Phenotypic Mutation 'paul' (pdf version)
Allelepaul
Mutation Type missense
Chromosome5
Coordinate24,577,702 bp (GRCm39)
Base Change C ⇒ T (forward strand)
Gene Nos3
Gene Name nitric oxide synthase 3, endothelial cell
Synonym(s) 2310065A03Rik, ecNOS, eNOS, Nos-3
Chromosomal Location 24,569,808-24,589,472 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] Nitric oxide is a reactive free radical which acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. Nitric oxide is synthesized from L-arginine by nitric oxide synthases. Variations in this gene are associated with susceptibility to coronary spasm. Alternative splicing and the use of alternative promoters results in multiple transcript variants. [provided by RefSeq, Oct 2016]
PHENOTYPE: Homozygotes for targeted null mutations exhibit reduced survival, hypertension, inhibited basal vasodilation, insulin resistance, fewer mitochondria, reduced heart rate, impaired ovulation and, in some, shortened limbs. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_008713; MGI:97362

MappedYes 
Amino Acid Change Threonine changed to Isoleucine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000030834] [ENSMUSP00000110742]
AlphaFold P70313
SMART Domains Protein: ENSMUSP00000030834
Gene: ENSMUSG00000028978
AA Change: T572I

DomainStartEndE-ValueType
low complexity region 11 27 N/A INTRINSIC
low complexity region 31 57 N/A INTRINSIC
Pfam:NO_synthase 118 480 1.7e-183 PFAM
Pfam:Flavodoxin_1 521 697 4.8e-54 PFAM
Pfam:FAD_binding_1 750 978 2.1e-82 PFAM
Pfam:NAD_binding_1 1010 1124 1.9e-18 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000030834)
SMART Domains Protein: ENSMUSP00000110742
Gene: ENSMUSG00000028978
AA Change: T572I

