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J Biol Chem, Vol. 274, Issue 17, 11469-11472, April 23, 1999
§,,,,,,,,,,, and¶
From the Beijing Institute of Radiation Medicine,
Chinese National Human Genome Center, 27 Taiping Road, Beijing
100850, People's Republic of China and § Southwest Hospital
of the Third Military Medical College, ChongQing 400042, People's
Republic of China
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Hepatopoietin (HPO) is a novel polypeptide
mitogen specific for hepatocytes and hepatoma cell lines, which is
derived from liver and supports its regeneration. To determine whether
HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its
binding activity by specific displacement test and Scatchard analysis
in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells.
The binding was saturable and specific because it was replaceable by
HPO but not by epidermal growth factor, transforming growth factor- Previous studies implicate that a small molecule derived from
liver itself specifically stimulates hepatocytes proliferation and
supports liver regeneration (1-3). In 1975, LaBrecque et al. (3) first reported that in the liver of a weaning rat and the
regenerating liver of a partially hepatectomized rat, there existed
hepatic stimulator substance
(HSS)1 that could
specifically stimulate DNA synthesis in hepatic cells. Other groups
have also carried out extensive research on HSS derived from other
species (2). At the same time, experiments and clinical research on
human fetal liver cells demonstrated its therapeutic effect on
hematopoietic diseases and severe liver diseases (2, 4). Since the
1980s, we began to isolate and purify the effective component from
fetal liver. We identified hepatic stimulatory activity in the fraction
with molecular size ranging from 10 to 30 kDa of human fetal liver
lysate (5-7). The activity was target-specific, which was different
from various well known nonspecific hepatic stimulators such as
insulin, EGF, insulin-like growth factor, and TGF- Materials--
Chemicals were purchased from Sigma Chemical
Company. Sodium-125I and hypercoat emulsions were purchased
from Amersham Pharmacia Biotech. Reagents for SDS-polyacrylamide gel
electrophoresis (PAGE) was obtained from Bio-Rad. Tissue culture
reagents were purchased from Life Technologies, Inc. EGF and TGF- HPO Biological Activity Test--
5 × 104
HepG2 cells were plated on a 96-well plastic culture plate and cultured
in Dulbecco's modified Eagle's medium containing 10% calf serum, 10 nM insulin, and 10 nM dexamethasone under
5% CO2 and 30% O2 in air at 37 °C. After
12 h, various concentrations of human HPO was added with EGF and
TGF- Isolation and Primary Culture of Rat
Hepatocytes--
Paraenchymal hepatocytes were isolated from adult
male Wistar rats weighing 150~200 g by in situ perfusion
of the liver with collagenase (18-20), further purified by Percoll
density gradient centrifugation. Cell viability was measured by testing
exclusion of trypan blue, and cell preparations showing over 90%
viability were used to culture. Cells were plated at a density of
105 cells/ml and cultured in Dulbecco's modified Eagle's
medium containing 10% calf serum, 10 nM insulin, and 10 nM dexamethasone under 5% CO2 and 30%
O2 in air at 37 °C.
Radioiodination of HPO--
rhHPO was iodinated by chloramine-T
methods (21-24). Briefly, 15 µl of 50 mM sodium
phosphate buffers (pH 7.0) and 0.5 mCi sodium-125I were
added to a siliconized tube containing 5 µg of HPO. The reaction was
started by adding 10 µl of chloramine-T solution (1 mg/ml) for 1 min
under room temperature. After halting the reaction using 20 µl of
ending solution (50 mM
N-acetyl-L-tyrosine (Sigma), 0.01 M
sodium metabisulfite, 10% glycerol, 0.1% xylene cyanole, 0.1 M sodium phosphate buffer), 125I-HPO was
separated from free iodines by gel filtration on a column (20 × 11.0 cm) of Sephadex G-25 (Amersham Pharmacia Biotech) equilibrated with PBS and 0.1% bovine serum albumin (Sigma), and the fractions containing 125I-HPO were pooled.
