Vol.
176,
No.
April
15,
1991
1, 1991
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
Hepatocyte Human Kunio
Growth Factor is a Potent Stimulator Melanocyte
Matsumoto,
Department
Received
AND
February
DNA Synthesis
Hisao Tajima,
23,
of
and Growth
and Toshikazu
of Biology, Faculty of Science, Fukuoka 812, Japan
45-51
Nakamura
Kyushu
University.
1991
SUiIfiWARX Hepatocyte growth factor (HGF) is a potent mitogen for adult rat hepatocytes in primary culture. HGF stimulates growth and DNA synthesis of normal human epidermal melanocytes in culture. The maximal stimulation of DNA synthesis by 4.0-fold occurred with 10 rig/ml HGF. This stimulatory effect was additive with both acidic and basic fibroblast growth factors, while it was inhibited by transforming growth factor-pl. Melanocytes expressed a single class of specific, high-affinity receptors for HGF with a Kd of 22 pM and approximately 120 receptors/cell. Thus, HGF is a potent mitogen for normal human epidermal melanocytes. 0 1991Rcademic Pre.55,Inc.
Hepatocyte partially
hepatectomized
rat platelets
composed
HGF
cDNAs
structures.
another
group
derived
from
to form HGF
hepatocytes markedly
has the highest in primary
increased
plasma
(6) and
amino
culture.
in the liver
(4) and rabbit
serum
polypeptide
of 728 amino
mature
HGF. Among
potential
to promote
HGF
activity
following
45
we their
independently that
by HGF is
acids and proteolytically known
peptide
the growth
the development
cells in the rat liver are non-parenchymal
factor
determined
revealed
and HGF
(11) and cell fractionation
growth
(3). Recently,
(7). and
acid sequence
of from
p-subunit
rat
serum
homogeneity
cDNA was also cloned
precursor
a two-chain
10). In situ hybridization producing
HGF
in the
to apparent
and the 34kD
human
(8). The predicted chain
identified
rat HGF is a 82kD
Human
a single
first
HGF in human
a-subunit from
(HGF)
(1) was purified
The purified
primary
factors,
rats
of the 69kD
processed
factor
(2, 3). Thereafter,
(5) was purified.
cloned
growth
mRNA
growth
of mature levels
were
of hepatitis
(10) revealed
that
(9, HGF-
liver cells, presumably
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
176,
No.
Kupffer
BIOCHEMICAL
1, 1991
cells and
hepatotropic
sinusoidal
factor
which
We obtained tissues
other
stimulated
the
that
liver
(7, 11)
of normal
hepatocytes
became
apparent
restricted
and
(submitted
to targeting Epidermal
Elucidation
a wide
of growth
and
rabbit
HGF
seems
renal
found
epithelial
receptor
publication).
Therefore,
by melanocytes in
conditions
and
in
tissue
As melanocytes
phorbolfibroblast
growth
receptors
epidermal
Melanocytes
13-acetate
factor
(16, 17). We now report human
repair.
in uiuo (13) and constitute
12-myristate-
melanocytes
(PMA)
that and
normal
a minor in culture
(bFGF) is a potent evidence
is not
for
mitogen
require
the
cells
rarely
in epidermal the presence
for human mitogen express
to
pathological only
(14- 17)
HGF is a potent that
and
population
growth
melanin.
is important
are regulated
is difficult.
HGF
role in producing
melanocytes
cells, cultivation
receptor
hepatocytes.
how
mitosis
HGF
for HGF
of the HGF
determine
undergo
that
rat
cells in secondary
specific
distribution
requirement
epidermal
to be a
in various
we recently
have a important
factor
COMMUNICATIONS
is present
a high affinity
for
melanocytes
Thus,
mRNA
tissue
only mature
RESEARCH
for the liver regeneration.
