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Originally published In Press as doi:10.1074/jbc.M105403200 on February 7, 2002

J. Biol. Chem., Vol. 277, Issue 16, 14011-14019, April 19, 2002
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Analysis of Gene Expression Profile Induced by Hepatocyte Nuclear Factor 4alpha in Hepatoma Cells Using an Oligonucleotide Microarray*

Takafumi NaikiDagger , Masahito NagakiDagger §, Yoshihiro Shidoji, Hisanori Kojima||, Motoaki ImoseDagger , Tomohiro KatoDagger , Nobuko Ohishi**, Kunio Yagi**, and Hisataka MoriwakiDagger

From the Dagger  First Department of Internal Medicine and the || Department of Neurology and Psychiatry, Gifu University School of Medicine, Gifu, Gifu 500-8705, Japan, the  Laboratory of Cellular Biochemistry, Department of Nutrition and Health Science, Siebold University of Nagasaki, Nagayo, Nagasaki 851-2195, Japan, and the ** Institute of Applied Biochemistry, Yagi Memorial Park, Mitake, Gifu 505-0116, Japan

Received for publication, June 12, 2001, and in revised form, January 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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) alpha  (2); D-site-binding protein (3); and C/EBPbeta /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 liver-specific 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 basement membrane, such as that derived from the Engelbreth-Holm-Swarm (EHS) gel, they retain their normal cell polarity and liver-specific gene expression by up-regulation of hepatic transcription factors, namely by HNF-1 and HNF-4 expression (9). These results strongly imply that among the liver-enriched transcription factors, HNF-1 and HNF-4, play a crucial role in the determination and maintenance of hepatocyte-specific differentiation status. HNF-4 is expressed at the earliest stage in the developing hepatic diverticulum (10). This expression of HNF-4 early in the genesis of the hepatic lineage precedes that of HNF-1, which has also been implicated in the regulation of liver gene expression (11). Importantly, HNF-4 is a positive regulator and activator of HNF-1 expression (12). A series of independent findings has contributed to the idea that HNF-4 may act the furthest upstream, as a master gene in a transcription factor cascade that could drive the differentiation of hepatic lineage.

In this study, to address the question of which genes are influenced by HNF-4alpha gene overexpression, we used cDNA microarray technology. Recent technological advances in the production of cDNA microarrays have made it possible to profile the gene expressions of tens of thousands of genes (13, 14). We used over 9000 human cDNAs printed onto microarrays and analyzed for an expression profile of HNF-4alpha -overexpressed cells in comparison with that induced by the adenovirus-mediated lacZ gene as a virus control. Of 9182 genes, we identified 62 genes that reached a greater than 2.0-fold increase or decrease in expression level after the HNF-4alpha transfection. Fifty-six genes were induced by HNF-4alpha overexpression, and six genes were repressed. This profiling analysis of the expression level of genes offers useful information in great quantity. For organizational purposes, we adapted a previous functional classification (15) to profile the genes based on their encoding protein functions.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. A monkey renal blastocyte cell line, COS-7, and human uterus cervical cancer cell line, HeLa, obtained from the Japanese Collection of Research Bioresources, were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin and streptomycin, and 12.5 mM HEPES buffer. 2.0 × 107 cells were subcultured overnight in a 150-mm dish.

Recombinant Adenovirus Vector-- A recombinant adenovirus vector carrying rat HNF-4alpha 2 cDNA was constructed according to previously reported protocols (16). In brief, cDNA encoding a full-length HNF-4alpha 2 was inserted into the SwaI site of the cosmid vector pAxCAwt. The construct was co-transfected with EcoT221-digested Ad5-dIX DNA-tagged viral terminal protein into 293 cells from a human embryonic kidney cell line. The recombinant adenovirus (AdCAGHNF4) was grown in 293 cells, purified, and titrated according to the conventional protocols (16). A recombinant adenovirus vector, AdCAGLacZ, bearing the Escherichia coli beta -galactosidase gene was obtained from the Riken Gene Bank (Tsukuba, Japan). After plating overnight, the medium was changed, and the cells were infected with purified recombinant adenovirus at a multiplicity of infection (m.o.i.) of 10 for 1 h.

beta -Galactosidase Cytochemical Assay-- Determination of lacZ gene expression was carried out according to the method described by Miyake et al. (16). In brief, the fixed cells were rinsed twice with phosphate-buffered saline (PBS), incubated in a reaction mixture containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, and 1 mg/ml X-gal in PBS for 1 h at 37 °C, and then washed three times with PBS.