125I-HPO Binding Assay--
Adult rat hepatocytes
and human hepatoma cells were cultured for 24 h then the
monolayers were washed with binding buffer (20 mM HEPES,
0.1% bovine serum albumin/Hanks, pH 7.0) and pre-incubated in the
presence of the same buffer for 30 min at 25 °C. After equilibration, fresh ice-cold binding buffer containing various concentrations of 125I-HPO with or without excess amounts
of unlabeled HPO was added as indicated. Incubation was run for 1 h at 25 °C with constant shaking. The monolayer was washed 5 times
with ice-cold buffer. Radioactivity of 125I-HPO to cells
was measured in a Cross-linking of 125I-HPO to Its Receptor--
After
binding of 125I-HPO to hepatocytes, each well was washed 5 times with cold PBS. Freshly prepared bis-sulfosuccinimidyl suberate
(Sigma) was then added to the final concentration of 0.25 mM to each well containing 1 ml of PBS (26-28). The dishes were incubated at 4 °C for 15 min on a shaker platform. After cross-linking, each plate was washed twice with cold PBS then placed in
1 ml of cold detachment buffer consisting of 10 mM Tris (pH
7.4), 1 mM EDTA, 0.25 M sucrose, and 1 mM phenylmethanesulfonyl fluoride. The cells were scraped
from the plates and pelleted in a microcentrifuge at 10,000 × g for 1 min. Cell pellets were solved in 50 µl of
nonreducing SDS-PAGE sample buffer (50 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, and 0.002% bromphenol blue) containing 1%
Triton X-100, boiled for 5 min, and subjected to 12.5% SDS-PAGE. Gel
were dried and exposed to Kodak film at Biological Activity of 125I-HPO--
To rule out the
effect of iodination on rhHPO, molecular weight and biological activity
of 125I-HPO were detected. As shown in Fig.
1A, 125I-HPO had
the similar molecular weight as the unlabeled HPO measured by SDS-PAGE
and autoradiography. Otherwise, 125I-HPO retained the same
biological activity (stimulation of hepatocyte proliferation) as the
unlabeled human HPO as shown in Fig. 1B. Taken together, the
results above demonstrated that iodination did not change the
characteristics of natural human HPO, and also indicated the
feasibility that 125I-HPO can be used for identification of
HPO receptor.
Identification of a High Affinity Receptor for HPO--
Fig.
2, A and C show
typical saturation curves of 125I-HPO binding to cultured
hepatocytes. Specific binding of HPO was saturated at about 1.5 pM. Scatchard analysis resulted in a rectilinear plot,
thereby suggesting the presence of a single class of high affinity
binding sites, i.e. the existence of a receptor of HPO. The
Kd value and the number of HPO receptors calculated from the Scatchard plots were 2 pM and 10,000 sites/cell
for primarily cultured rat hepatocytes, and 0.7 pM and
55,000 sites/cell for HepG2 cells, respectively.
Specificity of 125I-HPO Binding to the
Receptor--
Fig. 3 shows typical
displacement curves of 125I-HPO binding to the HPO
receptor. Only unlabeled HPO could replace the binding of
125I-HPO to the receptor in a
concentration-dependent manner, and almost complete
replacement was achieved with 100 times the concentrations of
125I-HPO. EGF, TGF- Identification of 125I-HPO·Receptor Complex--
To
characterize the molecular weight of this receptor, hepatocytes were
chemically cross-linked with 125I-HPO by the
homobifunctional reagent bis-sulfosuccinimidyl suberate. The HPO
receptor was specifically labeled with 125I-HPO and the
complex of receptor-ligand could be identified by SDS-PAGE and
autoradiography. This resulted in the labeling of one cross-linked
species that migrated as the complex of HPO (molecular mass, 15 kDa)
with the molecular mass of its receptor being about 90 kDa. This band
was absent when an excess amount of unlabeled HPO were present. EGF
does not compete with 125I-HPO for binding to its receptor
and did not affect the labeling of the band with molecular mass of
about 90 kDa, as shown in Fig. 4. Thus,
the molecular mass of the HPO receptor was calculated to be about 75 kDa by subtracting molecular mass of 15 kDa of HPO from 90 kDa of the
complex.
This study shows the presence of high affinity receptor for HPO on
rat hepatocytes and human hepatoma cells. This is the first report
about the existence of a cellular receptor for HPO. It seems likely
that HPO stimulate hepatocyte proliferation by binding to the specific
receptor on the cell surface. This finding might initiate further
understanding of the molecular mechanism of the biological action of
HPO and of its effect on liver regeneration.
