The -786C/T single-nucleotide polymorphism in the promoter of the gene for endothelial nitric oxide synthase: insensitivity to physiologic stimuli as a risk factor for rheumatoid arthritis.
OBJECTIVE
Shear stress is the main physiologic stimulus for the expression of NOS3, the gene for human endothelial nitric oxide synthase. Interestingly, a promoter variant of the NOS3 gene, the -786C variant, is insensitive to shear stress, and individuals homozygous for this single-nucleotide polymorphism (SNP) have an increased risk of developing coronary artery disease. The cytokine interleukin-10 (IL-10) is also capable of up-regulating endothelial NOS3 expression through binding of the transcription factor STAT-3 to a nearby promoter sequence. The aim of this study was to explore the possibility that the -786C variant of the NOS3 gene is also insensitive to IL-10 and that individuals with the -786C/C genotype are more prone to developing rheumatoid arthritis (RA).


METHODS
Endothelial cells were isolated from human umbilical cord veins, clonally expanded, and analyzed for NOS3 and IL-12 expression by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Umbilical cord arteries and blood samples from RA patients were genotyped for the -786C/T SNP of the NOS3gene.


RESULTS
In contrast to cells of other genotypes, endothelial cells of the -786C/C genotype did not reveal an increase in NOS3 expression upon exposure to IL-10, and the cytokine failed to suppress IL-12 expression upon stimulation of CD40. Preincubation of these cells with a 16-mer C-type decoy oligonucleotide fully reconstituted the defective IL-10-induced suppression of IL-12 synthesis. The frequency of the -786C/C genotype was significantly higher in the 596 RA patients than in the general population (19.1% versus 12.1%; P < 0.0001).


CONCLUSIONS
Individuals with the -786C/C genotype have an increased risk of developing RA. This may be explained by the IL-10 insensitivity of the C-type NOS3 gene promoter and the resulting failure to subdue CD40-mediated proinflammatory gene expression.
Melchers
Inga
I
University Medical Center, Freiburg, Germany.
Blaschke
Sabine
S
Hecker
Markus
M
Cattaruzza
Marco
M
D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000235:genetics* / Q000378:metabolism; D019013:CD40 Antigens / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D020022:Genetic Predisposition to Disease; D005838:Genotype; D006801:Humans; D016753:Interleukin-10 / Q000494:pharmacology; D018664:Interleukin-12 / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D052250:Nitric Oxide Synthase Type III / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012307:Risk Factors
D019013:CD40 Antigens; D016753:Interleukin-10; D018664:Interleukin-12; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III