DomainStartEndE-ValueType
low complexity region 11 27 N/A INTRINSIC
low complexity region 31 57 N/A INTRINSIC
Pfam:NO_synthase 114 485 9e-214 PFAM
Pfam:Flavodoxin_1 521 697 3.8e-54 PFAM
Pfam:FAD_binding_1 750 978 1.6e-79 PFAM
Pfam:NAD_binding_1 1010 1091 5.6e-12 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000115090)
Meta Mutation Damage Score 0.9556 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(9) : Gene trapped(1) Targeted(8)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL00903:Nos3 APN 5 24574860 missense probably damaging 1.00
IGL02059:Nos3 APN 5 24573996 missense probably damaging 1.00
IGL02354:Nos3 APN 5 24572621 missense probably damaging 1.00
IGL02361:Nos3 APN 5 24572621 missense probably damaging 1.00
IGL02936:Nos3 APN 5 24585991 missense probably damaging 0.97
IGL03190:Nos3 APN 5 24588627 missense probably damaging 1.00
Peter UTSW 5 24582853 missense probably damaging 0.99
R0111:Nos3 UTSW 5 24577702 missense probably damaging 1.00
R0387:Nos3 UTSW 5 24572583 missense probably damaging 1.00
R0755:Nos3 UTSW 5 24572295 missense probably damaging 1.00
R1156:Nos3 UTSW 5 24582617 missense probably benign 0.21
R1597:Nos3 UTSW 5 24573995 missense probably damaging 1.00
R1671:Nos3 UTSW 5 24588838 missense probably damaging 1.00
R1743:Nos3 UTSW 5 24582310 missense probably benign 0.22
R1830:Nos3 UTSW 5 24575131 missense probably damaging 1.00
R1882:Nos3 UTSW 5 24573818 missense probably damaging 1.00
R2294:Nos3 UTSW 5 24569855 missense probably damaging 0.99
R3114:Nos3 UTSW 5 24577629 splice site probably benign
R3978:Nos3 UTSW 5 24582929 missense probably damaging 1.00
R3980:Nos3 UTSW 5 24582929 missense probably damaging 1.00
R4016:Nos3 UTSW 5 24576714 missense probably damaging 1.00
R4905:Nos3 UTSW 5 24572329 missense probably benign 0.01
R4947:Nos3 UTSW 5 24582853 missense probably damaging 0.99
R5017:Nos3 UTSW 5 24571717 intron probably benign
R5095:Nos3 UTSW 5 24573916 splice site probably benign
R5096:Nos3 UTSW 5 24576955 missense probably damaging 1.00
R5102:Nos3 UTSW 5 24576625 missense probably damaging 1.00
R5311:Nos3 UTSW 5 24582343 missense probably benign 0.19
R5330:Nos3 UTSW 5 24574902 missense probably damaging 1.00
R5367:Nos3 UTSW 5 24576942 missense probably benign 0.00
R5394:Nos3 UTSW 5 24588888 missense probably benign 0.00
R5574:Nos3 UTSW 5 24573859 missense possibly damaging 0.80
R5889:Nos3 UTSW 5 24573775 intron probably benign
R6032:Nos3 UTSW 5 24584809 missense probably benign
R6032:Nos3 UTSW 5 24584809 missense probably benign
R6401:Nos3 UTSW 5 24584809 missense probably benign
R6517:Nos3 UTSW 5 24588622 missense probably damaging 1.00
R6888:Nos3 UTSW 5 24588333 missense possibly damaging 0.86
R6972:Nos3 UTSW 5 24585241 missense probably benign
R6973:Nos3 UTSW 5 24585241 missense probably benign
R7432:Nos3 UTSW 5 24572613 missense probably damaging 0.98
R7434:Nos3 UTSW 5 24587633 missense probably damaging 0.99
R7507:Nos3 UTSW 5 24577642 missense probably damaging 1.00
R7553:Nos3 UTSW 5 24586715 missense possibly damaging 0.62
R7652:Nos3 UTSW 5 24588610 missense probably damaging 1.00
R8094:Nos3 UTSW 5 24572218 missense probably benign 0.13
R8686:Nos3 UTSW 5 24573841 missense possibly damaging 0.83
R8794:Nos3 UTSW 5 24576745 missense probably damaging 1.00
R9016:Nos3 UTSW 5 24588639 missense probably damaging 1.00
R9192:Nos3 UTSW 5 24582611 missense probably benign 0.04
R9336:Nos3 UTSW 5 24584761 missense probably benign
X0020:Nos3 UTSW 5 24575122 missense probably damaging 1.00
X0061:Nos3 UTSW 5 24587633 missense probably damaging 0.99
Z1176:Nos3 UTSW 5 24582652 missense probably benign 0.02
Z1177:Nos3 UTSW 5 24588948 missense probably benign 0.00
Mode of Inheritance Autosomal Recessive
Local Stock
MMRRC Submission 038230-MU
Last Updated 2019-09-04 9:46 PM by Diantha La Vine
Record Created 2015-02-15 10:29 AM by Emre Turer
Record Posted 2015-12-11
Phenotypic Description

Figure 1. Paul mice exhibit weight loss seven days after exposure to dextran sulfate sodium. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The paul phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R0111 in a screen for mutants susceptible to dextran sulfate sodium (DSS)-induced colitis. The screen uses weight-loss as an indication of colitis. The paul mice were susceptible to low doses of DSS (1%) and showed weight loss seven days after DSS treatment (Figure 1).

Nature of Mutation

Figure 2. Linkage mapping of the DSS-induced weight loss using a recessive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 62 mutations (X-axis) identified in the G1 male of pedigree R0111.  Normalized phenotype data are shown for single locus linkage analysis without consideration of G2 dam identity.  Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 62 mutations. The susceptibility to DSS-induced colitis phenotype was linked by continuous variable mapping to a mutation in Nos3: a C to T transition at base pair 24,372,704 (v38) on chromosome 5, or base pair 7,886 in the NC_000071 GenBank genomic region.  Linkage was found with a recessive model of inheritance (P = 8.33 x 10-4), wherein five variant homozygotes departed phenotypically from five homozygous reference mice and two heterozygous mice (Figure 2).  

The mutation corresponds to residue 1,734 in the mRNA sequence NM_008713 within exon 13 of 26 total exons.

