Vol. 189, No. 2, 1992 December 15, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 684-690
Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression Isabel Fabregatt, Carmen de Juant, Toshikazu Nakamurazand Manuel Benitol 1Departamento de Bioqufmica y Biologfa Molecular, Centro Mixto C.S.I.C./U.C.M. Facuhad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain 2Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan Received
October
23,
1992
SUMMARY: Hepatocyte growth factor, which is a potent growth factor for primary cultured adult hepatocytes, strongly stimulated DNA synthesis of rat fetal (20-day of gestation) hepatocytes. Its mitogenic capacity, measured as (3H)-thymidine incorporation into acid precipitable material was dose dependent, being detectable at 1 nglml and maximal at 5 rig/ml. Over 15% of the cells entered into S-phase and mitosis as judged by flow cytometric analysis of the cell cycle. HGF had additive effects with transforming growth factor-alpha , whereas transforming growth factor-beta strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF. HGF induced c-fos and c-myc expression in a time-dependent manner, with a maximum at 30 min for c-fos and 8 h for c-myc. These results suggest that HGF may act as a proliferative factor during fetal liver growth. 0 1992Academic We**, Inc.
Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes in vitro (1,2) and may act as an important regulator of liver regeneration in response to injury, such as partial hepatectomy and hepatitis (3-5). HGF is present in a variety of rabbit and rat tissues and is mitogenic for melanocytes, endothelial cells, renal tubular cells and keratinocytes (6- 10). Recently, it has been described that HGF has the properties of a paracrine mediator of epithelial morphogenesis (11). Finally, HGF is identical to scatter factor, a non-mitogenic factor that promotes cell migration (12). Because of its peculiar ability to act as a mitogen (1,2, 6-lo), a motogen (12) as well as a morphogen (ll), HGF appears to be uniquely suited to regulate the biological processes that culminate in organogenesis and tissue regeneration. In addition antiproliferative
to stimulating
of cell growth and tissue repair, HGF has an
effect on several tumor cells (13,14). Considering that serum HGF is
elevated in patients with chronic hepatitis and liver cirrhosis (15), it was initially considered that HGF was a likely candidate for autocrine stimulation of hepatocellular carcinoma (HCC) cells. Contrary to these expectations, endogenous HGF expression in Fao HCC cells and treatment of HCC cells with recombinant HGF produce a marked inhibition of cell growth (16). 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
684
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Considering the difference between the effect of HGF on adult hepatocytes and its effect on transformed cells of the same cell lineage, the aim of this work has been to test the possible role of HGF in fetal liver growth. We have found that upon HGF stimulation, fetal (20 day of gestation) hepatocytes in primary culture increased DNA synthesis and entered into S-phase and mitosis. Moreover, HGF induced c-myc and c-fos expression in these cells. MATERIALS
AND METHODS
Materials: EGF, TGF-a and TGF-P were purchased from Sigma Chemical Co. (St. Louis. MO). HGF was obtained as previously described (17). Collagenase was from Boehringer (Mannheim, Germany). (3H)-Thymidine was from Amersham (Buckinghamshire, U.K.). Fetal and neonatal calf serum and culture media were from Imperial Laboratories ( Hampshire, U.K). Cycle test DNA reagent kit was from BectonDickinson (San Jose, CA). Isolation and culture of rat hepatocytes: Hepatocytes from 20-day old fetuses of Wistar rats were isolated, purified by differential centrifugation (5 min, 100 rpm) and plated on rat tail collagen-coated 60 mm-dishes as described previously (18). After attachment of the cells (3-4 hours), media was changed and replaced by serum-free medium. 