A hepatocyte nuclear factor-3 site in ALR promoter is important for interleukin 6-induced expression

Abstract

Liver regeneration is an important survival process that occurs after liver injury e.g. viral infection, toxins or hepatectomy. This process involves a variety of specific cytokines, growth factors and transcription factors. In the liver, hepatocyte nuclear factors (HNFs) play a central role during liver regeneration. These include the HNF3 transcription factors, also termed forkhead box (Fox) family. Additionally, it has been shown that Foxa2 (HNF-3β) expression is induced in regenerating mouse livers after partial hepatectomy as well as in livers from patients with hepatocellular carcinoma HCC. Furthermore, we have shown that Augmenter of Liver Regeneration (ALR) is significantly increased in hepatocellular carcinoma. In addition, increased expression levels of ALR were shown in a Foxa2- transgenic mouse model. The aim of our study was to identify transciption factors that regulate the expression of ALR. Promoter studies of ALR revealed potential binding sites for Foxa2, IL-6 RE, CEBP/β, YY1 and HNF-4α. The promoter activity of ALR was significantly induced over the control after co-transfection of Foxa2 in HepG2 cells. To verify whether the putative Foxa2 element is capable of binding Foxa2 protein, Electrophoresis Mobility Shift Assays (EMSA) were performed. Supershift analysis using α-Foxa2 antibody indicates the specific binding of Foxa2 to ALR promoter. This binding was inducible when the cells were simultaneously stimulated with IL-6. This increased binding after Foxa2-transfection was confirmed by elevated ALR protein levels using Western Blot technique. On the other hand, no binding was observed with IL-6 RE, CEBP/β and YY1. In conclusion, we demonstrated that Foxa2 enhances the expression of ALR. We assumed that ALR might be regulated by Foxa2, and this regulation could be amplified by simultaneously activation using IL-6. Taken together, both IL-6 and Foxa2 induce the expression of ALR, and Foxa2 is necessary in the IL-6 response of ALR promoter.