybrid panel and fluorescence in situ hybridization to map the NOS2 gene to 17cen-q11.2. Xu et al. (1994) mapped the NOS2 gene to 17cen-q11 by Southern blotting analysis of DNAs obtained from a panel of human/rodent hybrid cell lines. By fluorescence in situ hybridization, they found signals in the 17p11-q11 pericentromeric region. Gerling et al. (1994) mapped the Nos2 gene to mouse chromosome 11.

Using Southern blot hybridization, Bloch et al. (1995) demonstrated that the NOS2A, NOS2B, and NOS2C genes are all located on chromosome 17 between bands 17p13.1 and 17q25.


Gene Function

Napolitano et al. (2000) investigated the interactions between ET1 (131240) and the NO system in the fetoplacental unit. They examined the mRNA expression of ET1, inducible NOS (iNOS), and endothelial NOS (eNOS; 163729) in human cultured placental trophoblastic cells obtained from preeclamptic (PE; 189800) and normotensive pregnancies. ET1 expression was increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, was decreased; conversely, eNOS expression was increased. ET1 was able to influence its own expression as well as NOS isoform expression in normal and PE trophoblastic cultured cells. The findings suggested the existence of a functional relationship between ET(s) and NOS isoforms that could constitute the biologic mechanism leading to the reduced placental blood flow and increased resistance to flow in the fetomaternal circulation that are characteristic of the pathophysiology of preeclampsia.

The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (see 161560), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL12 production by macrophages and the mechanisms by which they do so remain undefined. Brightbill et al. (1999) demonstrated that microbial lipoproteins are potent stimulators of IL12 production by human macrophages and that induction is mediated by toll-like receptors (TLRs; see 603030). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.

Diefenbach et al. (1999) studied the relationship of IL12 and nitric oxide synthase-2 (NOS2) to innate immunity to the parasite Leishmania in mice. In the absence of NOS2 activity, IL12 was unable to prevent spreading of Leishmania parasites, did not stimulate natural killer cells for cytotoxicity or interferon (IFN)-gamma (147570) release, and failed to activate TYK2 (176941) and to tyrosine-phosphorylate STAT4 (600558), the central signal transducer of IL12, in NK cells. Activation of TYK2 in NK cells by IFN-alpha/beta (type I interferon; see 107470) also required NOS2. Thus, NOS2-derived NO is a prerequisite for cytokine signaling and function in innate immunity.

Exposure of human pancreatic islets to a mixture of cytokines induces expression of iNOS, impairs beta-cell function, and induces apoptosis. Johannesen et al. (2001) scanned all 27 exons of the human NOS2 gene and carried out linkage transmission disequilibrium testing of the identified NOS2 polymorphisms in a Danish nationwide IDDM (222100) family collection. Transmission disequilibrium testing was performed using 257 Danish IDDM families; 154 families were affected sib pair families, and 103 families were simplex families. In total, 10 polymorphisms were identified in 8 exons, of which 4 were tested in the family material. A C/T single-nucleotide polymorphism in exon 16 resulting in an amino acid substitution, ser608 to leu, showed linkage to IDDM in human leukocyte antigen DR3/4-positive affected offspring (P = 0.008; corrected P = 0.024). No other distorted transmission patterns were found for any other tested single-nucleotide polymorphism or constructed haplotypes with the exception of those including data from exon 16. The authors concluded that linkage of the human NOS2 gene to IDDM in a subset of patients supports a pathogenic role of nitric oxide in human IDDM.

In a review of reactive oxygen and nitrogen intermediates, Nathan and Shiloh (2000) noted that infections, microbial products, and cytokines do not consistently induce expression of NOS2 when applied to human blood mononuclear phagocytes, although these stimuli do induce production in rodent tissue macrophages. The authors stressed the importance of evaluating tissue rather than blood macrophages for human NOS2 expression.

Vouldoukis et al. (1995) found that ligation of the low affinity IgE receptor (CD23; 151445) induces NOS production by human monocyte-derived macrophages in vitro. CD23 ligation led to the intracellular killing of Leishmania major parasites, which could be blocked by the NOS inhibitor, NG-monomethyl-L-arginine monoacetate (NMMA).

Nicholson et al. (1996) reported that lung macrophages from tuberculosis (TB) patients express NOS2 in potentially mycobactericidal amounts.

Nozaki et al. (1997) demonstrated by RT-PCR and immunofluorescence analyses that alveolar macrophages (AMs) obtained from pulmonary fibrosis patients, but not from lung cancer patients, infected with the avirulent vaccine strain Mycobacterium bovis (BCG) produced higher levels of iNOS. Colony forming assays showed that these infected AMs effectively killed BCG, but they were less able to do so after treatment with NMMA.

Using immunoblot and immunohistochemical analyses, Facchetti et al. (1999) showed expression of a 130-kD NOS2 protein in the cytoplasm of CD68 (153634)-positive macrophages in infectious granuloma tissues, as well as in sarcoidosis (181000) and Kikuchi disease, but not in foreign body granulomas or Omenn syndrome (603554).

Choi et al. (2002) examined resected lungs from 8 TB patients for the expression of NOS and nitrotyrosine, a marker of NO expression. Immunohistochemical and morphometric analyses revealed that iNOS, nitrotyrosine, eNOS, and TNFA (191160), but not nNOS (NOS1; 163731), were expressed in CD68-positive epithelioid macrophages and giant cells in the inflammatory zone of the granulomas of the patients, but not in histologically normal tissue obtained from cancer patient control subjects. TNF expression was highest in necrotic areas.

The 26S proteasome is the major pathway responsible for iNOS degradation. Targeting proteins for proteasomal degradation may require their covalent linkage to multiubiquitin chains (i.e., ubiquitination). Kolodziejski et al. (2002) reported results of experiments indicating that iNOS is subject to ubiquitination, which is required for its degradation.

NO generated from inducible NO synthase has been implicated in migraine (157300) based on pharmacologic evidence in animals and humans. In a rat model, Reuter et al. (2002) showed that the NO donor glyceryl trinitrate (GTN) caused NOS2A expression in macrophages, mediated by increased activity of the nuclear transcription factor kappa-B (NFKB1; 164011), resulting in generation of NO within rodent dura mater 6 hours later. Parthenolide, a lactone found in the medical herb 'feverfew' which has been used successfully in the treatment of inflammatory conditions and migraine, blocked NOS2A expression in dura mater by inhibiting NFKB1.

Kim et al. (2005) showed that iNOS specifically binds to cyclooxygenase-2 (COX2; 600262) and S-nitrosylates it, enhancing COX2 catalytic activity. Selectively disrupting iNOS-COX2 binding prevented NO-mediated activation of COX2. Kim et al. (2005) suggested that the molecular synergism between iNOS and COX2 may represent a major mechanism of inflammatory responses.

Tezuka et al. (2007) showed that IgA class switch recombination is impaired in inducible nitric oxide synthase-deficient (iNOS-null) mice. iNOS regulates the T-cell-dependent IgA class switch recombination through expression of transforming growth factor-beta receptor (see TGFBR1, 190181), and the T-cell-independent IgA class switch recombination through production of a proliferation-inducing ligand (APRIL, also called TNFSF13; 604472) and a B-cell-activating factor of the tumor necrosis factor family (BAFF; 603969). Notably, iNOS is preferentially expressed in mucosa-associated lymphoid tissue (MALT) dendritic cells in response to the recognition of commensal bacteria by toll-like receptor (TLR; see 603030). Furthermore, adoptive transfer of iNOS-positive dendritic cells rescued IgA production in iNOS-null mice. Further analysis revealed that the MALT dendritic cells are a TNFA/iNOS-producing dendritic-cell subset, originally identified in mice infected with Listeria monocytogenes. The presence of a naturally occurring TNFA/iNOS-producing dendritic cell subset may explain the predominance of IgA production in the MALT, critical for gut homeostasis.

Eyler et al. (2011) found that highly tumorigenic human glioma stem cells (GSCs), but not normal neural progenitor cells, produced elevated NO via upregulated NOS2 expression. Consumption of NO in GSCs by lentivirus-introduced bacterial flavohemoglobin, or knockdown of NOS2 via short hairpin RNA, abrogated GSC growth, neurosphere formation, and tumorigenicity. Knockdown of NOS2 in normal neural progenitor cells had little effect. Quantitative RT-PCR detected elevated NOS2 expression in GSCs from 3 different primary gliomas and a xenograft. Microarray analysis of NOS2-knockdown GSCs revealed upregulation of several genes, including the cell cycle inhibitor CDA1 (TSPYL2; 300564). Transfection of NOS2 in HEK293 cells inhibited CDA1 mRNA and protein expression, and RT-PCR of several glioma xenografts confirmed an inverse relationship between NOS2 and CDA1 expression. Pharmacologic inhibition of NOS2 in mice slowed the growth of human glioma xenografts and inhibited tumor angiogenesis. Eyler et al. (2011) concluded that NO production in GSCs due to NOS2 upregulation contributes primarily to GSC tumor maintenance.

