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Hepatocyte growth factor modulates migration and proliferation of human microvascular endothelial cells in culture
Vol. 179, No. 2, 1991 September 16, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1042-l 049
I-IEPATGCYTE GROWTH FACTOR MODULATES MIGRATION AND PROLIFERATION OF HUMAN MICROVASCULAR ENDOTHELIAL CELLS IN CULTURE
Akio
Morimotol, Nobuyuki
Kazuki Okamural , Ryoji Hamanakat , Yasufumi Sato2, Shima3, Kanji HigashioJ and Michihiko Kuwanol
Departments of ‘Biochemistry and 2 Medicine, Oita Medical School, Hasamamachi, Oita 879-55, Japan 3 Research Institute of Life Science, Snow Brand Milk Products Company, Tochigi 329-05, Japan Received
August
5, 1991
S umm arv : Epidermal growth factor (EGF) induces tubular formation of cultured human omental microvascular endothelial (HOME) cells and EGF also stimulates cell migration as well as expression of tissue type plasminogen activator (t-PA). Here we studied the effects of hepatocyte growth factor (HGF) on cell proliferation, cell migration and expression of t-PA and other related genes. Migration of confluent HOME cells into the denuded space was stimulated by HGF after being wounded with razor blade, but at a reduced rate in comparison with EGF. HOME cells could be proliferated in response to exogenous 100 rig/ml of HGF at rates comparableto that of 20 rig/ml EGF. The chemotactic activity of HOME cells was significantly stimulated by HGF in a dose-dependent manner when assayed by Boyden chamber. HGF did not efficiently enhance expression of both the t-PA gene and a tissue inhibitor of metalloproteinase gene whereas it stimulated expression of plasminogen activator inhibitor- 1. Our present study provides a new evidence that some of the biological effects of HGF on HOME cells in culture are similar to those of Press,Inc. EGF. 0 1991Academic
Hepatocyte growth factor (HGF) stimulates growth of hepatocytes in culture HGF also stimulates cell growth of melanocytes, keratinocytes and (12). various other epithelial cell lines (3-5). HGF appearsto act as a mitogen on a Higashio aA. (6) have independently broad spectrum of various cell types. purified a tumors cytotoxic factor (F-TCF) which is secreted from human diploid fibroblasts (IMR-90) and the sequence of this factor is highly homologous to that of HGF cloned from liver (7) and platelets (8).
F-TCF is a
potently mitogenic in rat hepatocytes and human umbilical endothelial cells and is cytotoxic in several tumor cell lines (9). HGF or its related factors might thus be mitogenic or anti-mitogenic against various cell types. The effects of HGF on vascular endothelial cells are controversial (3, 5). The amino acid sequence of HGF reveals that it is a plasminogen-like protein and highly homologous to scatter factor (10) which stimulates the movement ooo6-291x/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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179, No. 2, 1991
of epithelial cells (11).
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A recent study by Bottaro a A. (12) has demonstrated
that the HGF receptor is a 8-subunit of the c-m membrane-spanningtyrosine kinase.
The m
human osteogenic sarcoma cell line (13) is
proto-oncogene product, a oncogene which is found in a
also expressed in many tissues (14,
15). This finding suggests that HGF-induced signaling affects various biological activities including cell differentiation and carcinogenesis. However there are no reports that HGF affects cell motility.
In this study, we
examined the effect of HGF on human microvascular endothelial cells, and found for the first time that HGF was mitogenic and chemotactic on human omental microvascular endothelial (HOME) cells in culture.
