Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Gene. 2014 Apr 25;540(1):62-7. doi: 10.1016/j.gene.2014.02.026. Epub 2014 Feb 21.
NADPH oxidase 4 (NOX4) is a member of the NADPH oxidase gene family that regulates cellular differentiation, innate immunity and tissue fibrosis. Transforming growth factor-β (TGF-β1) is known to induce expression of NOX4 mRNA in mesenchymal cells. However, the mechanisms of transcriptional regulation of NOX4 are not well understood. In this study, we examined the transcriptional regulation of NOX4 in human lung fibroblasts by TGF-β1. Five promoter-reporter constructs containing DNA fragments of 0.74kb, 1.35kb, 1.84kb, 3.97kb and 4.76kb upstream from the transcriptional start site (TSS) of the human NOX4 gene were generated and their relative responsiveness to TGF-β1 analyzed. TGF-β1-induced NOX4 gene promoter activation requires a region between -3.97kb and -4.76kb. Bioinformatics analysis revealed a 15bp AP-1/Smad binding element in this region. Mutation or deletion of either the AP-1 or the Smad element attenuated TGF-β1 responsiveness of the -4.76kb NOX4 promoter. Furthermore, insertion of this AP-1/Smad box conferred TGF-β1 inducibility to the non-responsive -3.97kb NOX4 promoter construct. Chromatin immunoprecipitation analysis indicated that phospho-Smad3 and cJun associate with this element in a TGF-β1-inducible manner. These results demonstrate that the AP-1/Smad box located between 3.97kb and 4.76kb upstream of the TSS site of the NOX4 promoter is essential for NOX4 gene transcription induced by TGF-β1 in human lung fibroblasts. Our study provides insights into the molecular mechanisms of NOX4 gene expression, informing novel therapeutic approaches to interfere with upregulation of NOX4 in diseases characterized by activation of the TGF-β1/NOX4 pathway.
NADPH 氧化酶 4（NOX4）是 NADPH 氧化酶基因家族的一员，可调节细胞分化、先天免疫和组织纤维化。转化生长因子-β（TGF-β1）已知可诱导间充质细胞中 NOX4mRNA 的表达。然而，NOX4 的转录调控机制尚不清楚。在这项研究中，我们通过 TGF-β1 检查了人肺成纤维细胞中 NOX4 的转录调控。生成了包含人 NOX4 基因转录起始位点（TSS）上游 0.74kb、1.35kb、1.84kb、3.97kb 和 4.76kb 的 DNA 片段的五个启动子-报告基因构建体，并分析了它们对 TGF-β1 的相对反应性。TGF-β1 诱导的 NOX4 基因启动子激活需要 TSS 上游-3.97kb 至-4.76kb 之间的区域。生物信息学分析显示该区域存在一个 15bp 的 AP-1/Smad 结合元件。该 AP-1 或 Smad 元件的突变或缺失减弱了-4.76kb NOX4 启动子对 TGF-β1 的反应性。此外，该 AP-1/Smad 盒的插入赋予了-3.97kb 非反应性 NOX4 启动子构建体对 TGF-β1 的诱导能力。染色质免疫沉淀分析表明，磷酸化 Smad3 和 cJun 以 TGF-β1 诱导的方式与该元件结合。这些结果表明，NOX4 启动子 TSS 位点上游 3.97kb 至 4.76kb 之间的 AP-1/Smad 盒对于 TGF-β1 诱导的人肺成纤维细胞中 NOX4 基因转录是必需的。我们的研究为 NOX4 基因表达的分子机制提供了深入了解，为通过干扰 TGF-β1/NOX4 途径激活来抑制疾病中 NOX4 的上调提供了新的治疗方法。