1718 TTGGTGGTGACCAGCACATTTGGCAATGGGGAT
567  -L--V--V--T--S--T--F--G--N--G--D-
 
The mutated nucleotide is indicated in red.  The mutation results in a threonine (T) to isoleucine (I) substitution at position 572 (T572I) in NOS3 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.999) (1).
Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 3. Protein domain organization of NOS3.  NOS3 has an N-terminal oxygenase domain that interacts with Heme (designated as HEME), a calmodulin (CaM)-binding region), a flavodoxin region, a flavin adenine dinucleotide (FAD)-binding region (FBR), and a nicotinamide adenine dinucleotide phosphate (NADPH)-binding region (NBR). The mutation in paul results in a threonine (T) to isoleucine (I) substitution at position 572. This image is interactive. Other mutations found in NOS3 are noted in red. Click on each mutation for more information.

Nitric oxide synthase 3 [NOS3; alternatively, endothelial NOS (eNOS)] is one of three nitric oxide synthase (NOS) enzymes. The other two members of the NOS family are NOS1 (alternatively, neuronal NOS (nNOS)] and inducible NOS (iNOS; alternatively, NOS2). NOS1 is constitutively expressed in neuronal cells, while iNOS is produced upon exposure to certain stimuli (e.g., cytokines).  The NOS isoforms require five cofactors to function: flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, BH4 [(1'R, 2'S, 6R)-5,6,7,8-tetrahydrobiopterin], and calmodulin (CaM) (2;3).

All of the NOS proteins share similar protein domains including an N-terminal oxygenase domain and a C-terminal reductase domain connected by a CaM-binding region (amino acids 490-509 in mouse NOS3) (Figure 3). Upon calcium-induced binding of CaM, an increased rate of electron transfer from NADPH to the reductase domain flavins and from the reductase domain to the heme center for the oxidation of the L-arginine substrate is induced. The NOS proteins differ in the region N-terminal to the oxygenase domain; that N-terminal peptide has an undefined function in NOS3.

The oxygenase domain of NOS3 (amino acids 39-482) has binding sites for heme, L-arginine, and BH4. The crystal structure of the bovine NOS3 heme domain has been solved (PDB:4NSE) (4). The heme domain is a tightly packed dimer and is a member of the α/β protein class. A zinc ion coordinates with Cys residues (Cys 96 and Cys101) from both monomers within a conserved Cys-(X)4-Cys motif. The Cys-(X)4-Cys motif is proposed to maintain the structure of the BH4-binding site. The metal center in NOS3 is located at the bottom of the dimer interface. Val106 forms a direct nonbonded contact with BH4. Without BH4, NO synthesis is reduced and superoxide is generated.

The NOS3 reductase domain (amino acids 519-1001) has binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), FMN (amino acids 519-702), and FAD (amino acids 755-1001). The reductase domain is homologous to NADPH:cytochrome P450 reductase and other flavoproteins.

NOS3 activity is regulated by several factors including lipid modification (e.g., N-myristoylation at Gly2 and S-palmitoylation at Cys15 and Cys26), phosphorylation, O-linked glycosylation, and S-nitrosylation as well as protein-protein interactions and substrate and cofactor availability (5-7). N-Myristoylation is essential for the proper localization of NOS3 to the plasma membrane (5;6).  Palmitoylation regulates the trafficking of NOS3 from the Golgi to the plasma membrane; mutants that are not palmitoylated are membrane-bound, but do not traffic to the plasma membrane (7;8). Association of NOS3 with some agonists [e.g., bradykinin (9), estradiol (10), ceramide (11), and vascular endothelial growth factor (VEGF) (12)] results in depalmitoylation, mediating NOS3 translocation from the plasma membrane to the cytoplasm. S-glutathionylation (i.e., formation of a disulfide bond between the reactive Cys-thiol and reduced glutathione) of NOS3 reduces NOS activity with a concomitant increase in superoxide production (13).

