TGF-β is a potent inhibitor of hepatocyte growth factor secretion by human fibroblasts

Cell Biology International Reports, Vol. 16, No. 9, 1992 TGF-~

917

IS A POTENT INHIBITOR OF H E P A T O C Y T E GROWTH F A C T O R S E C R E T I O N BY HUMAN F I B R O B L A S T S

Eiichi Gohda,

T e t s u h i k o Matsunaga, Hirotoshi K a t a o k a and Itaru Yamamoto*

Department of Immunochemistry, Faculty of Pharmaceutical Sciences, O k a y a m a University, O k a y a m a 700, Japan

ABSTRACT Transforming growth factor-~ 1 ( T G F - ~ 1) inhibited s e c r e t i o n of h u m a n h e p a t o c y t e g r o w t h factor (hHGF), which is also k n o w n as scatter factor or f i b r o b l a s t - d e r i v e d tumor c y t o t o x i c factor, by MRC-5 cells. The effect was d e t e c t a b l e at as little as i0 pg/ml and was m o r e potent than that of dexamethasone. C o m p l e t e i n h i b i t i o n was o b s e r v e d after 12 h in the presence of 5 ng/ml of T G F - ~ 1. Phorbol 12-myristate 1 3 - a c e t a t e - i n d u c e d s e c r e t i o n of hHGF from h u m a n skin fibroblasts was also s u p p r e s s e d by T G F - ~ i. T G F - ~ 2 inhibited hHGF s e c r e t i o n by MRC-5 cells to the same extent as T G F - ~ 1, but other g r o w t h factors such as e p i d e r m a l g r o w t h factor and acidic and basic fibroblast g r o w t h factors had only a slight or null i n h i b i t o r y effect. INTRODUCTION Hepatocyte growth factor (HGF) is a glycoprotein of about 85,000 daltons w h i c h was purified from p l a s m a of p a t i e n t s with fulminant hepatic failure (Gohda et al., 1988), rat p l a t e l e t s (Nakamura et al., 1987), rabbit serum and normal h u m a n plasma (Zarnegar and Michalopoulos, 1989) as a growth-stimulating factor for adult rat hepatocytes in p r i m a r y culture. HGF is a h e t e r o d i m e r c o m p o s e d of a heavy and a light chain, of about 60,000 and 35,000 daltons, respectively, which are probably linked by a single d i s u l f i d e bond. The p r i m a r y structure of the human HGF (hHGF) precursor deduced from its cDNA has a considerable homology (about 40%) with that of human plasminogen and suggests the presence of four kringle s t r u c t u r e s in a h e a v y chain (Miyazawa et al., 1989; N a k a m u r a et al., 1989). HGF is believed to play an important role in liver regeneration after partial hepatectomy and hepatic damage. Levels of HGF or an HGF-like factor in the plasma and liver of rats and mice, that w e r e treated w i t h h e p a t o t o x i n s or p a r t i a l l y hepatectomized, increased markedly prior to hepatic r e g e n e r a t i o n (Gohda et al., 1990a; Asami et al., 1991; Lindroos et al., 1991; Gohda et al., 1992a). HGF is also the most potent m i t o g e n for a d u l t rat h e p a t o c y t e s in p r i m a r y culture reported so far (Gohda et al., 1990b). However, w h i c h cells p r o d u c e HGF

*To whom c o r r e s p o n d e n c e should be addressed.

0309-1651/92/090917-10/$08.00/0

© 1992 Academic Press Ltd

918

Cell Bioiogy International Reports, Vol. 16, No. 9, 1992

and how its production are largely unknown.