Nairz et al. (2013) found that macrophages from mice lacking Nos2 displayed reduced expression of ferroportin-1 (FPN1, or SLC40A1; 604653). Nitric oxide upregulated FPN1 expression in mouse and human cells. Nos2-null mouse macrophages had increased iron content due to reduced Fpn1 activity. Reduced Fpn1 expression allowed enhanced iron acquisition by the intracellular bacterium Salmonella typhimurium. Mice lacking Nos2 or mice in which Nos2 activity was inhibited had increased iron accumulation in spleen and spleen macrophages. Lack of nitric oxide formation resulted in impaired Nrf2 (NFE2L2; 600492) expression and, consequently, reduced Fpn1 transcription and cellular iron export. Infection of Nos2-null mice or macrophages with S. typhimurium led not only to increased iron accumulation, but also to reduced Tnf, Il2 (147680), and Ifng expression and impaired pathogen control, all of which could be restored by treatment with iron chelators or overexpression of Fpn1 or Nrf2. Nairz et al. (2013) concluded that iron accumulation in Nos2-null macrophages counteracts a proinflammatory host response and that the protective effects of nitric oxide partially result from its ability to prevent iron overload in macrophages.

Yang et al. (2015) showed that, in the setting of obesity, inflammatory input through increased iNOS activity causes S-nitrosylation of a key unfolded protein response (UPR) regulator, IRE1-alpha (604033), which leads to a progressive decline in hepatic IRE1-alpha-mediated XBP1 (194355) splicing activity in both genetic (ob/ob) and dietary (high-fat diet-induced) models of obesity. Finally, in obese mice with liver-specific IRE1-alpha deficiency, reconstitution of IRE1-alpha expression with a nitrosylation-resistant variant restored IRE1-alpha-mediated XBP1 splicing and improved glucose homeostasis in vivo. Yang et al. (2015) concluded that their data described a mechanism by which inflammatory pathways compromise UPR function through iNOS-mediated S-nitrosylation of IRE1-alpha, which contributes to defective IRE1-alpha activity, impaired ER function, and prolonged ER stress in obesity.

By treating mouse bone marrow-derived macrophages (BMDMs) with IFN-gamma followed by the TLR4 (603030) agonist lipopolysaccharide (LPS), Simpson et al. (2022) found that IFN-gamma activated macrophages and triggered cell death via TLR signaling and Fasl (FASLG; 134638) expression. Knockout analysis revealed that efficient IFN-gamma/LPS-induced cell death required caspase-8 (CASP8; 601763) and the mitochondrial apoptosis effector proteins Bax (600040) and Bak (BAK1; 600516). Activation of Bax and Bak was not triggered by caspase-8 cleavage of its substrate Bid (601997). Instead, caspase-8 mediated transcriptional programming in macrophages to increase proapoptotic Noxa (604959) and reduce prosurvival Bcl2 (151430), thereby reducing prosurvival proteins Mcl1 (159552) and A1 to facilitate Bax/Bak activation and subsequent apoptotic cell death upon stimulation with IFN-gamma and LPS. Caspase-8 enzymatic activity was required for IFN-gamma/LPS-mediated activation of Bax/Bak and subsequent apoptotic cell death. Bax/Bak activation resulted in irreversible damage to mitochondria and caused cell death even when the functions of other downstream caspases were eliminated. Treatment with IFN-gamma/LPS induced robust expression of iNos and generation of nitric oxide in macrophages, upstream of Bax/Bak activation and cell death. However, toxicity of nitric oxide was not the direct cause of cell death. Instead, iNos expression played a role in reducing Mcl1 and A1 to sensitize macrophages for Bax/Bak activation and mitochondrial apoptosis. In agreement, both iNos and caspase-8 contributed to disease severity of SARS-CoV-2 infection in mice, as deletion of iNos or caspase-8 limited SARS-CoV-2-induced disease, whereas caspase-8 caused lethality through hemophagocytic lymphohistiocytosis independently of iNos.


Molecular Genetics

Resistance to Malaria

Kun et al. (1998) examined whether high plasma concentrations of NO found in severe malaria (see 611162) were due to variation in the promoter region of NOS2. Heterozygosity for a -969G-C SNP (163730.0002) was present in 30 of 100 Gambian children with mild malaria, but in only 17 of 100 Gambian children with severe malaria. The SNP was not found in any of 100 Germans. Heterozygous individuals were also at a significantly lower risk of reinfection.

Hobbs et al. (2002) postulated that NOS2 promoter polymorphisms could affect resistance to severe malaria as manifested by cerebral malaria, severe malarial anemia, and respiratory distress or metabolic acidosis (Marsh et al., 1995). From studies in Tanzania and Kenya, Hobbs et al. (2002) identified a novel single-nucleotide polymorphism, -1173C-T (163730.0001), in the NOS2 promoter that was significantly associated with protection from symptomatic malaria and severe malarial anemia.

Associations Pending Confirmation

Rutherford et al. (2001) provided evidence for the location of at least 1 hypertension susceptibility locus on chromosome 17. Analysis of 177 affected sib pairs gave evidence for significant excess allele sharing to marker D17S949 on chromosome 17q22-q24, with significant allele sharing also indicated for an additional marker, D17S799, located close to the centromere. Since these 2 genomic regions are well separated, the results indicated that there may be more than 1 chromosome 17 locus affecting blood pressure. Moreover, further investigation using a polymorphism within the promoter of the NOS2A candidate gene revealed both increased allele sharing among sib pairs and positive association of NOS2A to essential hypertension. Morris (2002) called into question the conclusions of Rutherford et al. (2001); Griffiths (2002), the senior author of the paper by Rutherford et al. (2001), responded.

Hao et al. (2004) conducted a large-scale case-control study exploring the associations of 426 single-nucleotide polymorphisms (SNPs) with preterm delivery (PTD) in 300 mothers with PTD and 458 mothers with term deliveries. Twenty-five candidate genes were included in the final haplotype analysis. Gene haplotypes at IL1R2 (147811) in blacks, NOS2A in whites, and OPRM1 (600018) in Hispanics were only associated with PTD in these specific ethnic groups.

In a subset of 73 families with Parkinson disease (PD; 168600) in which at least 1 member had disease onset before age 40 years, Hancock et al. (2006) found a significant association between PD and 2 SNPs in the NOS2A gene: the more frequent T allele of rs2255929 and the less frequent A allele of rs1060826 (p = 0.000059 and 0.0062, respectively). The 2-SNP haplotype showed an even stronger association with PD (p = 0.000013). Similar associations were not observed in a larger group of 286 multiplex families including all ages at onset. Epidemiologic studies have consistently reported an inverse association between Parkinson disease and cigarette smoking. In an overlapping group of 243 families, Hancock et al. (2006) found a significant association between cigarette smoking and the 2 risk alleles; however, when stratified by PD status, the presence of these alleles attenuated the protective inverse association between smoking and PD. Hancock et al. (2006) postulated that an alteration in NOS2A regulation could result in prolonged activity and cellular damage, and further concluded that NOS2A may be a risk factor in PD by influencing age at onset and by modifying the inverse association between PD and smoking.

Among 340 German PD patients with disease onset at a median of 52 years, Schulte et al. (2006) found no association between the disorder and 12 polymorphisms in the NOS2A gene.

Eumycetoma is a tumorous fungal infection, typically of the hands or feet, characterized by the infiltration of large numbers of neutrophils. It is caused by Madurella mycetomatis, a pathogen that is abundant in the soil and on the vegetation of Sudan, where the disease is common. Van de Sande et al. (2007) noted that ELISA has shown near universal IgG seropositivity in mycetoma patients and controls from endemic areas, but no seropositivity in European controls, implying that most individuals in endemic areas are exposed to the pathogen, but only a small percentage develop disease. Van de Sande et al. (2007) studied 11 SNPs in genes involved in neutrophil function in 125 Sudanese mycetoma patients and 140 ethnically and geographically matched controls and found significant differences in allele distributions for SNPs in IL8 (146930), IL8RB (146928), TSP4 (THBS4; 600715), NOS2, and CR1 (120620). Serum IL8 was significantly higher in patients compared with controls, while nitrite/nitrate levels were lower in patients and seemed to be associated with delayed wound healing. Van de Sande et al. (2007) concluded that there is a genetic predisposition toward susceptibility to mycetoma.