NOS3 can be phosphorylated on several serine, threonine, and tyrosine residues including Tyr81, Ser114, Thr495, Ser615, Ser633, Tyr657, and Ser1177 (human NOS3). Src-mediated phosphorylation of Tyr81 occurs upon acetylcholine, angiopoietin, ATP, BK, estrogen, S1P, thapsigargin, and VEGF stimulation, but does not alter the activity of NOS3 (14-16). Ser114 phosphorylation is constitutive and results in reduced NOS3 activity (17;18). Thr495 is consitutively phosphorylated by protein kinase C in response to Ox-LDL and O2, resulting in reduced NOS3 activity (19-22). Ser615 phosphorylation by AMPK, PKA, and/or Akt occurs upon VEGF, hypoxia, and adiponectin stimulation, resulting in increased NOS3 activity (18;23). PKA-mediated Ser633 phosphorylation occurs upon fluid shear stress, VEGF, and bradykinin stimulation, but does not change NOS3 activity (23;24). Pyk2-mediated Tyr657 phosphorylation occurs upon fluid shear stress, insulin, or angiotensin stimulation, resulting in reduced NOS3 activity (14-16). Akt-mediated Ser1177 phosphorylation occurs upon stimulation with VEGF, insulin, or estrogen as well as upon shear stress (22;25;26). Ser1177 can also be phosphorylated by CaMKII upon stimulation with bradykinin (19) and by PKA upon stimulation with fluid shear stress (17;22;25-27) . Phosphorylation of Ser1177 results in increased activity, while loss of phosphorylation in mutant mice results in hypertension, decreased vascular activity, insulin resistance, hyperinsulinemia, adiposity, and increased weight gain.

NOS3 is a member of an enzyme complex that consists of a NOS3 dimer, each monomer bound to a CaM, and several adaptor and regulatory proteins (Table 1).

Table1. NOS3-interacting proteins

NOS3-associated protein

Brief protein description

NOS3 regulation

References

Calmodulin

Calcium-binding protein

CaM binding to NOS3 is modulated by phosphorylation/dephosphorylation of Thr495; CaM binding aligns the oxygenase and reductase domains, resulting in efficient NO synthesis

(19;28)

Caveolin-1

Scaffold protein

Associates with amino acids 350-358 in NOS3 to antagonize CaM binding, subsequently inhibiting enzyme activity, and is essential for the intracellular localization of NOS3 to caveolae

(16;29)

Hsp90

Chaperone

Hsp90 association with NOS3 is dependent on phosphorylation of both Hsp90 and NOS3 in conditions of sheer stress; Hsp90 regulates the folding of NOS3 and the insertion of heme into immature protein

(30)

Platelet-endothelial cell adhesion molecule-1 (PECAM-1)

Cell adhesion molecule that regulates

leukocyte transmigration, cell migration, cell adhesion, and angiogenesis

Regulates NOS3 activity in response to shear stress. NOS3 regulation by PECAM-1 occurs through STAT3-mediated transcriptional control of NOSTRIN

(31-33)

Gab1 and SHP2

Adaptor protein (Gab1); tyrosine phosphatase (SHP2)

NOS3 activation upon fluid shear stress is dependent on association with Gab1 and SHP2

(34)

Dynamin

GTPase in endocytosis

Binds the reductase domain of NOS3 to regulate enzyme activity and subcellular localization; increases NO production

(35;36)

NOSIP

NOS3-interacting protein

Binds the C-terminus of the oxygenase domain to regulate the localization of NOS3 and calcium-induced activation

(37)

NOSTRIN

NOS3 traffic inducer)

(38)

Actin

Protein that forms microfilaments

Polymerization state of actin regulates NOS3 binding to Hsp90; formation of an actin-NOS3-Hsp90 complex leads to increased NOS activity

(39)

The paul mutation results in a threonine (T) to isoleucine (I) substitution at position 572 (T572I) within the flavodoxin region of the reductase domain.

Expression/Localization

NOS3 is constitutively expressed by vascular endothelial cells as well as endothelial cells in the brain, lung, liver, kidney, spleen, gastrointestinal tract, testis, and cervix (40-45). NOS3 is expressed in the embryonic heart of the mouse as early as embryonic day 9.5 (E9.5) (46). After E19.5, the expression level of NOS3 is low and remains low through adulthood primarily in the myocardium. In the kidney, NOS3 is expressed in the outer medulla and the thick ascending limb of the loop of Henle. In the epithelial cells of the lung, NOS3 is localized to the apical membrane in the basal body of the microtubules of the cilia (45). NOS3 is also expressed in hippocampal neurons, platelets, and cardiac myoctyes (47;48). In the brain, NOS3 localizes to the ciliated epithelium of the ependyma (45). NOS3 is predominantly localized to the plasma membrane and in the Golgi. At the plasma membrane, NOS3 is localized to caveolae (49).

