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

Abstract

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). 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. 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). 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.