cDNA Microarray-- 2.0 × 107 HuH-7 cells were inoculated into a 150-mm dish and cultured overnight. The cells were infected with AdCAGHNF4 or AdCAGLacZ as a control at 10 m.o.i. for 1 h. Forty-eight 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. Poly(A) RNA was purified using an Oligotex-dT30 mRNA purification kit (Takara, Osaka, Japan). One µg 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). In brief, isolated mRNA is reverse-transcribed with 5'-Cy3- or Cy5-labeled random 9-mers (Operon Technologies, Inc., Alameda, CA). The paired reactions were combined and purified with a TE-30 column (CLONTECH, Palo Alto, CA). The fluorescently labeled probe was then applied to the array for hybridization at 60 °C for 6.5 h. After hybridization, the glass was washed with buffer at decreasing ionic strength. The microarray was scanned at a resolution of 10 microns in order to detect Cy3 and Cy5 fluorescence. After scanning the signal, Incyte GEMtoolsTM software (Incyte Genomics) was used for image analysis. The area surrounding each element image was used to calculate a local background, which was then subtracted from the total element signal. Background-subtracted element signals are used to calculate Cy3:Cy5 ratios. The average of the resulting total Cy3 and Cy5 signal gives a ratio that is used to balance or normalize the signals.

Northern Blot Analysis-- Total RNA (10-40 µg) was separated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to positively charged nylon membranes, GeneScreen Plus (PerkinElmer Life Sciences). The membranes were hybridized with cDNA probes for human HNF-4alpha , HNF-1, ApoAI, ApoCIII, albumin, and TTR, each of which was created by the PCR method using a digoxigenin luminescent labeling kit (Roche Diagnostics, Mannheim, Germany). Scanning densitometry and signal intensity quantification were performed on an NIH Image program (version 1.61, developed at the National Institutes of Health, Bethesda, MD). The cellular levels of the transcripts were normalized by 28 S ribosomal RNA and shown as a fold of control values in the same experiment.

Western Blot Analysis-- Western blot analyses of HNF-4alpha and HNF-1alpha were performed according to the method described previously, with some modifications (17). In brief, 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). Protein concentration in homogenates was measured using a DC-protein assay (Bio-Rad) employing bovine serum albumin as the standard. The extracted proteins (20 µg) were subjected to 8% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions and then transferred onto a nitrocellulose membrane (Bio-Rad). Goat polyclonal anti-human HNF-4alpha and rabbit polyclonal anti-human HNF-1 antibodies (Santa Cruz; final dilution 1:200-1000) were used as the primary antibodies, and horseradish peroxidase-labeled anti-goat immunoglobulin (Santa Cruz; final dilution 1:1500) or horseradish peroxidase anti-rabbit immunoglobulin (Amersham Biosciences; final dilution 1:1500) was used as the secondary reagent. Detection was performed using an ECL system (Amersham Biosciences), and the data were processed on an NIH Image program to measure the signal intensity.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adenovirus-mediated HNF-4alpha Gene Transfer-- To determine the viral infection in hepatoma cells, we first assessed the expression of beta -galactosidase in the cells infected by the control adenovirus, AdCAGLacZ (Fig. 1B). AdCAGLacZ infection at 10 m.o.i. induced beta -galactosidase expression in 60-70% of the HuH-7 cells. Other cell lines also expressed beta -galactosidase to the same extent after AdCAGLacZ infection (data not shown). Phase-contrast photomicrographs revealed no apparent changes in cell shape in HuH-7 cells transfected at 10 m.o.i. by AdCAGLacZ (data not shown) or AdCAGHNF4 (Fig. 1D) over a 48-h culture period.


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  		Fig. 1.   X-gal staining (A and B) and phase-contrast photomicrographs (C and D) of HuH-7 cells infected with adenovirus vectors. Cells are not infected (A) or infected (B) with recombinant adenovirus carrying the lacZ gene at an m.o.i. of 10. At 48 h post-transfection, the cells were stained with X-gal in order to visualize expression of the transferred lacZ gene. The morphological phenotypes of HuH-7 cells not infected (C) or infected (D) with the adenovirus carrying HNF-4alpha were determined by phase-contrast microscopy. Original magnification is ×200.

