Advanced Institute for Medical Sciences, Dalian Medical University, Dalian, Liaoning, 116044, China; Department of Endocrinology, Dalian Municipal Central Hospital, Dalian Medical University, Dalian, Liaoning, 116033, China.
Advanced Institute for Medical Sciences, Dalian Medical University, Dalian, Liaoning, 116044, China.
Exp Cell Res. 2020 May 1;390(1):111949. doi: 10.1016/j.yexcr.2020.111949. Epub 2020 Mar 5.
Farnesoid X receptor (FXR) is a metabolic nuclear receptor, which protects liver from many endogenous and exogenous injuries. Metallothioneins (MTs) belong to a low-molecular-weight protein family involved in metal homeostasis and the regulation of hepatic oxidative stress. In the present study, we aimed to investigate the effect of FXR on hepatic MT1 expression and the underlying mechanism. C57BL/6 mice or primary cultured mouse hepatocytes were treated with the synthetic FXR ligand GW4064 or natural ligand CDCA. RNA-Sequencing (RNA-seq) analysis was performed to identify gene expression profile in the livers of mice treated with GW4064. Real-time PCR and Western blot were applied to determine the expression of MT1 and other FXR target genes in the livers of mice and primary hepatocytes treated with GW4064 and CDCA. Cellular and subcellular locations of MT1 in the livers of mice treated with GW4064 were examined using immunohistochemistry assay. FXR small interfering RNAs (siRNA) was transfected to silence FXR. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were utilized to confirm the regulation of MT1 gene promoter activity by FXR. RNA-seq analysis revealed that GW4064 treatment significantly induced MT1 expression in mouse liver. Consistently, MT1 expression in the hepatocytes of mouse livers and cultured hepatocytes was upregulated by GW4064 as well as CDCA. In addition, adenovirus-mediated overexpression of FXR markedly increased, while siRNA-mediated FXR silencing significantly suppressed MT1 expression in cultured hepatocytes. Luciferase reporter and ChIP assays further confirmed that the MT1 gene was under the direct control of FXR. Collectively, our findings demonstrate that MT1 is a novel target gene of FXR and may contribute to antioxidative capacity of FXR in liver diseases.
法尼醇 X 受体 (FXR) 是一种代谢核受体，可保护肝脏免受许多内源性和外源性损伤。金属硫蛋白 (MTs) 属于参与金属稳态和肝氧化应激调节的低分子量蛋白家族。在本研究中，我们旨在研究 FXR 对肝 MT1 表达的影响及其潜在机制。用合成 FXR 配体 GW4064 或天然配体 CDCA 处理 C57BL/6 小鼠或原代培养的小鼠肝细胞。进行 RNA 测序 (RNA-seq) 分析以鉴定用 GW4064 处理的小鼠肝脏中的基因表达谱。实时 PCR 和 Western blot 用于测定用 GW4064 和 CDCA 处理的小鼠和原代肝细胞中 MT1 和其他 FXR 靶基因的表达。用免疫组织化学检测用 GW4064 处理的小鼠肝脏中 MT1 的细胞和亚细胞定位。用 FXR 小干扰 RNA (siRNA) 转染沉默 FXR。利用荧光素酶报告基因和染色质免疫沉淀 (ChIP) 测定来证实 MT1 基因启动子活性受 FXR 的调节。RNA-seq 分析显示，GW4064 处理可显著诱导小鼠肝脏中 MT1 的表达。一致地，GW4064 和 CDCA 也可上调小鼠肝脏和培养的肝细胞中 MT1 的表达。此外，腺病毒介导的 FXR 过表达显著增加，而 siRNA 介导的 FXR 沉默则显著抑制培养的肝细胞中 MT1 的表达。荧光素酶报告基因和 ChIP 测定进一步证实 MT1 基因受 FXR 的直接调控。总之，我们的研究结果表明 MT1 是 FXR 的新靶基因，可能有助于 FXR 在肝脏疾病中的抗氧化能力。
