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

内皮型一氧化氮合酶(NOS3)基因启动子(T-786C，rs2070744)内的单核苷酸多态(SNP)对CC-型个体的内皮细胞(EC)对剪切力或典型的抗1型T辅助细胞因子IL-10的反应产生不利影响。C等位基因纯合性在大约12%的高加索人中出现，是冠心病、风湿性多肌痛或类风湿性关节炎的有力预测因素。在之前DFG资助的一个项目中，我们确定了一种涉及锰依赖的超氧化物歧化酶的代偿机制，有助于维持EC来源的NO的生物利用度。随着剪应力刺激CC-型而不是TT-型内皮细胞释放15-脱氧-β-12，14-前列腺素J2(15d-PGJ2)，我们表征了另一种代偿机制，不仅支持CC-型内皮细胞的抗炎能力，还可能作为一种新的一般防御机制来对抗慢性炎症。此外，从那时起获得的数据表明，可能通过染色质重塑对NOS3的表达进行表观遗传控制，这可能在CC和TT基因型的ECs之间至少有两个方面的不同。根据这一建议，我们计划：(I)建立STAT3引导的组蛋白-乙酰转移酶p300/CBP和/或组蛋白-赖氨酸N-甲基转移酶SETD7到NOS3基因(CC-基因型)-786位的CpG-二核苷酸的招募，以及与之相关的组蛋白H3乙酰化和/或甲基化的程度导致NOS3远端启动子的不同可及性，例如通过STAT3。或者，p300/CBP可以作为支架，协调例如STAT3与其位于-850到-842位的顺式作用元件的结合，从而实现和/或加速NOS3基因的反式激活。此外，通过(Ii)在疾病易感的遗传背景上产生相应的含有人类C-或T型NOS3启动子的敲入小鼠，我们计划验证人类NOS3基因的T-786C SNP如果存在，会促进这些动物的动脉硬化和/或关节炎的发展。这种复杂的实验方法的原因是，小鼠NOS3启动子包含5‘-GGCCAT-3’基序，而不是在人类中发现的5‘-GGC(T-&GT；C)GG-3’基序，因此不能形成可疑的关键CpG-二核苷酸。最后，我们计划(Iii)在模拟动脉硬化易发部位切应力条件的体外迁移模型中证实，人NOS3基因的T-786C SNP通过15d-PGJ2分别对Th1和Th17细胞-内皮细胞相互作用产生不同影响。此外，我们将把我们对15d-PGJ2血浆水平的分析扩展到类风湿关节炎患者，以证明这种前列腺素既是一种标志物，也是一种新的抗炎防御机制的关键成分。

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.