Forty-eight h after infection with AdCAGHNF4, Northern blot analysis demonstrated that lines of hepatoma-derived cells, HuH-7 and HepG2, and non-hepatocyte-derived cells, COS-7 and HeLa, showed increases in expression of HNF-4alpha mRNA (Fig. 2A). Western blot analysis constitutively detected the endogenous HNF-4alpha in HuH-7 and HepG2 cells (Fig. 3). AdCAGHNF4 infection increased the expression of HNF-4alpha protein in these cells. In COS-7 and HeLa cells, HNF-4alpha was detected after infection with AdCAGHNF4.


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  		Fig. 2.   A, Northern blot analysis of liver-specific transcription factor and transcripts in hepatocyte- and non-hepatocyte-derived cell lines. Total RNA were isolated from each cell line 48 h after infection with the indicated adenovirus and were then electrophoresed, blotted onto nylon membrane, and hybridized with the indicated digoxigenin-labeled cDNA probes. B, the hybridization signals were quantified using the NIH Image program and are shown as a fold of uninfected control cells. Data shown are the mean ± S.D. (n = 4). *, p < 0.05; **, p < 0.01 compared with AdCAGLacZ by Student's t test.


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  		Fig. 3.   Western blot analysis of HNF-4alpha and HNF-1 in hepatocyte- and non-hepatocyte-derived cell lines. Extracted protein were prepared from each cell line 48 h after infection with the indicated adenovirus at 10 or 50 m.o.i. and were then electrophoresed and blotted onto a nitrocellulose membrane. Blots were labeled with anti- HNF-4alpha and -HNF-1 antibodies. 54-kDa HNF-4alpha proteins were derived from full-length HNF-4alpha cDNA. The hybridization signals were quantified using the NIH Image program. Data shown are the mean ± S.D. (n = 4). *, p < 0.05; **, p < 0.01 compared with AdCAGLacZ by Student's t test

Re-expression of the Hepatic Phenotype Induced by HNF-4alpha -- It is well known that HNF-4 is an activator of the HNF-1 gene and that it functions in the regulation of several liver-specific genes (11, 12). The expression of HNF-1 and of various direct target genes of HNF-4 was analyzed by Northern blot analysis. As shown in Fig. 2, the intracellular levels of HNF-1, ApoAI, and ApoCIII mRNA were increased in the AdCAGHNF4-targeted hepatoma cells. On the other hand, the lacZ gene transfection failed to increase these transcripts. The results also showed that HNF-4alpha overexpressed by AdCAGHNF4 functioned well and could induce the liver-specific genes, ApoAI and ApoCIII. HNF-4alpha overexpression did not append the liver phenotype to that of nonhepatic cells (Fig. 2A). Further, HNF-4alpha overexpression failed to increase albumin and TTR mRNA, which are the targeted genes of HNF-1 and/or HNF-4.

cDNA Microarray Assay-- The human hepatoma-derived cell line, HuH-7, was infected by AdCAGHNF4 or AdCAGLacZ for 1 h. Forty-eight h later, a total of 600 ng of each purified poly(A) RNA from these cells were subjected to reverse transcription reaction, and the resultant cDNAs were labeled with either Cy3 or Cy5 fluorescence and then hybridized to the UniGEM Human V version 2.0 microarray containing 9182 sequence-verified human genes (analysis was performed by Incyte Genomics). These fluorescent cDNAs were simultaneously hybridized to probe sequences on the microarray, and the amount of fluorescence seen with the individual dyes was determined by confocal microscopy. The differential expression of each expressed sequence tag (EST) was then calculated from the relative intensity of the Cy3 versus Cy5 fluorescent signal. Three independent transfection experiments were conducted comparing AdCAGHNF4- and AdCAGLacZ- infected cells. Duplicate experiments were conducted on a single mRNA preparation from the cells. Recently, it has been reported that the coefficient of variation of the Incyte cDNA microarray technology platform for differential expression is 12% across the entire signal range (18). In the present study, about 80% of the top 50 genes identified by the first cDNA microarray experiment were also changed by more than 2.0-fold in the second analysis using the same mRNA (data not shown). There was slightly greater mRNA variation at the low signal levels. Fig. 4 shows the plots of the differential expression of the 9182 genes in these three experiments. Overall, the expression of most genes was not altered by HNF-4alpha .


