MOLECULAR CARCINOGENESIS 5:25-31(1992)
Expression of Transforming Growth Factor-Alpha in Regenerating Liver and During Hepatic Differentiation Ritva I? Evarts', Harushige Nakatsukasa, Elizabeth R. Marsden, Zongyi Hu, and Snorri 5. Thorgeirsson Laboratory of Experimental Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland Both t h e level of expression and cellular distribution of transcripts for transforming growth factor-alpha (TGF-a) were studied in adult rat liver after partial hepatectomy and during hepatic differentiation in fetal, neonatal, and adult livers by northern blot analysis and in situ hybridization. A marked increase in t h e expression o f TGF-a was observed in neonatal livers and in adult livers after partial hepatectomy and during hepatic regeneration following modification o f the Solt-Farber protocol. Quantitation of silver grains after in situ hybridization with a TGF-a riboprobe revealed a sixfold t o eightfold increase in fetal and neonatal hepatocytes. Moreover, the expression o f TGF-a in the liver 3 wk after birth was still fourfold higher than that o f the adult quiescent liver. Both proliferating oval cells and basophilic foci of hepatocytes generated by modification of the SoltFarber protocol were positive for TGF-a transcripts. The level o f TGF-a transcripts was sixfold higher in the basophilicfoci than in the surrounding liver. High concentrations o f TGF-a transcripts were observed in the oval cells that lined pseudoducts and in the transitional cells proliferating within the ducts. The combination of in situ hybridization and immunocytochemistry using cell-specific antibodies revealed the presence o f TGF-a transcripts in both oval cells and in perisinusoidal stellate cells. The observation that TGF-a transcripts were found b o t h in primitive liver epithelial cells and perisinusoidal stellate cells suggests that this growth factor, in addition to its mitogenic action, may also have other important functions in t h e liver. Key words: Liver, TGF-a, regeneration, differentiation INTRODUCTION Both extrahepatic-and intrahepatic-derivedgrowth factors are thought to be involved in the mitogenic stimulation of hepatocytes after partial hepatectomy. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) are the principalextrahepatic factors, while heparin-binding growth factor-I (HBGF-1) and transforming growth factoralpha (TGF-a) are the major intrahepatic growth factors. All these growth factors, with the exception of EGF, are significantly increased after partial hepatectomy [ 1-41, The nonparenchymal liver cells participate in the synthesis of HGF after major liver damage, such as that produced by treatment of animals with carbontetrachloride [41. The role of the mitogenic hepatic growth factors in the normal development and differentiation of the liver is still poorly understood. Of special interest are the intrahepatic growth factors, in particular TGF-a. Developmental expression of TGF-a mRNA has been studied in both rats and mice [5,6]. The transcripts were only observed between days 8 and 10 of fetal life. In contrast, immunologicalstudies in the developing rat revealed a significant amount of TGF-a in the liver a t birth, which then progressively decreased [7]. Increased expression of TGF-a is associated with neoplastictransformationof epithelial cells, and high levels of TGF-a expression have been found in a variety of tumors, especially in carcinomas [8,9]. In vitro transformation, by either retroviruses or chemicals, leads to increased expression of TGF-a [ 10-1 21. Overproductionof TGF-a in mice bearing a human TGF-a cDNA transgene 0 1992 WILEY-LISS,INC.
results in the appearance of uniform epithelial hyperplasia in several organs includingthe liver and in the late occurrence of hepatocellularcarcinomas 113,141. Other tissues preferentially affected by overproduction of TGF-a in transgenic mice include the pancreasand mammary glands [I 3-16]. The existence of a stem-cell compartment in adult rat liver has been demonstrated by several laboratories, including our own [ 17,181. The hepatic stem-cell compartment can be activated under conditionssuch as nutritional deficiency and during chemical hepatocarcinogenesis when lost liver mass can not be renewed from existing hepatocytes [ 191.The early stem cell-derived progeny, commonly referred to as oval cells, proliferatein close association with perisinusoidal stellate cells (It0 cells), and we have shown that these cells can differentiate into hepatocytes [ 17,181. This condition is similar to the early stages in liver development in which endodermal cells invade the septum transversum and differentiate into primitive hepatoblasts in close association with the mesenchymal cells 1201. In this study, we examined whether TGF-a expression was associated with growth and differentiation during both nor-
'Corresponding author: National Cancer Institute, Building 37, Room 3C28, Bethesda. MD 20892. Abbreviations: AAF, acetylaminofluorene; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; HGF, hepatocyte growth factor; HBGF-1, heparin-bindinggrowth factor-1; PBS, phosphate-bufferedsaline; TGF-a, transforming growth factoralpha.
