Tan Yongjun, Yoshida Yuichi, Hughes Douglas E, Costa Robert H
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60607-7170, USA.
Gastroenterology. 2006 Apr;130(4):1283-300. doi: 10.1053/j.gastro.2006.01.010.
BACKGROUND & AIMS: The hepatocyte nuclear factor 6 (HNF6 or ONECUT-1) protein is a cell-type specific transcription factor that regulates expression of hepatocyte-specific genes. Using hepatocytes for chromatin immunoprecipitation (ChIP) assays, the HNF6 protein was shown to associate with cell cycle regulatory promoters. Here, we examined whether increased levels of HNF6 stimulate hepatocyte proliferation during mouse liver regeneration.
Tail vein injection of adenovirus expressing the HNF6 complementary DNA was used to increase hepatic HNF6 levels during mouse liver regeneration induced by partial hepatectomy, and DNA replication was determined by bromodeoxyuridine incorporation. Cotransfection and ChIP assays were used to determine transcriptional target promoters.
Elevated expression of HNF6 during mouse liver regeneration causes a significant increase in the number of hepatocytes entering DNA replication (S phase), and mouse hepatoma Hepa1-6 cells diminished for HNF6 levels by small interfering RNA transfection exhibit a 50% reduction in S phase following serum stimulation. This stimulation in hepatocyte S-phase progression was associated with increased expression of the hepatocyte mitogen tumor growth factor alpha and the cell cycle regulators cyclin D1 and Forkhead box m1 (Foxm1) transcription factor. Cotransfection and ChIP assays show that tumor growth factor alpha, cyclin D1, and HNF6 promoter regions are direct transcriptional targets of the HNF6 protein. Coimmunoprecipitation assays with regenerating mouse liver extracts reveal an association between HNF6 and FoxM1 proteins, and cotransfection assays show that HNF6 stimulates Foxm1 transcriptional activity.
These mouse liver regeneration studies show that increased HNF6 levels stimulate hepatocyte proliferation through transcriptional induction of cell cycle regulatory genes.
肝细胞核因子6（HNF6或ONECUT-1）蛋白是一种细胞类型特异性转录因子，可调节肝细胞特异性基因的表达。利用肝细胞进行染色质免疫沉淀（ChIP）分析，结果显示HNF6蛋白与细胞周期调控启动子相关。在此，我们研究了HNF6水平升高是否会在小鼠肝脏再生过程中刺激肝细胞增殖。
通过尾静脉注射表达HNF6互补DNA的腺病毒，以在部分肝切除诱导的小鼠肝脏再生过程中提高肝脏HNF6水平，并通过溴脱氧尿苷掺入法测定DNA复制情况。采用共转染和ChIP分析来确定转录靶启动子。
在小鼠肝脏再生过程中，HNF6表达升高导致进入DNA复制（S期）的肝细胞数量显著增加，而通过小干扰RNA转染使HNF6水平降低的小鼠肝癌Hepa1-6细胞在血清刺激后S期减少了50%。肝细胞S期进展的这种刺激与肝细胞有丝分裂原肿瘤生长因子α以及细胞周期调节因子细胞周期蛋白D1和叉头框m1（Foxm1）转录因子的表达增加有关。共转染和ChIP分析表明，肿瘤生长因子α、细胞周期蛋白D1和HNF6启动子区域是HNF6蛋白的直接转录靶标。用再生小鼠肝脏提取物进行的免疫共沉淀分析揭示了HNF6与FoxM1蛋白之间的关联，共转染分析表明HNF6刺激Foxm1转录活性。
这些小鼠肝脏再生研究表明，HNF6水平升高通过转录诱导细胞周期调控基因来刺激肝细胞增殖。