Using a mixed case-control association analysis of 279 African American and 198 Caucasian tuberculosis (TB; see 607948) patients and 166 African American and 123 Caucasian controls, Velez et al. (2009) identified 10 SNPs in NOS2A that were associated with TB in African Americans but not Caucasians. Additionally, they identified gene-gene interactions between SNPs in NOS2A and IFNGR1 (107470) and TLR4 (603030). Velez et al. (2009) proposed that NOS2A variants may contribute to TB susceptibility in individuals of African descent and that these variants may act synergistically with SNPs in IFNGR1 and TLR4.

Drutman et al. (2020) reported a previously healthy 51-year-old man from Iran who after acute cytomegalovirus (CMV) infection had onset of progressive CMV disease that led to his death 29 months later. Whole-exome sequencing identified homozygosity for a frameshift variant in NOS2 (c.1436_1437insT; 163730.0003). Homozygosity for this variant segregated with the phenotype in the family; only the proband was homozygous. Drutman et al. (2020) cited Noda et al. (2001), who showed that mice deficient in NOS2 were highly vulnerable to the related murine CMV. The patient had titers against diphtheria and tetanus toxins, varicella, and various common viruses. The mechanistic connection between NOS2 deficiency and the patient's CMV phenotype required further investigation, although the authors favored the hypothesis that NOS2 expression in epithelial cells that are infected by CMV is required for control of the infection.


Animal Model

Noda et al. (2001) found that Nos2 -/- mice were more susceptible to lethal murine CMV (MCMV) infection than wildtype mice. Plaque assays showed that virus titer was markedly elevated in MCMV-infected Nos2 -/- mice, especially in salivary gland. Immune responses to MCMV infection were comparable with those of wildtype mice, as NK cell cytotoxicity, cytotoxic T-lymphocyte response, and IFN-gamma production after acute infection were not impaired. However, macrophages from Nos2 -/- mice exhibited lower antiviral activity, resulting in enhanced viral replication. In addition, absence of Nos2 prolonged MCMV-DNA latency after acute infection.

In immunodeficient mice inoculated with human peripheral blood mononuclear cells, Koh et al. (2004) examined transplanted human arteries for endothelial cell and vascular smooth muscle cell dysfunction. Within 7 to 9 days, transplanted arteries developed endothelial cell dysfunction but remained sensitive to exogenous NO. By 2 weeks, the grafts developed signs of vascular smooth muscle cell dysfunction, including impaired contractility and desensitization to NO. These T-cell dependent changes correlated with loss of endothelial NOS and expression of inducible NOS. Neutralizing IFN-gamma completely prevented both vascular dysfunction and changes in NOS expression; neutralizing TNF reduced IFN-gamma production and partially prevented dysfunction. Inhibiting iNOS partially preserved responses to NO at 2 weeks and reduced graft intimal expansion after 4 weeks in vivo. Koh et al. (2004) concluded that IFN-gamma is a central mediator of vascular dysfunction through dysregulation of NO production.

Colton et al. (2006) found that transgenic mice expressing mutant amyloid precursor protein (APP; 104760) on a Nos2-null background developed pathologic hyperphosphorylation of tau (MAPT; 157140) with aggregate formation in the brain. Lack of Nos2 increased insoluble APP levels, neuronal degeneration, caspase-3 (CASP3; 600636) activation, and tau cleavage, suggesting that nitric oxide may act at a junction point between the 2 main pathologies that characterize Alzheimer disease (AD; 104300).

Eyler et al. (2011) found that knockout of Nos2 in mice had no effect on neural development.

Mishra et al. (2013) infected Nos2 -/- mice with a strain of M. tuberculosis whose growth could be controlled exogenously. Using these mice, they found that Ifng and NO suppressed both bacterial growth in vivo and the continual production of Il1b by the Nlrp3 (606416) inflammasome, thereby inhibiting persistent neutrophil recruitment and preventing tissue damage. Mishra et al. (2013) concluded that NO has a dual role in promoting resistance to M. tuberculosis and in regulating inflammation, both of which are required for survival of this chronic infection.


ALLELIC VARIANTS ( 3 Selected Examples):

.0001 MALARIA, RESISTANCE TO

NOS2A, -1173C-T
  
RCV000015051

From studies in Tanzania and Kenya, Hobbs et al. (2002) identified a novel single-nucleotide polymorphism, -1173C-T, in the NOS2A gene that was significantly associated with protection from symptomatic malaria and severe malarial anemia (see 611162). The -1173C-T polymorphism was associated with increased fasting urine and plasma NO metabolite concentrations in Tanzanian children, suggesting that the polymorphism was functional in vivo.


.0002 MALARIA, SEVERE, RESISTANCE TO

NOS2A, -969G-C
  
RCV000015052

Kun et al. (1998) found that a SNP in the promoter region of NOS2A, -969G-C, was present significantly more often in Gambian children with mild malaria than in children with severe malaria (see 611192).


.0003 VARIANT OF UNKNOWN SIGNIFICANCE

NOS2A, 1-BP INS, 1436T
  
RCV002252323

This variant is classified as a variant of unknown significance because its contribution to fatal cytomegalovirus (CMV) infection has not been confirmed.

In a previously healthy 51-year-old man from Iran who after acute CMV infection had onset of progressive CMV disease that led to his death 29 months later, Drutman et al. (2020) reported homozygosity for a 1-bp insertion (c.1436_1437insT) in the NOS2A gene, predicted to result in a frameshift and a premature termination codon (Ile391IlefsTer26, I391fs). The truncated NOS2 protein was predicted to lack the entire C-terminal reductase domain required for the formation of nitric oxide, and thus be nonfunctional. NOS2 is intolerant to homozygosity for loss-of-function variants, and the I391fs variant was not found in 5,000 in-house controls, gnomAD, or in the Greater Middle Eastern (GME) Variome Database. Overexpression of the mutant protein in HEK293T cells resulted in no detectable nitric oxide production.


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  15. Hao, K., Wang, X., Niu, T., Xu, X., Li, A., Chang, W., Wang, L., Li, G., Laird, N., Xu., X. A candidate gene association study on preterm delivery: application of high-throughput genotyping technology and advanced statistical methods. Hum. Molec. Genet. 13: 683-691, 2004. [PubMed: 14976157, related citations] [Full Text]

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  18. Johannesen, J., Pie, A., Pociot, F., Kristiansen, O. P., Karlsen, A. E., Nerup, J., The Danish Study Group of Diabetes in Childhood, The Danish Insulin-Dependent Diabetes Mellitus Epidemiology and Genetics Group. Linkage of the human inducible nitric oxide synthase gene to type 1 diabetes. J. Clin. Endocr. Metab. 86: 2792-2796, 2001. [PubMed: 11397889, related citations] [Full Text]

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  20. Koh, K. P., Wang, Y., Yi, T., Shiao, S. L., Lorber, M. I., Sessa, W. C., Tellides, G., Pober, J. S. T cell-mediated vascular dysfunction of human allografts results from IFN-gamma dysregulation of NO synthase. J. Clin. Invest. 114: 846-856, 2004. [PubMed: 15372109, images, related citations] [Full Text]

  21. Kolodziejski, P. J., Musial, A., Koo, J.-S., Eissa, N. T. Ubiquitination of inducible nitric oxide synthase is required for its degradation. Proc. Nat. Acad. Sci. 99: 12315-12320, 2002. [PubMed: 12221289, images, related citations] [Full Text]

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  23. Lowenstein, C. J., Glatt, C. S., Bredt, D. S., Snyder, S. H. Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. Proc. Nat. Acad. Sci. 89: 6711-6715, 1992. [PubMed: 1379716, related citations] [Full Text]

  24. Marsden, P. A., Heng, H. H. Q., Duff, C. L., Shi, X.-M., Tsui, L.-C., Hall, A. V. Localization of the human gene for inducible nitric oxide synthase (NOS2) to chromosome 17q11.2-q12. Genomics 19: 183-185, 1994. [PubMed: 7514565, related citations] [Full Text]

  25. Marsh, K., Forster, D., Waruiru, C., Mwangi, I., Winstanley, M., Marsh, V., Newton, C., Winstanley, P., Warn, P., Peshu, N., Pasvol, G., Snow, R. Indicators of life-threatening malaria in African children. New Eng. J. Med. 332: 1399-1404, 1995. [PubMed: 7723795, related citations] [Full Text]