18-20 hours later , medium was again replaced for one of identical composition in the absence of serum, where growth factors were added. Assay of DNA synthesis: For this assay, cells were cultured in the absence (control) or in the presence of growth factors for 48 hours. DNA synthesis was determined by (3H)thymidine incorporation over the last 40 hours (0.5 mCi/ml, 0.5 mM thymidine). At the end of the incubation period, radioactivity in acid precipitable material was determined as previously described (19). Analysis of cell cycle by flow cytometry: The ploidy determination of hepatocyte nuclei was estimated by flow cytometry DNA analysis. Cells were detached from dishes by addition of 0.05% trypsin-0.02% EDTA After 2-3 min, trypsinization was stopped with 10% fetal calf serum in culture medium. The DNA content per nucleus was evaluated in a FAC SCAN flow cytometer (Becton-Dickinson) after staining nuclei with propidium iodide by using the Cycle test DNA reagent kit (Becton- Dickinson). Cell cycle analysis was performed using a Double Discriminator Module (DDM) which allows to make a distinction between the signals coming from a single nucleus and the ones produced by two or more agregated nuclei. For the computer analysis, only signals from single nuclei were considered (10,000 nuclei/assay). Northern blot analysis: Total cellular RNA was isolated essentially as described by Chomczynski and Sachi (20). For Northern blot analysis, samples containing 15 ug of total RNA were denatured as previously described (18), electrophoresed in 0.8% agarose gels containing 0.66M formaldehyde and blotted on Nylon filters (NY 13-N; Schleicher & Schuell). The membrane was hybridized with labeled c-myc DNA probe (American Type Culture Collection) (21), washed and rehybridized with labeled v-fos probe (Oncor, Gaithersburg, MD) (22). Labeling, hybridization and washing were performed as described before (18). Quantitation of autoradiograms was performed on a Molecular Dynamics scanning laser densitometer. RESULTS Effect of HGF on growth of fetal hepatocytes in primary culture. First we examined the effect of human recombinant HGF on DNA synthesis in fetal hepatocytes in primary culture by measuring the incorporation of (3H)-thymidine in TCA-precipitable material. HGF stimulated DNA synthesis of the fetal liver cells and its effect was dose-dependent, being detectable at 1 rig/ml and maximal at 5 @ml as shown in fig. 1. The concentration of HGF required for maximal stimulation in fetal liver cells is 685
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1012
HGF (NG/ML)
Fi . l.Effect
of HGF on the DNA synthesis of cultured fetal he?atocytes. Fetal were plated on 60 mm dishes at a density of 2x10“ cells/cm-. Cells, incubated for 48 h in serum-free medium and in the absence or the presence of different concentrations of HGF were analyzed for DNA synthesis measured as (3H)-thymidine incorporation over the last 40 hours of culture. A representative experiment is shown. I?-epatocytes
similar to that required for induction of growth of adult rat hepatocyte primary cultures (2, 14). The mitogenic stimulatoty effect of HGF on fetal hepatocytes was confirmed by flow cytometric analysis of the cell cycle. Twenty four hours after addition of 10 @ml of HGF, Gl-stated fetal hepatocytes decreased from 85 to 71%, increasing the proportion of cells stated in S and G2+M (Table I). So, upon stimulation of HGF, fetal rat hepatocytes entered into S phase and mitosis. Next, we compared the HGF-induced stimulations of DNA-synthesis in fetal hepatocytes to that produced by other known mitogens, as EGF or TGF-a. Fig. 2 shows that the stimulatory effect of HGF and EGF were similar and slightly lower than that observed with TGF-a. We also examined the additive effects of HGF and these other factors (fig. 2). The effect of HGF was additive or synergistic with that of TGF-a. In the presence of both HGF and TGF-a, DNA synthesis increased about 8-9 fold (fig. 2). As TGF-fi can inhibit the EGF-induced growth of fetal hepatocytes (23), we examined the effect of this inhibitory growth factor on the stimulation of DNA-synthesis produced by HGF. As shown in fig. 2, very low concentrations of TGF-p (0.5 rig/ml) totally inhibited HGF-induced growth in fetal hepatocytes in primary culture.