is

controlled

during

liver

regeneration

HGF is identical to both scatter factor (SF) (Gherardi and Stoker, 1990; Weidner et al., 1991), which stimulates the movement of some cultured epithelial cells, and fibroblastderived tumor cytotcxic factor (F-TCF) (Shlma et al., 1991), which is cytotoxic against several tumor cell lines. Human HGF/SF/F-TCF is actively secreted from human e m b r y o n i c lung fibroblast cell lines such as MRC-5 and IMR-90 (Stoker et al., 1987; Higashio et al., 1990) and vascular smooth muscle cells (Rosen et al., 1989). We have reported that human skin fibroblasts from a 4 month old baby secreted a small amount of hHGF and that it was remarkably stimulated by phorbol 12myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate, which are protein kinase C-activating phorbol esters (Gohda et al., 1992b). That hHGF is produced by PMA-treated HL-60, a promyelocytic leukemia cell line, has also been reported by Nishino et al. (1991). We have also found that d e x e m e t h a s c n e inhibited the PMA-induced secretion of hHGF by human skin fibroblasts (Gohda et al., 1992b). Since glucocorticoids stimulate the growth of some human fibroblast cell lines (Macieira-Coelho, 1966; Baker et al., 1978), in this study we tested the effects of well-known growth factors on hHGF secretion and found that transforming growth f a c t o r - ~ 1 (TGF1) and T G F - ~ 2 inhibited hHGF secretion by both MRC-5 cells and PMA-treated human skin fibroblasts. MATERIALS AND METHODS Reagents: Eagle's m i n i m u m essential m e d i u m (MEM) was purchased from Nissui Pharmaceutical Co. Ltd., Tokyo; fetal bovine serum (FBS) and human transforming growth factor-u (TGF-G , recombinant) were from GIBCO BRL, Gaithersburg, MD; human T G F - ~ 1 (recombinant) was from King Brewing Co. Ltd., Kakogawa, Japan; porcine TGF-~ 2 (porcine platelets), bovine acidic fibroblast growth factor (aFGF, bovine brain) and basic fibroblast growth factor (bFGF, bovine brain) were from R & D Systems, Minneapolis, MN; human epidermal growth factor (EGF, recombinant) Was from Wakunaga Pharmaceutical Co., Osaka; human platelet-derived growth factor (PDGF, BB homodimer, recombinant) was from Mallinckrodt Specialty Chemicals Co., Paris, KY; PMA and dexamethasone were from Sigma Chemical Co., St.Louls, MO; and [methyl-3H]thymidine (0.74 TBq/mmol) was from Du Pont-New England Nuclear, Boston, MA. Enzyme-linked immunosorbent assay (ELISA) kits for hHGF (Tsubouchi et al., 1991) was generously supplied by Otsnka A s s a y Laboratories, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan. Cell culture: MRC-5 human embryonic obtained from the Riken Cell Bank, abdominal skin fibroblasts, obtained