MATERIALS AND NETHODS HOME cell C&U-G : HGF was purified from IMR-90 cells conditioned medium as described previouily (6). EGF was purchased from Toyobo Co., Osaka, Japan. HOME cells were isolated from human omental adipose tissue and grown in M199 containing 10% fetal calf serum (FCS) as described previously (16, 17). Proliferation of HOME cells : The effect of HGF on the proliferation of HOME cells was examined as described (16). Briefly, HOME cells at 3 to 5 passages were harvested, and 5 x lo4 cells were plated on type 1 collagen coated 35 mm dish (CORNING). One day later, cells were incubated in M-199 containing 1% FCS and the indicated concentration of HGF or EGF. Cell number was determined after a 3 day incubation. MiPration and chemotaxis of HOME cells : The wound migration assay of HOME cells was performed as described previously (17). Confluent HOME cells on 35 mm plates were scraped with a razor blade, washed twice with phosphatebuffered saline (PBS) and incubated in M-199 without serum and with various dosesof HGF or 20 rig/ml EGF. After 24 hr, cells were fixed with methanol and stained with Giemsa. The numbers of cells crossing the starting line marked on the plate were counted in a 1000 x 100 urn square. Four different areas of the plates were counted and the mean values were determined. The chemotactic effect of HGF on HOME cells was examined using a Boyden chamber as describedpreviously (17). Briefly, 2.5 x 105 cells / ml of HOME cells suspended in M-199 with 1% FCS were plated in the upper chamber. Various concentrations of HGF were placed either in the upper or lower chamber. After a 24 hr incubation at 37’ C, cells that had migrated across the filter were counted. Five high-power fields per chamber were counted and the average values were determined. DNA orobes : Human tissue-type-plasminogen activator (t-PA) cDNA was obtained from Dr. W-D. Schleuning (Schering Akiengeselshaft Pharma Forshung, Berlin, Germany) (18), plasminogen activator inhibitor- 1 (PAI-1) cDNA was from D. J. Loskutoff (Research Institute of Scripps Clinic, La Jolla, CA) (19) and a human tissue inhibitor of metalloproteinase (TIMP) cDNA probe consisting of a 0.7 kb TIMP cDNA inserted into plasmid PUC9-F5 from Dr. D. Carmichael (Synergen, Boulder, Colo.) (20). No the n blot analysis : Northern blot analysis was performed as described pre:iourSly (17, 21). Briefly, HOME cells were incubated with 100 ng/ ml of HGF in M-199 containing 1% FCS. Total RNA was isolated, fractionated on 1% agarose gel containing 2.2M formaldehyde, and transferred to a Nytran filter (Schleicher and Schuell, Inc., Keene, NH). The filter-bound RNA was hybridized to 32P-labeled probes, and autoradiographed. 1043
Vol.
179, No. 2, 1991
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS AND DlSCUSSIoN To assessthe effects of HGF on cell growth, HOME cells were plated at 5 x 104 cells f35 mm dish and 1 day later various doses of HGF or 20 rig/ml EGF were added. determined.
After
3 days of incubation, the number of viable cells were
As seen as in Figure 1. the cell number increased as a function
of HGF concentration up to 100 @ml.
At 100 rig/ml of HGF, a 220% increase
in cell proliferation was observed over the control values (Fig. 1). Addition of EGF at 20 rig/ml stimulated cell number approximately 240% over the control which is in good agreement with our previous study (16). The migration of vascular endothelial cells is a critical
process in
angiogenesis.
Our previous study showed that EGF increased and tumor
necrosis factor-a
inhibited the formation of vessel-like structures by HOME
cells in collagen gels (16).
EGF stimulated cell migration in a migration assay
and tumor necrosis factor-a inhibited the migration (17). examined the effect of HGF on cell migration.
In this study we
Confluent monolayers of HOME
cells were wounded with a razor blade and further incubated in M-199 without serum and with 10 or 100 rig/ml of HGF or 20 rig/ml EGF for 24 hr at 37’ C (Fig.
I
None
Eie_L The effect of HGF on HOME cell proliferation. HOME cells were seeded at 5 x lo4 cells /35 mm plate in M-199 containing10% FCS. The next day, the cells were incubated in M-199 containing 1% FCS with the indicated doses of HGF or 20 rig/ml EGF. After 3 days, the cells were trypsinized and counted. Data are expressed as mean cell density (LSD) of triplicate dishes. (*) significantly (PC 0.05) different from the value in the absence of growth factor.
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u Wound migration assay. Confluent HOME cells were scraped with a razor blade, then washed with PBS. They were incubated for 24 hr in the absence of added factor (A). or in the presence of 20 rig/ml EGF (B), 10 rig/ml HGF (C) and 100 t&ml HGF (D), then photographed. The arrow indicates the wound edge.
HOME cells that migrated from the edge of the wound were fixed, stained 2). and counted as seen in Figure 2. HOME cell migration was stimulated 1.2- to 1.3-fold in the presence of 10 or 100 rig/ml HGF whereas EGF stimulated HOME cell migration 1.5fold over control (Fig. 3). We next examined the chemotactic activity of HOME cells using Boyden chambers. As seen as Figure 4, the chemotactic activity of HOME cells was stimulated by HGF in a dose-dependentmanner when HGF was present in the
30
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20 10 0 0
200
400
600
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Distance
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200
400
600
800
(urn)
u Quantitation of HGF or EGF on wound migration activity by HOME cells. Confluent HOME cells were scraped with a razor blade, then washed with PBS. They were incubated for 24 hr in the absence of added factor (A) or in the presence of 20 rig/ml EGF (B), 10 rig/ml HGF (C) and 100 rig/ml HGF (D) as seen in Fig. 2. Migration was quantitated by counting the number of cells within a 1000 xl00 urn area in four fields. The results are presented as mean number of cells per field.
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AND BIOPHYSICAL
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Fie. Effect of HGF on chemotactic activity of HOME cells in a Boyden chamber. HOME cells at 2.5 x105 cells /ml were placed in the upper chamber, and various concentrations of HGF were placed either in the upper or lower chamber for 24 hr. EGF at 20 n&ml was also placed in the lower chamber as a control. These data are presented as the mean number of cells from 15 different fields in three chambers. (*> significantly (PC 0.05) different from the value in the absence of growth factor.
lower
HGF
chamber.
higher
than
controls
HOME
cells
indicate
cells
HGF (Fig. 4). when
We previously cell
migration for
inhibitors
of HOME
fibroblast
genes
(16).
genes with
was
in HOME
cells
probe
(Fig.
of
was comparable
to
chamber.