TABLE I - CELL CYCLE ANALYSIS OF FETAL HEPATOCYTES CULTURED IN THE ABSENCE OR IN THE PRESENCE OF HGF Gl
S
G2 + M
CONTROL
84.8 + 3.4
9.8 f 1.6
5.4 + I.8
HGF
70.6 + 3. I
19.7 + 2.5
9.7 zi 0.6
Fetal hepatocytes were cultured in the absence (control) or in the presence of IO rig/ml HGF for 24 hours. Cells were collected and treated for analysis of cell cycle as described in Materials and Methods. Results represent the % of cells in each state and are the means+S.E.M. of three independent experiments.
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CONTROL HGF EGF TGFa HGF+EGF HGF+TGFa EGF+TGF-a HGF+TGF$ 0
10
DNA
20
SYNTHESIS
30
(DPM
40
X 1 ti3)
Fig. 2. Effect of various growth factors and combinations of HGF plus other growth factors on DNA synthesis of cultured fetal hepatocytes. Cells were cultured
for 48 h and pulse labeled with (3H)-thymidine for 40 h as described in fig. 1. The concentrations of growth factors were: HGF, EGF and TGF-alpha: 10 @ml; TGF-beta, 0.5 nglml. Resultsare the means+ S.E.M. of three independentexperiments.
Induction
of c-fos and c-myc
dependence
by HGF
in fetal
hepatocytes
in primary
culture:
of time.
Fetal hepatocytes were treated with HGF at 10 rig/ml. Total RNA was extracted at various times after peptide addition and analyzed for c-fos and c-myc mRNA levels by RNA blot transfer and hybridization to appropriate DNA probes. The 2.2 kilobase c-fos mRNA, which is at the limit of detection in non-stimulated cells, was increased by HGF 30 min after the addition of the factor, decreasing the expression after this time (fig.3 ). In contrast to the rapid induction of c-fos, HGF increased the 2.4 kilobase c-myc m-RNA level 2 hours after the addition of the factor, with a maximum at 8-24 hours, although control cells expressed considerable levels of c-myc mRNA.
DISCUSSION
The present results suggest that additionaly to the well known effect of HGF on the proliferation of mature adult hepatocytes (1,2) and its role as a regulator of liver regeneration (3-9, HGF may also be one of the factors implicated in fetal liver growth. HGF increased (3H)-thymidine incorporation into DNA in fetal rat hepatocyte primary cultures, reaching the maximum at 5 rig/ml (fig. 1). Cell cycle analysis by flow cytometry showed that 15% of the fetal hepatocytes entered into S-phase and mitosis in response to HGF (Table I). The proportion of HGF stimulated-cells was similar or even higher than that observed in incubations with EGF (23). Moreover, HGF effect was clearly synergistic or additive with that of TGF-a (fig. 2). TGF-a, that is produced at relatively high levels during development (24), is one of the most potent hepatotrophic mitogens in vitro (25). Considering that HGF is strongly expressed in human fetal liver as compared with adult liver which is not regenerating (26), both TGF-a and HGF could be the main mitogens responsible of the fetal liver growth. Moreover, TGF-p strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF (fig. 2). This growth inhibitory 687
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8 CH
24 CH
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HOURS
CH
‘FOS
C-FOS
Et.
Br.
staining
Fig. 3.Effect of HGF on the levels of c-fos and c-myc mRNAs in cultured fetal hepatocytes. Total RNA, extracted after 0.5,2,8,24 and 48 h was analyzed by Northern blotting as described in Materials and Methods. The radioactivity was visualized by autoradiography (left panel) and measured by scanning densitometry (right panel). A representative experiment is shown.