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  		Fig. 4.   Global comparison of gene expression in HNF-4alpha - and LacZ-infected cells. Each dot corresponds to the Cy3 and Cy5 fluorescent intensity of one single element on the microarray. A 2-fold change in expression is indicated as parallel broken lines.

Based on the observation that the expression level of a few genes changed by more than 2.0-fold between the control and the treated samples, we applied a threshold of a 2.0-fold change in expression level between AdHNF4- and AdLacZ-infected cells. As shown in Table I, in this analysis we identified 62 genes that were expressed at different levels with more than a 2.0-fold change. A total of 61 genes were identified as unique and were named in the GenBankTM, and the remaining single gene was identified as an unnamed EST in the GenBankTM. Of these 62 HNF-4alpha responsive genes, 56 genes (55 known genes and one unnamed EST) were up-regulated. The remaining six genes were down-regulated, and all were named in the GenBankTM.

                              
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Table I
Genes in which expression was altered more than 2-fold

HNF-4alpha is capable of binding to sequences present in the ApoAI, ApoCI, and ApoCIII gene promoter regions and has been proposed to regulate the expression of these genes (5, 19). In our cDNA microarray study, these expressions were induced in HuH-7 cells by HNF-4alpha , and the results were confirmed by Northern blot analysis (Fig. 2). Besides ApoAI and ApoCIII, most of the apolipoproteins, including ApoAII, ApoE, ApoCI, and lipoprotein(a), were induced by HNF-4alpha (Table I).

A mutation in HNF-4alpha can cause a form of early onset type 2 diabetes (maturity onset diabetes of the young, MODY1) (20). In the present study, the most pronounced induction in gene expression was shown in cysteine-rich intestinal protein-1. A study addressing the relationship between HNF-4alpha and cysteine-rich intestinal protein-1 has not, to our knowledge, been reported previously. This gene belongs to the LIM/double zinc finger protein family and is suggested to be important for cellular zinc transport (21). Low zinc absorption has been observed in diabetic animals and humans (22), and zinc plays a clear role in the synthesis, storage, and secretion of insulin (23). On the other hand, phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme in gluconeogenesis, was induced by HNF-4alpha in our microarray analysis. This enzyme is highly induced in the diabetic animal model as compensation for lack of insulin function (24). The HNF-1alpha binding site was found to be located in the promoter region of PEPCK (25). Moreover, a recent study has revealed that HNF-4alpha can bind the enhancer/promoter region of PEPCK directly (26). These findings suggest that in addition to HNF-1alpha , HNF-4alpha might be a major transcription factor of the PEPCK gene. We could not show the expression change of HNF-1alpha after overexpression of HNF-4alpha by cDNA microarray analysis because the former was not included in this microarray analysis. However, we did show that HNF-4alpha induced HNF-1 in HuH-7 cells by Northern and Western blot analysis (Figs. 2 and 3).

Some genes associated with the intracellular signaling pathway, such as cyclin-dependent kinase inhibitor, p21, protein kinase C, and peroxisome proliferator-activated receptor (PPAR) alpha , were induced by HNF-4alpha overexpression (Table I).

Functional Gene Classification-- Many of the genes that displayed differential expression encode proteins with known functions, but others, including novel and previously identified ESTs, correspond to genes with unknown functions. Genes were classified on the basis of the biological function of the encoded protein, using a modified version of a previously established classification scheme (15). The classification scheme was composed of seven major functional categories and several minor functional categories within the major categories. Genes observed in the present study were placed in a single major class if a function of the encoded protein had been well established. As shown in Table II, 45 genes were classified as known function genes; 4 genes were categorized as unclassified genes; and 13 genes (including ESTs) were not found on the list.

                              
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Table II
Summary of genes regulated by HNF-4alpha