26
EVARTS ETAL.
ma1 liver development and the activation of the hepatic stem cell compartment in adult liver. By using northern blot analysis and in situ hybridization together with immunocytochemistry, we are able to report here that this intrahepatic growth factor is important for the normal growth of primitive hepatic cells and may play an important role in the architecturalarrangement of differentiating cells both during fetal development and after lesions leading to the proliferation of oval cells and perisinusoidal stellate cells. MATERIALS AND METHODS Treatment of Animals
Fischer rats were used throughout the experiment. Animals were killed 24 h after partial hepatectomy. To produce proliferation of oval cells and perisinusoidal stellate cells, acetylaminofluorene (AAF) was administered by gavage (1.5 mg/d) for 4 d before and 4 d after partial hepatectomy. The animals were killed between 7 and 15 d after partial hepatectomy. Fetal and neonatal animals were killed at the times indicated in the figures. lmmunocytochernistry
A monoclonal antibody that recognizes both oval cells and bile duct cells was kindly provided by Dr. Harold Dunsford (The University of Texas Medical Branch, Galveston, TX). This was used to identify oval cells by the immunoperoxidase-staining method with a Vectastain ABC Elite kit (Vector Laboratories, St. Louis, MO). A monoclonal antibody against porcine desmin (Daco Corp., Santa Barbara, CA) was used to localize perisinusoidal stellate cells in the liver. Probes
Riboprobes of TGF-a for northern blot and in situ hybridization studies were produced by inserting a 1.4-kb fragment encoding the human TGF-a gene (kindly provided by Dr. Rik Derynk, Genentech Inc., South San Francisco, CA) into the multiple cloning site of pGEM-4Z. Northern blot hybridization was used to determine the antisense and sense orientation of the cDNA insert. 32P-and 35S-labeled riboprobes from Ncol (600 bp, antisense) and Nael (500 bp, sense) fragments were used for northern blot and in situ hybridization, respectively.
hybridized without dextran sulfate for 4 h. After two 30-min washes with 1 x SSC and with 30% formamide in 1 X SSC (final concentration) at 55"C, the slides were treated with 50 kg/mL RNase A for 30 min at 37°C. After an additional 1-h wash in 0.1 x SSC at room temperature, the immunocytochemical procedure was performed with pretreatment with hydrogen peroxide-methanol and then 1.5 kg/mL proteinase K for 20 min. The primary antibody was kept on the samples overnight at 4°C.The Vector kit directions were then followed. The slides were processed for autoradiography as previously described [231. Northern Blot Analysis
Poly (A)' RNA-selected mRNA from both fetal and adult livers was separated on 1YO agarose gels and transferred onto nitrocellulose. The blots were hybridized with 32P-labeled RNA probes a t 60°C overnight. After a high-stringency wash (0.1 YO SDS and 0.1 x SSC), the blots were exposed to Kodak XAR film. The stripped nitrocellulose filters were rehybridized with a 32P-labeledcDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal standard [24]. Automated Image Analysis
The Magiscan Image Analysis System (Joyce-Lobel, Gateshead, England) and a Nikon microscope equipped with a television videocamera were used to quantitate the areas covered by the silver grains. The number of silver grains on each sample was determined in five randomly selected fields of 0.04 mm2.The values obtained using the sense probe were subtracted from the values from corresponding samples probed with the antisense probe to correct for background. RESULTS Expression of TGF-a in Proliferating Hepatocytes
Rapidly proliferating hepatocytes can be divided into three groups: (1) fetal and neonatal hepatocytes; (2)hepatocytes immediately after partial hepatectomy; and (3) small basophilic hepatocytes that are derived from differentiating oval cells [ 181. Both in situ and northern hybridization revealed prominent expression of TGF-a. in both fetal and neonatal livers (Figures 1-3). Quantitation with image anal-
In Situ Hybridization
Serial frozen sections were fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7-8, and stored in 70% ethanol at 4°C until used. The slides were permeabilized for 10 min with 5 m M MgC12 in PBS followed by treatment with 0.2 M Tris, pH 7.4,containing 0.1 M glycine as described by Lawrence and Singer [2 I ] . After the permeabilization treatment, the slides were fixed in paraformaldehydefor 20 min, rinsed with PBS, and dehydrated in increasing concentrations of ethanol. Prehybridization and hybridization were performed at 42°C for 2 and 3 h, respectively. The compositions of the prehybridization and hybridization mixtures were described by Shivers et al. 1221. When a combination of in situ hybridization and immunocytochemistry was used, slides were
N I
PH
Time after birth
'ah 24h"Od 4d l w 2w 3wl
TG F- (Y
- 4.5kb
GAPDH
- 1.4kb
Figure 1. Northern blot hybridizationwith a 32P-labeledTGF-a riboprobe of poly(A)+ selected mRNA in normal (N) and 4 and 24 h after partial hepatectomy (PH), as well as at various time intervals after birth. A prominent increase in TGF-(r transcripts was observed, especially by day 4 after the birth. GAPDH was used as an internal standard.
