Yonago Acta medica 2003;46:103–108

Induction of Hepatocyte Nuclear Factor 3γ Gene Expression by the Extracellular Matrix Component from Fish Skin
Makoto Noguchi*, Yusuke Takahashi*†, Makoto Taniguchi†, Shun-ichi Isa†, Ichiro
Arifuku*, Akimichi Yamashita* and Kenzo Sato†*Industrial Research Institute of Tottori Prefecture, Sakaiminato 684-0041 and †Division of
Molecular Biology, Department of Molecular and Cellular Biology, Tottori University Faculty of
Medicine, Yonago 683-8503 JapanThe skin of the halibut (Reinhararditus hippoglossoides) has long been discarded as a waste
product by the food industry in large quantities; hence, we examined the biological activity
in the skin for utilization of the waste. In the present study, we determined induction ofhepatocyte nuclear factor 3γ (HNF3γ), which is involved in liver function, using humanhepatoma cell line HepG2 and extracellular matrix components extracted from the fishskin. Induction of the HNF3γ was detected by reporter plasmid constructed with thepromoter region of the HNF3γ gene and luciferase or green fluorescent protein genes.
The alkali extract containing proteoglycan showed HNF3γ induction, but the acid-extractenriched in collagen did not. Consequently, dermatan sulfate showed the strongest activity
in HNF3γ induction. These observations suggested that fish skin has some significant
biological activity, and indicates its possible function in the food and drug industry.
Key words: dermatan sulfate; fish skin; human hepatoma cell 3γ; liver function; reporter assay

The halibut species Reinhararditus hippoglossoides is a major marine-food product processed in
Tottori Prefecture, Japan. Fish skin in the marine
food industry, discarded as waste like bone and entrails, amounts to more than about 10% of the whole
fish, and is desired for utilization in some other products. The utilization of fish skin is expected tocontribute to the development of a new local industry. Fish skin contains 34% protein and related materials, 14% oil and 52% water (Noguchi et al., unpublished data). Proteins in the fish skin are mainlycomposed of collagens and extracellular matrix(ECM) components such as proteoglycans. In a recent study, we showed that hepatocyte growth fac-

tor (HGF) was induced in the human fibroblast cellline by treatment with dermatan sulfate (DS) purified from fish skin, which is one of the glycosaminoglycans composed of proteoglycans (Noguchi et al.,2003). Moreover, we observed that alkali-extractcontaining DS exhibited inhibition of tumor cell
growth using HepG2 (Noguchi et al., 2003).
On the other hand, hepatocyte nuclear factor
3γ (HNF3γ), a member of the forkhead protein
family is one of the liver-enriched transcription
factors for albumin and other liver-specific genes.
The mouse HNF3 gene family was isolated andcharacterized by Kaestner et al. (1994). HNF3
genes are important in early endoderm and liver

Abbreviations: C6S, chondoitin 6-sulfate; DS, dermatan sulfate; ECM, extracellular matrix; GFP, green fluorescentprotein; HGF, hepatocyte growth factor; HNF3γ, hepatocyte nuclear factor 3γ; HP, heparin; KPS, keratan
polysulfate; PBS, phosphate buffered saline103

M. Noguchi et al.

Likewise, GFP reporter plasmid was constructed byinsertion of the HNF3γ promoter segment (–775 bp
to +15 bp) adjacent upstream of the jellyfish GFP
coding sequence with poly A signal into pUC0CAT(Nakamura et al., 1989).

development in addition to regulation of hepatic
gene expression in the adult liver (Sasaki and
Hogan, 1993). HNF3γ was also found in the notochord and ventral epithelium, suggesting its involvement in mesoderm and neural axis formation(Monaghan et al., 1995). However, the knockout
mouse of the HNF3γ gene was reported to be fertile,
to develop normally, and to show no morphologicaldefects (Kaestner et al., 1998). Previously, we
showed that the HNF3γ transduced by an adenovirus vector had a hepatoprotective effect against
CCl4-liver injury and potency for HGF induction(Nakamura et al., 1999). Therefore, taking theseobservations together, it is quite interesting to see
whether the extracellular matrix component, especially DS, induces HNF3γ gene expression. In thisstudy, we determined the activity for induction of
HNF3γ gene expression using a reporter [the firefly
luciferase and the Aequorea victoria green fluorescent protein (GFP)] by extraction from the skin of
Reinhararditus hippoglossoides as industrial wastematerial from the viewpoint of utilization of an
unused resource.