  26. Mehrabian, M., Xia, Y.-R., Wen, P.-Z., Warden, C. H., Herschman, H. R., Lusis, A. J. Localization of murine macrophage inducible nitric oxide synthase to mouse chromosome 11. Genomics 22: 646-647, 1994. [PubMed: 7528168, related citations] [Full Text]

  27. Mishra, B. B., Rathinam, V. A. K., Martens, G. W., Martinot, A. J., Kornfeld, H., Fitzgerald, K. A., Sassetti, C. M. Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL-1-beta. Nature Immun. 14: 52-60, 2013. [PubMed: 23160153, images, related citations] [Full Text]

  28. Morris, B. J. Critique of 'Chromosome 17 and the inducible nitric oxide synthase gene in human essential hypertension' by Rutherford et al., Human Genetics, published online September 2001. (Letter) Hum. Genet. 110: 98-99, 2002. [PubMed: 11810304, related citations] [Full Text]

  29. Nairz, M., Schleicher, U., Schroll, A., Sonnweber, T., Theurl, I., Ludwiczek, S., Talasz, H., Brandacher, G., Moser, P. L., Muckenthaler, M. U., Fang, F. C., Bogdan, C., Weiss, G. Nitric oxide-mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection. J. Exp. Med. 210: 855-873, 2013. [PubMed: 23630227, images, related citations] [Full Text]

  30. Napolitano, M., Miceli, F., Calce, A., Vacca, A., Gulino, A., Apa, R., Lanzone, A. Expression and relationship between endothelin-1 messenger ribonucleic acid (mRNA) and inducible/endothelial nitric oxide synthase mRNA isoforms from normal and preeclamptic placentas. J. Clin. Endocr. Metab. 85: 2318-2323, 2000. [PubMed: 10852470, related citations] [Full Text]

  31. Nathan, C., Shiloh, M. U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Nat. Acad. Sci. 97: 8841-8848, 2000. [PubMed: 10922044, related citations] [Full Text]

  32. Nicholson, S., Bonecini-Almeida, M. D., Lapa e Silva, J. R., Nathan, C., Xie, Q., Mumford, R., Weidner, J. R., Calaycay, J., Geng, J., Boechat, N., Linhares, C., Rom, W., Ho, J. L. Inducible nitric oxide synthase in pulmonary alveolar macrophages from patients with tuberculosis. J. Exp. Med. 183: 2293-2302, 1996. [PubMed: 8642338, related citations] [Full Text]

  33. Noda, S., Tanaka, K., Sawamura, S., Sasaki, M., Matsumoto, T., Mikami, K., Aiba, Y., Hasegawa, H., Kawabe, N., Koga, Y. Role of nitric oxide synthase type 2 in acute infection with murine cytomegalovirus. J. Immunol. 166: 3533-3541, 2001. [PubMed: 11207313, related citations] [Full Text]

  34. Nozaki, Y., Hasegawa, Y., Ichiyama, S., Nakashima, I., Shimokata, K. Mechanism of nitric oxide-dependent killing of Mycobacterium bovis BCG in human alveolar macrophages. Infect. Immun. 65: 3644-3647, 1997. [PubMed: 9284131, related citations] [Full Text]

  35. Nussler, A. K., Di Silvio, M., Billiar, T. R., Hoffman, R. A., Geller, D. A., Selby, R., Madriaga, J., Simmons, R. L. Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin. J. Exp. Med. 176: 261-264, 1992. [PubMed: 1377225, related citations] [Full Text]

  36. Reuter, U., Chiarugi, A., Bolay, H., Moskowitz, M. A. Nuclear factor-kappa B as a molecular target for migraine therapy. Ann. Neurol. 51: 507-516, 2002. [PubMed: 11921057, related citations] [Full Text]

  37. Rutherford, S., Johnson, M. P., Curtain, R. P., Griffiths, L. R. Chromosome 17 and the inducible nitric oxide synthase gene in human essential hypertension. Hum. Genet. 109: 408-415, 2001. [PubMed: 11702222, related citations] [Full Text]

  38. Schulte, C., Sharma, M., Mueller, J. C., Lichtner, P., Prestel, J., Berg, D., Gasser, T. Comprehensive association analysis of the NOS2A gene with Parkinson disease. Neurology 67: 2080-2082, 2006. [PubMed: 17159127, related citations] [Full Text]

  39. Simpson, D. S., Pang, J., Weir, A., Kong, I. Y., Fritsch, M., Rashidi, M., Cooney, J. P., Davidson, K. C., Speir, M., Djajawi, T. M., Hughes, S., Mackiewicz, L., and 24 others. Interferon-gamma primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway. Immunity 55: 423-441, 2022. [PubMed: 35139355, images, related citations] [Full Text]

  40. Tezuka, H., Abe, Y., Iwata, M., Takeuchi, H., Ishikawa, H., Matsushita, M., Shiohara, T., Akira, S., Ohteki, T. Regulation of IgA production by naturally occurring TNF/iNOS-producing dendritic cells. Nature 448: 929-933, 2007. [PubMed: 17713535, related citations] [Full Text]

  41. van de Sande, W. W. J., Fahal, A., Verbrugh, H., van Belkum, A. Polymorphisms in genes involved in innate immunity predispose toward mycetoma susceptibility. J. Immun. 179: 3065-3074, 2007. [PubMed: 17709521, related citations] [Full Text]

  42. Velez, D. R., Hulme, W. F., Myers, J. L., Weinberg, J. B., Levesque, M. C., Stryjewski, M. E., Abbate, E., Estevan, R., Patillo, S. G., Gilbert, J. R., Hamilton, C. D., Scott, W. K. NOS2A, TLR4, and IFNGR1 interactions influence pulmonary tuberculosis susceptibility in African-Americans. Hum. Genet. 126: 643-653, 2009. [PubMed: 19575238, related citations] [Full Text]

  43. Vouldoukis, I., Riveros-Moreno, V., Dugas, B., Ouaaz, F., Becherel, P., Debre, P., Moncada, S., Mossalayi, M. D. The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc-epsilon-RII/CD23 surface antigen. Proc. Nat. Acad. Sci. 92: 7804-7808, 1995. [PubMed: 7544003, related citations] [Full Text]

  44. Xu, W., Charles, I. G., Liu, L., Moncada, S., Emson, P. Molecular cloning and structural organization of the human inducible nitric oxide synthase gene (NOS2). Biochem. Biophys. Res. Commun. 219: 784-788, 1996. [PubMed: 8645258, related citations] [Full Text]

  45. Xu, W., Charles, I. G., Moncada, S., Gorman, P., Sheer, D., Liu, L., Emson, P. Mapping of the genes encoding human inducible and endothelial nitric oxide synthase (NOS2 and NOS3) to the pericentric region of chromosome 17 and to chromosome 7, respectively. Genomics 21: 419-422, 1994. [PubMed: 7522210, related citations] [Full Text]

  46. Yang, L., Calay, E. S., Fan, J., Arduini, A., Kunz, R. C., Gygi, S. P., Yalcin, A., Fu, S., Hotamisligil, G. S. S-nitrosylation links obesity-associated inflammation to endoplasmic reticulum dysfunction. Science 349: 500-506, 2015. [PubMed: 26228140, images, related citations] [Full Text]


