Abstract
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.
Highlights
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 in embryonal liver development
HNF-4␣ has been found to play a significant role in liver and gastrointestinal organogenesis at the embryonal stage (12, 30)
HNF-4 has a limited tissue distribution. It is expressed at high levels in the liver, intestine, kidney, and pancreatic beta-islet cells, which are functionally relevant to the lipid and glucose metabolisms
Summary
Cell Lines and Culture Conditions—A human hepatoma cell line, HuH-7, and a human hepatoblastoma cell line, HepG2, obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan) were cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 1% heat-inactivated fetal bovine serum (Sigma-Aldrich), 1% penicillin and streptomycin (Invitrogen), 12.5 mM HEPES buffer (Sigma-Aldrich), 0.2% lactoalbumin (Sigma-Aldrich) or with 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin, and 12.5 mM HEPES buffer, respectively. The recombinant adenovirus (AdCAGHNF4) was grown in 293 cells, purified, and titrated according to the conventional protocols (16). Fortyeight h after the culture medium was replaced with fresh RPMI 1640 medium, the cells were washed twice with PBS, and total RNA was isolated from the AdCAGHNF4- and AdCAGLacZ-treated cells using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. One g of highly purified poly(A) RNA from the AdCAGHNF4- and AdCAGLacZ-treated cells was sent out for cDNA microarray analysis, including generation of cDNA, fluorescent labeling, and hybridization on the microarray glass, to Incyte Genomics (Human UniGEM V, version 2.0 microarray, Palo Alto, CA). Infected cells were rinsed twice with PBS, and protein preparation was performed according to the manufacturer’s instructions (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Detection was performed using an ECL system (Amersham Biosciences), and the data were processed on an NIH Image program to measure the signal intensity
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