Preparation of ECM
Standard glycosaminoglycans (AMPS KIT,
Seikagaku-Kogyo, Tokyo, Japan) contain bovine
keratan polysulphate (KPS), heparin (HP), hog dermatan sulfate (DS) and shark chondroitin 6-sulfate(C6S), and were used as described previously(Matsumoto et al., 1993; Tsuchiya et al., 1997).
GRGDSP peptide constructed from synthetic
tripeptids of arginine-glycin-aspartic acid was
obtained from Asahi Techno Glass, Chiba, Japan.
S-Palmitoyl coenzyme A potassium salt was from
Wako Pure Chemical, Osaka, Japan.
Fresh fish skins from Reinhararditus hippoglossoides were obtained as industrial waste products from a marine food processing market. Separation of ECM components was performed referring to the separation method of the collagen of Satoet al. (1986). Briefly, these skins were chopped anddegreased with acetone. Lipid was obtained by removing the acetone from the extract. Alkali extract,in which proteoglycan is enriched (Noguchi et al.,2003), was prepared from the degreased skin bycontinuous agitation in 0.5 M NaOH at 4˚C for 16 h.
Supernatant was neutralized with 0.5 M acetic acid,and low-molecular weight residues were removed
by dialysis with Cellofine Film (cutting molecularweight 8000) (Seikagaku-Kogyo) against H 2O.
Freeze-dried alkali extract was suspended and theconcentration adjusted to 50 mg/mL with H2O.
Acid extract enriched in collagen proteins was
prepared by gentle agitation of the remnants inalkali extraction with 0.5 M acetic acid at 4˚C for 3days, and the extract was dialyzed with Cellofine
Film (cutting molecular weight 8000) against H2O,
followed by freeze-drying. The acid extract wassuspended at a concentration of 50 mg/mL in 0.1 M
acetic acid, and neutralized with 0.1 M NaOH.

Materials and Methods

Cells
Human hepatoma cell line HepG2 was obtained
from Riken Cell Bank (Tsukuba, Japan). The HepG2/
GFP cell line was established by transformation
with reporter (GFP) plasmid, and by lifting the fluorescent colony. All these cell lines were maintainedin Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum.

Plasmid
Luciferase plasmids were constructed by insertionof the rat HNF3γ promoter segment (–1941 bp to+15 bp and –775 bp to +15 bp from the transcription
initiation site) into PicaGene Basic Vector (Nippon
Gene, Toyama, Japan) carrying the firefly luciferase gene (de Wet et al., 1985; Saito et al., 1999).

104

Induction of HNF3γ by glycosaminoglycan

DNA transfection and luciferase assay

A

Luciferase activity(RLU/µg protein)

SV40 EP

Luci

1500

Luci

125

-775 HNF-3γ

Luci

310

0

Luci

0

-1941 HNF-3γ

B(RLU/µg protein)

Luciferase activity

HepG2 cells were plated at a density of 1 × 105 cells/
mL on 24-well dishes. After incubation at 37˚C for
24 h, the cells were transfected with a reporter(luciferase) expression plasmid using calcium
phosphate co-precipitation (Chen and Okayama,
1987). The cells were administered with the fish
extract and further cultured at 37˚C for 48 h. The
luciferase activity was quantitated with a PicaGene
kit (Toyo-Inks, Tokyo) using the luminescencereader LF100 (Nissui Chemical, Tokyo) (Tamura
and Noda, 1994). Intensity of luminescence wasshown by relative light units (RLU) per µg protein
in assay.

Quantitative analysis of GFP expression
HepG2/GFP cells were inoculated at a density of 1
× 104 cells/0.1 mL in a 96 hole microplate, andincubated with glycosaminoglycan or other compounds at 37˚C for 72 h. Fluorescence from thetreated cells was determined by fluorescence microscopy and the intensity of fluorescence from individual cells quantitated by the Image Pro plus
analysis system (Nikon Co., Tokyo). The differences between values from experimental samples
and controls were assessed by Student’s t-test andexpressed as mean with SD. Differences with a Pvalue of < 0.05 were considered statistically significant.