inhibited EGF- induced proliferation of fetal cultured hepatocytes (23). Thus TGF-l3, whose role in the regulation of liver regeneration is well known (27), could also have an important role in arresting liver growth at the end of the development . In addition to these results, we have found that HGF increased c-fos and c-myc mRNA levels in fetal hepatocytes in culture (fig. 3). Time dependence of these inductions was similar to that found for other mitogens and other cultured cell systems (28), with a maximum for c-fos mRNA levels at 30 min and for c-myc mRNA levels at 8 hours. It is known that HGF stimulates production of inositol triphosphate inducing a prompt elevation of cytoplasmic free calcium level in adult rat hepatocytes (29) and it has been suggested that protein kinase C is fully activated in the HGF signal transduction pathway in hepatocytes (30). Since the induction of c-fos and c-myc expression seems to be a necessary step in the mitogenic response initiated by ligands that act through activation of the protein kinase C (28), our results, that show for the first time a regulation of c-fos and c-myc expression
by HGF in cultured
688
hepatocytes,
could
be in agreement
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with those previous results that implicate protein kinase C in the mitogenic signal transduction pathway elicited by HGF in hepatocytes (29,30). Finally, the results shown in this paper clearly demonstrate the mitogenic role of HGF in rat fetal liver cells, that differ of the antiproliferative effect found in several tumor cells (13, 14). Thus HGF role in growth of the normal (fetal or regenerating) liver seems to be inducing proliferation.
As has been suggested (16), the suppressive growth
regulatory capacity found in tumor cells could implicate the existence of intracellular signalling pathway downstream from tyrosine phosphorylation of HGF receptor that differ between normal (fetal or regenerating) and tumor cells, ACKNOWLEDGMENTS:
We are grateful to Dr. A. Alvarez for expert technical
assistance with the flow cytometer. This work has been supported by a grant from the Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, Spain.
REFERENCES 1. 3-.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
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Nakamura, T., Temmoto, H. and Ichihara, A. ( 1986) Proc. Natl. Acad. Sci. USA, 83,6489-6493. Nakamura, T., Nawa, K., Ichihara, A., Kaige, N. and Nishino, T. (1987) FEBS Lett., 224,311-316. Kinoshita, T., Hirao, S., Matsumoto, K. and Nakamura, T. (1991) Biochem. Biophys. Res. Commun., 177,330-335. Zamegar, R., DeFrances, M.C., Kost, D.P., Lindroos, P. and Michalopoulos, K. (1991) Biochem. Biophys. Res. Commun., 177,559-56.5. Nakamura, T. (1991) Prog. Growth Factor Res., 3,67-85. Zamegar, R., Muga, S., Rahija, R. and Michalopoulos, G. (1990). Proc. Natl. Acad. Sci. USA, 87, 1252-1256. Tashiro, K., Hagiya, M., Nishizawa, T., Seki, T., Shimonishi, M., Shimizu, S. and Nakamura, T. (1990) Proc. Natl. Acad. Sci. USA, 87,3200-3204. Kan, M., Zhang, G., Zarnegar, R., Michalopoulos, G., Myoken, Y ., Mckeehan, W. and, Stevens, J.I. (1991) B&hem. Biophys. Res. Commun., 174,33 l337. Igawa, T., Kanda, S., Kanetake, H., Saitoh, Y., Ichihara, A., Tomita, Y. and Nakamura, T. (1991) Biochem. Biophys. Res. Commun., 174,83 l-838. Rubin, J.S., Chan, A.M.L., Bottaro, D.P., 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, 88,415-419. Montesano, R., Matsumoto, K., Nakamura, T. and