HNF-4alpha has been considered an important regulator of genes of diverse functions, including those encoding metabolic proteins and serum proteins. Interestingly, the largest category of HNF-4alpha -induced genes was those involved in metabolism, including 26 of 40 HNF-4alpha -induced genes with known functions. Among the 56 HNF-4alpha -induced genes, 16 genes were not found on the list. Taken together, 65.0% of the overexpressed genes (26 of 40) were classified into the category of metabolism. We observed, however, that only a few serum protein-associated genes were regulated by HNF-4alpha . HNF-4alpha binds an essential region of TTR (5, 27). HNF-1, which can be induced by HNF-4alpha , has been shown to interact with the promoter regions of albumin, alpha -fetoprotein, TTR, and fibrinogen genes (28, 29). In our microarray analysis, none of these were induced in HuH-7 by HNF-4alpha (Table I). We have confirmed, using Northern blot analysis, that albumin and TTR mRNA were not induced in HuH-7 by HNF-4alpha overexpression (Fig. 2). Moreover, genes involved in cell division, cell signaling/cell communication, cell/organism defense, and gene/protein expression were significantly induced by HNF-4alpha in numbers greater than those of the genes repressed.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HNF-4alpha was identified initially as the binding protein to the upstream enhancer/promoter sequence of the ApoCIII gene (5). In recent studies, HNF-4alpha has been found to play a significant role in liver and gastrointestinal organogenesis at the embryonal stage (12, 30). In adults, 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. In fact, HNF-4alpha mutations have been reported to be causal for diabetes (31), suggesting that HNF-4alpha may participate in the regulation of glucose and lipid metabolisms. In our microarray analysis, about half of the overexpressed genes (26 of 40) induced by HNF-4alpha transfection were classified as metabolism genes (Table II).

Moreover, about half of the HNF-4alpha -induced metabolism genes were associated with lipid metabolism. The data presented here demonstrated that HNF-4alpha is important for the expression of several key hepatic genes involved in lipid transport and metabolism. High levels of ApoCIII correlate with increased fasting triacylglycerol levels in both clinical hypertriacylglycerolemic patients and murine models (32, 33), and overexpression of ApoCIII in mice causes hypertriacylglycerolemia and induces atherogenesis (34). On the other hand, recent studies suggest that high serum levels of triacylglycerol are inversely correlated with high density lipoprotein cholesterol levels (35) and that overexpression of ApoAI or ApoAIV increases cholesterol in plasma high density lipoprotein and protects against atherosclerosis (36, 37). Our microarray analysis showed that both ApoCIII and ApoAI were induced by HNF-4alpha . Mutation in the HNF-4alpha gene in MODY1 impaired triacylglycerol metabolism and insulin secretion. Shih et al. (38) reported that serum levels of ApoAII, ApoCIII, lipoprotein(a), and triacylgylcerol were significantly reduced in diabetic patients with HNF-4alpha mutations compared with these levels in matched diabetic patients. Mice lacking hepatic HNF-4alpha expression accumulated lipid in the liver and exhibited greatly reduced serum cholesterol and triacylglycerol levels (39). Moreover, overexpression of the ApoAI/CIII/AIV gene cluster in mice induces hyperlipidemia but reduces atherogenesis (40). These findings, taken together, suggest that modulation of apolipoprotein expression via HNF-4alpha may offer significant advantages in the treatment of hyperlipidemia.

The present cDNA microarray study revealed that lipid metabolism-associated genes other than the above mentioned apolipoprotein genes were induced by HNF-4alpha , demonstrating that carnitine/acylcarnitine translocase and carnitine acetyltransferase were induced by HNF-4alpha . These genes are important for lipid beta -oxidation, and their gene products carry free fatty acids or acyl-CoA into the mitochondrial inner membrane. These data suggest that HNF-4alpha may contribute to mitochondrial lipid oxidation. Moreover, our microarray data showed that PPARalpha was induced by HNF-4alpha . PPARalpha is well known as a transcriptional factor that can be activated by hypolipidemic drugs such as fibrates, and its activation enhances the catabolism of free fatty acids and leads to a decrease of serum triacylglycerol levels (41). These findings suggest that HNF-4alpha may be involved in lipid oxidation through induction of carnitine/acylcarnitine translocase or PPARalpha .

Heterozygous mutations in the HNF-4alpha gene are responsible for the early onset form of non-insulin-dependent diabetes mellitus, MODY1 (20). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF-4alpha protein, the precise mechanism by which it causes diabetes is unknown. Although it is likely that dysregulation of glucose homeostasis in MODY1 patients is due, in a large part, to reduced insulin secretion by pancreatic islets, loss of HNF-4alpha function in the liver may contribute to this disease. Using embryonal stem cells, Stoffel and Duncan (42) identified glucose transporter 2, aldolase B, glyceraldehyde-3-phosphate dehydrogenase, and liver-type pyruvate kinase as genes encoding components of the glucose-dependent insulin secretion pathway and in which expression is dependent on HNF-4alpha . HNF-1 interacts with the promoters and enhancers of aldolase B (43), as well as with liver pyruvate kinase genes (44). In the present study, several genes related to glucose metabolism were induced by HNF-4alpha . However, in contrast to findings in the aforementioned study, none of these genes were found to be induced in HuH-7 cells by HNF-4alpha overexpression. In two sugar-related genes, the expression of which was up-regulated by HNF-4alpha overexpression, PEPCK was found to play a crucial role in gluconeogenesis. Moreover, phosphorylase, a key enzyme of glycogenolysis, was up-regulated. Our data may be an interesting addition to the previous reports indicating that HNF-4alpha plays an important role in glucose metabolism.