600

*

400

200

0

t
t
oA )
ac ) BS ctionac )SP)
xtgr /mL GD mM yl CmM extrg/mL P
a de
xtr fluiid-(1 m GR (10 mito (2 kali-(5 m
E
c
l
l
A
A
pa
S-

Fig. 1. Transient expression of luciferase gene (Luci).
A: Luciferase reporter plasmids are constructed with simian virus early promoter (SV40 EP) as a positive control, –1941 bp to +15 bp and –775 bp to +15 bp of
HNF3γ DNA fragment (–1941 HNF3γ and –775
HNF3γ, respectively) and no DNA fragments (0) as
negative controls. Luciferase activity is shown in
the right panel in relative light units (RLU)/µg protein.
B: Luciferase activity was determined in the presence ofacid extract (1 mg/mL), synthetic hexapeptides
GRGDSP (10 mM), S-palmitoyle CoA (2 mM), alkali-extract (5 mg/mL), phosphate-buffered saline(PBS) and extraction fluid as controls. Concentrations of these samples were used as described by
Tsuchiya et al. (1997). Values are means with standard deviations from at least 3 independent experiments. *P < 0.01 (significant against PBS). HNF,
hepatocyte nuclear factor; CoA, coenzyme A.

Results
Previously we showed that overexpression of
HNF3γ gene in hepatocyte induces hepatoprotection against CCl4-induced liver injury (Nakamura etal., 1999). It evokes our interest as to whether the
extract from halibut skin is effective on induction of
the HNF3γ gene. Therefore, to establish an assay
system for expression of HNF3γ, we constructed aluciferase-reporter vector using the 2-kb promoter
region of the HNF3γ gene. Partially differentiatedfrom the human hepatoma cell line, HepG2 expresses the HNF3γ gene at low levels, as well as thealbumin gene, a hepatic marker (Saito et al., 1999).
Hence, the reporter plasmids were transfected to
HepG2 cells, and determined by luciferase assay.
As shown in Fig. 1A, luciferase plasmid containing
105

M. Noguchi et al.

the promoter region (–775 bp to +15 bp from transcription initiation site) showed a significant levelof transcriptional activity, rather than the reporter
carrying a longer promoter (–1941 bp to +15 bp).
Agents or chemical substances that are responsible for induction of HNF3γ gene expression
could have predictable hepatoprotective actions(Nakamura et al., 1999). For screening of inducer
substances for HNF3γ expression, we applied various extracts from the fish skin into the HepG2 cell
culture transfected with luciferase plasmid carrying–775 to +15 bp of the HNF3γ gene. As shown in
Fig. 1B, alkali extract from the fish skin exhibited astrong inducing activity for reporter plasmid shownby a 2-fold increase in luciferase activity. Interestingly, synthetic hexapeptids of GRGDSP constructed with a core sequence of arginine-glycin-aspartic
acid, which binds to integrins and antagonizes intracellular signal transmission, considerably inhibitedthe gene expression as well as S-Palmitoyl coenzyme A which is a HNF4 ligand. Meanwhile, administration of acid extract enriched in collagenscarcely increased luciferase activity comparedwith phosphate-buffered saline (PBS) and extraction fluid as controls. These observations suggested
that proteoglycans abundantly contained in alkali
extract may be involved in induction of HNF3γ.
Furthermore, to improve the assay system forextension of HNF3γ gene expression, we established the HepG2 cell line, which was transformed withthe green fluorescent protein (GFP) gene linked
with the –775 bp promoter region of the HNF3γ
gene. This cell line should express GFP with green
fluorescence responding to stimuli for HNF3γ gene
expression. This supposition was confirmed bysupplementation of alkali extract of the fish skin to
the cell culture. As shown in Fig. 2A, PBS-treatedcells were slightly fluorescent at the base level; incontrast, administration of alkali extract to the cells
generated strong fluorescence. Intensity of fluorescence from the individual cells (more than 50 cells)
was determined by an Image Pro plus analysis system, and alkali extract showed a 2.5-fold inductionof GFP in the transformed cell line compared with
control expression, suggesting utilization of the cell

