AIM: To investigate the role of hepatoma-derived growth factor (HDGF) in liver development, especially in the hepatocyte differentiation.

METHODS: We generated transgenic mice which overexpressed HDGF in hepatocytes under the transcriptional control of mouse albumin promoter/enhancer. To examine the effects of HDGF overexpression on hepatocyte differentiation, we investigated the expression patterns of the differentiation marker genes.

RESULTS: The HDGF transgenic mice developed normally and showed no apparent abnormality in the liver. However, the gene expression patterns of the liver in adult transgenic mice were similar to those of the neonatal liver in control mice.

CONCLUSION: These findings suggest that HDGF-overexpression partially suppresses hepatocyte maturation.

The liver is the major hematopoietic organ during the fetal period, and immature hepatocytes function as stromal cells which support hematopoiesis. During liver development, immature hepatocytes differentiate and acquire many functions in preparation for the metabolic conditions after birth[1,2]. The expression patterns of differentiation marker genes can represent the maturational stage of hepatocytes. Alpha-fetoprotein (AFP) is one of the early marker genes of the immature hepatocytes and its expression remarkably decreases after birth[3]. The expression of albumin, the most abundant protein synthesized by hepatocytes, begins during the mid-gestational stage, and this expression increases with the progression of liver development, especially after birth[4]. In the late-gestational stage hepatocytes begin to produce metabolic enzymes including tyrosine amino transferase (TAT) and glucose-6-phosphatase (G-6-Pase)[5,6]. Subsequently, hepatocytes gain a fully matured phenotype, characterized by the expression of tryptophan oxygenase (TO) within two weeks after birth[7]. The expression levels of TAT and G-6-Pase peak in the neonatal liver and decrease in the adult liver. In contrast, TO is barely expressed in the fetal and neonatal liver and is highly expressed in the adult liver. Although the gene expression patterns of hepatocytes continue to alter after birth, few studies have documented the growth and differentiation of post-natal hepatocytes.

Hepatoma- derived growth factor (HDGF) is a heparin-binding protein, which has been identified from the conditioned media of HuH-7 hepatoma cells[8,9]. HDGF stimulates the proliferation of fibroblasts, endothelial cells, vascular smooth muscle cells and hepatocytes[8-11]. Its primary sequence contains nuclear localization signals and the HDGF can be transported to the nucleus, thus indicating that HDGF is a unique nuclear/growth factor[12,13]. Recently, several novel genes have been identified for proteins which share a highly homologous amino terminal region consisting of about 100 amino acids; so-called HDGF- related proteins (HRPs)[14,15]. It is thought that HDGF and HRPs form a new gene family. Although HDGF was initially identified in human hepatoma-derived cells, HDGF mRNA is expressed in various normal adult tissues of mice and humans, thus suggesting that HDGF has some physiological functions in non-tumor cells[9].

Previous studies have suggested that HDGF participates in fetal organ development and adult tissue repair as an autocrine growth factor[10,11,16]. We have shown that HDGF is highly expressed in immature fetal hepatocytes and promotes their proliferation[16]. Furthermore, HDGF is induced in two animal models of liver regeneration[17], suggesting that HDGF plays an important role in the proliferation of both fetal and adult hepatocytes. Although the involvement of HDGF in cell differentiation has not been clarified, the suppressive effects of HDGF on gut cell maturation have been suggested[18]. We generated transgenic mice which overexpressed HDGF in hepatocytes under the control of the albumin promoter/enhancer, in order to examine the functional role of HDGF in liver development. The gene expression patterns of hepatocytes in adult transgenic mice resemble those of neonatal hepatocytes of wild-type mice, thus suggesting that HDGF-overexpression partially suppresses hepatocyte maturation.

An expression unit was constructed that contained the entire HDGF cDNA under the control of the mouse albumin promoter/enhancer (Alb-HDGF, Figure 1). Purified fragments were used for pronuclear injection and potential founders were analyzed for the genomic integration(s) of the transgene. Three founders containing the Alb-HDGF sequence were identified by Southern blot, and the transgenes were successfully transmitted in two lines (Figure 2A: Tg-48, and Tg-21). Northern blot analysis revealed that HDGF was highly expressed in the adult liver of Tg-48 mice, whereas HDGF expression in the liver of Tg-21 mice was almost equal to the wild-type mice (Figure 2B). We therefore used the Tg-48 mice to analyze the effects of HDGF overexpression in hepatocytes.

