JOURNAL OF CELLULAR PHYSIOLOGY 142:14Y-154 (1990)

Basic Fibroblast Growth Factor and Transforming Growth Factor-a are Hepatotrophic Mitogens In Vitro BlRClT HOFFMANN AND DIETER PAUL" Department of Cell Biology, Fraunhofer-lnstitute of Ioxirology and Aerosol Research, 3000 Hannover 6 I , Fcdcral Kcpublic of Germany Basic fibroblast growth factor (bFC;Fj and transforming growth factor-a (TCiFaj have been identified as potent hepatotrophic mitogens. bFGF and TGFa induce DNA synthesis in fetal and adult rat hepatocytes in primary culture and support fetal rat hcpatocyte multiplication in chemically defined medium. No additional exogenous growth or progression factors are required by the cells for traversing the cell cycle or for cell division. These mitogenic polypeptides, previously identified in various cell types including liver and endothelial cells, platelets, and macrophages may act locally in a paracrine mode in controlling hepatocyte multiplicalion in the liver during development and regeneration.

Basic fibroblast growth factor (bFGF) (Abraham et al., 1986) and transforming growth factor a (TGFa) (Marquardt et al., 1984) are angiogenic and are present during embryonal development and in several adult tissues. (Thomas and Gimenez-Gallego, 1986; Derynck, 1988). bFGF is related to acidic FGF (Thomas and Gimenez-Gallego, 1986) and is homologous to hsriKS3, int-2,FGF-5, and FGF-6 oncogenes (cf. Thomas, 1988, for review; Rifiin and Moscatelli, 1989; Marics et al., 1989). TGFa is related to epidermal growth factor (EGF) (Marquardt et al., 1984; Savage et al., 1972) and interacts with the EGF-receptor (Derynck, 1988). EGF stimulates DNA synthesis in hepatocytes in culture (Reid et al., 1988; Hoffmann et al., 1989 and citations therein) and in vivo following long-term administration to rats (Bucher et al., 1978). In adult rats, EGF blood levels seem to remain unchanged after partial hepatectomy, and EGF is not present in the liver (Rall et al., 1985), Kasselberg et al., 1 9 8 3 , suggesting that EGF is presumably not involved in controlling normal liver growth. Because EGF is not produced in rodents until after birth, (Popliker et al., 1987) TGFa presumably is the physiological ligand of the EGF receptor during development and early postnatal life (Derynck, 1988). We report here that TGFa or bFGF initiate DNA synthesis in cultured foetal and adult hepatocytes and support foetal hepatocyte multiplication in defined medium without the involvement of additional mitogens, and that TGFP, (Cheifetz et al., 1987) blocks G,iG, + S transition in growth-stimulated fetal hepatocytes.

MATERIALS AND METHODS Animals Wistar rats were obtained from Charles River Wiga, GmbH (Sulzfeld, FRG). C 1990 WILEY-LISS, INC.

Cell cultures and growth assays Pooled fetal livers of Wistar rats (day 19/20 of gestation) were minced and treated with collagenase (0.8 mgiml; Sigma 0130) on a magnetic stirrer a s described (Leffert and Paul, 1972). After the resulting cell SUSpension was filtered through sterile cheese cloth, hepatocytes were purified by centrifugation (5 min, 1,100 rpm), then at 2,000 rpm for 20 seconds. Cells were then suspended in arginine-free MX-82 medium (Hoffman e t al., 1989) with dialyzed newborn calf serum (5% viv; Gibco), seeded into 2 ml medium containing 0.4 mM ornithine and hydrocortisone (2 x 10 7M)in 3 cm tissue culture dishes (Nunc) a t 1 x lo5 cellsidish, and incubated in a humidified 10% C02/90% air incubator a t 37°C. After 2-3 hours the medium was removed, the cultures washed, and then incubated with serum-free medium. Hormones were added as indicated in the text. Mouse epidermal growth factor (EGF) and bovine basic fibroblast growth factor (bFGF) were obtained from Collaborative Research; recombinant IGF-I was from CIBA-Geigy (Easel, Switzerland). Bovine recombinant bFGF was purchased from Boehringer, human transforming growth factor-alpha (TGFa), synthetic, from Bachem (Easel, Switzerland), and bovine insulin from Sigma. TGFP, was supplied by R & D (Minneapolis, MN). Adult rat hepatocytes were obtained by in situ liver perfusion with collagenase a s described (Paul and Piasecki, 1984). Cells were seeded in culture medium with dialyzed serum as described above. For DNA synthesis initiation assays, 3H-thymidine (6 pM; 10 pCiidish) incorporation into TCA precipitable material was determined a s described (Hoffman et al., 1989). Autoradiographic analyses were performed Received June 26, 1989; accepted September 11, 1989. *To whom reprint requestsicorrespondence should be addressed.