TGF-a, HEPATIC PROLIFERATION, AND DIFFERENTIATION
I
T
L
1
3
27
4
5
6
7
3 8
Figure 2. Bar graph of the average number of silver grains per unit area of (1) adult liver; (2) 18-d fetal liver; (3) 4-d, (4) 7-d, and ( 5 ) 21-d-old animals; (6) adult liver 24 h after partial hepatectomy; (7) basophilic foci of AAF-treated animals 13-15 d after the operation; and (8) the hepatocytes surrounding the basophilic foci. Each bar graph representsfive fields (0.04 mm2)in the livers of three or four animals. The number of silver grains was normalized to the specificadivityof 5 x 1O8dpm/Fgof both antisense and sense probes. The number of grains obtained with the sense probe was subtracted from the number obtained with the antisense probe.
ysis demonstrated a sixfold to eightfold higher expression of TGF-a in the fetal and neonatal livers than in the control adult liver. This elevated expression was still evident 3 wk after birth and was fourfold higher than TGF-a in adult quiescent liver (Figure 2). A similar increase was observed 24 h after partial hepatectomy, when a majority of the cells undergo DNA synthesis (Figure 2). The combination of AAF treatment with partial hepatectomy prevented the normally observed proliferation of hepatocytes after partial hepatectomy, and the restoration of the liver mass was delayed. Instead, extensive proliferation of oval cells occurred, and later, rounded areas of small strongly basophilic hepatocytes derived from the differentiating primitive oval cells were seen. These cells play an important role in restoring the original liver mass and functions after partial hepatectomy of livers from AAF-treated rats [17,18]. In the very early stage of differentiation, these small basophilic hepatocytes show a weak staining for OV-6, an oval cell-positive antibody (Figure 4). The distribution of the silver grains for TGF-a was uneven in the earliest foci; the preexisting acidophilic hepatocytes lacked or had very few silver grains. A sixfold increase in the expression of TGF-a was observed in the basophilic foci compared with the surrounding liver, which showed expression similar to the untreated adult liver (Figures 2 and 5). TGF-a Expression in Oval and Transitional Cells TGF-a has been demonstrated to be present in the epithelial components in several normal tissues, whereas cells derived from mesenchyma express a low level of TGF-a [25]. Oval cells are of endodermal origin, whereas perisinusoidal
Figure 3. Expression of TGF-a in 18-d embryonic liver. (A) antisense probe; (B) sense probe. Note the lack of silver grains in the hemopoietic cells. Only a few silver grains are present where the sense TGF-a probe was used. Bars = 50 pm.
stellate cells, which coproliferate with oval cells, are of mesodermal origin. By using serial sections, the dense areas of OV-6-positive oval cells and desmin-positive perisinusoidal stellate cells could be overlayered when detected with appropriate antibodies (data not shown). Treatment of the animals by the modified Solt-Farber protocol produced a prominent increase in the expression of liver TGF-a (Figure 6). Cells in the areas of oval-cell proliferationwere positive for TGF-a transcripts (Figure 7). The combination of in situ hybridization and immunocytochemistry using a desmin antibody revealedthe presence of silver grains both in oval cells and in perisinusoidal stellate cells (Figure 8). A high dose of AAF combined with partial hepatectomy causes focal-cell necrosis. This results in the formation of pseudoducts that become lined either with basophilicoval cells, as shown in figure 9, or with metaplasticallytransformed cells. These empty luminal spaces can also become filled with transitional cells (Figure lo), which are W-6 positive (data not shown). Both the cells lining the pseudoducts and the transitional cells proliferating inside the ducts demonstrated a high expression of TGF-a (Figure 111.
DISCUSSION Increasedexpression of TGF-a is strongly associated with neoplastic development. It is present at high levels in a variety of human tumors as well as in tumors experimentally induced in animals [8,9]. TGF-a is also expressed in
28
EVARTS EJAL.