HGF
(12). We have also identified
on rat
cells.
acts as a trigger
DNA synthesis
cultures
BIOPHYSICAL
endothelial
evidence
than
AND
and
of
basic
melanocytes for normal high-affinity
for HGF. MATERIALS
AND METHODS
Cell Culture: Normal human epidermal melanocytes were obtained from Kurabo Co. (Osaka) and cultured as described (17). Briefly, these melanocytes were cultured in the nutrient medium MCDB 153 supplemented with ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), hydrocortisone (0.5 mM), insulin (5 mg/ml), PMA (10 rig/ml), and bovine pituitary extract (BPE, 25 pg protein/ml). In some experiments, BPE was deleted from the medium. Fourth or fifth passage cells were used. Measurement of Cell Growth and DNA Synthesis Melanocytes were plated at a density of lo* cells/well on a 12-well plate (Corning), and next day the medium was changed to fresh medium without BPE. An appropriate amount of growth factor was added to each culture and the cehs were cultured for 15 days. Cells were treated with trypsin and the number of cells was counted using a hematocytometer. DNA synthesis was determined by measuring the incorporation of (12511-deoxyuridine into the nuclei. Cells were plated at a density of 4 x 104 cells/well on a 24-well plate. The next day, the medium was changed to 46
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
fresh medium without BPE and an appropriate amount of growth factor was added. Cells were cultured for 24 h and pulse-labeled with 1 PCi of [125I]deoxyuridine (2200 Ci/mmol, New England Nuclear) for 4 h. These cultured cells were washed with PBS and then with ice-cold 10% (w/v) TCA. The cells were solubilized with 1 M NaOH and the radioactivity was counted in a gamma counter. Protein concentration was measured using the Micro BCA protein assay system (Pierce Chemical) with bovine serum albumin as the standard. Assay for [12sI]-HGF Binding: Human recombinant HGF was radioiodinated by the chloramine-T method. Details on the methods for the radioiodination and the binding of HGF to cells should be published elsewhere. Briefly, 1.5 M sodium phosphate buffer, pH 7.0 (10 ~1). 0.5 pg HGF (17 ~1). and 0.5 mCi Na[ 12511 (14 Ci/mg I. IMS 30. Amersham) were added to a siliconized tube and the reaction was started by adding of 5 ~1 of chloramine-T (100 pg/ml), four times at 30 set intervals. The reaction was stopped by adding 20 ~1 N-acetyl-L-tyrosine (50 mM), 200 ~1 KI (60 mM) and 200 ~1 urea solution (1.2 g/ml in 1 M acetic acid). ]12sI]-HGF was separated by molecular sieve chromatography on a Sephadex G-25 column (Pharmacia) equilibrated with 4 mM HCl, 75 mM NaCl, and one mg/ml bovine serum albumin. [1251]-HGF thus prepared had a specific activity of 70160 mCi/mg. Cells were cultured in 24-well plates and when subconfluency was reached, the cultures were refed with fresh medium without BPE. After a 24 h culture, the cells were washed once with binding buffer (Hanks’ solution containing 20 mM HEPES, 2 mg/ml bovine serum albumin, pH 7.0) and equilibrated in the buffer for 30 min at 10°C. Ice-cold binding buffer containing appropriate concentrations of unlabeled HGF was added and the cells were incubated at 10°C for 1 h. Cultures were washed three times with the ice-cold binding buffer and [ 12sI]-HGF specifically bound to cells was measured in a gamma counter after detaching the cells with trypsintreatment. Growth Factors: Human recombinant HGF was purified from culture medium of the COS-1 cells transfected with plasmid containing human HGF cDNA (6). Human recombinant bFGF and human recombinant acidic fibroblast growth factor (aFGF) were obtained from TOYOBO (Osaka). Transforming growth factor-p1 (TGF-Pl) was purified from human platelets, as described previously (18).
RESULTS We examined melanocytes factors absence
the
effect
in the presence
on the DNA
of HGF
of PMA,
synthesis
the cell growth
manner.
reached
stimulated
DNA
cell growth
promotion
was reached
with
about
Effects
with
by HGF, 10 rig/ml,
the
became with same
as shown a 4.0-fold 47
growth
of normal
the stimulatory
effect
is not usually
HGF stimulated
the maximum
synthesis
since
of melanocytes
of PMA. Fig.1 shows that
in a dose-dependent
on the
the growth
apparent 10 rig/ml
with
of growth
evident
in the
of melanocytes, 1.25 rig/ml
HGF. In addition,
dose-dependency
evident
in Fig. 2. The maximal stimulation
human
compared
and HGF in the
response to finding
Vol.
176,
No.