lung fibroblasts Tsukuba, Japan. from a normal

were Human baby

Cell Biology Intemational Reports, VoL 16, No. 9, 1992

919

(female, 4 months old) (Shindo et al., 1991), and kindly provided by the Department of Dermatology, Shinshu U n i v e r s i t y School of Medicine, Matsumoto, Japan, w e r e used at p o p u l a t i o n d o u b l i n g levels of 18-30. Both cell lines were c u l t u r e d as monolayers in MEM supplemented with 10% FBS, unless stated otherwise, at 37°C in a humidified a t m o s p h e r e of 5% COs in air. All experiments w e r e performed at least twice w i t h similar results. D e t e r m i n a t i o n of hHGF levels in c o n d i t i o n e d m e d i a and cell extracts: After reaching c o n f l u e n c e in 24-well p l a s t i c dishes (Nunc), the m e d i u m (i ml) in MRC-5 and human skin fibroblast cultures was replaced with that c o n t a i n i n g test clrtokines and compounds. The conditioned media were collected after i n c u b a t i o n for v a r i o u s times and w e r e i m m e d i a t e l y frozen at -30 for hHGF ELISA. Cell layers w e r e then washed 4 times w i t h p h o s p h a t e - b u f f e r e d saline (PBS) and solubilized in 1 ml of Lowry solution C (Lowry et al., 1951; Patterson, 1979) for c e l l u l a r protein d e t e r m i n a t i o n by the m e t h o d of Lowry et al. (1951). Some cell layers were w a s h e d w i t h PBS, then scraped into ice-cold PBS c o n t a i n i n g 1.5 M NaCI and 0.039% Triton X-lO0. An aliquot of the cell suspension was removed for p r o t e i n assay. B o v i n e serum a l b u m i n was then added at a ratio of 0.25%, then the cell suspension was sonicated and c e n t r i f u g e d at 2 0 , 0 0 0 X g for 20 min at 4 ~ . The s u p e r n a t a n t was stored at -30 ~ for hHGF ELISA. h H G F in the c o n d i t i o n e d m e d i a and cell e x t r a c t s was d e t e r m i n e d b y ELISA (Tsubouchi et al., 1991) and its levels were e x p r e s s e d as ng hHGF per mg of c e l l u l a r protein, as d e s c r i b e d p r e v i o u s l y (Gohda et al., 1992b). D e t e r m i n a t i o n of DNA synthesis in MRC-5 cells: Subconfluent MRC-5 cells, t r y p s i n i z e d and s u s p e n d e d in MEM s u p p l e m e n t e d w i t h 10% FBS were seeded in 24-well p l a s t i c dishes (Nunc) at a density of 1.2 X i0 ~ cells/cm z (i ml/well) and incubated overnight. Monolayers were then washed twice with MEM s u p p l e m e n t e d w i t h 2% FBS and cultured for a further 2 days in this medium. G r o w t h factors were then added to the cultures w i t h o u t a m e d i u m change. DNA synthesis, d e t e r m i n e d by labeling c u l t u r e d cells w i t h [3H]thymidine (37 kBq/ml, 37 GBq/mmol) for i0 h at 3 7 ~ b e t w e e n 20 and 30 h after the addition of g r o w t h factors as described previously (Gohda et al. 1990b), was e x p r e s s e d as i n c o r p o r a t e d [SH]thymidine per culture. RESULTS Confluent MRC-5 cells actively secreted hHGF/human SF as r e p o r t e d (Stoker et al., 1987). The hHGF c o n c e n t r a t i o n s in the c o n d i t i o n e d m e d i u m of 24, 48 and 72 h - c u l t u r e s were 1 2 ~ 3, 20~ 3 and 2 9 ~ 6 ng/ml (n=3), respectively. W e examined e f f e c t s of v a r i o u s growth factors on hHGF s e c r e t i o n by MRC-5 cells. As shown in Table i, it was r e m a r k a b l y inhibited by b o t h TGF- B 1 and T G F - ~ 2 and s l i g h t l y by bFGF and aFGF. T G F - a , EGF and

(

CellBiology lnternational Reports, Vol. 16, No. 9, 1992

920

Table 1 Effects of v a r i o u s g r o w t h factors o n h H G F secretion and D N A synthesis by MRC-5 cells Growth factor

Concentration (ng/ml)

None EGF

h H G F secreted (ng/mg protein)

1 10 1 i0 1 I0 1 i0 1 i0 1 5 1 5

PDGF aFGF bFGF TGF-G TGF-~ 1 TGF-~ 2

280 283 316 278 298 276 254 277 234 268 296 59 45 66 46

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

DNA s y n t h e s i s (cpm/culture)

22 19 7 15 4 i0 3 13 4 12 18 3 2 4 2

2721 5710 4975 6399 5706 7191 6575 5381 6869 5228 5410 7512 7514 7816 7204

~ ~ ~ ± ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

70 465 653 749 513 832 175 705 545 181 90 208 113 297 128

MRC-5 cells were incubated without or with the indicated c o n c e n t r a t i o n s of g r o w t h factors for 72 h (hHGF secretion) or 30 h (DNA synthesis), hHGF in the conditioned medium was d e t e r m i n e d by ELISA. DNA synthesis was d e t e r m i n e d by labeling cultured cells w i t h [3H]thymidine for the last 10 h. V a l u e s are m e a n s ~ S.D. for t r i p l i c a t e cultures.