Figure4. lntraductal proliferation of OV-6-positiveoval cells 2 wk after partial hepatectomy of an AAF-treated animal. Note
the slight positive staining of small hepatocytesfor OV-6 (arrow). Bar = 101 pm.
both chemically and virally transformed cell lines [I 0-1 21. This increased expression of TGF-a in neoplasia is commonly related to the increased growth that is observed in the tumor cell population. Our data demonstrated that TGF-a was expressed a t high levels both in prenatal and postnatal hepatocytes, and was also re-expressed, as has been shown by others [261, in hepatocytes after partial hepatectomy. It was also expressed a t high levels in replicating oval cells and perisinusoidalstellate cells, as well as in basophilic small hepatocytes. These findings support the notion that TGF-a is involved in growth regulation of both immature and fully differentiated hepatocytes. The presence of TGF-a in nonproliferating cells, such as brain cells, suggests that this growth factor might also have physiological functions unrelated to cell replication [27].Asignificant amount of TGF-a has been found in the embryonic brain and lung,
N
PH
AAF
nwfl l d after PH '
Figure 5. Dark-field illustration of a basophilicfocus 11 d after partial hepatectomy of an AAF-treated animal. (A) There is dense accumulation of silver grains in the basophilic area. Bar = 202 pm. (5) High magnification of TGF-a silver grains in the basophilicarea (surrounded by arrows). Bar = 50 prn.
TG F- (Y
- 4.5kb
GAPDH
- 1.4kb
Figure 6. Northern blot hybridizationwith 32P-labeledTGF-(U riboprobe of normal liver (N) and liver 24 h after partial hepatectomy (PH). Livers from five AAF-treated animals 11 d after partial hepatectomy (AAF) revealed marked expression of TGF-a. GAPDH was used as an internal standard.
TGF-a, HEPATIC PROLIFERATION,AND DlFFERENTlATlON
Figure 7. (A) Small cells composed of both oval cells and It0 cells in the oval-cell area are both positive for TGF-a. (6)Only a few silver grains are present where the sense probe for TGF-a was used. Bars = 50 bm.
Figure 8. A combination of in situ hybridization for TGF-a and immunohistochemistry for desmin-positive perisinusoidal stellate cells revealed the presence of TGF-a transcripts both in
29
and this level remained constant, whereas in the liver, the highest concentration was observed by embryonic day 20 and then progressively decreased after birth [7].Our data agrees with these findings, demonstrating high levels of transcripts for TGF-a in both prenatal and postnatal liver. Among the first replicating cell populationsthat appear after activation of the hepatic stem cell compartment are the oval cells and perisinusoidal stellate cells [I 71. Stellate cells may control, to a significant extent, the fate of oval cells by both paracrine means and the disposition of extracellular matrix. It has been established that stellate cells are the main source of TGF-p, and their production of extracellular matrix proteins can lead to fibrosis [28].These cells may, therefore, create a microenvironment that either promotes or inhibits oval-cell differentiation. They can effectively isolate the ductular oval cells by synthesizing extracellular matrix proteins, which then leads to the formation of adenofibrosis, or they can synthesize matrix proteins that are important for the further differentiation of the oval cells. In this study, we demonstrated that both oval cells and perisinusoidal stellate cells produced TGF-a transcripts. This indicates that TGF-a may play an important part in the proliferation of both mesenchymal cells and primitive hepatic epithelial cells and that TGF-a possibly contributes to their migration into the liver acini. In vitro studies have demonstrated that both TGF-a and extracellular matrix components are required for kidney tubulogenesis [29]. Therefore, it is conceivable that these factors are also required for the architecturalarrangementof both embyronic and adultliver. High levels Of TGF-a were also observed in the intraductally proliferating oval
desmin-positive (arrows) and -negative cells. Note the presence of large pseudoducts lined with oval cells. Bar = 50 pm.
30
EVARTS ETAL.
Figure 9. Hematoxylin-and-eosinstaining of a liver 13 d after partial hepatectomy. Short white arrows point t o dying hepatocytes. The long white arrow points t o intestinal metaplasia, while the short black arrow points to a pseudoduct. The long black arrow points t o a small basophilic hepatocyte, and the open arrows point t o a transitional cell. Bar = 33 pm.
Figure 1 1 . (A) Expression o f TGF-a in proliferating intraductal OV-6-positive cells. (B) Corresponding duct probed with sense probe. Bars = 33 km.