Bao Lige - updated : 03/23/2022
Bao Lige - updated : 12/09/2020
Ada Hamosh - updated : 06/04/2020
Ada Hamosh - updated : 12/2/2015
Paul J. Converse - updated : 11/10/2014
Paul J. Converse - updated : 7/2/2014
Patricia A. Hartz - updated : 3/9/2012
Paul J. Converse - updated : 7/27/2010
Paul J. Converse - updated : 5/4/2009
Cassandra L. Kniffin - updated : 11/20/2007
Ada Hamosh - updated : 11/7/2007
Cassandra L. Kniffin - updated : 9/12/2007
Paul J. Converse - updated : 7/5/2007
George E. Tiller - updated : 10/12/2006
Cassandra L. Kniffin - updated : 9/18/2006
Ada Hamosh - updated : 1/11/2006
Marla J. F. O'Neill - updated : 10/14/2004
Cassandra L. Kniffin - updated : 1/31/2003
Victor A. McKusick - updated : 12/27/2002
Paul J. Converse - updated : 9/16/2002
Victor A. McKusick - updated : 1/25/2002
Victor A. McKusick - updated : 12/27/2001
John A. Phillips, III - updated : 11/6/2001
John A. Phillips, III - updated : 5/10/2001
Ada Hamosh - updated : 7/28/1999
Ada Hamosh - updated : 5/5/1999
Creation Date:
Victor A. McKusick : 8/19/1992
carol : 05/06/2022
mgross : 03/23/2022
mgross : 12/09/2020
mgross : 10/12/2020
carol : 06/23/2020
alopez : 06/22/2020
alopez : 06/04/2020
carol : 04/25/2016
alopez : 12/2/2015
mgross : 11/10/2014
mcolton : 11/10/2014
mgross : 7/14/2014
mcolton : 7/2/2014
terry : 7/3/2012
terry : 6/6/2012
mgross : 3/12/2012
mgross : 3/12/2012
terry : 3/9/2012
mgross : 8/6/2010
terry : 7/27/2010
mgross : 5/5/2009
mgross : 5/5/2009
terry : 5/4/2009
alopez : 6/11/2008
wwang : 12/6/2007
ckniffin : 11/20/2007
alopez : 11/15/2007
terry : 11/7/2007
wwang : 9/21/2007
ckniffin : 9/12/2007
mgross : 7/5/2007
mgross : 7/5/2007
wwang : 12/1/2006
alopez : 10/12/2006
wwang : 10/11/2006
ckniffin : 9/18/2006
alopez : 1/13/2006
terry : 1/11/2006
carol : 11/5/2004
carol : 11/5/2004
carol : 11/5/2004
carol : 10/15/2004
terry : 10/14/2004
cwells : 11/10/2003
carol : 10/3/2003
carol : 10/2/2003
tkritzer : 5/8/2003
carol : 2/14/2003
ckniffin : 1/31/2003
cwells : 1/3/2003
terry : 12/27/2002
tkritzer : 12/20/2002
tkritzer : 12/10/2002
terry : 12/6/2002
tkritzer : 11/19/2002
mgross : 9/16/2002
carol : 1/30/2002
terry : 1/25/2002
cwells : 1/14/2002
cwells : 1/3/2002
terry : 12/27/2001
alopez : 11/6/2001
mgross : 5/10/2001
alopez : 7/30/1999
carol : 7/28/1999
alopez : 5/7/1999
terry : 5/5/1999
mark : 4/27/1996
terry : 4/22/1996
mark : 7/31/1995
terry : 9/12/1994
jason : 6/16/1994
carol : 4/15/1994
carol : 9/21/1993
carol : 9/13/1993

* 163730

NITRIC OXIDE SYNTHASE 2; NOS2


Alternative titles; symbols

NOS2A
NOS2A, INDUCIBLE, HEPATOCYTE
NITRIC OXIDE SYNTHASE, INDUCIBLE; INOS
NITRIC OXIDE SYNTHASE, MACROPHAGE


HGNC Approved Gene Symbol: NOS2

Cytogenetic location: 17q11.2     Genomic coordinates (GRCh38): 17:27,756,766-27,800,529 (from NCBI)


Gene-Phenotype Relationships

Location Phenotype Phenotype
MIM number
Inheritance Phenotype
mapping key
17q11.2 {Malaria, resistance to} 611162 3

TEXT

Description

Nitric oxide (NO) is a messenger molecule with diverse functions throughout the body. In the brain and peripheral nervous system, NO displays many properties of a neurotransmitter; it is implicated in neurotoxicity associated with stroke and neurodegenerative diseases, neural regulation of smooth muscle, including peristalsis, and penile erection. NO is also responsible for endothelium-derived relaxing factor (EDRF) activity regulating blood pressure. In macrophages, NO mediates tumoricidal and bactericidal actions, as indicated by the fact that inhibitors of NO synthase (NOS) block these effects. Neuronal NOS (163731) and macrophage NOS are distinct isoforms (Lowenstein et al., 1992). Both the neuronal and the macrophage forms are unusual among oxidative enzymes in requiring several electron donors: FAD, FMN, NADPH, and tetrahydrobiopterin.


Cloning and Expression

Lowenstein et al. (1992) cloned cDNA for the macrophage form of NOS and expressed the enzyme in human kidney cells. Macrophage enzyme showed 50% sequence identity to the neuronal enzyme. Like the neuronal form, NOS2 has recognition sites for FAD, FMN, and NADPH, and also has a consensus calmodulin binding site. Macrophage NOS mRNA was found to be strikingly inducible; it was absent in quiescent macrophages or spleen but prominent 2 to 6 hours after endotoxin treatment.

Nitric oxide is synthesized from L-arginine by the action of nitric oxide synthase(s), generating citrulline as a coproduct. The NO synthases, P450-type heme proteins, are all NADPH-, FMN-, and tetrahydrobiopterin-dependent. In endothelial cells and neuronal tissues, NO synthase activity is constitutively expressed and has a requirement for Ca(2+) and calmodulin. In contrast, NO synthase is inducible in macrophages and some other tissues. The inducible NO synthase in these cells does not require exogenous Ca(2+) or calmodulin for activity, and induction is inhibited by glucocorticoids. NO synthase is inducible in articular chondrocytes. Interleukin-1 beta (IL1B; 147720) induces the enzyme in human chondrocytes where induction is Ca(2+)-independent and marginally affected by glucocorticoids. Charles et al. (1993) isolated a cDNA clone that encoded a protein of 1,153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. The deduced amino acid sequence of the human chondrocyte inducible NO synthase showed 51% identity and 68% similarity with the endothelial NO synthase (163729) and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species. The induction of NO synthase in human cells had previously been shown only for hepatocytes (Nussler et al., 1992). Geller et al. (1993) cloned cDNA for an inducible NO synthase from human hepatocytes. The sequence differed from the human chondrocyte sequence at 7 amino acid positions.

Using a bovine NOS II-related cDNA to screen 2 human genomic libraries, Bloch et al. (1995) isolated clones containing 3 independent genes. One clone encoded the previously identified gene previously called NOS2 and called by them NOS2A. The 2 other genes specified amino acids homologous, but not identical, to those specified by the NOS2A gene; these were designated NOS2B (600719) and NOS2C (600720).


Gene Structure

Using both murine macrophage and human hepatocyte inducible NOS cDNAs as probes, Chartrain et al. (1994) isolated overlapping cosmid clones for the NOS2 gene from a human genomic library. The gene was estimated to be 37 kb long and consisted of 26 exons and 25 introns. They mapped the transcriptional initiation site 30 basepairs downstream of a TATA sequence. Xu et al. (1996) concluded that in fact the NOS2 open reading frame is encoded by 27 exons, with translation initiation and termination in exon 2 and exon 27, respectively.


Mapping

By Southern analysis of somatic cell hybrid lines, Marsden et al. (1994) mapped the NOS2 gene to chromosome 17; they refined the assignment to 17q11.2-q12 by a fluorescence in situ hybridization. Jenkins et al. (1994), who referred to the inducible NO synthase of mouse macrophages as Nos-1, found that the gene maps to mouse chromosome 11 in a region homologous to human chromosome 17q. They predicted that the human gene might lie in the region 17q11.2. Mehrabian et al. (1994) likewise mapped the mouse homolog to chromosome 11, using RFLVs in linkage analysis in an interspecific backcross. Chartrain et al. (1994) used polymerase chain reaction analysis of a human/rodent genomic DNA somatic cell hybrid panel and fluorescence in situ hybridization to map the NOS2 gene to 17cen-q11.2. Xu et al. (1994) mapped the NOS2 gene to 17cen-q11 by Southern blotting analysis of DNAs obtained from a panel of human/rodent hybrid cell lines. By fluorescence in situ hybridization, they found signals in the 17p11-q11 pericentromeric region. Gerling et al. (1994) mapped the Nos2 gene to mouse chromosome 11.

Using Southern blot hybridization, Bloch et al. (1995) demonstrated that the NOS2A, NOS2B, and NOS2C genes are all located on chromosome 17 between bands 17p13.1 and 17q25.


Gene Function

Napolitano et al. (2000) investigated the interactions between ET1 (131240) and the NO system in the fetoplacental unit. They examined the mRNA expression of ET1, inducible NOS (iNOS), and endothelial NOS (eNOS; 163729) in human cultured placental trophoblastic cells obtained from preeclamptic (PE; 189800) and normotensive pregnancies. ET1 expression was increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, was decreased; conversely, eNOS expression was increased. ET1 was able to influence its own expression as well as NOS isoform expression in normal and PE trophoblastic cultured cells. The findings suggested the existence of a functional relationship between ET(s) and NOS isoforms that could constitute the biologic mechanism leading to the reduced placental blood flow and increased resistance to flow in the fetomaternal circulation that are characteristic of the pathophysiology of preeclampsia.

