The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-β

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THE GENE OF HEPATOCYTE GROWTH FACTOR IS EXPRESSED IN FAT-STORING CELLS OF RAT LIVER AND IS DOWNREGULATED DURING CELL GROWTH AND BY TRANSFORMING GROWTH FACTOR-l3 G. Ramadoril,

K. Neubauer, M. Odenthal, T. Nakamura*, T. Knittel, S. Schwogler, K.-H. Meyer zum Btischenfelde

Department of Internal Medicine, Johannes Gutenberg University,

and

6.500 Mainz, Germany

*Department of Biology, Faculty of Science, Kyushu University, Fukuoka City, Japan Received

February

3,

1992

Summary: Hepatocyte growth factor (HGF) has beendetected in non-parenchymal cells but not in hepatocytes. We performed Northern blot analysisof total RNA extracted from rat hepatocytes, Kupffer cells, endothelial cells and fat-storing (Ito-) cells. Total RNA was extracted from fat-storing cells at different times after isolation and from cells treated with different amounts of transforming growth factor p. The RNA was hybridized with HGF, fibronectin-, and a-actin-specific cDNA probes, consecutively. We found an abundant amount of HGF mRNA in freshly isolated fat-storing cells, but not in other liver cells. The amount of the HGF transcripts decreasessignificantly in FSC during the time of culture, while fibronectin gene expression increasesand a-actin gene expressionas well. TGF+ dramatically inhibits HGF gene expression,but causesan enhancedtibronectin mRNA level. Northern blot hybridisation of total RNA from CClh-chronically damagedIiver with HGF cDNA shows a significant increaseof HGF mRNA during development of liver fibrosis. We suggestthat in damagedliver either non-parenchymalcells, others than FSC, becameable to expressthe HGF in vivo , or other mediatorsovercome the inhibitory effect of TGF-l3. Q1992Academic Press,Inc.

Hepatocyte growth factor (HGF) is a peptide growth factor that stimulatesDNA replication of hepatocytes in primary cultures (l-3). The factor has been purified from various sourcesand cloned (4-10). Although HGF was originally describedas hepatocyte specific, it is now clear that HGF stimulatesgrowth of many different cell types (11). HGF is detectablein plasmaand in serum (12,13). Blood HGF ltvels markedly increasein patients with fulminant hepatic failure and are reduced to near normal in patients,who had recovered from the disease(13). Serum levels of HGF rise after partial hepatectomy in the rat (14,159. HGF does also rise in the damagedliver tissueof rats after administrationof D-galactosamineor Ccl, (16,17). HGF messengerRNA (mRNA) and protein is expressedin many organs, as shown in human and in rat 1To whom correspondenceshouldbe addressed. Abbreviations: HGF, hepatocyte growth factor; FSC, fat-storing cell; TGF+, transforming growth factor -/3.

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0006-291X192 $1.50 Copyright 0 1992 by Academic Press. Iw. All rights of reproduction in any form reserved.

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(17,18). In the liver HGF-mRNA is present in non-parenchymal cells, but apparently not in hepatocytes (17,18). It has been supposed that endothelial cells and Kupffer cells are responsible for HGF synthesis in rat liver (17,18). By Northern blot analysis we show that HGF-specific transcripts are easily detectable in total RNA from freshly isolated rat fat-storing (Ito-) cells (FSC), but not in the RNA from freshly isolated endothelial cells or from isolated and cultured Kupffer cells. The HGF-mRNA decreases in FSC during the 7 days in primary culture. Transforming growth factor B (TGF-B), a key mediator of liver fibrosis, markedly decreases HGFmRNA-level

Material

in cultured FSC, but increases fibronectin-gene-expression,

and Methods

Animals: Adult male Wktur rats (retired breeder)weighing 400-600 g were usedfor isolation of the parenchymal and non-parenchymalliver cells. Isolation and culture of liver cells: Hepatocytes, Kupffer cells, encothelial cells FSC, skin fibroblastsand aortic smoothmusclecells were isolatedasdescribedelsewhere(19-21). FSC were kept in culture up to day 7 after isolation. Culture medium (DMEM supplementedwith 15 % FCS, 100 U/ml penicillin, 100 ug streptomycin and 1 % L-glutamine) was replaced the first time 3 days after inoculation and then every day. Cells were treated +vith human platelet derived TGF-f3 (1-5 rig/ml medium) for 20 h at day 0 and 3 after isolation. Induction of acute and chronic liver damage: Acute and chronic liver damagewere induced by oral administration of CClqas previously described(20). For induction of an acute liver damage 150 ~1 CCL, per 100 g body weight were once administratedto rats. A chronic liver damagewas induced by gas&al gavage of Ccl, weekly with an inital dosisof 40 ~1; subsequentdoseswere increasedabout two times of the previous gavagein considerationof weight responseof the animals (22). RNA extraction and Northern blot analysis: RNA was extracted from ceils and from liver according to Chirgwin (23) as describedelsewhere(19). RNA was separatedby agarosegel electrophoresis,blotted onto nitrocellulose membranesand hybridized with 32P-labeledcDNA probes specific for rat HGF (6), human fibronectin (24), chicken a-actin, which also contains B-, y-actin specific cDNA sequences(25), and with an oligonucleotide (AACGATCAGAGTAGTTGGTATITCACC) complementary to 28 S rRNA (26).