Orci, L. (1991) Cell, 67, 901908. Weidner, K.M., Arakaki, N., Hartmann, G., Vandekerckhove, J., Weingart, S., Hishida, T., Daikuhara, Y. and Bichmeier, W. (1991) Proc. Natl. Acad. Sci. USA, 88,7001-7005. Higashio, K., Shima, N., Goto, M., Itagaki, Y ., Nago, M., Yasuda, H. and Morinaga, T. (1990) Biochem. Biophys. Res. Commun., 170,397-404. Tajima, H., Matsumoto, K. and Nakamura, T. (1991) FEBS Lett., 291,229232. Tsubouchi, H., Nitani, Y ., Hirono, S., Nakayama, H., Gohda, E., Aralcaki, N., Sukiyama, O., Takahashi, K., Kimoto, M., Kawakami, S., Setoguchi, M., y-yhikawa, T., Shin, S., Arima, T. and Daikuham, Y. (1991) Hepatology, 13, Shiota, G., Rhoads, D.B., Wang, T.C., Nakamura, T. and Schmidt, E.V. (1992) Proc. Natl. Acad. Sci., USA, 89,373-377. Nakanmra, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K. and Shimizu, S. (1989) Nature, 342,440-443. 689
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De Juan, C., Benito, M. and Fabregat, I. (1992) J. Cell. Physiol., 152,95-101. Rozengurt, E. and Sinnett-Smith (1983) Proc. NatI. Acad. Sci. USA, 80, 2936-2940. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. van Straaten, R., Muller, R., Curran, T. van Beveren, C. and Verma, I.M. (1983) Proc. NatI. Acad. Sci. USA, 80,3X3-3187. AlitaIo, K., Schwab, M., Lin, C. C., Varmus, H. E. and Bishop, J. M. (1983) Proc. Natl. Acad. Sci., USA, 80, 1707-1711. De Juan, C., Benito, M., Alvarez, A. and Fabregat, I. (1992) Exp. Cell Res., In press. Derynck, R. (1988) Cell, 54,593-595. Hoffmann, B. and Paul, D. (1990) J. Cell. Physiol., 142, 149-154. Selden, C., Jones, M., Wade, D. and Hodgson, H. (1990) FEBS I&t., 270, 81-84. Braun, L., Mead, J. E., Panzica, M., Mikumo, R., Bell, G. I. and Fausto, N. (1988) Proc. Natl. Acad. Sci., USA, 85, 1539-1543. Rozengurt, E. and Sinnett-Smith, J. W. (1987) J. Cell. Physiol., 131, 218-225. Mine, T., Kojima, I., Ogata, E. and Nakamura, T. (1991) B&hem. Biophys. Res. Commun., 181, 1173-l 180. Arakaki, N., Hirono, S., Kawakani, S., Tsubouchi, H., Ishii, T., Ham, H. and Daikuhara, Y. (1992) Biochem. Biophys. Res. Commun., 185,22-28.
690
1.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 684-690
Growth stimulation of rat fetal hepatocytes in response to hepatocyte growth factor: modulation of c-myc and c-fos expression Isabel Fabregatt, Carmen de Juant, Toshikazu Nakamurazand Manuel Benitol 1Departamento de Bioqufmica y Biologfa Molecular, Centro Mixto C.S.I.C./U.C.M. Facuhad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain 2Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan Received
October
23,
1992
SUMMARY: Hepatocyte growth factor, which is a potent growth factor for primary cultured adult hepatocytes, strongly stimulated DNA synthesis of rat fetal (20-day of gestation) hepatocytes. Its mitogenic capacity, measured as (3H)-thymidine incorporation into acid precipitable material was dose dependent, being detectable at 1 nglml and maximal at 5 rig/ml. Over 15% of the cells entered into S-phase and mitosis as judged by flow cytometric analysis of the cell cycle. HGF had additive effects with transforming growth factor-alpha , whereas transforming growth factor-beta strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF. HGF induced c-fos and c-myc expression in a time-dependent manner, with a maximum at 30 min for c-fos and 8 h for c-myc. These results suggest that HGF may act as a proliferative factor during fetal liver growth. 0 1992Academic We**, Inc.
Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes in vitro (1,2) and may act as an important regulator of liver regeneration in response to injury, such as partial hepatectomy and hepatitis (3-5). HGF is present in a variety of rabbit and rat tissues and is mitogenic for melanocytes, endothelial cells, renal tubular cells and keratinocytes (6- 10). Recently, it has been described that HGF has the properties of a paracrine mediator of epithelial morphogenesis (11). Finally, HGF is identical to scatter factor, a non-mitogenic factor that promotes cell migration (12). Because of its peculiar ability to act as a mitogen (1,2, 6-lo), a motogen (12) as well as a morphogen (ll), HGF appears to be uniquely suited to regulate the biological processes that culminate in organogenesis and tissue regeneration. In addition antiproliferative
to stimulating
of cell growth and tissue repair, HGF has an
effect on several tumor cells (13,14). Considering that serum HGF is
elevated in patients with chronic hepatitis and liver cirrhosis (15), it was initially considered that HGF was a likely candidate for autocrine stimulation of hepatocellular carcinoma (HCC) cells. Contrary to these expectations, endogenous HGF expression in Fao HCC cells and treatment of HCC cells with recombinant HGF produce a marked inhibition of cell growth (16). 0006-291X/92 Copyright All rights
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
684
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Considering the difference between the effect of HGF on adult hepatocytes and its effect on transformed cells of the same cell lineage, the aim of this work has been to test the possible role of HGF in fetal liver growth. We have found that upon HGF stimulation, fetal (20 day of gestation) hepatocytes in primary culture increased DNA synthesis and entered into S-phase and mitosis. Moreover, HGF induced c-myc and c-fos expression in these cells. MATERIALS
AND METHODS
Materials: EGF, TGF-a and TGF-P were purchased from Sigma Chemical Co. (St. Louis. MO). HGF was obtained as previously described (17). Collagenase was from Boehringer (Mannheim, Germany). (3H)-Thymidine was from Amersham (Buckinghamshire, U.K.). Fetal and neonatal calf serum and culture media were from Imperial Laboratories ( Hampshire, U.K). Cycle test DNA reagent kit was from BectonDickinson (San Jose, CA). Isolation and culture of rat hepatocytes: Hepatocytes from 20-day old fetuses of Wistar rats were isolated, purified by differential centrifugation (5 min, 100 rpm) and plated on rat tail collagen-coated 60 mm-dishes as described previously (18). After attachment of the cells (3-4 hours), media was changed and replaced by serum-free medium. 18-20 hours later , medium was again replaced for one of identical composition in the absence of serum, where growth factors were added. Assay of DNA synthesis: For this assay, cells were cultured in the absence (control) or in the presence of growth factors for 48 hours. DNA synthesis was determined by (3H)thymidine incorporation over the last 40 hours (0.5 mCi/ml, 0.5 mM thymidine). At the end of the incubation period, radioactivity in acid precipitable material was determined as previously described (19). Analysis of cell cycle by flow cytometry: The ploidy determination of hepatocyte nuclei was estimated by flow cytometry DNA analysis. Cells were detached from dishes by addition of 0.05% trypsin-0.02% EDTA After 2-3 min, trypsinization was stopped with 10% fetal calf serum in culture medium. The DNA content per nucleus was evaluated in a FAC SCAN flow cytometer (Becton-Dickinson) after staining nuclei with propidium iodide by using the Cycle test DNA reagent kit (Becton- Dickinson). Cell cycle analysis was performed using a Double Discriminator Module (DDM) which allows to make a distinction between the signals coming from a single nucleus and the ones produced by two or more agregated nuclei. For the computer analysis, only signals from single nuclei were considered (10,000 nuclei/assay). Northern blot analysis: Total cellular RNA was isolated essentially as described by Chomczynski and Sachi (20). For Northern blot analysis, samples containing 15 ug of total RNA were denatured as previously described (18), electrophoresed in 0.8% agarose gels containing 0.66M formaldehyde and blotted on Nylon filters (NY 13-N; Schleicher & Schuell). The membrane was hybridized with labeled c-myc DNA probe (American Type Culture Collection) (21), washed and rehybridized with labeled v-fos probe (Oncor, Gaithersburg, MD) (22). Labeling, hybridization and washing were performed as described before (18). Quantitation of autoradiograms was performed on a Molecular Dynamics scanning laser densitometer. RESULTS Effect of HGF on growth of fetal hepatocytes in primary culture. First we examined the effect of human recombinant HGF on DNA synthesis in fetal hepatocytes in primary culture by measuring the incorporation of (3H)-thymidine in TCA-precipitable material. HGF stimulated DNA synthesis of the fetal liver cells and its effect was dose-dependent, being detectable at 1 rig/ml and maximal at 5 @ml as shown in fig. 1. The concentration of HGF required for maximal stimulation in fetal liver cells is 685
Vol.