BIOCHEMICAL
1, 1991
01
AND
BIOPHYSICAL
02
RESEARCH
O’ b
HGF (ng I ml)
COMMUNICATIONS
I
I
5 HGF
10
f20
(ng i ml)
NJ?. 1. Effect of HGF on the growth of normal human epidermal melanocytes. Cells were plated at a density of 104 cells/well on a 12-well plate. The next day, the medium was changed to fresh medium without BPE. and HGF was added at the various concentrations. Cultures were refed on days 6 and 12 with the same medium and the culture was stopped on day 15. The average of duplicate measurements is shown. Fig. 2. Effect of HGF on DNA synthesis of normal human epidermal melanocytes. Cells were plated at a density of 4 x 104 cells/well on a 24-well plate, The next day, the medium was changed to fresh medium without BPE, and HGF was added at the various concentrations. Cells were cultured for 24 h and pulse-labeled with 1 PCi of [ 1251]-deoxyuridine for 4 h. The average of triplicate measurements is shown.
of HGF. The half-maximal
in the absence
stimulation
was seen with
3 rig/ml
HGF. the effects of HGF. aFGF. bFGF and the combinations
Fig. 3 shows
HGF and other (10 rig/ml) 8.0-fold,
growth
and aFGF respectively.
same potency
factors
on the DNA synthesis
(10 rig/ml) HGF
stimulated
(10 rig/ml)
as seen with
alone
DNA synthesis stimulated
can inhibit
the growth
including
human
epidermal
keratinocytes
inhibited
DNA
rig/ml)
strongly
stimulated
confirm
that
we searched
for a specific
shows
[1251)-radiolabeled
human
it with
of various (19.
of the
almost
the
with
those
epithelial
cells,
20).
human
and
TGF-pl
(10
melanocytes
by HGF.
To further
saturable
synthesis
bFGF
by 3%fold
bFGF. The effect of HGF was additive
of bFGF and aFGF. TGF-pl normal
of melanocytes.
of
that
manner. melanocytes
Scatchard express
human
receptor
melanocytes
are target
for HGF on human
HGF
specifically
analysis
approximately 48
melanocytes.
bound
of the specific
cells of HGF,
to the
binding
120 receptors/cell
Fig. 4
cells
showed with
in a that
a Kd of
Vol.
176,
No.
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
2.5 .E B e P E 2 b
2.0
1.5
X
E P z .i? $ r, E $
1.0
0.5
2 0 None HGF
03
bFGF
aFGF
HGF
HGF
HGF
bF+GF aF+GF TG&l
I
04 O
I 20
10 ‘=‘I-HGF
30
( pM )
Fig. 3. Effect of HGF, aFGF. bFGF, and combinations of HGF plus other growth factors on DNA synthesis of normal human epidermal melanocytes. Cells were plated at a density of 4 x 104 cells/well on 24-well plates. The next day, the medium was changed to fresh medium without BPE, and growth factors (10 rig/ml for each) were added. Cells were cultured for 24 h and pulse-labeled with 1 pC!i of [ 1251]-deoxyuridine for 4 h. The average of triplicate measurements & SD) is shown. Fig. 4. Analysis of [12sI]-HGF binding to normal human epidermal melanocytes. Binding of [1251]-HGF was performed as described in the text. Each point shows the amount of [ 125I]-HGF specifically bound to melanocytes exposed to increasing concentrations of HGF. Inset: Scatchard analysis of the data on binding.
22 pM. The very low Kd value is fairly for stimulation
of both
growth
consistent
with the half-maximal
and DNA synthesis
of 3 rig/ml
HGF
dose (35 pM
HGF). DISCUSSION
Evidence
that HGF acts as a potent
epidermal
melanocytes
synthesis
and
carried
high
those of bFGF for HGF,
is as follows:
cell growth
affinity and
distinct
aFGF.
from
hence
those
factor
1) HGF strongly
of human
receptors
growth
melanocytes:
for normal
enhanced 2) human
human
both
melanocytes
for HGF. The effect of HGF was additive HGF
probably
for other 49
acts
growth
through
factors.
a specific In fact,
DNA
with
receptor
Scatchard
Vol.
176,
No.
analysis
BIOCHEMICAL
1, 1991
revealed
seems
that
to differ
number
are similar identified
to findings
affinity
receptor
from
rat liver. Hepatocytes value
located
epidermal
at the epidermal-dermal
folicles.
the
melanocytes
culture.