Results and Discussion

In our in vivo experiments we confirm the data publishedby others (16,17) that HGF-mRNA increasesin the liver after acute or chronical damage(Figure 1). By in situ hybridisation as well as immunohistochemistry HGF-expression hasbeen shownin sinusoidal(endothelial and Kupffer cells), but not in parenchymal cells of the normal as well as of the damaged liver (17,18). However, no identification of the cells was performed. This is of importance as FSC representa third important population of the liver non-parenchymalliver cells. To further explore the sourceof HGF in normal liver we performed isolation of parenchymal and of non-parenchymal cells according to standardmethods. We cultured liver cells, skin fibroblasts, and aortic smooth musclecells as well. Northern blot analysisof the RNA extracted from the cells showedabundantamountsof HGF only in freshly isolatedFSC. We also found that HGF-geneexpressionin FSC decreasesduring the time in culture and almostdisappearsat day 7 after isolation. The reason for the lower amountsof the HGF-transcripts in FSC of one of the two experiments (Figure 2, left) is not known at the moment. In contrast an increasedexpressionof 740

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Figure 1. HGF gene expression m normal (c.), acutely (a.) and chronically damaged (2x;, 4x, 6x, 8x, 10x) rat liver. Northern blot anlysis of total liver RNA. Ftlters were hybridized with 32P-labeled cDNA probes (human fibronectin. rat HGF, and chicken B, y-actin) and with an oligonucleotide (28 S rRNA), consecuttvely. Figure 2. HGF gene expression In fat-storing cells (FSC). Comparison with hepatocytes (H), Kupffer cells (KC), endothelial cells (EC), aortic smooth-muscle cells (SMC), and skin fibroblasts (F). Northern Blot analysis of RNA extracted from freshly isolated (f) hepatocjtes, Kupffer cells and endothelial cells as well from freshly isolated (0) and cultured (day 3, day 7) fat-storing cells (the FSC were from two different isolations). The filter was hybridized with 32P-labeled cDNA probes (human fibronectin, rat HGF, chicken a, 8, y-actm and wrth an oligonucleotide (28 S rRNA), consecutively.

the a-actin-

and of the fibronectin-gene-expressionwas observed.The factor(s) responsible

for

such apparently spontaneouschangesare still unknown. In RNA from hepatocytes, Kupffer cells, endothelial cells, fibroblasts, and smooth-muscIecells HGF-transcripts were not detectable (Figure

3). As regeneration

always accompanies

acute as well as chronic

liver damage and

as it is known that TGF-b is a key mediator of liver fibrogenesiswe investigated the effect of TGF-P treatment on HGF- and on fibronectin-gene-expressionin primary culture of FSC. For this put-pose FSC were exposed to TGF-B for 20 h immediately after isolation and at day 3 of culture. While fibronectin synthesisis stimulated by TGF-P, a significant decreaseof HGFtranscripts in cultured FSC was induced (Figure 3). In freshly isolated cells no effect was observed after TGF-p treatment (data not shown).The lack of effect of TGF-P on freshly isolated FSC is explained by the lack of receptor expressionin “quiescentcells” (27). Taken together

Kb +

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Figure 3. TGF-B downregulates HGF gene expression m fat-storing cells (FSC). FSC (at day 2 after isolation) were cultured overnight in absence (3) or in presence (3+) of 5 nglml TGF-B (representative for three Independent experiments). Cells were lysed for RNA extraction. RNA from freshly isolated (0), from cultured (day 3 and day 7) FSC (the FSC were from a third isolation) from aortic smooth-muscle cells (SMC) and from skin fibroblasts (F) was studied by Northern blot analysis. Filters were hybridized with 32P-labeled cDNA probes (human fibronectin, rat HGF) and an oligonucleotide (28.5 rRNA), consecutively.

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our in viva and in vitro studies indicate that FSC should be responsible for HGF synthesis in normal liver and that spontaneous “activation” (20) of FSC in culture or treatment with TGF-f3 significantly.reduces One interpretation

HGF-gene-expression

while fibronectin-gene-expression

is increased.

of these findings could be that after liver damage, leading to FSC “activa-

tion” (20), non-parenchymal

cells others than FSC could start to express the HGF-gene.