Figure 10. lntraductal proliferation of oval cells. Black arrows point t o surrounding perisinusoidal stellate cells. Hematoxylinand-eosin staining 13 d after partial hepatectomy o f an AAFtreated animal. Bar = 33 km.
cells (Figure 1 l ) , which also remained positivefor the ovalcell antibody. Oval cells lining the pseudoducts that are derived from the spaces left by the dying hepatocytes were also strongly positive for TGF-a (Figure 8). Prolonged expression of TGF-a in transgenic mice leads to the hypertrophy of several organs, including the liver, and also results in neoplastic transformation 113-1 51. It is not known, however, whether TGF-a expression in the preneoplastic foci is elevated throughout their progression to hepatocellular carcinoma or whether this expression is restricted to the rapidly proliferating hepatocytes. In the hamster oral epithelium, TGF-a is abundantly expressed in the basal layer and increases in the hyperplastic lesions produced by carcinogen administration. However, transformation to carcinoma is not associated with further elevation of TGF-a [30]. Even though TGF-a is thought to play an important role in cellular proliferation and transformation, its role in the normal cellular physiology is poorly understood. However, its presence in preimplantation blastocysts [31] and in nonproliferating brain cells 1271seems to indicate other important functions for this polypeptide growth factor. Other liver-specificgrowth factors are involvedwith TGF-a
in the proliferation of the hepatocytes after partial hepatectomy. However, their role in the proliferation and differentiation of embryonic hepatocytes is entirely unknown. Our study suggests a prominent role for this cell-membrane protein during liver development and may also indicate other important functions in addition to its mitogenic action. Received June 1 1, 1991; revisedAugust 5,1991 ; accepted August 8, 1991.
REFERENCES 1. Kan M, Huang J. Mansson P-E, Yasumitsu H, Carr B, McKeehan WL. Heparin-bindinggrowth factor type 1 (acidic fibroblast growth factor): A potential biphasic autocrine and paracrine regulator of hepatocyte regeneration. Proc Natl Acad Sci USA86:7432-7436, 1989. 2. Nakamura T, Nishizawa T, Hagiya M, et al. Molecular cloning and expression of human hepatocyte growth factor. Nature 342:440443, 1989. 3. Zarnegar R, Muga S, Rahija R, Michalopoulos G. Tissue distribution of hepatopoietin-A: A heparin-binding polypeptide growth factor for hepatocytes. Proc Natl Acad Sci USA 87:1252-1256, 1990. 4. Kinoshita T, Tashiro K, Nakamura T. Marked increase of HGF mRNA in non-parenchymal liver cells of rats treated with hepatotoxins. Biochem Biophys Res Commun 165: 1229-1 234, 1989. 5. Lee DC, RochfordR, Todaro G, Villarreal LF! Developmental expression of rat transforming growth factor-a mRNA. Mol Cell Biol 5:3644-3646, 1985. 6. Wilcox 1. Derynck R. Developmental expression of transforming growth factors alpha and beta in mouse fetus. Mol Cell Biol B:3415-3422,1988. 7. Brown PI, Lam R, Laksmana 1, Fisher DA. Transforming growth factor alpha in developing rats. Am J Physiol259:€256-€260, 1990.
TGF-a, HEPATIC PROLIFERATION,AND DIFFERENTIATION
8 Derynck R, Goeddel DV, Ullrich A, et al. Synthesis of messenger
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12 13
14
15 16
17
18
RNAs for transforming growth factors a and p and the epidermal growth factor receptor by human tumors. Cancer Res 47:707-712, 1987. Luetteke NC, MichalopoulosGK, Teixido J, Gilmore R, Massague J, Lee DC. Characterization of high molecular weight transforming growth factor a produced by rat hepatocellularcarcinoma cells. Biochemistry 27:6487-6494, 1988. Raymond W, Lee DC, Grisham JW, Earp S . Regulation of transforming growth factor a messenger RNA expression in a chemically transformed rat hepatic epithelial cell line by phorbol esters and hormones. Cancer Res 49:3608-3612,1989. Hampton LL, Worland PJ, Thorgeirsson SS, Huggett AC. Expression of growth-related genes during tumor progression in v-raftransformed rat liver epithelial cells. Cancer Res 50:7460-7467, 1990. lgnotz FW,Kelly 6, Davis RJ, Massague J. Biologically active precursor for transforming growth factor type a, released by retrovirally transformed cells. Proc Natl Acad Sci USA 83:6307-6311, 1986. Sandgren EP. Luetteke NC, Palmiter RD, Brinster RL, Lee DC. Overexpressionof TGF-a in transgenic mice: Induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61 :1121-1 135, 1990. Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, Merlin0 GT. TGF-(r overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 61 :1137-1146, 1990. Matsui Y, Alter SA, Holt JT, Hogan ELM, Coffey RJ. Development of mammary hyperplasia and neoplasia in MTV-TGF-a transgenic mice. Cell61:1147-1155,1990. Cardiello F, McGeady ML, Kim N, et al. Transforming growth factor-a expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-rasprotooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-a complementary DNA leads to transformation. Cell Growth and Differentiation 1 :407-420, 1990. Evarts RP. Nakatsukasa H, Marsden ER, Hsia C-C, Dunsford HA, Thorgeirsson SS. Cellular and molecular changes in the early stages of chemical hepatocarcinogenesisin the rat. Cancer Res 50:34393444,1990. Evarts RR Nagy F: Marsden E, Thorgeirsson SS.A precursor-product relationshio exists between oval cells and heoatocvtes in rat liver. Carcinogenesis8: 1737-1 740, 1987. .