We have
on rat hepatocytes
have approximately (Higuchi
derived
junction
Since melanocytes
HGF may function conditions,
and
500-600
and
Nakamura,
radiation
mesoderm-origin: monocytes
or infiltrating
monocytes tissue,
to stimulate
A scatter
that HGF stimulates (12). There
quantity
the growth
stimulates
or highly
related
such
those
of
fibroblasts, under
(unpublished endothelial
pathological
cells,
conditions
to the basal layer of the
of renal tubular
keratinocytes
of epithelial
epithelial
of the HGF
an important
in development,
interactions
morphogenesis,
50
we found
cells in secondary
receptor
(to be published).
and carcinogenesis.
cells was
to HGF (22). In addition,
for epithelial-mesenchymal
regeneration,
stimuli
among
lung fibroblasts
the motility
well be a mediator role
pathological
to selected
may diffuse
is a wide distribution
epidermal
certain
elusive.
of melanocytes.
which
the growth
has remained
cells in the liver (1 l), blood
by dermal
macrophages
a certain
factor
to be identical
cells including
and
of
in uiuo (13), the
cells are found
and embryonic
if HGF is produced
and in wounded epidermis
and in response
crest are
at the matrix
mitosis
under
cells and endothelial
and macrophages,
and
melanocytes
(21). HGF producing
Kupffer
Therefore,
undergo
for melanocytes
process,
from the neural
of the skin
only rarely
as a mitogen
wound-healing
as ultraviolet
culture
in primary
pM
melanocytes
role HGF may play in uivo for epidermal
found
(22 PM) and
for publication).
The human
data).
on melanocytes
to human
for HGF
of 20-30
COMMUNICATIONS
receptor
in HGF-binding
adult rat hepatocytes
a Kd
RESEARCH
(17). The Kd value
high
with
submitted
for bFGF
with
membranes
receptors/cell
hair
that
BIOPHYSICAL
of the HGF
(120 sites/cell)
specific,
plasma
the character
from
of receptors
AND
in epithelial
Thus,
HGF
may
and would
play
wound-healing,
tissue
Vol.
176,
No.
Acknowledgments: manuscript.
BIOCHEMICAL
1, 1991
We thank
This work
Cancer from the Ministry
AND
BIOPHYSICAL
Dr. Mariko
was supported of Education,
Ohara
RESEARCH
COMMUNICATIONS
for critical
by a Research
Grant
Science and Culture
reading for Science
of the and
of Japan.
REFERENCES
1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Nakamura. T., Nawa, K. and Ichihara, A. (1984) Biochem. Biophys. Res. Commun. 122, 1450-1459. Nakamura, T., Teramoto, H. and Ichihara, A. (1986) Proc. NatI. Acad. Sci. USA. 83. 6489-6493. Nakamura, T., Nawa. K., Ichihara, A., Kaise, A. and Nishino, T. (1987) FEBS Lett. 224, 31 l-318. Gohda, E., Tsubouchi, H., Nakayama. H., Hirono, S., Sakiyama. 0.. Takahashi, K., Miyazaki, H., Hashimoto, S. and Daikuhara, Y. (1988) J. Clin. Invest. 81. 414-419. Zamegar, R. and Michalopoulos, G. (1989) Cancer Res. 49, 3314-3320. Nakamura, T., Nishizawa. T.. Hagiya, M., Seki. T., Shimonishi, M., Sugimura, A., Tashiro, K. and Shimizu, S. (1989) Nature 342. 440-443. Tashiro, K., Hagiya, M., Nishizawa, T., Seki, T., Shimonishi, M., Shimizu. S. and Nakamura, T. (1990) Proc. Nail. Acad. Sci. USA. 87, 3200-3204. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayama, H., Hirono, S., Sakiyama, 0.. Takahashi, K., Gohda, E., Daikuhara. Y. and Kitamura, N. (1989) Biochem. Biophys. Res. Commun. 163, 967-973. Asami. 0.. Ihara, I.. Shimizu. N., Shimizu, S.. Tomita, Y., Ichihara, A. and Nakamura, T. (1991) J. Biochem. 