The liver is the major site of plasma protein synthesis. HNF-4alpha is also known to play a role in the regulation of plasma protein genes, including albumin, TTR, and alpha -fetoprotein, directly or through the activation of HNF-1. However, in the present microarray experiment, expressions of these serum protein genes were not up-regulated by HNF-4alpha overexpression, which was confirmed by Northern blot analysis (Fig. 2). In agreement with our results, Späth and Weiss (7) demonstrated that many of the hepatocyte-specific genes, including TTR, failed to be activated in HNF-4-transfected rat hepatoma cells. In addition, they reported that the addition of dexamethasone resulted in a significant induction of TTR in the transfectant. It is difficult to adequately explain why HNF-4 protein did not lead to activation of a majority of the hepatocyte-specific marker genes, and in particular, of the serum proteins produced in the liver, whereas HNF-4 remarkably activated most of the apolipoprotein genes. This could be due to suboptimal concentrations, which were only able to activate serum protein genes, or to deficiencies of other transcription factors, such as HNF-3 and C/EBP, and accessory factors of the combinatorial control system that characterize liver gene transcription, based on the finding that HNF3 mRNA was shown to be repressed in HuH-7 cells after HNF-4alpha overexpression by reverse transcription PCR (data not shown).

Because a 2.0-fold difference in relative transcript level is a threshold commonly used for analysis and interpretation of microarray data (45), we applied a threshold of a 2.0-fold change. Some investigators have reported that less than a 2.0-fold change in relative transcript abundance can be biologically significant (46, 47). The criterion we set forth for defining HNF-4alpha -responsive genes appeared to be stringent. A greater than 2.0-fold difference in gene expression was required in the repeated comparisons of AdCAGHNF4- and AdCAGLacZ-infected cells. Performing repeated experiments was crucial because the multiple cDNA microarray restricted the candidate list to a limited number of genes (Fig. 5). If we had performed this experiment only once, we would have detected increased expression of 60-100 genes, about one-third of the identified genes, that would not have been replicated in further experiments (Fig. 5). However, this stringent inclusion criterion may exclude potentially real differences. Because the difference in one of the experiments is a <2.0-fold cut-off, genes were not considered positive.


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  		Fig. 5.   Venn diagram of the number of genes altered by more than 2.0-fold in each of three independent HNF-4alpha overexpression experiments.

The present microarray analysis indicates that HNF-4alpha target genes influence a variety of cellular processes including metabolism, protein synthesis, cell cycle progression, and signal transduction. However, the complex physiological effects of HNF-4alpha are likely to be recapitulated by metabolism, particularly by lipid metabolism genes.

    FOOTNOTES

* This work was supported in part by Grant-in-aid 10670462 from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Research Group of Intractable Liver Disease, sponsored by the Ministry of the Health, Labor, and Welfare of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: First Dept. of Internal Medicine, Gifu University School of Medicine 40 Tsukasa-machi, Gifu 500-8705, Japan. Tel.: 81-58-267-2843; Fax: 81-58-262-8484; E-mail address: mnagaki@cc.gifu-u.ac.jp.

Published, JBC Papers in Press, February 7, 2002, DOI 10.1074/jbc.M105403200

    ABBREVIATIONS

The abbreviations used are: HNF, hepatocyte nuclear factor; C/EBP, CCAAT/enhancer-binding protein; TTR, transthyretin; Apo, apolipoprotein; m.o.i., multiplicity of infection; X-gal, 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside; EST, expressed sequence tag; MODY, maturity onset diabetes of the young; PEPCK, phosphoenolpyruvate carboxykinase; PPAR, peroxisome proliferator-activated receptor; EST, expressed sequence tag; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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