BACKGROUND:Medial arterial calcification is a chronic systemic vascular disorder distinct from atherosclerosis and is commonly observed in patients with chronic kidney disease, diabetes, and aging individuals. We previously showed that NR4A3 (nuclear receptor subfamily 4 group A member 3), an orphan nuclear receptor, is a key regulator in apo (apolipoprotein) A-IV-induced atherosclerosis progression; however, its role in vascular calcification is poorly understood.METHODS:We generated NR4A3^−/− mice and 2 different types of medial arterial calcification models to investigate the biological roles of NR4A3 in vascular calcification. RNA-seq was performed to determine the transcriptional profile of NR4A3^−/− vascular smooth muscle cells under β-glycerophosphate treatment. We integrated CUT&Tag analysis and RNA-seq data to further investigate the gene regulatory mechanisms of NR4A3 in arterial calcification and target genes regulated by histone lactylation.RESULTS:NR4A3 expression was upregulated in calcified aortic tissues from chronic kidney disease mice, 1,25(OH)₂VitD₃ overload–induced mice, and human calcified aorta. NR4A3 deficiency preserved the vascular smooth muscle cell contractile phenotype, inhibited osteoblast differentiation-related gene expression, and reduced calcium deposition in the vasculature. Further, NR4A3 deficiency lowered the glycolytic rate and lactate production during the calcification process and decreased histone lactylation. Mechanistic studies further showed that NR4A3 enhanced glycolysis activity by directly binding to the promoter regions of the 2 glycolysis genes ALDOA and PFKL and driving their transcriptional initiation. Furthermore, histone lactylation promoted medial calcification both in vivo and in vitro. NR4A3 deficiency inhibited the transcription activation and expression of Phospho1 (phosphatase orphan 1). Consistently, pharmacological inhibition of Phospho1-attenuated calcium deposition in NR4A3-overexpressed vascular smooth muscle cells, whereas overexpression of Phospho1 reversed the anticalcific effect of NR4A3 deficiency in vascular smooth muscle cells.CONCLUSIONS:Taken together, our findings reveal that NR4A3-mediated histone lactylation is a novel metabolome-epigenome signaling cascade mechanism that participates in the pathogenesis of medial arterial calcification.

中文翻译：

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孤儿核受体 NR4A3 通过组蛋白乳酰化促进血管钙化

背景：内侧动脉钙化是一种不同于动脉粥样硬化的慢性全身性血管疾病，常见于慢性肾病、糖尿病和老年人中。我们之前表明，NR4A3（核受体亚家族 4 A 组成员 3）是一种孤儿核受体，是 apo（载脂蛋白）A-IV 诱导的动脉粥样硬化进展的关键调节因子。方法：我们构建了NR4A3 ^−/−小鼠和2种不同类型的内侧动脉钙化模型，以研究NR4A3在血管钙化中的生物学作用。进行 RNA-seq 以确定 β-甘油磷酸盐处理下 NR4A3 ^−/−血管平滑肌细胞的转录谱。我们整合CUT&Tag分析和RNA-seq数据，进一步研究NR4A3在动脉钙化中的基因调控机制以及组蛋白乳酰化调控的靶基因。结果：慢性肾病小鼠钙化主动脉组织中NR4A3表达上调，1,25(OH) ) ₂ VitD ₃超载诱导的小鼠和人类主动脉钙化。 NR4A3 缺乏保留了血管平滑肌细胞的收缩表型，抑制了成骨细胞分化相关基因的表达，并减少了脉管系统中的钙沉积。此外，NR4A3 缺乏会降低钙化过程中的糖酵解速率和乳酸生成，并减少组蛋白乳酰化。机理研究进一步表明，NR4A3 通过直接结合 2 个糖酵解基因ALDOA和PFKL的启动子区域并驱动其转录起始来增强糖酵解活性。此外，组蛋白乳酰化在体内和体外均促进内侧钙化。 NR4A3 缺陷会抑制 Phospho1（磷酸酶孤儿 1）的转录激活和表达。一致地，对 NR4A3 过表达的血管平滑肌细胞中 Phospho1 的药理学抑制作用减弱了钙沉积，而 Phospho1 的过表达逆转了血管平滑肌细胞中 NR4A3 缺乏的抗钙作用。 结论：综上所述，我们的研究结果表明，NR4A3 介导的组蛋白乳酰化是一种新型的代谢组-表观基因组信号级联机制，参与内侧动脉钙化的发病机制。