109, 8-13. Kinoshita. T., Tashiro. K. and Nakamura. T. (1989) Biochem. Biophys. Res. Commun. 165. 1229-1234. Noji, S., Tashiro, K., Kayama. E., Nohno, T.. Ohyama. K., Taniguchi, S. and Nakamura, T. (1990) Biochem. Biophys. Res. Commun. 173. 42-47. Igawa, T., Kanda. S., Kanetake, H., Saitoh, Y., Ichihara, A., Tomita, Y. and Nakamura, T. (1991) Biochem. Biophys. Res. Commun. 174, 831-838. Jimbow, K., Roth, S.I., Fitzpatrick, T.B. and Szabo, G. (1975) J. Cell Biol. 66, 663-670. Eisinger, M. and Marko, 0. (1982) Proc. Natl. Acad. Sci. USA. 79, 20182022. Eisinger. M.. Marko. 0.. Ogata, S. and Old, L.J. (1985) Science 229, 984-986. Halaban. R.. Ghosh. S. and Baird, A. (1987) In Vitro Cell. Dev. Biol. 23. 37-52. Pittelkow, M.R. and Shipley, G.D. (1989) J. Cell. Physiol. 140, 565-576. Nakamura, T., Teramoto. H., Tomita. Y. and Ichihara, A. (1986) Biochem. Biophys. Res. Commun. 134. 755-763. Shipley, G.D., Pittelkow, M.R.. Wille, J. J., Jr., Scott, R.E. and Moses, H.L. (1986) Cancer Res. 46. 2068-2071. Matsumoto. K., Hashimoto, K.. Hashiro, M., Yoshimasa, H. and Yoshikawa, K. (1990) J. Cell. Physiol. 145, 95101. Rosdahl. I. and Szabo, G. (1978) J. Invest. Dermatol. 70, 143-148. Gherardi, E. and Stoker, M. (1990) Nature 346. 228.
51
176,
No.
April
15,
1991
1, 1991
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
Hepatocyte Human Kunio
Growth Factor is a Potent Stimulator Melanocyte
Matsumoto,
Department
Received
AND
February
DNA Synthesis
Hisao Tajima,
23,
of
and Growth
and Toshikazu
of Biology, Faculty of Science, Fukuoka 812, Japan
45-51
Nakamura
Kyushu
University.
1991
SUiIfiWARX Hepatocyte growth factor (HGF) is a potent mitogen for adult rat hepatocytes in primary culture. HGF stimulates growth and DNA synthesis of normal human epidermal melanocytes in culture. The maximal stimulation of DNA synthesis by 4.0-fold occurred with 10 rig/ml HGF. This stimulatory effect was additive with both acidic and basic fibroblast growth factors, while it was inhibited by transforming growth factor-pl. Melanocytes expressed a single class of specific, high-affinity receptors for HGF with a Kd of 22 pM and approximately 120 receptors/cell. Thus, HGF is a potent mitogen for normal human epidermal melanocytes. 0 1991Rcademic Pre.55,Inc.
Hepatocyte partially
hepatectomized
rat platelets
composed
HGF
cDNAs
structures.
another
group
derived
from
to form HGF
hepatocytes markedly
has the highest in primary
increased
plasma
(6) and
amino
culture.
in the liver
(4) and rabbit
serum
polypeptide
of 728 amino
mature
HGF. Among
potential
to promote
HGF
activity
following
45
we their
independently that
by HGF is
acids and proteolytically known
peptide
the growth
the development
cells in the rat liver are non-parenchymal
factor
determined
revealed
and HGF
(11) and cell fractionation
growth
(3). Recently,
(7). and
acid sequence
of from
p-subunit
rat
serum
homogeneity
cDNA was also cloned
precursor
a two-chain
10). In situ hybridization producing
HGF
in the
to apparent
and the 34kD
human
(8). The predicted chain
identified
rat HGF is a 82kD
Human
a single
first
HGF in human
a-subunit from
(HGF)
(1) was purified
The purified
primary
factors,
rats
of the 69kD
processed
factor
(2, 3). Thereafter,
(5) was purified.
cloned
growth
mRNA
growth
of mature levels
were
of hepatitis
(10) revealed
that
(9, HGF-
liver cells, presumably
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
176,
No.