PDGF had no i n h i b i t o r y effect. The g r o w t h - p r o m o t i n g these factors on MRC-5 cells w e r e also examined. g r o w t h factors s t i m u l a t e d DNA synthesis (Table i).

effects of All seven

The d o s e - r e s p o n s e for the T G F - ~ 1 - p r o d u c e d inhibition in 72 h-cultures of MRC-5 cells is shown in Fig. 1. It i n h i b i t e d hHGF secretion at as little as 10 pg/ml and by 84% at 5 ng/ml. The hHGF levels in cell layers u n t r e a t e d and t r e a t e d w i t h 5 ng/ml of T G F - ~ 1 for 72 h were 89 and 10 ng/mg c e l l u l a r protein, respectively. Thus cellular hHGF levels were also r e d u c e d by T G F - ~ 1. Figure 1 also shows the effect of TGF-~ 1 on hHGF secretion by PMA-treated human skin fibroblasts. T r e a t m e n t of the cells w i t h 0.1 and 5 ng/ml of T G F - ~ 1 for 72 h resulted in 52 and 93% inhibition, respectively. TGF-~ 2 p r o d u c e d a similar e f f e c t o n hHGF s e c r e t i o n by the P M A - t r e a t e d fibroblasts (data not shown). Figure

2A

and

B

shows

time

courses

of

the

T G F - ~ 1-produced

921

Cell Biology International Reports, Vol. 16, No. 9, 1992

300 I-{,_., "~ 2 0 0

O"

'

0

.'I

I

1

I

I

10 10 = TGF-/~I (pg/ml)

I

I

103

10 `=

Fig. i. D o s e - r e s p o n s e for T G F - ~ 1 - p r o d u c e d i n h i b i t i o n of h H G F secretion by MRC-5 cells and PMA-treated human skin fibroblasts. C o n f l u e n t M R C - 5 c e l l s ( • ) a n d I0 n M P M A - t r e a t e d h u m a n s k i n f i b r o b l a s t s ( • ) w e r e i n c u b a t e d w i t h o u t or w i t h t h e indicated concentrations of TGF-~ 1 for 72 h. hHGF in the c o n d i t i o n e d m e d i u m w a s d e t e r m i n e d by ELISA. V a l u e s are m e a n s ~ S.D. for t r i p l i c a t e c u l t u r e s . The h H G F level in the c o n d i t i o n e d medium of human skin fibroblasts incubated without PMA and T G F - ~ 1 was 1 6 ~ 2 n g / m g c e l l u l a r p r o t e i n .

i n h i b i t i o n of h H G F s e c r e t i o n by b o t h c e l l lines. The i n h i b i t i o n was apparent in 12 or 24 h-cultures and was complete t h e r e a f t e r . We r e p o r t e d p r e v i o u s l y t h a t d e x a m e t h a s o n e i n h i b i t e d P M A - i n d u c e d s e c r e t i o n of h H G F f r o m h u m a n s k i n f i b r o b l a s t s a n d that maximal i n h i b i t i o n w a s o b s e r v e d at 1 ~ M d e x a m e t h a s o n e ( G o h d a et al., 1992b). The t i m e c o u r s e of i n h i b i t i o n by T G F - ~ 1 w a s t h e n c o m p a r e d w i t h t h a t b y d e x a m e t h a s o n e u s i n g d o s e s of e a c h t h a t e x e r t e d m a x i m a l e f f e c t s . As s h o w n in Fig. 2B, T G F - ~ 1 suppressed the induced hHGF secretion from human skin fibroblasts more than dexamethasone at a n y t i m e in c u l t u r e . Secretion of hHGF from MRC-5 cells was also inhibited by d e x a m e t h a s o n e (Fig. 2A). The e f f e c t w a s a g a i n l e s s p o t e n t t h a n that of T G F - ~ i. The inhibition by dexamethasone was not c o m p l e t e : the t r e a t e d c e l l s s e c r e t e d h H G F c o n t i n u o u s l y , though at a r e d u c e d rate, d u r i n g 7 2 - h in c u l t u r e (Fig. 2A).

922

Ceil Biology International Reports, Vol. 16, No. 9, 1992 i

3001 A

~200

100

I

0

500

•"

C G)

o ~

|

24 48 Time after addition (h)

f

72

B

400

2 300

E C

"" 200 IJ..

,.I,,1¢

100

0

24 48 72 96 Time after addition (h)

120

Fig. 2. T i m e c o u r s e of TGF- ~ 1 - p r o d u c e d i n h i b i t i o n of h H G F s e c r e t i o n by M R C - 5 c e l l s (A) and I0 n M P M A - t r e a t e d h u m a n s k i n f i b r o b l a s t s (B). C o n f l u e n t c e l l s w e r e i n c u b a t e d w i t h o u t ( O ) or with 5 ng/ml (A) o r 1 n g / m l (B) of TGF-~ 1 (•) or I~ M d e x a m e t h a s o n e (m) for t h e i n d i c a t e d p e r i o d s . V a l u e s a r e means+_ S.D. for t r i p l i c a t e c u l t u r e s .