These
is closely is a most
by
of PAI-
study
also
expression
Northern
demonstrated
5), which
results
correlated
angiogenic
the
collagenase
and
demonstrated of
blot
analysis.
the expression with
was consistent
with
potent
that
basic
interleukin-6
The effect of HGF on expression
examined cDNA
recent
activity
cells.
which
expression
stimulated
cells (22).
a t-PA
EGF, Our
4-fold
HGF had no effect on the migration
in the upper gene expression
induces
factor
in HOME
TIMP
analysis mRNA
also
growth
that t-PA
activity
chemotactic
and the effect
factor for HOME
cells (17).
cells,
The
EGF,
However,
was present
reported
HOME (TIMP)
collagenase and
HGF
the chemotactic
of serum.
by 20 rig/ml
that HGF is a chemotactic
factor
stimulated
in the presence
was enhanced
that of 100 rig/ml of HOME
at 100 rig/ml
and
of t-PA,
PAI-
Northern
blot
of a 2.6 kb t-PA our previous
report
EGF stimulated the expression of t-PA mRNA, whereas 100 n&ml (17). had no effect on the expression of the t-PA gene in HOME cells (Fig. 5). contrast,
, the expression
in HOME TIMP
HGF
cells.
gene (Fig.
of the PAIhowever
gene was enhanced
had no stimulatory
5). 1046
by both HGF
effect
HGF By
and EGF
on the expression
of
Vol.
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TIMP-
4 20s
rRNA 018s u Time kinetics of t-PA, PAI-I and TIMP mRNA synthesis in HOME cells treated with 100 @ml HGF. Confluent HOME cells were incubated for 0, 3, 6, 12 and 24 hr with 100 rig/ml HGF and harvested. As control experiment, HOME cells were treated for 12 hr with 20 @ml EGF. Total RNA was fractionated on 1% agarose gel and 15 pg of RNA was transferred to a Nytran filter. Northern blot hybridization was performed with 32P-labeled tPA cDNA, PAI- cDNA and TIMP cDNA. Ribosomal RNA (rRNAs) loaded on the gels under various conditions are shown after staining with ethidium promide: arrows indicate 28s and 18s rRNAs.
In this study, on
the
growth
cells.
we demonstrated
for the first time that HGF has potent
and chemotactic activity
HGF is a heparin
binding
effects
of human microvascular endothelial
polypeptide
growth
factor
(23)
which
consists
of a heavy and light chain with molecular weights of 70,000 and 35,000 Daltons
(1. 2, 24). adult
rat
HGF was first isolated from plasma that stimulates the growth of hepatocytes
in vitro.
HGF is considered to be a hormone-like
substance and to have a defined target, namely hepatocytes.
It
is
widely
distributed in a variety of organs including the pancreas, brain, thyroid, submaxillary gland, small intestine and non-parenchymal liver cells such as Kupfer cells and sinusoid endothelial cells (25, 26).
The growth-modulation
effect of HGF is not restricted to hepatocytes, but is observed in a broad spectrum of cell types including melanocytes, keratinocytes and a variety of epithelial cell lines (3-S).
HGF appearsto be widely expressed,and it acts on a
wide variety of cell types in a paractine fashion. on vascular endothelial cells are controversial
Although the effects of HGF (3, 5) our present study
support the hypothesis that HGF is mitogenic and chemotactic to HOME cells. 1047
Vol.
179, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Scatter factor enhances the movement of epithelial cells, but had no effect on cell proliferation (11). The amino acid sequenceof scatter factor is highly homologous to that of HGF. In this study we demonstrated that HGF has a lesser effect on cell migration than that of EGF (Fig. 2), whereas it is potently chemotactic to HOME cells. EGF, a potent mitogenic factor for HOME cells, stimulates t-PA gene expression as well as cell migration, suggesting a close coupling of cell migration and angiongenesisof HOME cells with expression of t-PA (17) (Sato, Y., Okamura, K., Morimoto, A., Kuwano, M., unpublished data). Angiogenesis of HOME cells is thus a phenomenon coupled with protease production, cell migration and chemotactic activity.
HGF can stimulate
chemotaxis and cell proliferation of HOME cells, but not expression of plasminogen activator gene. HGF has a lesser effect on cell migration than EGF. This data presents the notion that HGF itself is insufficient to complete Further study should the process of angiogenesis of HOME cells in vitro. provide
more details
concerning
the
angiogenic
potential
of
HGF
in
comparison with EGF.