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19. Wilson JW, Leduc EH. Role of cholangioles in restoration of the liver of the mouse after dietary injury. J Pathol Bacteriol 76:441449, 1958. 20. Houssaint E. Differentiationof the mouse hepatic primordium: I. An analysis of tissue interactions in hepatocyte differentiation. Cell Differentiation9:269-279, 1980. 21. Lawrence JB, Singer RH. Quantitative analysis of in situ hybridization methods for the detection of actinogene expression. Nucleic Acids Res 13:1777-1799, 1985. 22. Shivers BD, Schachter BS, Pfaff DW. In situ hybridization for the study of geneexpressionin the brain. Methods Enzymol 124:497510,1986. 23. Evarts RP. Nagy P. Marsden E, Thorgeirsson SS. In situ hybridization studies on expression of albumin and a-fetoprotein during the early stage of neoplastic transformation in rat liver. Cancer Res47:5469-5475,1987. 24. Fort P, Marty L, Piechaczyk M, et al. Various rat tissues express only one major mRNA species from the glyceraldehyde-3-phosphate dehydrogenase multigenic family. Nucleic Acids Res 13: 1431-1442,1985, 25. Bauknecbt T, Kommos F, Wintzer 0, Walker R. TGF-a expression and cellular localization in nonmalignant and malignant tissues. J Cell Biochem Suppl 14E:68, 1990. 26. Mead JE, Fausto N. Transforming growth factor a may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc Natl Acad Sci USA 86: 1558-1 562,1989. 27. Wilcox JN, Derynck RJ. Localization of cells synthesizing transforming growth factor-alpha mRNA in the mouse brain. Neuroscience8:1901-1904, 1988. 28. Nakatsukasa H, Evarts RP, Hsia C-C. ThorgeirssonSS.Transforming growth factor-pl and type I procollagentranscriptsduring regeneration and earlyfibrosis of rat liver. Lab Invest 63: 171-180, 1990. 29. Taub M, Wang Y, Szczesny TM, Kleinman HK. Epidermal growth factor or transforming growth factor a is required for kidney tubulogenesis in matrigel cultures in serum-free medium. Proc Natl Acad Sci USA87:4002-4006, 1990. 30. Chang L-C, Chou MY, Chow P. et al. Detection of transforming growth factor-a messenger RNA in normal and chemically transformed hamster oral epithelium by in situ hybridization. Cancer Res 49:6700-6707,1989, 31. Rappolee DA, Brenner CA, Schulz R, Mark D, Werb Z. Developmental exoression of PDGF. TGF-a. and TGF-B aenes in oreirnplantationmouse embryos.Science241:182i-f825, 1988.
Expression of Transforming Growth Factor-Alpha in Regenerating Liver and During Hepatic Differentiation Ritva I? Evarts', Harushige Nakatsukasa, Elizabeth R. Marsden, Zongyi Hu, and Snorri 5. Thorgeirsson Laboratory of Experimental Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland Both t h e level of expression and cellular distribution of transcripts for transforming growth factor-alpha (TGF-a) were studied in adult rat liver after partial hepatectomy and during hepatic differentiation in fetal, neonatal, and adult livers by northern blot analysis and in situ hybridization. A marked increase in t h e expression o f TGF-a was observed in neonatal livers and in adult livers after partial hepatectomy and during hepatic regeneration following modification o f the Solt-Farber protocol. Quantitation of silver grains after in situ hybridization with a TGF-a riboprobe revealed a sixfold t o eightfold increase in fetal and neonatal hepatocytes. Moreover, the expression o f TGF-a in the liver 3 wk after birth was still fourfold higher than that o f the adult quiescent liver. Both proliferating oval cells and basophilic foci of hepatocytes generated by modification of the SoltFarber protocol were positive for TGF-a transcripts. The level o f TGF-a transcripts was sixfold higher in the basophilicfoci than in the surrounding liver. High concentrations o f TGF-a transcripts were observed in the oval cells that lined pseudoducts and in the transitional cells proliferating within the ducts. The combination of in situ hybridization and immunocytochemistry using cell-specific antibodies revealed the presence o f TGF-a transcripts in both oval cells and in perisinusoidal stellate cells. The observation that TGF-a transcripts were found b o t h in primitive liver epithelial cells and perisinusoidal stellate cells suggests that this growth factor, in addition to its mitogenic action, may also have other important functions in t h e liver. Key words: Liver, TGF-a, regeneration, differentiation INTRODUCTION Both extrahepatic-and intrahepatic-derivedgrowth factors are thought to be involved in the mitogenic stimulation of hepatocytes after partial hepatectomy. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) are the principalextrahepatic factors, while heparin-binding growth factor-I (HBGF-1) and transforming growth factoralpha (TGF-a) are the major intrahepatic growth factors. All these growth factors, with the exception of EGF, are significantly increased after partial hepatectomy [ 1-41, The nonparenchymal liver cells participate in the synthesis of HGF after major liver damage, such as that produced by treatment of animals with carbontetrachloride [41. The role of the mitogenic hepatic growth factors in the normal development and differentiation of the liver is still poorly understood. Of special interest are the intrahepatic growth factors, in particular TGF-a. Developmental expression of TGF-a mRNA has been studied in both rats and mice [5,6]. The transcripts were only observed between days 8 and 10 of fetal life. In contrast, immunologicalstudies in the developing rat revealed a significant amount of TGF-a in the liver a t birth, which then progressively decreased [7]. Increased expression of TGF-a is associated with neoplastictransformationof epithelial cells, and high levels of TGF-a expression have been found in a variety of tumors, especially in carcinomas [8,9]. In vitro transformation, by either retroviruses or chemicals, leads to increased expression of TGF-a [ 10-1 21. Overproductionof TGF-a in mice bearing a human TGF-a cDNA transgene 0 1992 WILEY-LISS,INC.
results in the appearance of uniform epithelial hyperplasia in several organs includingthe liver and in the late occurrence of hepatocellularcarcinomas 113,141. Other tissues preferentially affected by overproduction of TGF-a in transgenic mice include the pancreasand mammary glands [I 3-16]. The existence of a stem-cell compartment in adult rat liver has been demonstrated by several laboratories, including our own [ 17,181. The hepatic stem-cell compartment can be activated under conditionssuch as nutritional deficiency and during chemical hepatocarcinogenesis when lost liver mass can not be renewed from existing hepatocytes [ 191.The early stem cell-derived progeny, commonly referred to as oval cells, proliferatein close association with perisinusoidal stellate cells (It0 cells), and we have shown that these cells can differentiate into hepatocytes [ 17,181. This condition is similar to the early stages in liver development in which endodermal cells invade the septum transversum and differentiate into primitive hepatoblasts in close association with the mesenchymal cells 1201. In this study, we examined whether TGF-a expression was associated with growth and differentiation during both nor-
'Corresponding author: National Cancer Institute, Building 37, Room 3C28, Bethesda. MD 20892. Abbreviations: AAF, acetylaminofluorene; EGF, epidermal growth factor; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; HGF, hepatocyte growth factor; HBGF-1, heparin-bindinggrowth factor-1; PBS, phosphate-bufferedsaline; TGF-a, transforming growth factoralpha.
26
EVARTS ETAL.
ma1 liver development and the activation of the hepatic stem cell compartment in adult liver. By using northern blot analysis and in situ hybridization together with immunocytochemistry, we are able to report here that this intrahepatic growth factor is important for the normal growth of primitive hepatic cells and may play an important role in the architecturalarrangement of differentiating cells both during fetal development and after lesions leading to the proliferation of oval cells and perisinusoidal stellate cells. MATERIALS AND METHODS Treatment of Animals
Fischer rats were used throughout the experiment. Animals were killed 24 h after partial hepatectomy. To produce proliferation of oval cells and perisinusoidal stellate cells, acetylaminofluorene (AAF) was administered by gavage (1.5 mg/d) for 4 d before and 4 d after partial hepatectomy. The animals were killed between 7 and 15 d after partial hepatectomy. Fetal and neonatal animals were killed at the times indicated in the figures. lmmunocytochernistry
A monoclonal antibody that recognizes both oval cells and bile duct cells was kindly provided by Dr. Harold Dunsford (The University of Texas Medical Branch, Galveston, TX). This was used to identify oval cells by the immunoperoxidase-staining method with a Vectastain ABC Elite kit (Vector Laboratories, St. Louis, MO). A monoclonal antibody against porcine desmin (Daco Corp., Santa Barbara, CA) was used to localize perisinusoidal stellate cells in the liver. Probes
Riboprobes of TGF-a for northern blot and in situ hybridization studies were produced by inserting a 1.4-kb fragment encoding the human TGF-a gene (kindly provided by Dr. Rik Derynk, Genentech Inc., South San Francisco, CA) into the multiple cloning site of pGEM-4Z. Northern blot hybridization was used to determine the antisense and sense orientation of the cDNA insert. 32P-and 35S-labeled riboprobes from Ncol (600 bp, antisense) and Nael (500 bp, sense) fragments were used for northern blot and in situ hybridization, respectively.