Cell Biology International Reports, Vol. 16, No. 9, 1992

923

DISCUSSION This study d e m o n s t r a t e d that the secretion of hHGF by b o t h MRC5 h u m a n embryonic lung fibroblasts and P M A - t r e a t e d h u m a n skin fibroblasts was remarkably inhibited by T G F - ~ 1 and T G F - ~ 2. The effect of TGF-~ 1 was more potent than that of dexamethasone, which reportedly inhibits hHGF secretion by PMA-treated human skin fibroblasts (Gohda et al., 1992b). The inhibitory effect of both T G F - ~ 1 and T G F - ~ 2 is u n l i k e l y to be related to their growth-promoting activity, since o t h e r growth factors had slight or null inhibitory effect. While this paper was preparing, Ramadori et al. (1992) reported that TGFinhibited HGF gene expression in cultured rat fat-storing (Ito) cells, indicating that the inhibitory effect of T G F - ~ on HGF p r o d u c t i o n is not limited to its effect on human fibroblasts. It is not k n o w n at present w h e t h e r TGF-~ acts as an inhibitor of hHGF secretion also in vivo. However, time courses of changes in the HGF and TGF-~ mRNA levels in the rat liver remnant after partial hepatectomy are under a reciprocal relationship (Zarnegar et al., 1991; Selden et al., 1990; Braun et al., 1988). The amount of HGF mRNA in regenerating rat liver increased rapidly after partial hepatectomy, peaked at 12 h and g r a d u a l l y returned to normal levels after 72 h (Zarnegar et al., 1991; Selden et al., 1990). Cells that express the HGF gene in partially hepateotomized rat livers have not yet been identified, but those in h e p a t o t o x i n - d a m a g e d rat livers are r e p o r t e d l y Kupffer and endothelial cells (Noji et al., 1990). O n the other hand, e x p r e s s i o n of the TGF-~ gene in rat liver remained low until 12 h after partial hepatectomy, increased sharply thereafter and peaked at 72 h (Braun et al., 1988). We are now examining the effect of T G F - ~ on the elevation of HGF m R N A and HGF levels in regenerating rat liver. M a n y lines of evidence support the concept that T G F - ~ is a physiological inhibitor of liver regeneration, which ceases after enlargement of the liver remnant to its original mass. This factor inhibits DNA synthesis by adult rat hepatocytes in p r i m a r y culture (Nakamura et al., 1985; Carr et al., 1986; Russell, 1988) and by regenerating rat liver (Russell et al., 1988). DNA synthesis in regenerating rat liver peaked at 24 h post partial hepatectomy, then gradually declined and was negligible at 72 h (Zarnegar et al., 1991; Bucher, 1963) when the maximal level of TGF-~ m R N A was reached. Thus it is possible that T G F - ~ acts as a dual inhibitor of D N A synthesis in liver regeneration: it not only directly inhibits DNA synthesis of hepatocytes, but also m a y suppress secretion of HGF which is believed to play an important role in hepatocyte proliferation during liver regeneration. ACKNOWLEDGMENTS We

are

grateful

to

Otsuka

Assay

Laboratories

for

supplying

924

Cell Biology lntemational Reports, VoL 16, No. 9, 1992

hHGF ELISA kits. This work was in-Aid for Scientific Research Science and Culture of Japan.