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hours after the medium change the cells were incubated with either Fig. 1. Induction of DNA synthesis in foetal hepatocytes in primary culture by mouse EGF, human TGFu, bovine pituitary FGF, and bo- EGF, synthetic TGFa, recombinant bFGF (Rec), FGF (from bovine vine recombinant bFGF. Foetal rat hepatocytes were prepared as de- pituitary), recombinant insulin growth factor I (human), or with boscribed elsewhere (Hoffman et al., 1989). Cells were plated (1 x lo5 vine insulin at the indicated concentrations. Eight hours after induccells per 3 cm NUNC tissue culture dish) in 2 ml arginine-free MX-82 tion, r3H1-thymidine was added to the cultures for 40 hr (see A). The + 10% dialyzed newborn calf serum. After 2 hr the cultures were incubated cultures were washed with PBS, the cells removed with PBSiEDTA and added into 2 ml of 10% TCA. Radioactivity retained washed and incubated in arginine-free MX-82 medium containing ornithine (0.4 mM) hydrocortisone (2 X 10-7M). A Twenty-four on 2.4 cm Whatrnan GF/C glass fibre filters was determined in a hours after the medium change EGF (100ngiml) and insulin (10 pg/ Beckman scintillation counter. Induction of DNA synthesis in foetal ml) were added to the culture I.-( or they remained untreated (0-0). rat hepatocytes by IFG I(100 ngiml) resulted in 1,245 i 82 cpmidish Every 6 h cultures were pulsed for 6 h r with [3H1-thymidine (Amer- and induction by insulin (10pgiml) in 1,832 ? 107 cpmidish. Results sham) (6 pM, 10 pCi per dish). The cells were fixed with formalin are expressed as net cpmiculture over background ( = 1,000 cpm (3.7%in PBS) and processed for autoradiography as described (Hoff- 80). Experiments were conducted in triplicate dishes. Bars indicate ?SD. mann et al., 1989). Each point is the result of one triplicate experiments. The experimental error did not exceed ?8%. B, C: Twenty-four

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(Hoffman et al., 1989) using Kodak AR-10 autoradiographic stripping film. For growth assays, cells were removed from the dish with 0.05% trypsini0.01 M EDTA and counted in a Coulter Counter. The experimental error for cell number determinations in triplicate dishes did not exceed +lo%.

RESULTS Fetal and adult hepatocytes in primary cultures were prepared as indicated in Materials and Methods for standard DNA synthesis initiation assays in chemically defined arginine-free culture medium (Fig. 1;Ta-

151

BFGF & TGF-u ARE HEPATOTROPHIC MITOGENS IN VITRO

Fig. 2. Photomicrographs of autoradiographic analyses of fetal rat hepatocytes in primary cultures after incubation with [‘HI-thymidine in the presence of TGF-or (100 ngiml) (A) or bFGF (100 ngiml) (B). After 40 hr, cells were fixed and prepared for autoradiography as described in Materials and Methods. Phase contrast. x 325.

ble I). These culture conditions have previously been shown to maintain fetal rat hepatocyte proliferation and the expression of liver-specific genes (e.g., albumin, a-fetoprotein, tyrosine amino transferase) and the biosynthesis of arginine for at least 10 days (Hoffman et al., 1989). Mitogenic responses of quiescent hepatocytes t o EGF as determined by C3H1-thymidine incorporation into DNA were highest when cells were allowed to rest immediately after seeding for 1 day prior to induction (Fig. la). Autoradiographic analyses showed that quiescent hepatocytes were recruited t o enter S phase 612 h r following stimulation with EGF, reaching maximal levels of induction a t 18-24 hr. The response of hepatocytes to EGF is clearly dose-dependent (Fig. l b ) and similar to that of synthetic TGFa at levels of 0.217 nM. Results shown in Figure l c demonstrate that either purified bovine pituitary FGF or bovine recombinant bFGF was similarly active in inducing DNA synthesis in foetal hepatocytes at levels of 0.1-6 nM. Either recombinant insulin growth factor I (IGF I) (220 nM) or insulin (up to 1.7 pM) elicited weak mitogenic responses when added alone to the cultures and did

TABLE 1. Induction of DNA-synthesis in adult rat hepatocytes in primary culture bv growth factors Peutide none EGF

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cDmiculture 1,356 ? 103 7.151 2 2 -1 -1-4 >---

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Adult rat hepatmytes were obtained by liver perfusion as described Waul and Piasecki 1984). Cells were seeded (see legend of Fig. 1) and induced with either EGF (100 ng/ml), synthetic TGFa (100 ng/mlj or recombinant bFGF (100 ng/ mll. [3Hl-thymidine incorporation assays were conducted as indicated in Figure 1. The results are presented in terms of cpm (mean) per experiment using triplicate dishes ? S.D.