A

PBS

Alkali-extract

B

Relative fluorescenceintensity(5 mg/L)

*
200

100

0

S
PB

t
ac
xtr mL)
e
li- /

ka mg
Al (5

Fig. 2. Fluorescence from HepG2/GFP cells treatedwith alkali extract.
A: HepG2/GFP cells were administered with 5 mg/mL
alkali extract from fish skin for 3 days, and determined by fluorescent microscopy (original magnification, × 200).
B: Intensity of fluorescence from individual cells quantitated by the Image Pro plus analysis system, andrelative fluorescence intensity is shown by counting
the intensity from PBS-treated cells as 100. Values
are means with SDs from at least 50 independent
cells. *P < 0.01 (against PBS). GFP, green fluorescent protein; PBS, phosphate-buffered saline.

line carrying a HNF3γ-promoted GFP gene as ascreening system for hepatoprotective agents.
In a recent study, we showed what compound
included in alkali extract of the fish skin operates as
an inducer of HNF3γ gene expression: we fractionated the extract by ion-exchange chromatography,
and identified dermatan sulfate (DS) as a main component in the skin glycosaminoglycan (Noguchi etal., 2003). Hence, we assessed inducing activity for

106

Relative fluorescenceintensity

Induction of HNF3γ by glycosaminoglycan

injured liver (Nakamura et al., 1999). This hepatoprotection by HNF3γ turned out to be due to superinduction of hepatocyte growth factor HGF. Similarly, forced expression of HNF3γ in primary hepatocyte culture by adenovirus vector showed conservation effects on liver function such as albumin gene
expression, as well as overexpression of HNF3γinduced proliferation arrest in the cells (Nakamura
et al., 1999). These observations indicate that induction of HNF3γ gene expression is involved in apivotal role in the maintenance of hepatocyte differentiation.
Previously, we reported that DS dose-dependently increased the HGF concentration in the culture medium of MRC-5 human fibroblast cells(Noguchi et al., 2003). This stimulatory effect of
DS on HGF production was evident in various typesof cells, such as MRC-9, IMR-90, WI-38 and HL60 (Matsumoto et al., 1995). Furthermore, we observed that alkali extract from fish skin has a growth
inhibitory function that is possibly mediated by HGF
induction in various cell types, such as HepG2, human hepatoma, HL60 and U937, histiocytic lymphoma cells (Noguchi et al., 2003).
Taken together, these observations suggested
that DS from the fish skin should be involved in induction of HNF3γ expression mediated by intracellular signal transduction, followed by enhancingof HGF expression, and that HNF3γ also induced
liver-specific gene expression and HGF-encouraged liver function or hepatoprotective function.
In this study, we feel we have shown that proteoglycan or glycosaminoglycan, especially DS
derived from fish skin, can be used successfully in
medications and functional foods for the maintenance of human liver function.

*

300

*

200

100

0

PBS

KPS

HP

DS

C6S

Fig. 3. Fluorescence from HepG2/GFP cells treatedwith glycosaminoglycans. HepG2/GFP cells were treated with 50 µg/mL keratan polysulphate (KPS), heparin(HP), dermatan sulfate (DS) and condoroitin 6 sulfate(C6S) for 3 days. Relative intensity of fluorescence isexhibited as in Fig. 2. Values are means with SDs from atleast 50 independent cells. *P < 0.01 (significant against
PBS). GFP, green fluorescent protein; PBS, phosphatebuffered saline.

the HNF3γ gene by expression of GFP reporter using purified DS and other glycosaminoglycans. As
shown in Fig. 3, DS showed strong activity for induction of expression of GFP, as well as KPS which
was also expressed at a considerably high level. In
heparin, it increased a little. In comparison to theseglycosaminoglycans, C6S hardly induced GFP expression. These results indicated that DS and KPS
had an inducing activity to HNF3γ gene expression.

