Functional significance of a single nucleotide polymorphism in the gene encoding endothelial nitric oxide synthase

• Applicant: Professor Markus Hecker, Ph.D.
• Subject Area: Anatomy and Physiology ; Cardiology, Angiology ; Rheumatology
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 321001878

A single nucleotide polymorphism (SNP) within the promoter of the endothelial nitric oxide (NO) synthase (NOS3) gene (T-786C, rs2070744) adversely affects the response of endothelial cells (EC) from CC-genotype individuals to shear stress or the prototypic anti-type 1 T-helper (Th1) cell cytokine interleukin-10. Homozygosity for the C-allele, which occurs in about 12% of Caucasians, is a strong predictor for coronary heart disease, polymyalgia rheumatica or rheumatoid arthritis. In a preceding DFG-funded project, we identified a compensatory mechanism involving manganese-dependent superoxide dismutase that helps to maintain the bioavailability of EC-derived NO. With the shear stress-stimulated release of 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) from CC- but not TT-genotype ECs, we have characterised another compensatory mechanism that may not only support the anti-inflammatory capacity of the CC-genotype ECs but also act as a novel general defence mechanism against chronic inflammation. Moreover, data acquired since then point to a possible epigenetic control of NOS3 expression through chromatin remodelling that may differ in at least two aspects between CC- and TT-genotype ECs. With this proposal we plan to (i) establish that STAT3-guided recruitment of the histone-acetyltransferase p300/CBP and/or the histone-lysine N-methyltransferase SETD7 to the CpG-dinucleotide at position -786 in the NOS3 gene (CC-genotype) and the degree of histone H3 acetylation and/or methylation associated therewith results in a different accessibility, e.g. by STAT3, of the distal NOS3 promoter. Alternatively, p300/CBP may serve as a scaffold coordinating the binding of, e.g. STAT3 to its cis-acting element at position -850 to -842, thereby enabling and/or accelerating trans-activation of the NOS3 gene. Moreover, by (ii) generating the corresponding knock-in mice harbouring the human C- or T-type NOS3 promoter on a disease-susceptible genetic background, we plan to verify that the T-786C SNP of the human NOS3 gene, if present, boosts the development of arteriosclerosis and/or arthritis in these animals. The reason for this complex experimental approach is that instead of the 5'-GGC(T->C)GG-3' motif found in humans the murine Nos3 promoter contains a 5'-GGCCAT-3'motif so that the suspected critical CpG-dinucleotide cannot form. Finally, we plan to (iii) corroborate in an in vitro transmigration model mimicking shear stress conditions at arteriosclerosis predilection sites that the T-786C SNP of the human NOS3 gene differentially affects Th1 and Th17 cell-endothelial cell interaction, respectively, through 15d-PGJ2. Furthermore, we will extend our analysis of the plasma levels of 15d-PGJ2 to patients with rheumatoid arthritis to prove that this prostanoid is both a marker and a pivotal constituent of a novel anti-inflammatory defence mechanism.

• DFG Programme: Research Grants