Vollmar Johanna, Kim Yong Ook, Marquardt Jens Uwe, Galle Peter R, Schuppan Detlef, Zimmermann Tim
Department of Internal Medicine II, Hospital of Worms, D-67550 Worms, Germany.
Institute of Translational Immunology, Fibrosis and Metabolism Centre, Johannes Gutenberg-University Mainz, D-55131 Mainz, Germany.
Exp Ther Med. 2021 Apr;21(4):349. doi: 10.3892/etm.2021.9780. Epub 2021 Feb 11.
Organic cation transporters (human, OCT; mouse, Oct) are responsible for the intracellular uptake and detoxification of a broad spectrum of endogenous and exogenous substrates. The OCT1 gene SLC22A1 (human; mouse, Scl22a1) is transactivated by hepatocyte nuclear factor 4α (human, HNF4α; mouse, Hnf4α). HNF4α is a master regulator of hepatocyte differentiation and is frequently associated with hepatocellular carcinoma (HCC). In addition, the downregulation of HNF4α is associated with enhanced fibrogenesis. Our recent study revealed that hepatocarcinogenesis and fibrosis were enhanced with the loss of Oct3 (gene, Slc22a3). Notably, differences in Hnf4α expression, and in cholestasis and fibrosis were also detected in Oct3-knockout (FVB.Slc22a3tm10pb, Oct3) mice. To the best of our knowledge, no data exists on an interaction between Oct3 and Hnf4α. We hypothesised that loss of Oct3 may have an impact on Hnf4α expression. In the present study, gene expression analyses were performed in liver tissue from untreated Oct3 and wild type (FVB, WT) mice. C57BL/6, Oct3 and WT mice were treated with pro-fibrotic carbon tetrachloride (CCl) or thioacetamide (TAA) for 6 weeks to chemically induce liver fibrosis. Cholestasis-associated fibrosis was mechanically generated in Oct3 and WT mice by bile duct ligation (BDL). Finally, stably OCT1- and OCT3-transfected tumour cell lines and primary murine hepatocytes were treated with the non-selective OCT inhibitor quinine and Hnf4α expression was quantified by qPCR and immunofluorescence. The results revealed that Hnf4α is one of the top upstream regulators in Oct3 mice. Hnf4α mRNA expression levels were downregulated in Oct3 mice compared with in WT mice during cholestatic liver damage as well as fibrogenesis. The downregulation of Hnf4α mRNA expression in fibrotic liver tissue was reversible within 4 weeks. In stably OCT1- and OCT3-transfected HepG2 and HuH7 cells, and primary murine hepatocytes, functional inhibition of OCT led to the upregulation of Hnf4α mRNA expression. Hnf4α was revealed to be located in the cytosol of WT hepatocytes, whereas Oct3 hepatocytes exhibited nuclear Hnf4α expression. In conclusion, Hnf4α was downregulated in response to cholestasis and fibrosis, and functional inhibition of Oct may lead to the upregulation of Hnf4α.
有机阳离子转运体（人类为OCT；小鼠为Oct）负责细胞内摄取和解毒多种内源性和外源性底物。OCT1基因SLC22A1（人类；小鼠为Scl22a1）由肝细胞核因子4α（人类为HNF4α；小鼠为Hnf4α）反式激活。HNF4α是肝细胞分化的主要调节因子，且常与肝细胞癌（HCC）相关。此外，HNF4α的下调与纤维化增强有关。我们最近的研究表明，Oct3（基因Slc22a3）缺失会增强肝癌发生和纤维化。值得注意的是，在Oct3基因敲除（FVB.Slc22a3tm10pb，Oct3）小鼠中也检测到Hnf4α表达、胆汁淤积和纤维化方面的差异。据我们所知，尚无关于Oct3与Hnf4α之间相互作用的数据。我们推测Oct3缺失可能会影响Hnf4α表达。在本研究中，对未处理的Oct3和野生型（FVB，WT）小鼠的肝脏组织进行了基因表达分析。用促纤维化的四氯化碳（CCl）或硫代乙酰胺（TAA）处理C57BL/6、Oct3和WT小鼠6周，以化学诱导肝纤维化。通过胆管结扎（BDL）在Oct3和WT小鼠中机械性地产生胆汁淤积相关性纤维化。最后，用非选择性OCT抑制剂奎宁处理稳定转染OCT1和OCT3的肿瘤细胞系和原代小鼠肝细胞，并通过qPCR和免疫荧光对Hnf4α表达进行定量。结果显示，Hnf4α是Oct3小鼠中上游调节因子之首。在胆汁淤积性肝损伤和纤维化过程中，与WT小鼠相比，Oct3小鼠中Hnf4α mRNA表达水平下调。纤维化肝组织中Hnf4α mRNA表达的下调在4周内是可逆的。在稳定转染OCT1和OCT3的HepG2和HuH7细胞以及原代小鼠肝细胞中，OCT的功能抑制导致Hnf4α mRNA表达上调。结果显示，Hnf4α位于WT肝细胞的细胞质中，而Oct3肝细胞中Hnf4α表达于细胞核。总之。Hnf4α在胆汁淤积和纤维化时下调，Oct的功能抑制可能导致Hnf4α上调。