We thank Drs. K. Kohno and M. Ono in our laboratory for fruitful discussion. REFEREN-
1. Nakamura, T., Nawa, K., Ichihara, A., Kaise, N. and Nishino, T. (1987) FEBS Lett. 224, 311-316. 2. Gohda, E., Tsuboushi,H., Nakayarna,H., Hirono, S., Sakuyama,O., Takahashi, K., Miyazaki, H., Hashimoto, S. and Daikuhara, Y. (1988) J. Clin. Invest. 81, 414-419. 3. Rubin J. S., Chan, A. M. L. Bottaro. D. P. B., Burgess,W. H., Taylor, W. G., Cech, A. C., Hirschfield, D. W., Wong, J., Miki, T., Finch, P. W., and Aaronson, S. A. (1991) Proc. Natl. Acad. Sci. USA. 88, 415419. 4. Matsumoto, K., Tajima, H., and Nakamura,T. (1991) Biochem. Biophys. Res. Commun. 176, 45-51. 5. Kan, M.. Zhang. G., Zamegar, R., Michalopoulos. G., Myoken, Y., Mckeehan, W. L. and Stevens, J. I. (1991) Biochem. Biophys. Res. Commun. 174, 331-337. 6. Higashio, K., Shima, N., Goto, M., Itagaki, Y., Nagao, M., Yasuda, H. and Morinaga, T. (1990) Biochem. Biophys. Res. Commun. 170, 397-404. 7. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi,K., Okigaki, M.. Arakaki, N., Nakayama, H., Hirono. S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y. and Kitamura, N. (1989) Biochem. Biophys. Res. Commun. 163, 967-973. 8. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A.. Tashiro. K. and Shimizu, S. (1989) Nature 342. 440443. 9. Shima, N., Itagaki. Y., Nagao. M., Yasuda, H., Morinaga, T., and Higashio, K. (1991) Cell Biolo. Intern. Rep. 15, 397408. 10. Gherardi. E. and Stoker, M. (1990) Nature 346. 228. 11. Stoker, M. and Perryman, M. (1985) J. Cell Sci. 77, 209-223. 12. Bottaro, D. P., Rubin, J. S.. Faletto, D. L., Chan. A. M. L.. Kmiecik, T. E., Vande Woude, G. F., and Aaronson. S. A. (1991) Science 251, 802-804. 13. Cooper, C. S., Park, M., Blair. D. G., Tainsky, M. A., Huebner, K., Crove, C. M. and Vande Woude. G. F. (1984) Nature 311, 29-33. 1048
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AND BIOPHYSICAL
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14. Testa, J. R., Park, M., Blair, D. G., Kalbakji, A., Arden, K. and Vande Woude, G. F. (1990) Oncogene5, 156 5-1571. 15. Iyer. A., Kmiecik, T. E., Park, M., Daar, I. Blair, D. G., DUM, K. J., Sutrave, P., Ihle, J. N., Bodescot, M. and Vande Woude, G. F. (1990) Cell Growth & Diff. 1, 87-95. 16. Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Aso, K., Van Damme, J., Welgus, H. G. and Kuwano, M. (1989). J. Immunol. 143, 1619-1627. 17. Mawatari, M., Okamura, K., Matsuda, T., Hamanaka,R., Mizoguchi, H., Higashio, K. and Kuwano, M. (1991) Exp. Cell Res. 192, 574-580. 18. Fisher, R., Wailer E. K. Grossi, G., Thompson,D., Tizard, R. and Schleuning, W. D., (1985) J. Biol. Chem. 260, 11223-11230. 19. Raymond, R. S., Bevilacqua, M. P., Sawdey, M., Gimbrone, M. A. and Lostukoff, D. J. (1988) J. Biol. Chem. 263, 5797-5803. 20. Carmichael,D. F., Sommer,A., Thompson,R. C., Anderson, D. C., Smith, C. G., Welgus, H. G. and Stricklin, G. P. (1986) Proc. Natl. Acad. Sci. USA. 83, 2407-2412. 21. Mizoguchi, H., Uchiumi, T., Ono, M., Kohno, K. and Kuwano, M. (1990) Biochim. Biophys. Acta. 1052, 475-482. 22. Okamura, K., Sato, Y., Matsuda, T., Hamanaka,R., Ono, M., Kohno, K. and Kuwano, M. (1991) J. Biol. Chem. in press. 23. Matsumoto, A. and Yamamoto, N. (1991) Biochem. Biophys. Res. Commun. 174, 90-95. 24. Zamegar, R. and Michalopoulos, G. (1989) Cancer Res. 49, 33143320. 25. Noji, S., Tashiri, K., Koyama, E., Nohno, T., Oyama, K., Taniguchi, S. and Nakamura, T. (1990) Biobhem. Biophys. Res. Commun. 173, 42-47. 26. Zameger, R., Muga, S., Rahija, R. and Michalopoulos, G. (1990) Proc. Natl. Acad. Sci. USA. 87, 1252-1256.