HPO can obviously stimulate hepatocyte proliferation and liver
regeneration. The biological effect of HPO on the stimulation of DNA
synthesis in hepatocytes is half-maximal at 0.5-1.0 pM concentration (13). The half-maximum dosage for HPO activity was in
good accord with the Kd value (0.8-2.0
pM) of the HPO receptor, thereby indicating that this high
affinity receptor may play an important physiological role in the
signaling system. 125I-HPO induced a time- and
dose-dependent effect on binding to cell surface sites of
the primarily cultured rat hepatocytes and human hepatoma cells. The
binding reaction is reversible and saturable. When an excess amount of
unlabeled HPO was added, it could replace the cell surface sites that
HPO has been binding to. In addition, the binding is specific because a
great amount of EGF, TGF- By binding test and specific replacement test, we also found that the
distribution of HPO receptor was certainly specific. Whether normal
(primarily cultured rat hepatocytes, L02 cells, and primarily cultured
human fetal hepatocytes) or abnormal hepatocytes (e.g.
hepatoma cells such as HepG2, HTC, SMMC7721 cell lines) were derived
from liver, all of them contained the receptor of HPO (data not shown).
But for the nonhepatocytes cells derived from other tissue, such as
COS-7 cells (kidney), GLC-82 cells (lung), K562 cells (hematopoietic
tissue), Hep2 cells (larynx), and Chinese hamster ovary cells (ovary),
none of them had a dose-dependent effect or receptor (data
not shown). These results demonstrated that the tissue distribution and
location of the cells of the HPO receptor was unique but correlated
with the responsiveness of the unique target cells of HPO.
Although primarily cultured rat hepatocytes, human hepatoma cells, and
human fetal hepatocytes all have the receptor for HPO, they are
different in the number and the affinity of the receptor for HPO. Of
primarily cultured rat hepatocytes and hepatoma cells, the receptor
number of the former is less and the receptor affinity (Kd value) is lower than that of the latter. The
receptor number and affinity of fetal hepatocyte are median among the
three kinds of cells above. The differences in HPO receptor number and affinity of those cells are in good accord with the degree of their
response to HPO. The facts pointed out that HPO acts biologically on
the target cells via its specific receptor and that the number and
affinity of the HPO receptor represent the physiological modulation of
its target cells.
,
or insulin. Scatchard analysis indicated the presence of a single class
of high affinity receptor with dissociation constant
(Kd) of 2 and 0.7 pM, and a receptor
density of about 10,000 sites/cell and 55,000 sites/cell in the rat
hepatocytes and human hepatoma cells, respectively. The
Kd values were consistent with the half-maximum
dose of HPO activity. Affinity cross-linking of the receptor with
125I-HPO revealed a polypeptide of molecular mass
approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus,
the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in
hepatocytes and hepatoma cells, which may account for biological effect.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. The
characteristics of the effective component derived from human fetal
liver were consistent with those of HSSs derived from other species,
suggesting that the effective component could be the human-derived
homologue of the animal's HSS. Then, we purified this activity and
demonstrated that the biological activity of its pure form is identical
to those of the crude form and consistent with those of animal-derived
HSSs, but evidently different from those of serum-derived hepatocyte
growth factor (8). The factor was named as hepatopoietin (HPO). Later,
we proved that HPO is encoded by mRNA of fetal liver (9) and
further cloned (10) its full-length cDNA, encoding a 15.1-kDa
protein from the cDNA library of human fetal liver, which is of
87% homology with rat augmenter of liver regeneration cDNA (11)
and is identical to the human homologue of yeast ERV1 (essential for
respiration and viability) cDNA (12). Recombinant human
hepatopoietin (rhHPO) can stimulate proliferation of hepatocytes and
hepatoma cells in vitro (13). Furthermore, in animal models,
rhHPO promotes regeneration and recovery of damaged hepatocytes and
rescues acute hepatic failure in vivo (13, 14). Thus, HPO is
a growth factor important in liver regeneration. However, the signaling
mechanism of HPO has been unclear. It remained unknown whether HPO bind to a specific receptor in cell membrane then initiate a corresponding cytoplasmic signal transduction pathway and mediate its biological effect on hepatocytes. We report here the identification and
characterization of the receptor for HPO, which may give some insight
into the mechanisms of its biological action.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were purchased from Earth Chemical Corp. rhHPO was expressed in
Escherichia coli and prepared with high purity (>95%) as
described previously (15). HepG2 was from the human hepatoma cell line.
as the positive controls. The incubation time was for 24 h, then 0.5 µCi [3H]thymidine deoxyribose was added to
each plate well for 3 h. Radioactivity of cells was measured in a
-counter after detachment of the monolayer by incubation for 10~20
min with 200 µl of 0.25% trypsin solution (16, 17).