The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (see 161560), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL12 production by macrophages and the mechanisms by which they do so remain undefined. Brightbill et al. (1999) demonstrated that microbial lipoproteins are potent stimulators of IL12 production by human macrophages and that induction is mediated by toll-like receptors (TLRs; see 603030). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.

Diefenbach et al. (1999) studied the relationship of IL12 and nitric oxide synthase-2 (NOS2) to innate immunity to the parasite Leishmania in mice. In the absence of NOS2 activity, IL12 was unable to prevent spreading of Leishmania parasites, did not stimulate natural killer cells for cytotoxicity or interferon (IFN)-gamma (147570) release, and failed to activate TYK2 (176941) and to tyrosine-phosphorylate STAT4 (600558), the central signal transducer of IL12, in NK cells. Activation of TYK2 in NK cells by IFN-alpha/beta (type I interferon; see 107470) also required NOS2. Thus, NOS2-derived NO is a prerequisite for cytokine signaling and function in innate immunity.

Exposure of human pancreatic islets to a mixture of cytokines induces expression of iNOS, impairs beta-cell function, and induces apoptosis. Johannesen et al. (2001) scanned all 27 exons of the human NOS2 gene and carried out linkage transmission disequilibrium testing of the identified NOS2 polymorphisms in a Danish nationwide IDDM (222100) family collection. Transmission disequilibrium testing was performed using 257 Danish IDDM families; 154 families were affected sib pair families, and 103 families were simplex families. In total, 10 polymorphisms were identified in 8 exons, of which 4 were tested in the family material. A C/T single-nucleotide polymorphism in exon 16 resulting in an amino acid substitution, ser608 to leu, showed linkage to IDDM in human leukocyte antigen DR3/4-positive affected offspring (P = 0.008; corrected P = 0.024). No other distorted transmission patterns were found for any other tested single-nucleotide polymorphism or constructed haplotypes with the exception of those including data from exon 16. The authors concluded that linkage of the human NOS2 gene to IDDM in a subset of patients supports a pathogenic role of nitric oxide in human IDDM.

In a review of reactive oxygen and nitrogen intermediates, Nathan and Shiloh (2000) noted that infections, microbial products, and cytokines do not consistently induce expression of NOS2 when applied to human blood mononuclear phagocytes, although these stimuli do induce production in rodent tissue macrophages. The authors stressed the importance of evaluating tissue rather than blood macrophages for human NOS2 expression.

Vouldoukis et al. (1995) found that ligation of the low affinity IgE receptor (CD23; 151445) induces NOS production by human monocyte-derived macrophages in vitro. CD23 ligation led to the intracellular killing of Leishmania major parasites, which could be blocked by the NOS inhibitor, NG-monomethyl-L-arginine monoacetate (NMMA).

Nicholson et al. (1996) reported that lung macrophages from tuberculosis (TB) patients express NOS2 in potentially mycobactericidal amounts.

Nozaki et al. (1997) demonstrated by RT-PCR and immunofluorescence analyses that alveolar macrophages (AMs) obtained from pulmonary fibrosis patients, but not from lung cancer patients, infected with the avirulent vaccine strain Mycobacterium bovis (BCG) produced higher levels of iNOS. Colony forming assays showed that these infected AMs effectively killed BCG, but they were less able to do so after treatment with NMMA.

Using immunoblot and immunohistochemical analyses, Facchetti et al. (1999) showed expression of a 130-kD NOS2 protein in the cytoplasm of CD68 (153634)-positive macrophages in infectious granuloma tissues, as well as in sarcoidosis (181000) and Kikuchi disease, but not in foreign body granulomas or Omenn syndrome (603554).

Choi et al. (2002) examined resected lungs from 8 TB patients for the expression of NOS and nitrotyrosine, a marker of NO expression. Immunohistochemical and morphometric analyses revealed that iNOS, nitrotyrosine, eNOS, and TNFA (191160), but not nNOS (NOS1; 163731), were expressed in CD68-positive epithelioid macrophages and giant cells in the inflammatory zone of the granulomas of the patients, but not in histologically normal tissue obtained from cancer patient control subjects. TNF expression was highest in necrotic areas.

The 26S proteasome is the major pathway responsible for iNOS degradation. Targeting proteins for proteasomal degradation may require their covalent linkage to multiubiquitin chains (i.e., ubiquitination). Kolodziejski et al. (2002) reported results of experiments indicating that iNOS is subject to ubiquitination, which is required for its degradation.

NO generated from inducible NO synthase has been implicated in migraine (157300) based on pharmacologic evidence in animals and humans. In a rat model, Reuter et al. (2002) showed that the NO donor glyceryl trinitrate (GTN) caused NOS2A expression in macrophages, mediated by increased activity of the nuclear transcription factor kappa-B (NFKB1; 164011), resulting in generation of NO within rodent dura mater 6 hours later. Parthenolide, a lactone found in the medical herb 'feverfew' which has been used successfully in the treatment of inflammatory conditions and migraine, blocked NOS2A expression in dura mater by inhibiting NFKB1.

Kim et al. (2005) showed that iNOS specifically binds to cyclooxygenase-2 (COX2; 600262) and S-nitrosylates it, enhancing COX2 catalytic activity. Selectively disrupting iNOS-COX2 binding prevented NO-mediated activation of COX2. Kim et al. (2005) suggested that the molecular synergism between iNOS and COX2 may represent a major mechanism of inflammatory responses.

Tezuka et al. (2007) showed that IgA class switch recombination is impaired in inducible nitric oxide synthase-deficient (iNOS-null) mice. iNOS regulates the T-cell-dependent IgA class switch recombination through expression of transforming growth factor-beta receptor (see TGFBR1, 190181), and the T-cell-independent IgA class switch recombination through production of a proliferation-inducing ligand (APRIL, also called TNFSF13; 604472) and a B-cell-activating factor of the tumor necrosis factor family (BAFF; 603969). Notably, iNOS is preferentially expressed in mucosa-associated lymphoid tissue (MALT) dendritic cells in response to the recognition of commensal bacteria by toll-like receptor (TLR; see 603030). Furthermore, adoptive transfer of iNOS-positive dendritic cells rescued IgA production in iNOS-null mice. Further analysis revealed that the MALT dendritic cells are a TNFA/iNOS-producing dendritic-cell subset, originally identified in mice infected with Listeria monocytogenes. The presence of a naturally occurring TNFA/iNOS-producing dendritic cell subset may explain the predominance of IgA production in the MALT, critical for gut homeostasis.

Eyler et al. (2011) found that highly tumorigenic human glioma stem cells (GSCs), but not normal neural progenitor cells, produced elevated NO via upregulated NOS2 expression. Consumption of NO in GSCs by lentivirus-introduced bacterial flavohemoglobin, or knockdown of NOS2 via short hairpin RNA, abrogated GSC growth, neurosphere formation, and tumorigenicity. Knockdown of NOS2 in normal neural progenitor cells had little effect. Quantitative RT-PCR detected elevated NOS2 expression in GSCs from 3 different primary gliomas and a xenograft. Microarray analysis of NOS2-knockdown GSCs revealed upregulation of several genes, including the cell cycle inhibitor CDA1 (TSPYL2; 300564). Transfection of NOS2 in HEK293 cells inhibited CDA1 mRNA and protein expression, and RT-PCR of several glioma xenografts confirmed an inverse relationship between NOS2 and CDA1 expression. Pharmacologic inhibition of NOS2 in mice slowed the growth of human glioma xenografts and inhibited tumor angiogenesis. Eyler et al. (2011) concluded that NO production in GSCs due to NOS2 upregulation contributes primarily to GSC tumor maintenance.

Nairz et al. (2013) found that macrophages from mice lacking Nos2 displayed reduced expression of ferroportin-1 (FPN1, or SLC40A1; 604653). Nitric oxide upregulated FPN1 expression in mouse and human cells. Nos2-null mouse macrophages had increased iron content due to reduced Fpn1 activity. Reduced Fpn1 expression allowed enhanced iron acquisition by the intracellular bacterium Salmonella typhimurium. Mice lacking Nos2 or mice in which Nos2 activity was inhibited had increased iron accumulation in spleen and spleen macrophages. Lack of nitric oxide formation resulted in impaired Nrf2 (NFE2L2; 600492) expression and, consequently, reduced Fpn1 transcription and cellular iron export. Infection of Nos2-null mice or macrophages with S. typhimurium led not only to increased iron accumulation, but also to reduced Tnf, Il2 (147680), and Ifng expression and impaired pathogen control, all of which could be restored by treatment with iron chelators or overexpression of Fpn1 or Nrf2. Nairz et al. (2013) concluded that iron accumulation in Nos2-null macrophages counteracts a proinflammatory host response and that the protective effects of nitric oxide partially result from its ability to prevent iron overload in macrophages.