1049
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1042-l 049
I-IEPATGCYTE GROWTH FACTOR MODULATES MIGRATION AND PROLIFERATION OF HUMAN MICROVASCULAR ENDOTHELIAL CELLS IN CULTURE
Akio
Morimotol, Nobuyuki
Kazuki Okamural , Ryoji Hamanakat , Yasufumi Sato2, Shima3, Kanji HigashioJ and Michihiko Kuwanol
Departments of ‘Biochemistry and 2 Medicine, Oita Medical School, Hasamamachi, Oita 879-55, Japan 3 Research Institute of Life Science, Snow Brand Milk Products Company, Tochigi 329-05, Japan Received
August
5, 1991
S umm arv : Epidermal growth factor (EGF) induces tubular formation of cultured human omental microvascular endothelial (HOME) cells and EGF also stimulates cell migration as well as expression of tissue type plasminogen activator (t-PA). Here we studied the effects of hepatocyte growth factor (HGF) on cell proliferation, cell migration and expression of t-PA and other related genes. Migration of confluent HOME cells into the denuded space was stimulated by HGF after being wounded with razor blade, but at a reduced rate in comparison with EGF. HOME cells could be proliferated in response to exogenous 100 rig/ml of HGF at rates comparableto that of 20 rig/ml EGF. The chemotactic activity of HOME cells was significantly stimulated by HGF in a dose-dependent manner when assayed by Boyden chamber. HGF did not efficiently enhance expression of both the t-PA gene and a tissue inhibitor of metalloproteinase gene whereas it stimulated expression of plasminogen activator inhibitor- 1. Our present study provides a new evidence that some of the biological effects of HGF on HOME cells in culture are similar to those of Press,Inc. EGF. 0 1991Academic
Hepatocyte growth factor (HGF) stimulates growth of hepatocytes in culture HGF also stimulates cell growth of melanocytes, keratinocytes and (12). various other epithelial cell lines (3-5). HGF appearsto act as a mitogen on a Higashio aA. (6) have independently broad spectrum of various cell types. purified a tumors cytotoxic factor (F-TCF) which is secreted from human diploid fibroblasts (IMR-90) and the sequence of this factor is highly homologous to that of HGF cloned from liver (7) and platelets (8).
F-TCF is a
potently mitogenic in rat hepatocytes and human umbilical endothelial cells and is cytotoxic in several tumor cell lines (9). HGF or its related factors might thus be mitogenic or anti-mitogenic against various cell types. The effects of HGF on vascular endothelial cells are controversial (3, 5). The amino acid sequence of HGF reveals that it is a plasminogen-like protein and highly homologous to scatter factor (10) which stimulates the movement ooo6-291x/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1042
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179, No. 2, 1991
of epithelial cells (11).
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A recent study by Bottaro a A. (12) has demonstrated
that the HGF receptor is a 8-subunit of the c-m membrane-spanningtyrosine kinase.
The m
human osteogenic sarcoma cell line (13) is
proto-oncogene product, a oncogene which is found in a
also expressed in many tissues (14,
15). This finding suggests that HGF-induced signaling affects various biological activities including cell differentiation and carcinogenesis. However there are no reports that HGF affects cell motility.
In this study, we
examined the effect of HGF on human microvascular endothelial cells, and found for the first time that HGF was mitogenic and chemotactic on human omental microvascular endothelial (HOME) cells in culture.
MATERIALS AND NETHODS HOME cell C&U-G : HGF was purified from IMR-90 cells conditioned medium as described previouily (6). EGF was purchased from Toyobo Co., Osaka, Japan. HOME cells were isolated from human omental adipose tissue and grown in M199 containing 10% fetal calf serum (FCS) as described previously (16, 17). Proliferation of HOME cells : The effect of HGF on the proliferation of HOME cells was examined as described (16). Briefly, HOME cells at 3 to 5 passages were harvested, and 5 x lo4 cells were plated on type 1 collagen coated 35 mm dish (CORNING). One day later, cells were incubated in M-199 containing 1% FCS and the indicated concentration of HGF or EGF. Cell number was determined after a 3 day incubation. MiPration and chemotaxis of HOME cells : The wound migration assay of HOME cells was performed as described previously (17). Confluent HOME cells on 35 mm plates were scraped with a razor blade, washed twice with phosphatebuffered saline (PBS) and incubated in M-199 without serum and with various dosesof HGF or 20 rig/ml EGF. After 24 hr, cells were fixed with methanol and stained with Giemsa. The numbers of cells crossing the starting line marked on the plate were counted in a 1000 x 100 urn square. Four different areas of the plates were counted and the mean values were determined. The chemotactic effect of HGF on HOME cells was examined using a Boyden chamber as describedpreviously (17). Briefly, 2.5 x 105 cells / ml of HOME cells suspended in M-199 with 1% FCS were plated in the upper chamber. Various concentrations of HGF were placed either in the upper or lower chamber. After a 24 hr incubation at 37’ C, cells that had migrated across the filter were counted. Five high-power fields per chamber were counted and the average values were determined. DNA orobes : Human tissue-type-plasminogen activator (t-PA) cDNA was obtained from Dr. W-D. Schleuning (Schering Akiengeselshaft Pharma Forshung, Berlin, Germany) (18), plasminogen activator inhibitor- 1 (PAI-1) cDNA was from D. J. Loskutoff (Research Institute of Scripps Clinic, La Jolla, CA) (19) and a human tissue inhibitor of metalloproteinase (TIMP) cDNA probe consisting of a 0.7 kb TIMP cDNA inserted into plasmid PUC9-F5 from Dr. D. Carmichael (Synergen, Boulder, Colo.) (20). No the n blot analysis : Northern blot analysis was performed as described pre:iourSly (17, 21). Briefly, HOME cells were incubated with 100 ng/ ml of HGF in M-199 containing 1% FCS. Total RNA was isolated, fractionated on 1% agarose gel containing 2.2M formaldehyde, and transferred to a Nytran filter (Schleicher and Schuell, Inc., Keene, NH). The filter-bound RNA was hybridized to 32P-labeled probes, and autoradiographed. 1043
Vol.
179, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
RESULTS AND DlSCUSSIoN To assessthe effects of HGF on cell growth, HOME cells were plated at 5 x 104 cells f35 mm dish and 1 day later various doses of HGF or 20 rig/ml EGF were added. determined.
After
3 days of incubation, the number of viable cells were
As seen as in Figure 1. the cell number increased as a function
of HGF concentration up to 100 @ml.
At 100 rig/ml of HGF, a 220% increase
in cell proliferation was observed over the control values (Fig. 1). Addition of EGF at 20 rig/ml stimulated cell number approximately 240% over the control which is in good agreement with our previous study (16). The migration of vascular endothelial cells is a critical
process in
angiogenesis.
Our previous study showed that EGF increased and tumor
necrosis factor-a
inhibited the formation of vessel-like structures by HOME
cells in collagen gels (16).
EGF stimulated cell migration in a migration assay
and tumor necrosis factor-a inhibited the migration (17). examined the effect of HGF on cell migration.
In this study we
Confluent monolayers of HOME
cells were wounded with a razor blade and further incubated in M-199 without serum and with 10 or 100 rig/ml of HGF or 20 rig/ml EGF for 24 hr at 37’ C (Fig.
I
None
Eie_L The effect of HGF on HOME cell proliferation. HOME cells were seeded at 5 x lo4 cells /35 mm plate in M-199 containing10% FCS. The next day, the cells were incubated in M-199 containing 1% FCS with the indicated doses of HGF or 20 rig/ml EGF. After 3 days, the cells were trypsinized and counted. Data are expressed as mean cell density (LSD) of triplicate dishes. (*) significantly (PC 0.05) different from the value in the absence of growth factor.
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u Wound migration assay. Confluent HOME cells were scraped with a razor blade, then washed with PBS. They were incubated for 24 hr in the absence of added factor (A). or in the presence of 20 rig/ml EGF (B), 10 rig/ml HGF (C) and 100 t&ml HGF (D), then photographed. The arrow indicates the wound edge.
HOME cells that migrated from the edge of the wound were fixed, stained 2). and counted as seen in Figure 2. HOME cell migration was stimulated 1.2- to 1.3-fold in the presence of 10 or 100 rig/ml HGF whereas EGF stimulated HOME cell migration 1.5fold over control (Fig. 3). We next examined the chemotactic activity of HOME cells using Boyden chambers. As seen as Figure 4, the chemotactic activity of HOME cells was stimulated by HGF in a dose-dependentmanner when HGF was present in the
30
5 CJ
20
si 10 = v8 0 t II 40 E c’ 30 z v
20 10 0 0
200
400
600
800
Distance
0
200
400
600
800
(urn)
u Quantitation of HGF or EGF on wound migration activity by HOME cells. Confluent HOME cells were scraped with a razor blade, then washed with PBS. They were incubated for 24 hr in the absence of added factor (A) or in the presence of 20 rig/ml EGF (B), 10 rig/ml HGF (C) and 100 rig/ml HGF (D) as seen in Fig. 2. Migration was quantitated by counting the number of cells within a 1000 xl00 urn area in four fields. The results are presented as mean number of cells per field.
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Fie. Effect of HGF on chemotactic activity of HOME cells in a Boyden chamber. HOME cells at 2.5 x105 cells /ml were placed in the upper chamber, and various concentrations of HGF were placed either in the upper or lower chamber for 24 hr. EGF at 20 n&ml was also placed in the lower chamber as a control. These data are presented as the mean number of cells from 15 different fields in three chambers. (*> significantly (PC 0.05) different from the value in the absence of growth factor.
lower
HGF
chamber.
higher
than
controls
HOME
cells
indicate
cells
HGF (Fig. 4). when
We previously cell
migration for
inhibitors
of HOME
fibroblast
genes
(16).
genes with
was
in HOME
cells
probe
(Fig.
of
was comparable
to
chamber.