Kupffer
BIOCHEMICAL
1, 1991
cells and
hepatotropic
sinusoidal
factor
which
We obtained tissues
other
stimulated
the
that
liver
(7, 11)
of normal
hepatocytes
became
apparent
restricted
and
(submitted
to targeting Epidermal
Elucidation
a wide
of growth
and
rabbit
HGF
seems
renal
found
epithelial
receptor
publication).
Therefore,
by melanocytes in
conditions
and
in
tissue
As melanocytes
phorbolfibroblast
growth
receptors
epidermal
Melanocytes
13-acetate
factor
(16, 17). We now report human
repair.
in uiuo (13) and constitute
12-myristate-
melanocytes
(PMA)
that and
normal
a minor in culture
(bFGF) is a potent evidence
is not
for
mitogen
require
the
cells
rarely
in epidermal the presence
for human mitogen express
to
pathological only
(14- 17)
HGF is a potent that
and
population
growth
melanin.
is important
are regulated
is difficult.
HGF
role in producing
melanocytes
cells, cultivation
receptor
hepatocytes.
how
mitosis
HGF
for HGF
of the HGF
determine
undergo
that
rat
cells in secondary
specific
distribution
requirement
epidermal
to be a
in various
we recently
have a important
factor
COMMUNICATIONS
is present
a high affinity
for
melanocytes
Thus,
mRNA
tissue
only mature
RESEARCH
for the liver regeneration.
HGF
(12). We have also identified
on rat
cells.
acts as a trigger
DNA synthesis
cultures
BIOPHYSICAL
endothelial
evidence
than
AND
and
of
basic
melanocytes for normal high-affinity
for HGF. MATERIALS
AND METHODS
Cell Culture: Normal human epidermal melanocytes were obtained from Kurabo Co. (Osaka) and cultured as described (17). Briefly, these melanocytes were cultured in the nutrient medium MCDB 153 supplemented with ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM), hydrocortisone (0.5 mM), insulin (5 mg/ml), PMA (10 rig/ml), and bovine pituitary extract (BPE, 25 pg protein/ml). In some experiments, BPE was deleted from the medium. Fourth or fifth passage cells were used. Measurement of Cell Growth and DNA Synthesis Melanocytes were plated at a density of lo* cells/well on a 12-well plate (Corning), and next day the medium was changed to fresh medium without BPE. An appropriate amount of growth factor was added to each culture and the cehs were cultured for 15 days. Cells were treated with trypsin and the number of cells was counted using a hematocytometer. DNA synthesis was determined by measuring the incorporation of (12511-deoxyuridine into the nuclei. Cells were plated at a density of 4 x 104 cells/well on a 24-well plate. The next day, the medium was changed to 46
Vol.
176,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
fresh medium without BPE and an appropriate amount of growth factor was added. Cells were cultured for 24 h and pulse-labeled with 1 PCi of [125I]deoxyuridine (2200 Ci/mmol, New England Nuclear) for 4 h. These cultured cells were washed with PBS and then with ice-cold 10% (w/v) TCA. The cells were solubilized with 1 M NaOH and the radioactivity was counted in a gamma counter. Protein concentration was measured using the Micro BCA protein assay system (Pierce Chemical) with bovine serum albumin as the standard. Assay for [12sI]-HGF Binding: Human recombinant HGF was radioiodinated by the chloramine-T method. Details on the methods for the radioiodination and the binding of HGF to cells should be published elsewhere. Briefly, 1.5 M sodium phosphate buffer, pH 7.0 (10 ~1). 0.5 pg HGF (17 ~1). and 0.5 mCi Na[ 12511 (14 Ci/mg I. IMS 30. Amersham) were added to a siliconized tube and the reaction was started by adding of 5 ~1 of chloramine-T (100 pg/ml), four times at 30 set intervals. The reaction was stopped by adding 20 ~1 N-acetyl-L-tyrosine (50 mM), 200 ~1 KI (60 mM) and 200 ~1 urea solution (1.2 g/ml in 1 M acetic acid). ]12sI]-HGF was separated by molecular sieve chromatography on a Sephadex G-25 column (Pharmacia) equilibrated with 4 mM HCl, 75 mM NaCl, and one mg/ml bovine serum albumin. [1251]-HGF thus prepared had a specific activity of 70160 mCi/mg. Cells were cultured in 24-well plates and when subconfluency was reached, the cultures were refed with fresh medium without BPE. After a 24 h culture, the cells were washed once with binding buffer (Hanks’ solution containing 20 mM HEPES, 2 mg/ml bovine serum albumin, pH 7.0) and equilibrated in the buffer for 30 min at 10°C. Ice-cold binding buffer containing appropriate concentrations of unlabeled HGF was added and the cells were incubated at 10°C for 1 h. Cultures were washed three times with the ice-cold binding buffer and [ 12sI]-HGF specifically bound to cells was measured in a gamma counter after detaching the cells with trypsintreatment. Growth Factors: Human recombinant HGF was purified from culture medium of the COS-1 cells transfected with plasmid containing human HGF cDNA (6). Human recombinant bFGF and human recombinant acidic fibroblast growth factor (aFGF) were obtained from TOYOBO (Osaka). Transforming growth factor-p1 (TGF-Pl) was purified from human platelets, as described previously (18).