The other posstbility could be, that cytoktnes others than TGF-P could overcome the inhibitory effect of TGF-fl on HGF-gene-expresston

in FSC. In vitro experiments

using different cyto-

kines together with TGF-P for treatment of FSC and other non-parenchymal

liver cells in vitro

are underway in our laboratory. References 1. Michalopoulus, G. (1990) FASEB J. 4. 176-187 2. Selden, C., and Hodgon, H.J.F. (1991) GUT 32,602~603 3. Nakamura, T. (1991) Prog. Growth Fa.:tor Res. 3,67-86 4. Miyazawa, K., Tsubouchi, H., Naka, 0. Takahashi, K., Okigaki, M. Araraki, N., and Nakamyama, H. (1989) Biochem. Biophys. Res. Commun. 163,967-973 5. Nakamura, T., Nishizwa, T., Hagiya, M., S&i, T., Shimonishi, M., Sugimura, A., Tashiro, K., and Shimizu, S. (1989) Nature (lond) ;42,440-443 6. Tashiro, K., Hagiya, M., Nishizawa, T., Seki, T., Shimonishi, M., Shimizu, S., and Nakamu ra,T. (1990) Proc. Natl. Acad. Sci. USA 87,3200-3204 7. Wolf, H.K., Zamegar, R., Oliver, L., and Michalopoulus, G.K. (1991) Am. J. Pathol. 138, 1035-1043 8. Seki, T., Ihara, I., Sugimura, A., Shimonishi, M., Nishizawa, T., Asami, O., Hagiya, M., Nakamura, T., and Shimizu, S. (1990) Biochem. Biophys. Res. Commun. 172,321-327 9. Tsubouchi, H., Hirona, S., Gohda, E., Nakayama, N., Takahashim, K., Sakiyama, O., Miyazaki, H., et al. (1989) Hepatology 9,875881 lO.Okajima, A., Miyazawa, K., and Kitamura, N. (1990) Eur. J. B&hem. 193,375381 1l.Kan, M., Zhang, G., Zamegar, R., Michalopoulus, G., Myoken, Y ., MC Keehan, W.I., and Stevens, J.I. (1991) Biochem. Biophys. Res. Commun. 174,331-337 12.Michalopoulus, G., Houck, K.A., Dolan, M.L., and Luetteke, N.C. (1984) Cancer Res. 44, 44144419 13.Tsubouchi, H., Niitani, Y., Hirona, S., Nakayama, N., et al. (1991) Hepatology 13, l-5 14.Nakamura, T., Nawa, K., and Ichihara, A. (1984) B&hem. Biophys. Res. Commun. 122, 1450-1459 15.Okajima, A., Miyazawa, K., and Kitamiura,N. (1990) Eur. J. Biochem. 193,275-281 16,Kinoshita, T. Tashiro, K., and Nakamura, N. (1989) Biochem. Biophys. Res. Commun. 165, 1229- 1234 17.Noji, S., Tashiro, K., Koyama, E., Nohmo, T., Ohyama, K., Tamguchi, S., and Nakamura, T. (1990) Btochem. Biophys. Res. Commun. 173,42-47 18. Wolf, H.K., Zamegar, R., and Michalopoulus, G. (1991) Hepatology 14,488494 19.Ramadori, G., Sipe, J.D., Dinarello, C.A., Mizel, S.B., and Colten, H.R. (1985) J. Exp. Med. 162,930~942 20,Ramadori, G., Veit, T., Schwogler, S., Dienes, H.P., Knittel, T., Rieder, H., and Meyer zum Btischenfelde, K.H. (1990) Virchows Archiv B 59,349-357 21.Ramadot-i, G., Schwogler, S., Veit, T., Rieder, H.. Chiquet-Ehrismann, R., Maclcie, E.J., and Meyer zum Biischenfelde, K.H., (1991) Virchows Arch. B 60, 145-155 22.Proctor, E. and Chatamam, K. (1982) Gastroenterology 83, 1183-l 190 23.Chirgwin J.M., Przybyla, A.E., MC Donald, R.J., and Rutter, W.J. (1979) Biochemistry 18, 52945230 24.Umezawa, K., Kombliht, A.R., and Bamlle, F.E. (1985) FEBS Lett. 186,31-34 %Schwarz, R.J., Haron, J.A., Rothblum, K.N., and Dugaiczyk, A. (1980) Biochemistry 19, 5883-5890 26. Barbu, V. and Dautry, F. (1989) Nucl. Acid Res. 17,7115-7118 27.Friedman S.L. and Yamasaki, G. (1991) Hepatology 14, 133 A 742