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BIOCHEMICAL
2
AND
BIOPHYSICAL
I 0
RESEARCH
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I 2
4
6
8
1012
HGF (NG/ML)
Fi . l.Effect
of HGF on the DNA synthesis of cultured fetal he?atocytes. Fetal were plated on 60 mm dishes at a density of 2x10“ cells/cm-. Cells, incubated for 48 h in serum-free medium and in the absence or the presence of different concentrations of HGF were analyzed for DNA synthesis measured as (3H)-thymidine incorporation over the last 40 hours of culture. A representative experiment is shown. I?-epatocytes
similar to that required for induction of growth of adult rat hepatocyte primary cultures (2, 14). The mitogenic stimulatoty effect of HGF on fetal hepatocytes was confirmed by flow cytometric analysis of the cell cycle. Twenty four hours after addition of 10 @ml of HGF, Gl-stated fetal hepatocytes decreased from 85 to 71%, increasing the proportion of cells stated in S and G2+M (Table I). So, upon stimulation of HGF, fetal rat hepatocytes entered into S phase and mitosis. Next, we compared the HGF-induced stimulations of DNA-synthesis in fetal hepatocytes to that produced by other known mitogens, as EGF or TGF-a. Fig. 2 shows that the stimulatory effect of HGF and EGF were similar and slightly lower than that observed with TGF-a. We also examined the additive effects of HGF and these other factors (fig. 2). The effect of HGF was additive or synergistic with that of TGF-a. In the presence of both HGF and TGF-a, DNA synthesis increased about 8-9 fold (fig. 2). As TGF-fi can inhibit the EGF-induced growth of fetal hepatocytes (23), we examined the effect of this inhibitory growth factor on the stimulation of DNA-synthesis produced by HGF. As shown in fig. 2, very low concentrations of TGF-p (0.5 rig/ml) totally inhibited HGF-induced growth in fetal hepatocytes in primary culture.
TABLE I - CELL CYCLE ANALYSIS OF FETAL HEPATOCYTES CULTURED IN THE ABSENCE OR IN THE PRESENCE OF HGF Gl
S
G2 + M
CONTROL
84.8 + 3.4
9.8 f 1.6
5.4 + I.8
HGF
70.6 + 3. I
19.7 + 2.5
9.7 zi 0.6
Fetal hepatocytes were cultured in the absence (control) or in the presence of IO rig/ml HGF for 24 hours. Cells were collected and treated for analysis of cell cycle as described in Materials and Methods. Results represent the % of cells in each state and are the means+S.E.M. of three independent experiments.
686
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189,
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CONTROL HGF EGF TGFa HGF+EGF HGF+TGFa EGF+TGF-a HGF+TGF$ 0
10
DNA
20
SYNTHESIS
30
(DPM
40
X 1 ti3)
Fig. 2. Effect of various growth factors and combinations of HGF plus other growth factors on DNA synthesis of cultured fetal hepatocytes. Cells were cultured
for 48 h and pulse labeled with (3H)-thymidine for 40 h as described in fig. 1. The concentrations of growth factors were: HGF, EGF and TGF-alpha: 10 @ml; TGF-beta, 0.5 nglml. Resultsare the means+ S.E.M. of three independentexperiments.
Induction
of c-fos and c-myc
dependence
by HGF
in fetal
hepatocytes
in primary
culture:
of time.