RESULTS We examined melanocytes factors absence
the
effect
in the presence
on the DNA
of HGF
of PMA,
synthesis
the cell growth
manner.
reached
stimulated
DNA
cell growth
promotion
was reached
with
about
Effects
with
by HGF, 10 rig/ml,
the
became with same
as shown a 4.0-fold 47
growth
of normal
the stimulatory
effect
is not usually
HGF stimulated
the maximum
synthesis
since
of melanocytes
of PMA. Fig.1 shows that
in a dose-dependent
on the
the growth
apparent 10 rig/ml
with
of growth
evident
in the
of melanocytes, 1.25 rig/ml
HGF. In addition,
dose-dependency
evident
in Fig. 2. The maximal stimulation
human
compared
and HGF in the
response to finding
Vol.
176,
No.
BIOCHEMICAL
1, 1991
01
AND
BIOPHYSICAL
02
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O’ b
HGF (ng I ml)
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I
I
5 HGF
10
f20
(ng i ml)
NJ?. 1. Effect of HGF on the growth of normal human epidermal melanocytes. Cells were plated at a density of 104 cells/well on a 12-well plate. The next day, the medium was changed to fresh medium without BPE. and HGF was added at the various concentrations. Cultures were refed on days 6 and 12 with the same medium and the culture was stopped on day 15. The average of duplicate measurements is shown. Fig. 2. Effect of HGF on DNA synthesis of normal human epidermal melanocytes. Cells were plated at a density of 4 x 104 cells/well on a 24-well plate, The next day, the medium was changed to fresh medium without BPE, and HGF was added at the various concentrations. Cells were cultured for 24 h and pulse-labeled with 1 PCi of [ 1251]-deoxyuridine for 4 h. The average of triplicate measurements is shown.
of HGF. The half-maximal
in the absence
stimulation
was seen with
3 rig/ml
HGF. the effects of HGF. aFGF. bFGF and the combinations
Fig. 3 shows
HGF and other (10 rig/ml) 8.0-fold,
growth
and aFGF respectively.
same potency
factors
on the DNA synthesis
(10 rig/ml) HGF
stimulated
(10 rig/ml)
as seen with
alone
DNA synthesis stimulated
can inhibit
the growth
including
human
epidermal
keratinocytes
inhibited
DNA
rig/ml)
strongly
stimulated
confirm
that
we searched
for a specific
shows
[1251)-radiolabeled
human
it with
of various (19.
of the
almost
the
with
those
epithelial
cells,
20).
human
and
TGF-pl
(10
melanocytes
by HGF.
To further
saturable
synthesis
bFGF
by 3%fold
bFGF. The effect of HGF was additive
of bFGF and aFGF. TGF-pl normal
of melanocytes.
of
that
manner. melanocytes
Scatchard express
human
receptor
melanocytes
are target
for HGF on human
HGF
specifically
analysis
approximately 48
melanocytes.
bound
of the specific
cells of HGF,
to the
binding
120 receptors/cell
Fig. 4
cells
showed with
in a that
a Kd of
Vol.
176,
No.
