Hepcidin expression in liver cells: evaluation of mRNA levels and transcriptional regulation
Hepcidin expression in liver cells: evaluation of mRNA levels and transcriptional regulation
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DOI：
10.1016/j.gene.2014.05.040
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发表时间：
2014-08-01
期刊：
影响因子：
3.5
通讯作者：
Funaba, Masayuki
中科院分区：
文献类型：
--
作者：
Kanamori, Yohei;Murakami, Masaru;Funaba, Masayuki
肝中产生的铁调素负调节肠铁吸收，并且骨形态发生蛋白（BMP）途径众所周知刺激铁调素表达。然而，铁调素表达的调控尚未完全阐明。在这项研究中，我们评估了不同的系统，可用于确定如何hepcidin表达的调节。肝细胞系如HepG 2细胞和Hepa 1 -6细胞中铁调素的基础表达低于肝和原代肝细胞中的表达;细胞系中铁调素的表达水平接近RT-PCR和RT-qPCR分析的检测限。用抑制素A、RNAlater或MG-132处理增强HepG 2细胞中的铁调素表达，表明组蛋白脱乙酰化、mRNA的不稳定性或正调节铁调素表达的蛋白质的蛋白质体降解可能是导致HepG 2细胞中铁调素表达降低的原因。在基于内切酶的报告基因测定中，BMP诱导HepG 2细胞和Hepa 1 -6细胞中报告基因hepcidin（-2018）-luc的转录，该报告基因包含hepcidin启动子的nt - 2018至nt - 35。然而，BRE-luc，一种用于评估BMP信号传导的代表性报告基因，在HepG 2细胞中对BMP无反应。这些结果表明，铁调素转录可以在肝细胞系中最好地评估，并且铁调素启动子以高灵敏度感测BMP信号传导。本研究表明，在mRNA水平上的铁调素表达的调控研究应在原代肝细胞中进行评估，肝细胞系非常适合研究铁调素的转录调控。(C)2014爱思唯尔有限公司版权所有。
Hepcidin produced in the liver negatively regulates intestinal iron absorption, and the bone morphogenetic protein (BMP) pathway is well-known to stimulate hepcidin expression. However, the regulation of hepcidin expression has not been fully elucidated. In this study, we evaluate different systems that can be used to determine how hepcidin expression is regulated. The basal expression of hepcidin in liver cell lines, such as HepG2 cells and Hepa1-6 cells, was lower than that in the liver and primary hepatocytes; the expression levels of hepcidin in the cell lines were near the limit of detection for RT-PCR and RT-qPCR analyses. Treatment with trichostatin A, RNAlater, or MG-132 enhanced the expression of hepcidin in HepG2 cells, suggesting that histone deacetylation, instability of mRNA, or proteosomal degradation of the protein(s) that positively regulate hepcidin expression may be responsible for the decreased expression of hepcidin in HepG2 cells. In luciferase-based reporter assays, BMP induced the transcription of a reporter, hepcidin(-2018)-luc, that contains nt - 2018 through nt - 35 of the hepcidin promoter in HepG2 cells and Hepa1-6 cells. However, BRE-luc, a representative reporter used to evaluate BMP signaling, was unresponsive to BMP in HepG2 cells. These results suggest that hepcidin transcription can be best evaluated in liver cell lines and that the hepcidin promoter senses BMP signaling with high sensitivity. The present study demonstrates that studies regarding the regulation of hepcidin expression at the mRNA level should be evaluated in primary hepatocytes, and liver cell lines are well-suited for studies examining the transcriptional regulation of hepcidin. (C) 2014 Elsevier B.V. All rights reserved.