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Figure 2 Genomic integration and mRNA expression of the HDGF-transgene. A: Southern blot analysis with HDGF cDNA probe. Genomic DNA was isolated from individual mouse tails and Southern blot analysis was performed according to standard methods. The bands representing endogenous HDGF and transgenes are shown. Genomic DNA of a transgenic mice (line number 21: Tg-21) contains high copy numbers of the transgene. Genomic DNA of the other transgenic mice (line number 48: Tg-48) contains low copy numbers of the transgene; B: Northern blot analysis with HDGF cDNA probe. Total RNA was isolated from liver tissues of both transgenic (Tg) and wild-type (WT) mice. Twenty micrograms of total RNA extracted was loaded and hybridized with mouse cDNA of the HDGF-specific sequence. HDGF expression was high in the liver of the Tg-48 mouse. The expression level of HDGF in the liver of Tg-21 was almost equal to the level expressed in the control liver. Ribosomal RNA of 18S is shown in the lower panel.

HDGF overexpressing mice (Tg-48) developed normally and did not show any abnormality in appearance. In addition, no obvious histological abnormality associated with the expression of HDGF was detected in these mice up to 12 mo of age (data not shown). The expression patterns of genes related to hepatocyte differentiation were investigated by Northern blotting to examine the effects of HDGF overexpression on liver development in detail. In normal mice, consistent with the previous studies, G-6-Pase expression was high in the neonatal liver and relatively low in adult (8 wk old) liver, whereas TO expression was higher in the adult liver in comparison to the neonatal liver (Figure 3A). Conversely, the expression of G-6-Pase was high, while the TO expression was low in the adult livers of transgenic mice (Figure 3B and D). These gene expression patterns observed in the livers of adult transgenic mice, were similar to the patterns observed in the neonatal stage of control livers. These findings have indicated that HDGF overexpressing hepatocytes in adult (8 wk old) transgenic mice appear to have the characteristics of hepatocytes in neonatal wild-type mice, suggesting the possible maturational retardation of hepatocytes in the transgenic mice. However, G-6-Pase expression in the transgenic liver in 24 wk-old mice decreased to the level of the normal liver, and the expression level of TO in the transgenic liver was increased almost to the level of the control liver, although small differences were still observed (Figure 3C and D). Therefore, HDGF overexpression did not completely block hepatocyte maturation and HDGF overexpressing hepatocytes could acquire almost fully differentiated phenotypes. These findings suggest that HDGF-overexpression partially suppresses the hepatocyte differentiation observed in the post-natal stage and thus causes the maturational delay of the hepatocytes.

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Figure 3 The expression of differentiation marker genes of hepatocytes. A: The expression of differentiation marker genes of hepatocytes in normal mice. RNA was extracted from fetal mice of E (embryonic day) 13.5 and 15.5, and postnatal mice of zero weeks (new born) and 8 wk after birth. Twenty micrograms of total RNA was loaded and hybridized with mouse cDNA probes. Ribosomal RNA of 18S is shown in the lowest panel; B: The expression of differentiation marker genes of hepatocytes in adult (8 wk) transgenic mice. In the control liver of 8-wk-old mouse, G-6-Pase expression is low and TO expression is high. In contrast, G-6-Pase expression is high and TO expression is low in the transgenic liver. Ribosomal RNA of 18S is shown in the lowest panel; C: The expression of differentiation marker genes of hepatocytes in adult (24w) transgenic mice. Unlike the 8-wk-old mouse, expression levels of G-6-Pase and TO show small differences between the transgenic liver and the normal liver. Ribosomal RNA of 18S is shown in the lowest panel. D: Densitometry measurements of Northern blot analysis. Northern blot signals in Figure 3B and C were measured and normalized by the internal control bands (18S). The expression levels of G-6-Pase and TO in the liver of Tg-48 mouse were compared with the levels in the control liver. The ratios of band intensities (Tg-48/WT) were graphically shown. Two independent experiments were conducted in duplicate, and similar results were obtained.

