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

内皮一氧化氮合酶基因编码单核苷酸多态性的功能意义

• 批准号: 321001878
• 负责人: Professor Dr. Markus Hecker, Ph.D.
• 金额: --
• 依托单位: Institut für Physiologie und Pathophysiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/321001878?language=en
• 关键词: Functional; significance; single; nucleotide; polymorphism

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.

内皮一氧化氮（NO）合成酶（NOS3）基因启动子（T-786C, rs2070744）内的单核苷酸多态性（SNP）对cc基因型个体内皮细胞（EC）对剪切应激或原型抗1型t辅助（Th1）细胞因子白介素-10的反应产生不利影响。约12%的白种人存在c等位基因的纯合性，这是冠心病、风湿性多肌痛或类风湿性关节炎的一个强有力的预测指标。在之前dfg资助的一个项目中，我们确定了一种涉及锰依赖性超氧化物歧化酶的补偿机制，该机制有助于维持ec衍生NO的生物利用度。随着剪切应力刺激15-deoxy-delta12,14-前列腺素J2 （15d-PGJ2）从CC-而不是tt -基因型ec中释放，我们发现了另一种代偿机制，它不仅支持CC-基因型ec的抗炎能力，而且还作为一种新的抗慢性炎症的一般防御机制。此外，从那时起获得的数据指出，CC-和tt -基因型ECs之间可能存在至少两个方面的差异，即通过染色质重塑对NOS3表达的表观遗传控制。根据这一建议，我们计划(i)确定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对NOS3基因（cc基因型）-786位cpg -二核苷酸的募集，以及与之相关的组蛋白H3乙酰化和/或甲基化程度，会导致NOS3远端启动子的不同可及性，例如STAT3。或者，p300/CBP可以作为一个支架，协调STAT3与其位于-850至-842位置的顺式作用元件的结合，从而启用和/或加速NOS3基因的反式激活。此外，通过（ii）产生具有疾病易感遗传背景的人类C型或t型NOS3启动子的相应敲入小鼠，我们计划验证人类NOS3基因的T-786C SNP，如果存在，会促进这些动物动脉硬化和/或关节炎的发展。采用这种复杂的实验方法的原因是，在人类中发现的5‘- ggc (T->C)GG-3’基序中，小鼠Nos3启动子含有5‘- ggccat -3’基序，因此无法形成可疑的关键cpg -二核苷酸。最后，我们计划（iii）在模拟动脉硬化易感部位剪切应力条件的体外迁移模型中证实，人类NOS3基因的T-786C SNP分别通过15d-PGJ2差异地影响Th1和Th17细胞与内皮细胞的相互作用。此外，我们将对类风湿关节炎患者的血浆15d-PGJ2水平进行分析，以证明这种前列腺素既是一种标志物，也是一种新型抗炎防御机制的关键组成部分。