These
is closely is a most
by
of PAI-
study
also
expression
Northern
demonstrated
5), which
results
correlated
angiogenic
the
collagenase
and
demonstrated of
blot
analysis.
the expression with
was consistent
with
potent
that
basic
interleukin-6
The effect of HGF on expression
examined cDNA
recent
activity
cells.
which
expression
stimulated
cells (22).
a t-PA
EGF, Our
4-fold
HGF had no effect on the migration
in the upper gene expression
induces
factor
in HOME
TIMP
analysis mRNA
also
growth
that t-PA
activity
chemotactic
and the effect
factor for HOME
cells (17).
cells,
The
EGF,
However,
was present
reported
HOME (TIMP)
collagenase and
HGF
the chemotactic
of serum.
by 20 rig/ml
that HGF is a chemotactic
factor
stimulated
in the presence
was enhanced
that of 100 rig/ml of HOME
at 100 rig/ml
and
of t-PA,
PAI-
Northern
blot
of a 2.6 kb t-PA our previous
report
EGF stimulated the expression of t-PA mRNA, whereas 100 n&ml (17). had no effect on the expression of the t-PA gene in HOME cells (Fig. 5). contrast,
, the expression
in HOME TIMP
HGF
cells.
gene (Fig.
of the PAIhowever
gene was enhanced
had no stimulatory
5). 1046
by both HGF
effect
HGF By
and EGF
on the expression
of
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RESEARCH
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TIMP-
4 20s
rRNA 018s u Time kinetics of t-PA, PAI-I and TIMP mRNA synthesis in HOME cells treated with 100 @ml HGF. Confluent HOME cells were incubated for 0, 3, 6, 12 and 24 hr with 100 rig/ml HGF and harvested. As control experiment, HOME cells were treated for 12 hr with 20 @ml EGF. Total RNA was fractionated on 1% agarose gel and 15 pg of RNA was transferred to a Nytran filter. Northern blot hybridization was performed with 32P-labeled tPA cDNA, PAI- cDNA and TIMP cDNA. Ribosomal RNA (rRNAs) loaded on the gels under various conditions are shown after staining with ethidium promide: arrows indicate 28s and 18s rRNAs.
In this study, on
the
growth
cells.
we demonstrated
for the first time that HGF has potent
and chemotactic activity
HGF is a heparin
binding
effects
of human microvascular endothelial
polypeptide
growth
factor
(23)
which
consists
of a heavy and light chain with molecular weights of 70,000 and 35,000 Daltons
(1. 2, 24). adult
rat
HGF was first isolated from plasma that stimulates the growth of hepatocytes
in vitro.
HGF is considered to be a hormone-like
substance and to have a defined target, namely hepatocytes.
It
is
widely
distributed in a variety of organs including the pancreas, brain, thyroid, submaxillary gland, small intestine and non-parenchymal liver cells such as Kupfer cells and sinusoid endothelial cells (25, 26).
The growth-modulation
effect of HGF is not restricted to hepatocytes, but is observed in a broad spectrum of cell types including melanocytes, keratinocytes and a variety of epithelial cell lines (3-S).
HGF appearsto be widely expressed,and it acts on a
wide variety of cell types in a paractine fashion. on vascular endothelial cells are controversial
Although the effects of HGF (3, 5) our present study
support the hypothesis that HGF is mitogenic and chemotactic to HOME cells. 1047
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Scatter factor enhances the movement of epithelial cells, but had no effect on cell proliferation (11). The amino acid sequenceof scatter factor is highly homologous to that of HGF. In this study we demonstrated that HGF has a lesser effect on cell migration than that of EGF (Fig. 2), whereas it is potently chemotactic to HOME cells. EGF, a potent mitogenic factor for HOME cells, stimulates t-PA gene expression as well as cell migration, suggesting a close coupling of cell migration and angiongenesisof HOME cells with expression of t-PA (17) (Sato, Y., Okamura, K., Morimoto, A., Kuwano, M., unpublished data). Angiogenesis of HOME cells is thus a phenomenon coupled with protease production, cell migration and chemotactic activity.
HGF can stimulate
chemotaxis and cell proliferation of HOME cells, but not expression of plasminogen activator gene. HGF has a lesser effect on cell migration than EGF. This data presents the notion that HGF itself is insufficient to complete Further study should the process of angiogenesis of HOME cells in vitro. provide
more details
concerning
the
angiogenic
potential
of
HGF
in
comparison with EGF.