Yang et al. (2015) showed that, in the setting of obesity, inflammatory input through increased iNOS activity causes S-nitrosylation of a key unfolded protein response (UPR) regulator, IRE1-alpha (604033), which leads to a progressive decline in hepatic IRE1-alpha-mediated XBP1 (194355) splicing activity in both genetic (ob/ob) and dietary (high-fat diet-induced) models of obesity. Finally, in obese mice with liver-specific IRE1-alpha deficiency, reconstitution of IRE1-alpha expression with a nitrosylation-resistant variant restored IRE1-alpha-mediated XBP1 splicing and improved glucose homeostasis in vivo. Yang et al. (2015) concluded that their data described a mechanism by which inflammatory pathways compromise UPR function through iNOS-mediated S-nitrosylation of IRE1-alpha, which contributes to defective IRE1-alpha activity, impaired ER function, and prolonged ER stress in obesity.

By treating mouse bone marrow-derived macrophages (BMDMs) with IFN-gamma followed by the TLR4 (603030) agonist lipopolysaccharide (LPS), Simpson et al. (2022) found that IFN-gamma activated macrophages and triggered cell death via TLR signaling and Fasl (FASLG; 134638) expression. Knockout analysis revealed that efficient IFN-gamma/LPS-induced cell death required caspase-8 (CASP8; 601763) and the mitochondrial apoptosis effector proteins Bax (600040) and Bak (BAK1; 600516). Activation of Bax and Bak was not triggered by caspase-8 cleavage of its substrate Bid (601997). Instead, caspase-8 mediated transcriptional programming in macrophages to increase proapoptotic Noxa (604959) and reduce prosurvival Bcl2 (151430), thereby reducing prosurvival proteins Mcl1 (159552) and A1 to facilitate Bax/Bak activation and subsequent apoptotic cell death upon stimulation with IFN-gamma and LPS. Caspase-8 enzymatic activity was required for IFN-gamma/LPS-mediated activation of Bax/Bak and subsequent apoptotic cell death. Bax/Bak activation resulted in irreversible damage to mitochondria and caused cell death even when the functions of other downstream caspases were eliminated. Treatment with IFN-gamma/LPS induced robust expression of iNos and generation of nitric oxide in macrophages, upstream of Bax/Bak activation and cell death. However, toxicity of nitric oxide was not the direct cause of cell death. Instead, iNos expression played a role in reducing Mcl1 and A1 to sensitize macrophages for Bax/Bak activation and mitochondrial apoptosis. In agreement, both iNos and caspase-8 contributed to disease severity of SARS-CoV-2 infection in mice, as deletion of iNos or caspase-8 limited SARS-CoV-2-induced disease, whereas caspase-8 caused lethality through hemophagocytic lymphohistiocytosis independently of iNos.


Molecular Genetics

Resistance to Malaria

Kun et al. (1998) examined whether high plasma concentrations of NO found in severe malaria (see 611162) were due to variation in the promoter region of NOS2. Heterozygosity for a -969G-C SNP (163730.0002) was present in 30 of 100 Gambian children with mild malaria, but in only 17 of 100 Gambian children with severe malaria. The SNP was not found in any of 100 Germans. Heterozygous individuals were also at a significantly lower risk of reinfection.

Hobbs et al. (2002) postulated that NOS2 promoter polymorphisms could affect resistance to severe malaria as manifested by cerebral malaria, severe malarial anemia, and respiratory distress or metabolic acidosis (Marsh et al., 1995). From studies in Tanzania and Kenya, Hobbs et al. (2002) identified a novel single-nucleotide polymorphism, -1173C-T (163730.0001), in the NOS2 promoter that was significantly associated with protection from symptomatic malaria and severe malarial anemia.

Associations Pending Confirmation

Rutherford et al. (2001) provided evidence for the location of at least 1 hypertension susceptibility locus on chromosome 17. Analysis of 177 affected sib pairs gave evidence for significant excess allele sharing to marker D17S949 on chromosome 17q22-q24, with significant allele sharing also indicated for an additional marker, D17S799, located close to the centromere. Since these 2 genomic regions are well separated, the results indicated that there may be more than 1 chromosome 17 locus affecting blood pressure. Moreover, further investigation using a polymorphism within the promoter of the NOS2A candidate gene revealed both increased allele sharing among sib pairs and positive association of NOS2A to essential hypertension. Morris (2002) called into question the conclusions of Rutherford et al. (2001); Griffiths (2002), the senior author of the paper by Rutherford et al. (2001), responded.

Hao et al. (2004) conducted a large-scale case-control study exploring the associations of 426 single-nucleotide polymorphisms (SNPs) with preterm delivery (PTD) in 300 mothers with PTD and 458 mothers with term deliveries. Twenty-five candidate genes were included in the final haplotype analysis. Gene haplotypes at IL1R2 (147811) in blacks, NOS2A in whites, and OPRM1 (600018) in Hispanics were only associated with PTD in these specific ethnic groups.

In a subset of 73 families with Parkinson disease (PD; 168600) in which at least 1 member had disease onset before age 40 years, Hancock et al. (2006) found a significant association between PD and 2 SNPs in the NOS2A gene: the more frequent T allele of rs2255929 and the less frequent A allele of rs1060826 (p = 0.000059 and 0.0062, respectively). The 2-SNP haplotype showed an even stronger association with PD (p = 0.000013). Similar associations were not observed in a larger group of 286 multiplex families including all ages at onset. Epidemiologic studies have consistently reported an inverse association between Parkinson disease and cigarette smoking. In an overlapping group of 243 families, Hancock et al. (2006) found a significant association between cigarette smoking and the 2 risk alleles; however, when stratified by PD status, the presence of these alleles attenuated the protective inverse association between smoking and PD. Hancock et al. (2006) postulated that an alteration in NOS2A regulation could result in prolonged activity and cellular damage, and further concluded that NOS2A may be a risk factor in PD by influencing age at onset and by modifying the inverse association between PD and smoking.

Among 340 German PD patients with disease onset at a median of 52 years, Schulte et al. (2006) found no association between the disorder and 12 polymorphisms in the NOS2A gene.

Eumycetoma is a tumorous fungal infection, typically of the hands or feet, characterized by the infiltration of large numbers of neutrophils. It is caused by Madurella mycetomatis, a pathogen that is abundant in the soil and on the vegetation of Sudan, where the disease is common. Van de Sande et al. (2007) noted that ELISA has shown near universal IgG seropositivity in mycetoma patients and controls from endemic areas, but no seropositivity in European controls, implying that most individuals in endemic areas are exposed to the pathogen, but only a small percentage develop disease. Van de Sande et al. (2007) studied 11 SNPs in genes involved in neutrophil function in 125 Sudanese mycetoma patients and 140 ethnically and geographically matched controls and found significant differences in allele distributions for SNPs in IL8 (146930), IL8RB (146928), TSP4 (THBS4; 600715), NOS2, and CR1 (120620). Serum IL8 was significantly higher in patients compared with controls, while nitrite/nitrate levels were lower in patients and seemed to be associated with delayed wound healing. Van de Sande et al. (2007) concluded that there is a genetic predisposition toward susceptibility to mycetoma.

Using a mixed case-control association analysis of 279 African American and 198 Caucasian tuberculosis (TB; see 607948) patients and 166 African American and 123 Caucasian controls, Velez et al. (2009) identified 10 SNPs in NOS2A that were associated with TB in African Americans but not Caucasians. Additionally, they identified gene-gene interactions between SNPs in NOS2A and IFNGR1 (107470) and TLR4 (603030). Velez et al. (2009) proposed that NOS2A variants may contribute to TB susceptibility in individuals of African descent and that these variants may act synergistically with SNPs in IFNGR1 and TLR4.

Drutman et al. (2020) reported a previously healthy 51-year-old man from Iran who after acute cytomegalovirus (CMV) infection had onset of progressive CMV disease that led to his death 29 months later. Whole-exome sequencing identified homozygosity for a frameshift variant in NOS2 (c.1436_1437insT; 163730.0003). Homozygosity for this variant segregated with the phenotype in the family; only the proband was homozygous. Drutman et al. (2020) cited Noda et al. (2001), who showed that mice deficient in NOS2 were highly vulnerable to the related murine CMV. The patient had titers against diphtheria and tetanus toxins, varicella, and various common viruses. The mechanistic connection between NOS2 deficiency and the patient's CMV phenotype required further investigation, although the authors favored the hypothesis that NOS2 expression in epithelial cells that are infected by CMV is required for control of the infection.