NOS3 mRNA and protein levels can be regulated by viscous drag of blood flowing over the endothelial cell surface both in cultured endothelial cells and in intact arteries. NOS3 is also regulated by several hormones including estradiol, bradykinin, and VEGF in addition to elevation in cytosolic calcium levels (10;50;51). NOS3 activation by shear stress and isometric vessel contraction is independent of changes in the levels of intracellular calcium (52;53). Tumor necrosis factor-α (TNF-α) activates NOS3 in HeLa and microvascular endothelial cells (54;55).

Background
Figure 4. Endothelium and NO. Several stimuli, including acetylcholine, shear stress, and bradykinin activate NOS3 through the release of intracellular calcium. L-Arginine is converted to L-citrulline by NOS3 with subsequent production of NO. NO diffuses into vascular smooth muscle cells and stimulates guanylate cyclase, leading to cGMP production and vasodilation.

The NOS proteins catalyze the conversion of L-arginine and oxygen into L-citrulline and nitric oxide (NO) in a NADPH-dependent reaction (Figure 4). The conversion of L-arginine to L-citrulline occurs in a two-step oxidation process. The reaction consumes 1.5 mol of NADPH and 2 mol of oxygen per mol of L-citrulline formed. In the initial reaction, L-arginine is hydroxylated, leading to the formation of NG-hydroxy-l-arginine, an alternative substrate of NOS. The intermediate is subsequently oxidated using one electron from NADPH, forming L-citrulline and NO. NOS can also catalyze the production of superoxide.

NO is a signaling molecule that functions in several physiological processes including cell growth, apoptosis, neurotransmission, and immune system regulation. During normoxic cellular conditions, NOS-derived NO is oxidized to nitrite and nitrate. In immune system regulation, NO reduces oxidative stress by inhibiting the recruitment of neutrophils to sites of inflammation. NO also reacts with, and inactivates, superoxide. NO inhibits the function of NADPH oxidase, a superoxide generating system found in phagocytes (56).

Endothelial cells found in the microvasculature line blood and lymphatic vessels, regulating vascular supply and immune cell emigration. Upon exposure to cytokines (e.g., IL-6, IL-23, IL-12 and TNF-α) and growth factors, endothelial cells undergo rapid changes. NO reduces leukocyte and platelet adhesion to the endothelium as well as endothelial permeability (57;58) and vasodilation (59). NO-mediated changes to endothelial permeability is due to increased guanylate cyclase and phospholipase C activity leading to increased intracellular calcium as well as activation of the Ras/Raf/PKC/ERK pathway, which leads to increased actin contractility (Figure 4) (57;60;61). NOS3 constitutively produces NO in the vasculature. NOS3-produced NO mediates neovascularization, vessel wall tension, platelet aggregation, vascular permeability and the interaction between leuckocytes and endothelial cells [reviewed in (62)].

NOS3 has several known functions. In the heart, NOS3-derived NO mediates vasodilation, vascular homeostasis, morphogenesis of coronary arteries, myocardial capillary development, and angiogenesis [(59); reviewed in (63)]. NOS3-derived NO regulates voltage-dependent L-type calcium currents in cardiomyocytes as well as cardiomyogenesis during embryonic development (46;64;65). NO is proposed to function in cardiac cells to alter basal contractility by activating the nitrosylation of the ryanodine receptor 2 (RyR2) (66), by directing S-nitrosylatin of the L-type calcium channel (67), by mediating cGMP-independent activation of adenylyl cyclase (68), by mediating a cGMP-dependent increase in cAMP (69), and by assisting in a PKG-mediated activation of the RyR (70). Transgenic mice that overexpress NOS3 in the vascular wall exhibited low blood pressure as well as reduced vascular reactivity compared to wild-type littermates (71). In the kidney, NOS3-produced NO inhibited NaCl and NaHCO3 absorption through the inhibition of transporters including the Na/K/2Cl co-transporter and Na/H exchangers (44;72;73). In the lung, NOS3-produced NO stimulates ciliary beat frequency to regulate the amount and composition of airway surface liquid (47). In the bone marrow, NOS3-derived NO regulates the mobilization of bone marrow-derived hematopoietic stem and progenitor cells (74;75).