BIOCHEMICAL
1, 1991
AND
BIOPHYSICAL
RESEARCH
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2.5 .E B e P E 2 b
2.0
1.5
X
E P z .i? $ r, E $
1.0
0.5
2 0 None HGF
03
bFGF
aFGF
HGF
HGF
HGF
bF+GF aF+GF TG&l
I
04 O
I 20
10 ‘=‘I-HGF
30
( pM )
Fig. 3. Effect of HGF, aFGF. bFGF, and combinations of HGF plus other growth factors on DNA synthesis of normal human epidermal melanocytes. Cells were plated at a density of 4 x 104 cells/well on 24-well plates. The next day, the medium was changed to fresh medium without BPE, and growth factors (10 rig/ml for each) were added. Cells were cultured for 24 h and pulse-labeled with 1 pC!i of [ 1251]-deoxyuridine for 4 h. The average of triplicate measurements & SD) is shown. Fig. 4. Analysis of [12sI]-HGF binding to normal human epidermal melanocytes. Binding of [1251]-HGF was performed as described in the text. Each point shows the amount of [ 125I]-HGF specifically bound to melanocytes exposed to increasing concentrations of HGF. Inset: Scatchard analysis of the data on binding.
22 pM. The very low Kd value is fairly for stimulation
of both
growth
consistent
with the half-maximal
and DNA synthesis
of 3 rig/ml
HGF
dose (35 pM
HGF). DISCUSSION
Evidence
that HGF acts as a potent
epidermal
melanocytes
synthesis
and
carried
high
those of bFGF for HGF,
is as follows:
cell growth
affinity and
distinct
aFGF.
from
hence
those
factor
1) HGF strongly
of human
receptors
growth
melanocytes:
for normal
enhanced 2) human
human
both
melanocytes
for HGF. The effect of HGF was additive HGF
probably
for other 49
acts
growth
through
factors.
a specific In fact,
DNA
with
receptor
Scatchard
Vol.
176,
No.
analysis
BIOCHEMICAL
1, 1991
revealed
seems
that
to differ
number
are similar identified
to findings
affinity
receptor
from
rat liver. Hepatocytes value
located
epidermal
at the epidermal-dermal
folicles.
the
melanocytes
culture.
We have
on rat hepatocytes
have approximately (Higuchi
derived
junction
Since melanocytes
HGF may function conditions,
and
500-600
and
Nakamura,
radiation
mesoderm-origin: monocytes
or infiltrating
monocytes tissue,
to stimulate
A scatter
that HGF stimulates (12). There
quantity
the growth
stimulates
or highly
related
such
those
of
fibroblasts, under
(unpublished endothelial
pathological
cells,
conditions
to the basal layer of the
of renal tubular
keratinocytes
of epithelial
epithelial
of the HGF
an important
in development,
interactions
morphogenesis,
50
we found
cells in secondary
receptor
(to be published).
and carcinogenesis.
cells was
to HGF (22). In addition,
for epithelial-mesenchymal
regeneration,
stimuli
among
lung fibroblasts
the motility
well be a mediator role
pathological
to selected
may diffuse
is a wide distribution
epidermal
certain
elusive.
of melanocytes.
which
the growth
has remained
cells in the liver (1 l), blood
by dermal
macrophages
a certain
factor
to be identical
cells including
and
of
in uiuo (13), the
cells are found
and embryonic
if HGF is produced
and in wounded epidermis
and in response
crest are
at the matrix
mitosis
under
cells and endothelial
and macrophages,
and
melanocytes
(21). HGF producing
Kupffer
Therefore,
undergo
for melanocytes
process,
from the neural
of the skin
only rarely
as a mitogen
wound-healing
as ultraviolet
culture
in primary
pM
melanocytes
role HGF may play in uivo for epidermal
found
(22 PM) and
for publication).
The human
data).
on melanocytes
to human
for HGF
of 20-30
COMMUNICATIONS
receptor
in HGF-binding
adult rat hepatocytes
a Kd
RESEARCH
(17). The Kd value
high
with
submitted
for bFGF
with
membranes
receptors/cell
hair
that
BIOPHYSICAL
of the HGF
(120 sites/cell)
specific,
plasma
the character
from
of receptors
AND
in epithelial
Thus,
HGF
may
and would
play
wound-healing,
tissue
Vol.
176,
No.
Acknowledgments: manuscript.
BIOCHEMICAL
1, 1991
We thank
This work
Cancer from the Ministry
AND
BIOPHYSICAL
Dr. Mariko
was supported of Education,
Ohara
RESEARCH
COMMUNICATIONS
for critical
by a Research
Grant
Science and Culture
reading for Science
of the and
of Japan.
REFERENCES
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