Animal Model

Noda et al. (2001) found that Nos2 -/- mice were more susceptible to lethal murine CMV (MCMV) infection than wildtype mice. Plaque assays showed that virus titer was markedly elevated in MCMV-infected Nos2 -/- mice, especially in salivary gland. Immune responses to MCMV infection were comparable with those of wildtype mice, as NK cell cytotoxicity, cytotoxic T-lymphocyte response, and IFN-gamma production after acute infection were not impaired. However, macrophages from Nos2 -/- mice exhibited lower antiviral activity, resulting in enhanced viral replication. In addition, absence of Nos2 prolonged MCMV-DNA latency after acute infection.

In immunodeficient mice inoculated with human peripheral blood mononuclear cells, Koh et al. (2004) examined transplanted human arteries for endothelial cell and vascular smooth muscle cell dysfunction. Within 7 to 9 days, transplanted arteries developed endothelial cell dysfunction but remained sensitive to exogenous NO. By 2 weeks, the grafts developed signs of vascular smooth muscle cell dysfunction, including impaired contractility and desensitization to NO. These T-cell dependent changes correlated with loss of endothelial NOS and expression of inducible NOS. Neutralizing IFN-gamma completely prevented both vascular dysfunction and changes in NOS expression; neutralizing TNF reduced IFN-gamma production and partially prevented dysfunction. Inhibiting iNOS partially preserved responses to NO at 2 weeks and reduced graft intimal expansion after 4 weeks in vivo. Koh et al. (2004) concluded that IFN-gamma is a central mediator of vascular dysfunction through dysregulation of NO production.

Colton et al. (2006) found that transgenic mice expressing mutant amyloid precursor protein (APP; 104760) on a Nos2-null background developed pathologic hyperphosphorylation of tau (MAPT; 157140) with aggregate formation in the brain. Lack of Nos2 increased insoluble APP levels, neuronal degeneration, caspase-3 (CASP3; 600636) activation, and tau cleavage, suggesting that nitric oxide may act at a junction point between the 2 main pathologies that characterize Alzheimer disease (AD; 104300).

Eyler et al. (2011) found that knockout of Nos2 in mice had no effect on neural development.

Mishra et al. (2013) infected Nos2 -/- mice with a strain of M. tuberculosis whose growth could be controlled exogenously. Using these mice, they found that Ifng and NO suppressed both bacterial growth in vivo and the continual production of Il1b by the Nlrp3 (606416) inflammasome, thereby inhibiting persistent neutrophil recruitment and preventing tissue damage. Mishra et al. (2013) concluded that NO has a dual role in promoting resistance to M. tuberculosis and in regulating inflammation, both of which are required for survival of this chronic infection.


ALLELIC VARIANTS 3 Selected Examples):

.0001   MALARIA, RESISTANCE TO

NOS2A, -1173C-T
SNP: rs9282799, gnomAD: rs9282799, ClinVar: RCV000015051

From studies in Tanzania and Kenya, Hobbs et al. (2002) identified a novel single-nucleotide polymorphism, -1173C-T, in the NOS2A gene that was significantly associated with protection from symptomatic malaria and severe malarial anemia (see 611162). The -1173C-T polymorphism was associated with increased fasting urine and plasma NO metabolite concentrations in Tanzanian children, suggesting that the polymorphism was functional in vivo.


.0002   MALARIA, SEVERE, RESISTANCE TO

NOS2A, -969G-C
SNP: rs1800482, gnomAD: rs1800482, ClinVar: RCV000015052

Kun et al. (1998) found that a SNP in the promoter region of NOS2A, -969G-C, was present significantly more often in Gambian children with mild malaria than in children with severe malaria (see 611192).


.0003   VARIANT OF UNKNOWN SIGNIFICANCE

NOS2A, 1-BP INS, 1436T
SNP: rs1908748329, ClinVar: RCV002252323

This variant is classified as a variant of unknown significance because its contribution to fatal cytomegalovirus (CMV) infection has not been confirmed.

In a previously healthy 51-year-old man from Iran who after acute CMV infection had onset of progressive CMV disease that led to his death 29 months later, Drutman et al. (2020) reported homozygosity for a 1-bp insertion (c.1436_1437insT) in the NOS2A gene, predicted to result in a frameshift and a premature termination codon (Ile391IlefsTer26, I391fs). The truncated NOS2 protein was predicted to lack the entire C-terminal reductase domain required for the formation of nitric oxide, and thus be nonfunctional. NOS2 is intolerant to homozygosity for loss-of-function variants, and the I391fs variant was not found in 5,000 in-house controls, gnomAD, or in the Greater Middle Eastern (GME) Variome Database. Overexpression of the mutant protein in HEK293T cells resulted in no detectable nitric oxide production.


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Contributors:
Bao Lige - updated : 03/23/2022
Bao Lige - updated : 12/09/2020
Ada Hamosh - updated : 06/04/2020
Ada Hamosh - updated : 12/2/2015
Paul J. Converse - updated : 11/10/2014
Paul J. Converse - updated : 7/2/2014
Patricia A. Hartz - updated : 3/9/2012
Paul J. Converse - updated : 7/27/2010
Paul J. Converse - updated : 5/4/2009
Cassandra L. Kniffin - updated : 11/20/2007
Ada Hamosh - updated : 11/7/2007
Cassandra L. Kniffin - updated : 9/12/2007
Paul J. Converse - updated : 7/5/2007
George E. Tiller - updated : 10/12/2006
Cassandra L. Kniffin - updated : 9/18/2006
Ada Hamosh - updated : 1/11/2006
Marla J. F. O'Neill - updated : 10/14/2004
Cassandra L. Kniffin - updated : 1/31/2003
Victor A. McKusick - updated : 12/27/2002
Paul J. Converse - updated : 9/16/2002
Victor A. McKusick - updated : 1/25/2002
Victor A. McKusick - updated : 12/27/2001
John A. Phillips, III - updated : 11/6/2001
John A. Phillips, III - updated : 5/10/2001
Ada Hamosh - updated : 7/28/1999
Ada Hamosh - updated : 5/5/1999

Creation Date:
Victor A. McKusick : 8/19/1992

Edit History:
carol : 05/06/2022
mgross : 03/23/2022
mgross : 12/09/2020
mgross : 10/12/2020
carol : 06/23/2020
alopez : 06/22/2020
alopez : 06/04/2020
carol : 04/25/2016
alopez : 12/2/2015
mgross : 11/10/2014
mcolton : 11/10/2014
mgross : 7/14/2014
mcolton : 7/2/2014
terry : 7/3/2012
terry : 6/6/2012
mgross : 3/12/2012
mgross : 3/12/2012
terry : 3/9/2012
mgross : 8/6/2010
terry : 7/27/2010
mgross : 5/5/2009
mgross : 5/5/2009
terry : 5/4/2009
alopez : 6/11/2008
wwang : 12/6/2007
ckniffin : 11/20/2007
alopez : 11/15/2007
terry : 11/7/2007
wwang : 9/21/2007
ckniffin : 9/12/2007
mgross : 7/5/2007
mgross : 7/5/2007
wwang : 12/1/2006
alopez : 10/12/2006
wwang : 10/11/2006
ckniffin : 9/18/2006
alopez : 1/13/2006
terry : 1/11/2006
carol : 11/5/2004
carol : 11/5/2004
carol : 11/5/2004
carol : 10/15/2004
terry : 10/14/2004
cwells : 11/10/2003
carol : 10/3/2003
carol : 10/2/2003
tkritzer : 5/8/2003
carol : 2/14/2003
ckniffin : 1/31/2003
cwells : 1/3/2003
terry : 12/27/2002
tkritzer : 12/20/2002
tkritzer : 12/10/2002
terry : 12/6/2002
tkritzer : 11/19/2002
mgross : 9/16/2002
carol : 1/30/2002
terry : 1/25/2002
cwells : 1/14/2002
cwells : 1/3/2002
terry : 12/27/2001
alopez : 11/6/2001
mgross : 5/10/2001
alopez : 7/30/1999
carol : 7/28/1999
alopez : 5/7/1999
terry : 5/5/1999
mark : 4/27/1996
terry : 4/22/1996
mark : 7/31/1995
terry : 9/12/1994
jason : 6/16/1994
carol : 4/15/1994
carol : 9/21/1993
carol : 9/13/1993