Inhibition of NO results in increased neutrophil recruitment, increased oxidative stress, mast cell degranulation, and increased microvascular and epithelial permeability (76-79). NOS3 functions during pro-inflammatory reactions in immune cells by regulating iNOS expression in response to endotoxin in an animal model of sepsis through the regulation of nuclear factor κB (NF-κB) (80;81). In Nos3-/- mice, iNOS expression was reduced in the liver, lung, heart, and aorta compared to that in wild-type mice (82). In addition, sepsis-associated systemic hypotension and mortality were reduced in the Nos3-/- mice compared to wild-type mice with experimentally-induced sepsis.

Nos3-/- mice exhibit developmental vascular defects in certain organs including heart, aorta, lung, and kidney with a concomitant increased rate of postnatal mortality compared to wild-type mice (83-89). Nos3 deficiency leads to several vascular disorders including vasodilation and hypertension as well as accelerates vascular diseases (87;90-95). Nos3-/-  mice exhibited pulmonary congestion with focal alveolar edema as well as congenital atrial and ventricular septal defects (85). Loss of Nos3 expression results in cardiac hypertrophy, congenital septal defects, and postnatal heart failure [85% by postnatal day 7 (P7)] (85). NOS3 also has a role in non-endothelial sites including cardiomyocytes, osteoblasts, adipocytes, renal tubules, and erythrocytes (96-101). Nos3-/- female mice exhibited longer estrous cycles and reduced ovulation rate than wild-type mice (102;103). Nos3-/- mice exhibited less activity than wild-type mice in open field habituation as well as accelaeratued place learning the water maze due to changes in sensorimotor capacities (104). Compared to wild-type mice, the Nos3-/- mice had higher dopamine turnover in the ventral striatum as well as increased concentrations of the serotonin metabolite 5-HIAA in the cerebellum and an accelerated serotonin turnover in the frontal cortex (104). Nos3-/- mice also exhibit defects in lung morphogenesis, leading to respiratory distress and death in most of the animals (83). The lungs of the neonatal animals had thickening of saccular septae and reduced surfactant material as well as regions of capillary hypoperfusion and misalignment of pulmonary veins (83). Loss of Nos3 expression results in increased glomerulosclerosis, mesangiolysis, and tubular damage of the kidney as well as loss of endothelial cells due to reduced endothelial cell proliferation and increased apoptosis. Glomerular and tubulointersitial injury with a loss of glomerular capillaries and peritubular capillaries was also observed upon loss of Nos3 expression (84). Nos3-/- mice exhibit fasting hyperinsulinemia, hyperlipidemia and reduced (by 40%) insulin-stimulated glucose uptake compared to wild-type mice due to impaired NO synthesis (105).

In humans, NOS3 deficiency can result in bicuspid aortic valves leading to aortic valve stenosis, endocarditis, aortic aneurysm, and aortic dissection. Loss of NOS3 function inhibits tumor initiation and maintenance (106). NOS3 mediates an increase in the nitrosylation and activation of Ras, a required factor throughout tumorigenesis, indicating that activation of a PI3K-AKT-NOS3-Ras pathway by oncogenic Ras in cancer cells mediates tumor growth. The region of chromosome 7q36 encoding NOS3 is a locus for susceptibility to familial pregnancy-induced hypertension syndrome (OMIM: 189800) (107), susceptibility to late-onset Alzheimer’s disease (OMIM: 104300), susceptibility to coronary artery spasm 1, susceptibility to hypertension (OMIM:145500), and placental abruption. In the German population, the Glu298Asp (894G>T) polymorphism in NOS3 is linked to increased risk of ischemic stroke onset [OMIM:601367; (108)]. The Glu298Asp mutation is common, but is not associated with the occurrence or severity of coronary artery disease, in the Australian (109) and United Kingdom (110) populations. Patients homozygous for the Glu298Asp allele are at a moderately increased risk of ischemic heart disease (111). Homozygosity of the Glu298Asp allele are at higher risk for developing late-onset Alzheimer’s disease (112). In addition, the Glu298Asp variant is common in patients with Fabry disease, a lysosomal storage disease that causes progressive renal failure, cardiac disease, and skin lesions, among other symptoms (113). The Glu298Asp variant has been linked to South African pre-eclamptic patients that are predisposed to developing abruption placentae (114).