Fetal hepatocytes were treated with HGF at 10 rig/ml. Total RNA was extracted at various times after peptide addition and analyzed for c-fos and c-myc mRNA levels by RNA blot transfer and hybridization to appropriate DNA probes. The 2.2 kilobase c-fos mRNA, which is at the limit of detection in non-stimulated cells, was increased by HGF 30 min after the addition of the factor, decreasing the expression after this time (fig.3 ). In contrast to the rapid induction of c-fos, HGF increased the 2.4 kilobase c-myc m-RNA level 2 hours after the addition of the factor, with a maximum at 8-24 hours, although control cells expressed considerable levels of c-myc mRNA.
DISCUSSION
The present results suggest that additionaly to the well known effect of HGF on the proliferation of mature adult hepatocytes (1,2) and its role as a regulator of liver regeneration (3-9, HGF may also be one of the factors implicated in fetal liver growth. HGF increased (3H)-thymidine incorporation into DNA in fetal rat hepatocyte primary cultures, reaching the maximum at 5 rig/ml (fig. 1). Cell cycle analysis by flow cytometry showed that 15% of the fetal hepatocytes entered into S-phase and mitosis in response to HGF (Table I). The proportion of HGF stimulated-cells was similar or even higher than that observed in incubations with EGF (23). Moreover, HGF effect was clearly synergistic or additive with that of TGF-a (fig. 2). TGF-a, that is produced at relatively high levels during development (24), is one of the most potent hepatotrophic mitogens in vitro (25). Considering that HGF is strongly expressed in human fetal liver as compared with adult liver which is not regenerating (26), both TGF-a and HGF could be the main mitogens responsible of the fetal liver growth. Moreover, TGF-p strongly inhibited DNA synthesis of fetal hepatocytes stimulated by HGF (fig. 2). This growth inhibitory 687
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Fig. 3.Effect of HGF on the levels of c-fos and c-myc mRNAs in cultured fetal hepatocytes. Total RNA, extracted after 0.5,2,8,24 and 48 h was analyzed by Northern blotting as described in Materials and Methods. The radioactivity was visualized by autoradiography (left panel) and measured by scanning densitometry (right panel). A representative experiment is shown.
inhibited EGF- induced proliferation of fetal cultured hepatocytes (23). Thus TGF-l3, whose role in the regulation of liver regeneration is well known (27), could also have an important role in arresting liver growth at the end of the development . In addition to these results, we have found that HGF increased c-fos and c-myc mRNA levels in fetal hepatocytes in culture (fig. 3). Time dependence of these inductions was similar to that found for other mitogens and other cultured cell systems (28), with a maximum for c-fos mRNA levels at 30 min and for c-myc mRNA levels at 8 hours. It is known that HGF stimulates production of inositol triphosphate inducing a prompt elevation of cytoplasmic free calcium level in adult rat hepatocytes (29) and it has been suggested that protein kinase C is fully activated in the HGF signal transduction pathway in hepatocytes (30). Since the induction of c-fos and c-myc expression seems to be a necessary step in the mitogenic response initiated by ligands that act through activation of the protein kinase C (28), our results, that show for the first time a regulation of c-fos and c-myc expression
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with those previous results that implicate protein kinase C in the mitogenic signal transduction pathway elicited by HGF in hepatocytes (29,30). Finally, the results shown in this paper clearly demonstrate the mitogenic role of HGF in rat fetal liver cells, that differ of the antiproliferative effect found in several tumor cells (13, 14). Thus HGF role in growth of the normal (fetal or regenerating) liver seems to be inducing proliferation.
As has been suggested (16), the suppressive growth
regulatory capacity found in tumor cells could implicate the existence of intracellular signalling pathway downstream from tyrosine phosphorylation of HGF receptor that differ between normal (fetal or regenerating) and tumor cells, ACKNOWLEDGMENTS:
We are grateful to Dr. A. Alvarez for expert technical
assistance with the flow cytometer. This work has been supported by a grant from the Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, Spain.
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