-counter after detachment of the monolayer with
0.25% trypsin solution (24-27). All binding experiments were done in triplicate.
70 °C for 5-7days.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
[in a new window]
Fig. 1.
Characteristics of 125I-HPO.
A, SDS gel electrophoresis; molecular weight standards,
left lane; 125I-HPO, middle lane;
unlabeled HPO, right lane. B, stimulation
of [3H]thymidine incorporation into DNA of HepG2 cell by
125I-HPO, unlabeled HPO, EGF (10 ng/ml), and TGF- (10 ng/ml).
View larger version (21K):
[in a new window]
Fig. 2.
A, saturation curves of
125I-HPO binding to its receptor on adult rat primarily
cultured hepatocytes. 105 adult rat monolayer hepatocytes
were incubated for 1 h at 25 °C with 125I-HPO or
unlabeled HPO. 
, total binding; 
, specific binding; 
,
nonspecific binding. A Scatchard plot of the HPO receptor on rat
hepatocytes is shown in Fig. 2B. B, Scatchard
plot of the HPO receptor on rat hepatocytes. C. Saturation curves of
125I-HPO binding to its receptor on HepG2 cells.
105 human monolayer hepatoma cells were incubated for
1 h at 25 °C with 125I-HPO or unlabeled HPO. ,
total binding; , specific binding; 
, nonspecific binding. A
Scatchard plot of HPO receptor on human hepatoma cell lines is shown in
Fig. 2D. D, Scatchard plot of HPO receptor on
human hepatoma cell lines.
, and insulin could not replace
125I-HPO to the receptor even at 100 times the
concentrations (1 nM) of unlabeled HPO. These results
confirmed the specificity of 125I-HPO binding to the
receptor and indicated that HPO receptor is different from the
receptors of EGF, TGF-, or insulin.
View larger version (15K):
[in a new window]
Fig. 3.
Displacement of 125I-HPO on its
receptor of hepatocytes by various concentrations of various
ligands. Hepatocytes were incubated in the presence of 1 pM 125I-HPO and the indicated concentration of
unlabeled HPO ( 
), EGF(
), TGF- (×), and insulin (). Results
are expressed as a percentage of 125I-HPO bound to control
without unlabeled HPO.
View larger version (145K):
[in a new window]
Fig. 4.
Affinity cross-linking of
125I-HPO to its cell surface receptor. Hepatocytes
(106) were incubated with 1 pM
125I-HPO for 1 h at 25 °C in the presence
(lane D) or absence (lane C) of 100 pM unlabeled HPO or in the presence of 200 pM
EGF (lane E). After binding, cells were washed extensively
with cold PBS, and cross-linked to bound 125I-HPO with 0.25 mM bis-sulfosuccinimidyl suberate as described under
"Experimental Procedures." Following cross-linking, cells were
washed and scraped off the dishes, sedimented, and solubilized in 50 µl of SDS-PAGE sample buffer containing 1% Triton-100. Aliquots (25 µl) were boiled and subjected to 12.5% SDS-PAGE under nonreducing
conditions. The gel was dried and subjected to autoradiography. No
cross-linker was used in lane B. In lane A,
bis-sulfosuccinimidyl suberate was added to 125I-HPO
solution in the absence of cells. The migration positions of molecular
weight standards are indicated on the left side.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and insulin make no effect on
125I-HPO binding to the cell surface sites. Therefore, it
is reasonable to conclude that the hepatocytes surface sites are just
the receptors for HPO, which have typical characteristics of a receptor
such as high affinity, high specificity, reversibility, and saturation.
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ACKNOWLEDGEMENTS |
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We thank Dr. YongTao Yu for technical help, and Professors Lin Mei, Bai Lu, Haifan Lin, and Mingyao Liu for helpful suggestions.
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FOOTNOTES |
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* This work was supported by Chinese National Distinguished Young Scholar Awards (39620514) and Chinese National Natural Science Foundation (39780005).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: Tel.: 8610-68159479; Fax: 8610-68213044; E-mail: hefc{at}nic.bmi.ac.cn.
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ABBREVIATIONS |
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The abbreviations used are:
HSS, hepatic
stimulator substance;
HPO, hepatopoietin;
rhHPO, recombinant human HPO;
HGF, hepatocyte growth factor;
EGF, epidermal growth factor;
TGF-, transforming growth factor-;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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