We thank Drs. K. Kohno and M. Ono in our laboratory for fruitful discussion. REFEREN-
1. Nakamura, T., Nawa, K., Ichihara, A., Kaise, N. and Nishino, T. (1987) FEBS Lett. 224, 311-316. 2. Gohda, E., Tsuboushi,H., Nakayarna,H., Hirono, S., Sakuyama,O., Takahashi, K., Miyazaki, H., Hashimoto, S. and Daikuhara, Y. (1988) J. Clin. Invest. 81, 414-419. 3. Rubin J. S., Chan, A. M. L. Bottaro. D. P. B., Burgess,W. H., Taylor, W. G., Cech, A. C., Hirschfield, D. W., Wong, J., Miki, T., Finch, P. W., and Aaronson, S. A. (1991) Proc. Natl. Acad. Sci. USA. 88, 415419. 4. Matsumoto, K., Tajima, H., and Nakamura,T. (1991) Biochem. Biophys. Res. Commun. 176, 45-51. 5. Kan, M.. Zhang. G., Zamegar, R., Michalopoulos. G., Myoken, Y., Mckeehan, W. L. and Stevens, J. I. (1991) Biochem. Biophys. Res. Commun. 174, 331-337. 6. Higashio, K., Shima, N., Goto, M., Itagaki, Y., Nagao, M., Yasuda, H. and Morinaga, T. (1990) Biochem. Biophys. Res. Commun. 170, 397-404. 7. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi,K., Okigaki, M.. Arakaki, N., Nakayama, H., Hirono. S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y. and Kitamura, N. (1989) Biochem. Biophys. Res. Commun. 163, 967-973. 8. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A.. Tashiro. K. and Shimizu, S. (1989) Nature 342. 440443. 9. Shima, N., Itagaki. Y., Nagao. M., Yasuda, H., Morinaga, T., and Higashio, K. (1991) Cell Biolo. Intern. Rep. 15, 397408. 10. Gherardi. E. and Stoker, M. (1990) Nature 346. 228. 11. Stoker, M. and Perryman, M. (1985) J. Cell Sci. 77, 209-223. 12. Bottaro, D. P., Rubin, J. S.. Faletto, D. L., Chan. A. M. L.. Kmiecik, T. E., Vande Woude, G. F., and Aaronson. S. A. (1991) Science 251, 802-804. 13. Cooper, C. S., Park, M., Blair. D. G., Tainsky, M. A., Huebner, K., Crove, C. M. and Vande Woude. G. F. (1984) Nature 311, 29-33. 1048
Vol.
179, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14. Testa, J. R., Park, M., Blair, D. G., Kalbakji, A., Arden, K. and Vande Woude, G. F. (1990) Oncogene5, 156 5-1571. 15. Iyer. A., Kmiecik, T. E., Park, M., Daar, I. Blair, D. G., DUM, K. J., Sutrave, P., Ihle, J. N., Bodescot, M. and Vande Woude, G. F. (1990) Cell Growth & Diff. 1, 87-95. 16. Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Aso, K., Van Damme, J., Welgus, H. G. and Kuwano, M. (1989). J. Immunol. 143, 1619-1627. 17. Mawatari, M., Okamura, K., Matsuda, T., Hamanaka,R., Mizoguchi, H., Higashio, K. and Kuwano, M. (1991) Exp. Cell Res. 192, 574-580. 18. Fisher, R., Wailer E. K. Grossi, G., Thompson,D., Tizard, R. and Schleuning, W. D., (1985) J. Biol. Chem. 260, 11223-11230. 19. Raymond, R. S., Bevilacqua, M. P., Sawdey, M., Gimbrone, M. A. and Lostukoff, D. J. (1988) J. Biol. Chem. 263, 5797-5803. 20. Carmichael,D. F., Sommer,A., Thompson,R. C., Anderson, D. C., Smith, C. G., Welgus, H. G. and Stricklin, G. P. (1986) Proc. Natl. Acad. Sci. USA. 83, 2407-2412. 21. Mizoguchi, H., Uchiumi, T., Ono, M., Kohno, K. and Kuwano, M. (1990) Biochim. Biophys. Acta. 1052, 475-482. 22. Okamura, K., Sato, Y., Matsuda, T., Hamanaka,R., Ono, M., Kohno, K. and Kuwano, M. (1991) J. Biol. Chem. in press. 23. Matsumoto, A. and Yamamoto, N. (1991) Biochem. Biophys. Res. Commun. 174, 90-95. 24. Zamegar, R. and Michalopoulos, G. (1989) Cancer Res. 49, 33143320. 25. Noji, S., Tashiri, K., Koyama, E., Nohno, T., Oyama, K., Taniguchi, S. and Nakamura, T. (1990) Biobhem. Biophys. Res. Commun. 173, 42-47. 26. Zameger, R., Muga, S., Rahija, R. and Michalopoulos, G. (1990) Proc. Natl. Acad. Sci. USA. 87, 1252-1256.
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