Functional activity of human hepatoma cells transfected with adenovirus-mediated hepatocyte nuclear factor (HNF)-4 gene.

Fulminant hepatic failure (FHF) is still associated with high mortality despite recent advances in medical management. There is need of an effective and safe bioartificial liver (BAL) support to help keep patients with FHF alive until an organ becomes available for transplantation or the native liver recovers. The aim of this study was to establish highly functional liver cells by means of transfecting hepatocyte nuclear factor (HNF)-4 gene for the development of BAL. We constructed adenovirus vector carrying rat HNF-4 cDNA, and transfected to hepatoma-derived cell lines, HepG2 and HuH-7, to enforce expression of the exogenous HNF-4 gene. We analyzed expression of HNF-4, HNF-1, and liver-specific genes in cells infected by the adenovirus vector expressing HNF-4. Adenovirus-mediated HNF-4 gene transfer resulted in increases in expressions of HNF-4, HNF-1, and liver-specific genes such as apolipoproteins, alpha1-antitrypsin (alpha1-AT), phosphoenolpyruvate carboxy-kinase, cytochrome P450 families, and glutamine synthetase in transfected hepatoma cells. Cells overexpressing HNF-4 removed ammonia from medium supplemented with NH4Cl to a greater extent than control cells. These findings demonstrated that transfected cell lines restored differentiated gene expressions and liver-specific function by the overproduction of HNF-4. HNF-4-overexpressing hepatocyte cell lines are useful for bioreactor of BAL systems.

Analysis of gene expression profile induced by hepatocyte nuclear factor 4alpha in hepatoma cells using an oligonucleotide microarray.

Hepatocyte nuclear factor 4alpha (HNF-4alpha), a liver-specific transcription factor, plays a significant role in many liver-specific functions, including lipid, glucose, drug, and ammonia metabolism, and also in embryonal liver development. However, its functions and regulation are not yet clearly understood. In this study, we constructed an adenovirus vector carrying rat HNF-4alpha cDNA and transfected the adenovirus to human hepatoma cells, HuH-7, to enforce expression of the exogenous HNF-4alpha gene. We analyzed HNF-4alpha-induced genes using cDNA microarray technology, which included over 9000 genes. As a result, 62 genes showed a greater than 2.0-fold change in expression level after the viral transfection. Fifty-six genes were consistently induced by HNF-4alpha overexpression, and six genes were repressed. To assess HNF-4alpha function, we attempted to classify the genes, which had been classified by their encoding protein functions in a previous report. We could classify 45 genes. The rest of the HNF-4alpha-sensitive genes were unclassified (4 genes) or not identified (13 genes). Among the classified genes, almost half of the induced genes (26 of 40) were related to metabolism genes and particularly to lipid metabolism-related genes. This cDNA microarray analysis showed that HNF-4alpha is one of the central liver metabolism regulators.