Analysis of Gene Expression Profile Induced by Hepatocyte Nuclear Factor 4α in Hepatoma Cells Using an Oligonucleotide Microarray

Abstract

Hepatocyte nuclear factor 4␣ (HNF-4␣), 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-4␣ cDNA and transfected the adenovirus to human hepatoma cells, HuH-7, to enforce expression of the exogenous HNF-4␣ gene. We analyzed HNF-4␣-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-4␣ overexpression, and six genes were repressed. To assess HNF-4␣ 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-4␣-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-4␣ is one of the central liver metabolism regulators.Differentiation of mammalian cells is associated with changes in gene expression that are primarily controlled at the level of transcription. Tissue-specific gene transcription is regulated based on the recognition of cis-elements of the target genes, accomplished by transcription factors that have restricted tissue distributions. Transcription factors that control embryonic cell differentiation are often required to maintain and regulate gene expression in the adult cell. Liver-specific gene expression is governed by the combinatorial action of a small set of liver-enriched transcription factors as follows: hepatocyte nuclear factor-1 (HNF-1), 1 a member of the POU homeobox gene family (1); the leucine zipper dimerization family, including CCAAT/enhancer-binding protein (C/EBP) ␣ (2); Dsite-binding protein (3); and C/EBP␤/liver activator protein (4); HNF-4, a member of the steroid hormone receptor superfamily (5); and HNF-3, the DNA binding domain, which is very similar to that of the Drosophila homeotic forkhead gene (6). Although many of these factors have been shown to be important components of the differentiation process that culminates in the fully functional liver, the manner in which different members of these families participate in the determination of cell phenotypes is poorly understood. Dedifferentiated hepatoma variants (7) and intertypic rat hepatoma-human fibroblast hybrids that show extinction of liver-specific gene expression (8) are deficient for the expression only of HNF-4 and HNF-1, and re-expression of liverspecific genes in revertants correlates with the re-expression of both liver-enriched transcriptional factors. We have demonstrated that when hepatocytes are plated onto a model ba...

Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4α gene

“…19,20 In short, HNF4␣-forced reexpression decreased the population of stem/progenitor cells in hepatoma cells and destroyed self-renewal of CSCs by targeting against these important signaling pathways. Consistent with results reported by Naiki et al, 21 we witnessed a list of elevated genes that are indicators for hepatocyte maturation along with proliferative suppression. Moreover, the ammonia detoxification function was also enhanced by HNF4␣ overexpression.…”

“…24 HNF4␣ has already been shown to increase expression of the p21 gene by antagonizing cMyc activation. 21,25,26 Furthermore, for control of apolipoprotein C III expression, a competition is present between c-Myc and HNF4␣, indicating that HNF4␣ and c-Myc compete for control of the genes involved in cell proliferation as well as differentiation. 27 In our study, we demonstrated that both protein p21 and apolipoprotein C III were raised in hepatoma cells along with increased HNF4␣ expression.…”

Prenatal programming of hepatocyte nuclear factor 4α in the rat: a key mechanism in the ‘foetal origins of hyperglycaemia’?

“…HNF4A is produced from an early stage in hepatocyte differentiation and plays a key role in regulation of liver development and metabolic function; nearly half of the transcribed genes in the liver, including Pck2, are thought to be controlled by HNF4A [34][35][36]. Overexpression of Hnf4a in cell culture results in stimulation of Pck2 transcription [37]. Conversely, Hnf4a liver-specific knockout mice fail to induce PCK2 during fasting [35].…”

Embryonic development of the liver

“…137 Combined with available genetic, biochemical, and gene array data, these results suggest that HNF4␣ is a central determinant of hepatocyte function and differentiation and is crucial for establishing normal liver architecture during development. 139,145,146 In mammals, maturation of the liver is further complicated by the fact that the fetal hepatic environment is also the site of hematopoiesis between E11.5 and E16.5. In this regard, there appears to be a close relationship between hematopoietic cells and the developing hepatic parenchyma, which is governed by cytokine signaling.…”