not act synergistically with either TGFa of bFGF in DNA initiation assays (unpublished observations). Also adult rat hepatocytes were responsive to the mitogenic stimulation by either TGFa or bFGF (Table I). Autoradiograms (Fig. 2) show that the induction of DNA synthesis by TGF-a or bFGF occurred in hepatocytes and not in non-parenchymal cells. When foetal hepatocytes were incubated for several days in the

HOFFMANN AND PAUI.

requiring additional mitogens and that they lead to the multiplication of fetal hepatocytes. These observations contrast with previous results which demonstrated that several cell types including fibroblasts (Pledger e t al., 1978) and lymphocytes (Neckers and Cossman, 1983) require distinct commitment and progression factors acting sequentially during G,/G, to effect G, + S transition. An early response of foetal hepatocytes to mitogenic stimulation by either TGFa or bFGF involves the transient expression of the competence gene c-fos in analogy to EGF-stimulated adult rat hepatocytes (Kruijer et al., 1986) (unpublished results). The observation that EGF is not produced in rodents until >2 weeks after birth led to the suggestion t h a t the physiological stimulus for EFG receptors during development is TGFa (Popliker et al., 1987). The results shown in Figure 1 suggest that both TGFa and bFGF, which are produced a t relatively high levels during development (Thomas and Gimenez-Gallego, 1986, Derynck, 1988), could be directly involved in the control of liver size in the developing animal (Mead and Fausto, 1989; Brenner et al., 1989). Furthermore, the observed responsiveness of adult hepatocytes to mitogenic stimulation by either TGFa or bFGF (Table I) suggests that these growth factors, which are present in macrophages or platelets (Baird et al., 1985; JosephSilverstein et al., 1988; Rappolee et al., 1988), might also be physiological regulators of liver growth in adult 10 DAYS CULTURE rats. Although humoral factors have been implicated in the control of liver regeneration after partial hepaFig. 3. Multiplication of foetal rat hepatocytes in primary culture in tectomy (Michalopoulos et al., 1984; Michalopoulos et response to growth factors. Foetal rat hepatocytes were obtained as al., 1982; Paul et al., 1972; Moolten and Bucher, 1967), described (Fig. 1).One portion of the hepatocytes was purified by differential centrifugation (Hoffman et al., 1989) before seeding. Pu- no hepatotrophic mitogens have a s yet been purified rified hepatocytes and conventional liver cell suspensions were seeded from blood of partially hepatectomized rats. Blood leva t 4.8 x lo4 cells per 3 cm dish in MX-82 medium + 10% dialyzed els of EGF seem not to change after partial hepatecnewborn calf serum. After 2 hours the medium was replaced by fresh tomy and EGF is not produced in the liver (Rall et al., medium MX-82 t ornithine + hydrocortisone 0plus either 1985; Kasselberg et al., 1985), suggesting that this mi(100 ngiml) or synthetic recombinant bFGF TGFu (100 ng/ml After 10 days the cells were trypsinized and counted togen is not directly involved in the control of liver in a Coulter counter. Bars indicate i SD. growth. However, bFGF, which lacks a classic signal peptide (Abraham et al., 1986), has been identified in endothelial cells (Vlodavsky et al., 1987) and also in presence of either bFGF or TGFa, the cells multiplied, adult liver tissue, from which it is presumably delivundergoing 1-2 rounds of division without the require- ered to adjacent target cells by leakage from damaged ment of additional growth factors (Fig. 3). hepatocytes (Ueno et al., 1986). Recent observations have indicated that type p The observations reported here also bear on questransforming growth factor (TGFP) (Popliker et al., tions related to tumorigenesis in the liver. Recently, 19871, which has been shown to be present in platelets, hepatoma cell lines were described which constitumacrophages, and in other cell types, is a negative reg- tively produce either bFGF (Klagsbrun et al., 1986) or ulator of adult hepatocyte proliferation by demonstrat- TGFa (Luetteke et al., 1988), suggesting a role of these ing that it inhibited DNA synthesis in EGF-stimulated mitogens in hepatoma angiogenesis as well as in autoadult r a t hepatocytes in culture (Braun et al., 1988, crine tumour growth. Furthermore, the findings that and citations therein) and in vivo in regenerating liver overproduction of either bFGF or TGFa as specified by after partial hepatectomy (Russell et al., 1988). The appropriate recombinant plasmids transforms cultured results of our studies (Fig. 4) demonstrate that in cells (Derynck, 1988; Neufeld et al., 1988; Rogelj et al., TGFa- or bFGF-stimulated fetal hepatocyte TGFP, in- 1988), and that FGF-related DNA sequences are oncohibits G,/G, + S transition in a dose-dependent man- genic upon transfection into NIH3T3 cells (ref. Thomas ner (>0.1 pM), blocking entry of stimulated cells into 1988), lend support to this notion. S-phase at a level of >4 pM. In contrast to TGFa or bFGF, which are released from macrophages or platelets as mitogenically active DISCUSSION peptides (Derynck, 1988; Neckers and Cossman, 1983; The results shown in this communication indicate Kruijer e t al., 1986), TFGp is released in a latent, bithat bFGF and TGFa are potent hepatotrophic mito- ologically inactive form and acquires its normal recepgens in rat hepatocytes in primary cultures. It is nota- tor-binding properties after activation in the circulable that each individual peptide induced DNA synthe- tion (Wakefield et al., 1988). Thus, after release, sis in quiescent fetal or adult r a t hepatocytes without biologically active stimulators and inhibitors of hepatic