Discussion
Reinhararditus hippoglossoides skin, which is usually discarded as industrial waste, contains abundant
collagens and proteoglycans, and its utilization as a
biofunctional material can be expected. In thisstudy, we determined the biological function ofthese extracellular matrix components by inductionof the HNF3γ gene using GFP and luciferase reporter assays. DS showed a significant amount of activity for the induction of HNF3γ in the HepG2 humanhepatoma cell culture. Previously, we showed thatoverexpression of HNF3γ mediated by adenovirus
vector revealed some hepatoprotective ability to the

Acknowledgments: We thank Mr. Shinobu Yoshioka
and Mr. Koji Kashiwagi, Daimatsu Co., Yonago, Japan,
for the offer of the fish skin. We are grateful to Dr.
Yoshikazu Ayagi, Yonago National College of Technology, for the helpful comments.

107

M. Noguchi et al.

acute liver injury caused by CCl4. FEBS Lett 1999;
459:1–4.
10 Nakamuta M, Furuichi M, Takahashi K, Suzuki N,
Endo H, Yamamoto M. Isolation and characterization of a family of rat endogenous retroviral sequences. Virus Genes 1989;3:69–83.
11 Noguchi M, Arifuku I, Yamashita A, Sato K. Induction of hepatocyte growth ffactor and inhibition of
proliferation by extracellular matrix component derived from fish skin of Reinhararditus hippoglossoides. Fisheries Sci 2003;69:401–407.
12 Saito K, Nakamura T, Komoda H, Hori N, Adachi K,
Ito K, et al. Isolation and characterization of the rat
hepatocyte nuclear factor-3γ (HNF-3γ) gene. Res
Commun Biochem Cell Mol Biol 1999;3:157–169.
13 Sasaki K, Hogan BL. Differential expression of multiple fork head related genes during gastrulation andaxial pattern formation in the mouse embryo. Development 1993;118:47–59.
14 Sato K, Yoshinaka R, Sato M. A simple method for
determining collagen in fish muscle. Bull Japan Soc
Sci Fish 1986;52:889–893
15 Tamura M, Noda M. Identification of a DNA sequence involved in osteoblast-specific gene expression via interaction with helix-loop-helix (HLH)type transcriptional factors. J Cell Biol 1994;126:
773–782.
16 Tsuchiya T, Sakaki T, Fuchino T. Glycosaminoglycan. In: Kimura S, ed. The extracellular matrixof fish and marine invertevbrates. Tokyo: Koseisha
Koseikaku; 1997. p.61–72.

Reference
1 Chen C, Okayama, H. High-efficiency transformation of mammalian cells by plasmid DNA.
Mol Cell Biol 1987;7:2745–2752.
2 de Wet JR, Wood KV, Helinski DR, DeLuca M.
Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc
Natl Acad Sci U S A 1985;82:7870–7873.
3 Inouye S, Tsuji FI. Aequorea green fluorescent protein: expression of the gene and fluorescent characteristics of the recombinant protein. FEBS Lett
1994;341:277–280.
4 Kaestner KH, Hiemisch H, Luckow B, Schutz G.
The HNF-3 gene family of transcription factors in
mice: gene structure, cDNA sequence, and mRNA
distribution. Genomics 1994;20:377–385.
5 Kaestner KH, Hiemisch H, Schutz G. Targeted disruption of the gene encoding hepatocyte nuclear factor 3γ results in reduced transcription of hepatocytespecific genes. Mol Cell Biol 1998;18:4245–4251.
6 Matsumoto K, Tajima H, Okazaki H, Nakamura T.
Heparin as an inducer of hepatocyte growth factor. J
Biochem 1993;114:820–826
7 Matsumoto K, Okazaki H, Nakamura T. Novelfunction of prostaglandins as inducers of gene expression of HGF and putative mediators of tissue regeneration. J Biochem 1995;117:458–464.
8 Monaghan AP, Grau E, Bock D, Schutz G. The mouse
homologue of the orphan nuclear receptor tailless isexpressed in the developing forebrain. Development
1995;121:839–853.
9 Nakamura T, Akiyoshi H, Shiota G, Isono M,
Nakamura K, Moriyama M, et al. Hepatoprotective
action of adenovirus-transferred HNF-3γ gene in

Received August 26, 2003; accepted October 1, 2003
Corresponding author: Kenzo Sato, PhD

108