Putative Mechanism
Figure 5. NOS function in inflammatory bowel disease. In IBD, angiogenesis of the intestinal microvasculature maintains inflammation by altering the endothelial lining of the vessels. The endothelium mediates the recruitment of inflammatory cells, tissue damage, and the production of inflammatory mediators. During cases of inflammation, the endothelial changes of the intestinal vasculature occurs in response to cytokines, chemokines and growth factors released by immune cells. In addition to angiogenesis, the changes in the vasculature leads to adhesion molecule expression, leukocyte extravasation, decreased endothelial barrier function and increased coagulation. Figure is adapted from Cromer, W.E. et al. 2011.

During normal conditions, NOS3-derived NO is a scavenger that absorbs O2- and also functions to generate the oxidant peroxynitrite (ONOO-). In inflammatory bowel disease (IBD), NOS3 expression is reduced, leading to reduced endothelium-dependent vasodilation and increased oxidant formation (Figure 5) (115). Loss of Nos3 expression leads to an increased severity of IBD (116;117). Treatment of Nos3-/- mice with 3% DSS led to an increased rate of weight loss, bloody stool, and histopathology including disrupted tissue architecture, edema, and immune cell infiltration compared to DSS-treated wild-type mice (117). The Nos3-/- mice exhibited more gut injury and higher expression levels of the mucosal addressin MAdCAM-1 compared to DSS-treated wild-type mice. Similar to the Nos3-/- mice , homozygous paul mice also exhibit weight loss after treatment with low dose (1.5%) DSS, indicating loss of NOS3 function as the result of the mutation. Exposure of HUVECs to serum from patients with Crohn’s disease led to a reduction in NOS3, while exposure to serum from patients with ulcerative colitis led to an increase in NOS3 expression (118). Chronic administration of NOS inhibitors can cause intestinal inflammation (119;120). Studies using NOS inhibitors N(G)-nitro-L-arginine-methyl ester (L-NAME) and NG-Monomethyl-L-Arginine (L-NMMA) indicated that loss of NO leads to increased mucosal permeability (121). The increased permeability can be reversed by treatment with NO donors, L-arginine, or cGMP donors.

Primers PCR Primer
paul_pcr_F: ACAAGGATTCAGCGTGCAATCAGG
paul_pcr_R: AGAAGTGCCGTCCCCATATCAAAAG

Sequencing Primer
paul_seq_F: GGCCTTGTCTTGCCTAAGTG
paul_seq_R: TACAGGAATCAAGGTTCCCAG
Genotyping

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold


The following sequence of 428 nucleotides is amplified (chromosome 5, + strand):


1   acaaggattc agcgtgcaat caggccttgt cttgcctaag tgaggacaga gacaggagga
61  agacaagcgt caacacccat cttctcaccg caggtcctgt gcatggatga gtatgatgtg
121 gtgtccctag agcacgaggc actggtgttg gtggtgacca gcacatttgg caatggggat
181 cctccggaga atggagaggt aaggctttca ggagaaaaga ttcaaaacag ggccagctag
241 acaggtggat gcaaacacac acacacacac tcacacacac actcacatat acacagcagc
301 atggaaagtt aatatgctaa gtagccccac ttcctgatgg ggatgtgaag tgctgggaac
361 cttgattcct gtatccccag ccatctccat tatctgtgca actcttttga tatggggacg
421 gcacttct


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsEmre Turer and Bruce Beutler