McNeill Eileen, Crabtree Mark J, Sahgal Natasha, Patel Jyoti, Chuaiphichai Surawee, Iqbal Asif J, Hale Ashley B, Greaves David R, Channon Keith M
Division of Cardiovascular Medicine, British Heart Foundation Centre of Research Excellence, John Radcliffe Hospital, University of Oxford, Oxford, UK; Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
Free Radic Biol Med. 2015 Feb;79:206-16. doi: 10.1016/j.freeradbiomed.2014.10.575. Epub 2014 Nov 6.
Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1(fl/fl)) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1(fl/fl)Tie2cre). Macrophages from Gch1(fl/fl)Tie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1(fl/fl)Tie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1(fl/fl)Tie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1(fl/fl)Tie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1(fl/fl)Tie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1(fl/fl)Tie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.
诱导型一氧化氮合酶（iNOS）是巨噬细胞炎症反应中的关键酶，它是在促炎刺激下强烈诱导产生的一氧化氮（NO）的来源。然而，在巨噬细胞炎症反应中，与iNOS诱导不同的NO产生的具体作用仍未得到证实。我们构建了一种新型小鼠模型，该模型条件性缺失编码GTP环化水解酶1（GTPCH）的Gch1，GTPCH是四氢生物蝶呤（BH4）生物合成中的一种必需酶，而BH4是iNOS产生NO所需的辅助因子。将带有floxed Gch1等位基因的小鼠（Gch1(fl/fl)）与Tie2cre转基因小鼠杂交，导致白细胞中Gch1缺失（Gch1(fl/fl)Tie2cre）。Gch1(fl/fl)Tie2cre小鼠的巨噬细胞缺乏GTPCH蛋白和从头生物蝶呤生物合成。当用脂多糖（LPS）和干扰素γ（IFNγ）激活时，Gch1(fl/fl)Tie2cre小鼠的巨噬细胞诱导iNOS蛋白的方式与野生型对照无明显差异，但通过L-瓜氨酸产生、NO的电子顺磁共振自旋捕获以及亚硝酸盐积累判断，未产生可检测到的NO。用二氢乙锭孵育Gch1(fl/fl)Tie2cre巨噬细胞发现，在iNOS表达的情况下超氧化物产生显著增加，并且在其他活性氧（ROS）种类中存在不依赖iNOS、依赖BH4的增加。通过补救途径产生BH4的前体物质蝶酰三谷氨酸可恢复正常的BH4水平、一氧化氮产生和细胞氧化还原状态，这表明BH4缺乏的影响是可逆的。Gch1(fl/fl)Tie2cre巨噬细胞在细胞因子产生方面仅表现出轻微改变，细胞迁移正常，基础基因表达变化最小。然而，iNOS诱导后的基因表达分析确定了野生型和Gch1(fl/fl)Tie2cre巨噬细胞之间有78个基因发生了改变。通路分析发现NRF2激活减少，在BH4缺乏的Gch1(fl/fl)Tie2cre巨噬细胞中，典型的NRF2基因（gclm、prdx1、gsta3、nqo1和过氧化氢酶）的诱导减少。这些发现确定了BH4依赖的iNOS调节和NO产生是巨噬细胞炎症激活中NRF2依赖反应的特定要求。