A number of studies have suggested that HDGF is involved in the development of various organs[10,11,16,18,21]. We have demonstrated that HDGF is a unique growth factor, which is highly expressed in fetal liver and promotes fetal hepatocyte proliferation[16]. However, familial genes often compensate for the functions of other family members, and HDGF-null mice have been reported to show no obvious phenotype, perhaps as a result of the redundant functions of HDGF related genes[22]. We therefore generated the HDGF transgenic mice and examined the functional role of HDGF in vivo according to the gain-of-function method.

Although several other groups have also reported the involvement of HDGF in the development of various organs through its growth stimulating activity, little is known about the role of HDGF in cellular maturation. As for hepatocyte differentiation, Kamiya et al[20] established a primary culture system of murine fetal hepatocytes to investigate the mechanism that controls late fetal liver development. In the culture system, the administration of Oncostain M and dexamethasone can induce hepatocyte differentiation and recapitulate the maturational process of hepatocytes ranging from mid-gestation to new-born stage. This culture system was used to clarify the involvement of HDGF in hepatocyte differentiation although down-regulation of HDGF could not induce the cellular differentiation process of the late gestation stage[16].

In the present study, the overexpression of HDGF under the control of the albumin promoter did not cause any apparent morphological abnormalities in the liver. However, the gene expression patterns showed the possibility that the maturational process of hepatocytes during the post-natal stage was disturbed. This result is consistent with the report by Lepourcelet et al[18], which documented that overexpression of HDGF in the mouse fetal gut explants retards epithelial differentiation, suggesting a suppressive role of HDGF in epithelial differentiation.

Several proteins strongly expressed in both tumors and fetal organs, such as carcinoembryonic antigen and AFP, are known as oncofetal proteins[23,24]. HDGF is expressed exclusively in both fetal and cancer tissues, indicating that HDGF can also be regarded as an oncofetal protein. Although several oncofetal proteins are clinically used as tumor markers, there are few proteins whose functional roles in cancer cells have so far been demonstrated. HDGF expression is strongly associated with the prognosis of many malignant diseases including pancreatic cancer, esophageal cancer, colorectal cancer, gastrointestinal stromal tumor, gastric cancer and heptocellular carcinoma (HCC)[25-31]. Recently, Lee et al[32] showed that individuals with HCC who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis. The gene expression pattern that distinguished this subtype from other types of HCC contained the markers of oval cells (hepato-cholangio progenitor cells), thus suggesting that the HCC of this subtype may be derived from hepatic progenitor/stem cells. Two groups have shown that high expression of HDGF is closely related to the poor prognosis of HCC[30,31] and HDGF stimulates the growth of immature fetal hepatocytes[16]. Recently, we have found that HDGF is highly expressed in oval cells and promotes their proliferation (Iwamoto et al. in preparation), thereby suggesting the involvement of HDGF in the proliferation of immature hepatic cells. Therefore, HDGF may stimulate the proliferation of HCC cells derived from hepatic progenitor/stem cells and thereby cause the poor prognosis. HDGF expression may maintain the characteristics of immature cells and be associated with high growth activity of malignant cells. HDGF not only promotes hepatocyte proliferation but also inhibits their differentiation, indicating that HDGF is an oncofetal protein which participates both in the cellular growth and differentiation. Clarifying the functional role of HDGF would give us new insights into molecular mechanisms common to normal and malignant hepatic cell growth.

Since HDGF-null mice did not show any remarkable abnormalities, perhaps as a result of the compensation by HDGF-related genes, the down-regulation of HDGF should inhibit the growth of cancer cells without any serious side effects on normal organs. Therefore, HDGF is considered to be a candidate therapeutic target. Although little is known about the regulation of HDGF expression, we recently have found that Vitamin K2 negatively controls the transcription of HDGF in hepatoma cells[33]. However, the suppressive effects of the Vitamin K2 are limited and it is necessary to elucidate the whole regulation of the HDGF expression in hepatic cells, especially in hepatoma cells.

In conclusion, HDGF overexpressing transgenic mice showed the possible inhibitory role of HDGF on hepatocyte differentiation. The identification of both the regulation and signal transduction of HDGF makes it possible to obtain a better understanding of liver development, regeneration, and carcinogenesis.

We thank T Komori (Nagasaki University) for the helpful discussion.