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concentrations of TGFp, as indicated in the diagram. Eight hours after addition of the peptides, I3H1-thymidine was added to the cultures for 40 h r and TCA precipitable material determined (see legend to Fig. 1). Results are expressed in percent of maximal stimulation (without TGFp, = 1008).

growth may become available to liver tissue sequentially, exerting positive and negative signals in controlling liver cell proliferation. In summary, TFGru, bFGF, and presumably additional mitogens such a s platelet-derived hepatocyte growth factorb) (Paul and Piasecki, 1984) (e.g., HGF) (Nakamura et al., 1986) as well as TGFP, may participate in concert a s hepatotrophic factors in triggering and modulating liver growth during development and during regeneration in response t o either surgical or toxic liver injury.

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ACKNOWLEDGMENTS We thank Dr. G. Kay for helpful comments on the manuscript. This study forms part of a doctoral thesis (B.H., Dept. of Biology, University of Hamburg). We are grateful to Ms. G. Marchelleck for expert preparation of the manuscript. This work was supported by research grants from the Deutsche Forschungsgemeinschaft and the Bundesminister fiir Forschung und Technologie (Bonn) and by the Jung-Stiftung fur Wissenschaft und Forschung (Hamburg).

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growth factor synthesis by the kidney and other tissues, Nature, 313~228-231. Rappolee, D.A., Mark, D., Banda, M.J., and Werb, Z. (1988) Wound macrophages express TGFa and other growth factors in vivo: analysis by mRNA phenotyping. Science, 241:708-712. Reid, L.M., Abreu, SL., and Montgomery, K. (1988) Extracellular matrix and hormonal regulation of synthesis and abundance of mRNAs in cultured liver cells. In: The Liver: Biology and Pathology. I.M. Arias ed. Raven Press, New York, pp. 717-723. Rifkin, D.B., and Moscatelli, D. (1989) Recent development in the cell cycle biology of basic fibroblast growth factor. J. Cell. Biol., 109: 1-8. Rogelj, S., Weinberg, R.A., Fanning, P., and Klagsbrun, U. (1988) Basic fibroblast growth factor fused to a signal peptide transforms cells. Nature, 331r173-175. Russell, W.E., Coffey, R.J., Quellette, A.J., and Moses, H.L. (1988) Type P-transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proc. Natl. Acad. Sci. USA, 85.5126-5130. Savage, C.R., Inagami, T., and Cohen, S. (1972) The primary structure of epidermal growth factor. J. Biol. Chem., 247:7612-7617. Thomas, K.A. (1988) Transforming potential of fibroblast growth factor genes. Trends Biochem. Sci., 13:327-328. Thomas, K.A., and Gimenez-Gallego,G. (1986) Fibroblast growth factors: broad spectrum mitogens with potent mitogenic activity. Trends Biochem. Sci., 11.3144. Ueno, N., Baird, A,, Esch, F., Shimasaki, S., Ling, N., and Guillemin, R. (198ti) Purification and partial characterization of a mitogenic factor from bovine liver: structural homology with basic fibroblast growth factor. Regul. Pept., 16r135-145. Vlodavski, I., Folkman, J.,Sullivan, R., Fridman, R., Ishai-Michaeli, R., Sasse, J., and Klagsbrun, M. (1987) Endothelial cell-derived basic fibroblast growth factor: Synthesis and deposition into subendothelial extracellular matrix. Proc. Natl. Acad. Sci. USA, 84: 2292-2296. Wakefield. L.M.. Smith. D.M.. Flanders. K.C.. and Suorn. M.B. (1988) Latent transforming growth factor P from humanBlatelets J Biol Chem ,263 7646-7654