supported, in part, by Grantsfrom the Ministry of Education,

REFERENCES

Asami, 0., lhara, I., Shimidzu, N., Shimizu, S., Tomita, Y., Ichihara, A. and Nakamura, T. (1991). Purification and characterization of hepatocyte growth factor from injured liver of carbon tetrachloride-treated rats. J. Biochem. 109: 8-13. Baker, J.B., Barsh, G.S., Carney, D.H. and Cunningham, D.D. (1978). Dexamethasone modulates binding and action of epidermal growth factor in serum-free cell culture. Proc. Natl. Acad. Sci. USA 75: 1882-1886. Braun, L. , Mead, J.E., Panzica, M., Mikumo, R., Bell, G.I. and Fausto, N. (1988). Transforming g r o w t h factor ~ mRNA increases during liver regeneration: A possible paracrine mechanism of growth regulation. Proc. Natl. Acad. Sci. USA 85: 1539-1543. Bucher, N.L.R. (1963). Regeneration of mammalian liver, in: Bouene, G.H. and Danielli, J.F. (eds.). International review of cytology. Vol. 15. N e w York: Academic Press. Carr, B.I., Hayashi, I., Branum, E.L. and Moses, H.L. (1986). Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type transforming growth factor. Cancer Res. 46: 2330-2334. Gherardi, E. and Stoker, M. (1990). Hepatocytes and scatter factor. Nature 346: 228. Gohda, E., Hayashi, Y., Kawaida, A., Tsubouchi, H. and Yamamoto, I. (1990a). Hepatotrophic growth factor in blood of mice treated with carbon tetrachloride. Life Sci. 46: 1801-1808. Gohda, E., Kataoka, H., Tsubouchi, H., Daikuhara, Y. and Yamamoto, I. (1992b). Phorbol e s t e r - i n d u c e d secretion of human hepatocyte growth factor by human skin fibroblasts and its inhibition by dexamethasone. FEBS. Lett. 301: 107-110. Gohda, E., Matsunaga, T., Kataoka, H., Kasada, C. and Yamamoto, I. (1992a). Purification and characterization of a mouse hepatocyte growth-stimulating factor from the liver of carbon tetrachloride-treated mice. J. Pharmacobio-Dyn. 15, 131-137. Gohda, E., Tsubouchi, H., Nakayama, H., Hirono, S., Sakiyama, 0., Takahashi, K., Miyazaki, H., Hashimoto, S. and Daikuhara, Y. (1988). Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. J. Clin. Invest. 81: 414-419. Gohda, E., Yamasaki, T., Tsubouchi, H., Kurobe, M., Sakiyama, 0., Aoki, H., Niidani, N., Shin, S., Hayashi, K., Hashimoto, S., Daikuhara, y. and Yamamoto, I. (1990b).

Cell Biology international Reports, Vol. 16, No. 9, 1992

925

Biological and immunological properties of human hepatocyte growth factor from plasma of patients with fulminant hepatic failure. Biochim. Biophys. Acta 1053: 21-26. Higashio, K., Shima, N., Goto, M., Itagaki, Y., Nagao, M., Yasuda, H. and Morinaga, T. (1990). Identity of a tumor cytotoxic factor from human fibroblasts and hepatocyte growth factor. Biochem. Biophys. Res. Commun. 170: 397-404. Lindroos. P.M., Zarnegar, R. and Michalopoulos, G.K. (1991). Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration. Hepatology 13: 743-749. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Macieira-Coelho, A. (1966). Action of cortisone on human fibroblasts in vitro. Experientia 22: 390-391. Miyazawa, K., Tsubouchi, H., Naka, D., Takahashi, K., Okigaki, M., Arakaki, N., Nakayema, H., Hirono, S., Sakiyama, O., Takahashi, K., Gohda, E., Daikuhara, Y. and Kitamura, N. (1989). Molecular cloning and sequence analysis of cDNA for human h e p a t o c y t e growth factor. Biochem. Biophys. Res. Commun. 163: 967-973. Nakamura, T., Nawa, K., Ichihara, A., Kaise, N. and Nishino, T. (1987). Purification and subunit structure of hepatocyte growth factor from rat platelets. FEBS Lett. 224: 311-316. Nakamura, T., Nishizawa, T., Hagiya, M., Seki, T., Shimonishi, M., Sugimura, A., Tashiro, K. and Shimizu, S. (1989). Molecular cloning and expression of human hepatocyte growth factor. Nature 342: 440-443. Nakamura, T., Tomita, Y., Hirai, R., Yamaoka, K., Kaji, K. and Ichihara, A. (1985). Inhibitory effect of transforming growth factor-~ on DNA synthesis of adult rat hepatocytes in primary culture. Biochem. Biophys. Res. Commun. 133: 1042-1050. Nishino, T., Kaise, N., Shindo, Y., Nishino, N., Nishida, T., Yasuda, S. and Masui, Y. (1991). Promyelocytic leukemia cell line, HL-60, produces human hepatocyte growth factor. Biochem. Biophys. Res. Commun. 181: 323-330. Noji, S., Tashiro, K., Koyema, E., Nohno, T., Ohyama, K., Taniguchi, S. and Nakamura, T. (1990). Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers, as revealed by in situ hybrydization. Biochem. Biophys. Res. Commun. 173: 42-47. Patterson, M.K., Jr. (1979). Measurement of growth and v i a b i l i t y of cells in culture. Methods Enzymol. 58: 141-152. Ramadori, G., Neubauer, K., Odenthal, M., Nakamura, T., Knittel, T., Schw6gler, S. and M e y e r zum BUschenfelde, K.-H. (1992). The gene of h e p a t o c y t e growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming t

926

Cell Biology International Reports, VoL 16, No. 9, 1992

growth factor-B. Biochem. Biophys. Res. Commun. 183: 739-742. Rosen, E.M., Goldberg I.D., Kacinski, B.M., Buckholz, T. and Vinter, D.W. (1989). Smooth muscle releases an epithelial cell scatter factor which binds to heparin. In Vitro Cell. Dev. Biol. 25: 163-173. Russell, W.E. (1988). Transforming growth factor beta (TGF-B ) inhibits hepatocyte DNA synthesis independently of EGF binding and EGF receptor autophosphorylation. J. Cell. Physiol. 135: 253-261. Russell, W.E., Coffey, R.J., Jr., Ouellette, A.J. and Moses, H.L. (1988). Type ~ transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proc. Natl. Acad. Sci. USA 85: 5126-5130. Selden, C., Jones, M., Wade, D. and Hodgson, H. (1990). Hepatotropin mRNA expression in human foetal liver development and in liver regeneration. FEBS Lett. 270: 81-84. Shima, N., Nagao, M., Ogaki, F., Tsuda, E., Murakami, A. and Higashio, K. (1991). Tumor cytotoxic factor/hepatocyte growth factor from human fibroblasts: Cloning of its cDNA, purification and characterization of recombinant protein. Biochem. Biophys. Res. Commun. 180: 1151-1158. Shindo, Y., Akiyama, J., Yamazaki, Y., Saito, K. and Takase, Y. (1991). Changes in enzyme activities in skin fibroblasts derived from persons of various ages. Exp. Gerontol. 26: 29-35. Stoker, M., Gherardi, E., Perryman, M. and Gray, J. (1987). Scatter factor is a fibroblast-derived modulator of epithelial cell motility. Nature 327: 239-242. Tsubouchi, H., Niitani, Y., Hirono, S., Nakayama, H., Gohda, E., Arakaki, N., Sakiyama, 0., Takahashi, K., Kimoto, M., Kawakami, S., Setoguchi, M., Tachikawa, T., Shin, S., Arima, T. and Daikuhara, Y. (1991). Levels of the human hepatocyte growth factor in serum of patients w i t h various liver diseases determined by an enzyme-linked immunosorbent assay. Hepatology 13: 1-5. Weidner, K.M., Arakaki, N., Hartmann, G., Vandekerckhove, J., Weingart; S., Rieder, H., Fonatsch, C., Tsubouchi, H., Hishida, T., Daikuhara, Y. and Birchmeier, W. (1991). Evidence for the identity of human scatter factor and human hepatocyte growth factor. Proc. Natl. Acad. Sci. U S A 88: 7001-7005. Zarnegar, R., DeFrances, M.C., Kost, D.P., Lindroos, P. and Michalopoulos, G.K. (1991). Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy. Biochem. Biophys. Res. Commun. 177: 559-565. Zarnegar, R. and Michalopoulos, G. (1989). Purification and biological characterization of h u m a n hepatopeietin A, a polypeptide growth factor for hepatoclrtes. Cancer Res. 49: 3314-3320. Paper received 18.06.92.

Paper accepted 08.07.92.