hybridized without dextran sulfate for 4 h. After two 30-min washes with 1 x SSC and with 30% formamide in 1 X SSC (final concentration) at 55"C, the slides were treated with 50 kg/mL RNase A for 30 min at 37°C. After an additional 1-h wash in 0.1 x SSC at room temperature, the immunocytochemical procedure was performed with pretreatment with hydrogen peroxide-methanol and then 1.5 kg/mL proteinase K for 20 min. The primary antibody was kept on the samples overnight at 4°C.The Vector kit directions were then followed. The slides were processed for autoradiography as previously described [231. Northern Blot Analysis
Poly (A)' RNA-selected mRNA from both fetal and adult livers was separated on 1YO agarose gels and transferred onto nitrocellulose. The blots were hybridized with 32P-labeled RNA probes a t 60°C overnight. After a high-stringency wash (0.1 YO SDS and 0.1 x SSC), the blots were exposed to Kodak XAR film. The stripped nitrocellulose filters were rehybridized with a 32P-labeledcDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal standard [24]. Automated Image Analysis
The Magiscan Image Analysis System (Joyce-Lobel, Gateshead, England) and a Nikon microscope equipped with a television videocamera were used to quantitate the areas covered by the silver grains. The number of silver grains on each sample was determined in five randomly selected fields of 0.04 mm2.The values obtained using the sense probe were subtracted from the values from corresponding samples probed with the antisense probe to correct for background. RESULTS Expression of TGF-a in Proliferating Hepatocytes
Rapidly proliferating hepatocytes can be divided into three groups: (1) fetal and neonatal hepatocytes; (2)hepatocytes immediately after partial hepatectomy; and (3) small basophilic hepatocytes that are derived from differentiating oval cells [ 181. Both in situ and northern hybridization revealed prominent expression of TGF-a. in both fetal and neonatal livers (Figures 1-3). Quantitation with image anal-
In Situ Hybridization
Serial frozen sections were fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7-8, and stored in 70% ethanol at 4°C until used. The slides were permeabilized for 10 min with 5 m M MgC12 in PBS followed by treatment with 0.2 M Tris, pH 7.4,containing 0.1 M glycine as described by Lawrence and Singer [2 I ] . After the permeabilization treatment, the slides were fixed in paraformaldehydefor 20 min, rinsed with PBS, and dehydrated in increasing concentrations of ethanol. Prehybridization and hybridization were performed at 42°C for 2 and 3 h, respectively. The compositions of the prehybridization and hybridization mixtures were described by Shivers et al. 1221. When a combination of in situ hybridization and immunocytochemistry was used, slides were
N I
PH
Time after birth
'ah 24h"Od 4d l w 2w 3wl
TG F- (Y
- 4.5kb
GAPDH
- 1.4kb
Figure 1. Northern blot hybridizationwith a 32P-labeledTGF-a riboprobe of poly(A)+ selected mRNA in normal (N) and 4 and 24 h after partial hepatectomy (PH), as well as at various time intervals after birth. A prominent increase in TGF-(r transcripts was observed, especially by day 4 after the birth. GAPDH was used as an internal standard.
TGF-a, HEPATIC PROLIFERATION, AND DIFFERENTIATION
I
T
L
1
3
27
4
5
6
7
3 8
Figure 2. Bar graph of the average number of silver grains per unit area of (1) adult liver; (2) 18-d fetal liver; (3) 4-d, (4) 7-d, and ( 5 ) 21-d-old animals; (6) adult liver 24 h after partial hepatectomy; (7) basophilic foci of AAF-treated animals 13-15 d after the operation; and (8) the hepatocytes surrounding the basophilic foci. Each bar graph representsfive fields (0.04 mm2)in the livers of three or four animals. The number of silver grains was normalized to the specificadivityof 5 x 1O8dpm/Fgof both antisense and sense probes. The number of grains obtained with the sense probe was subtracted from the number obtained with the antisense probe.
ysis demonstrated a sixfold to eightfold higher expression of TGF-a in the fetal and neonatal livers than in the control adult liver. This elevated expression was still evident 3 wk after birth and was fourfold higher than TGF-a in adult quiescent liver (Figure 2). A similar increase was observed 24 h after partial hepatectomy, when a majority of the cells undergo DNA synthesis (Figure 2). The combination of AAF treatment with partial hepatectomy prevented the normally observed proliferation of hepatocytes after partial hepatectomy, and the restoration of the liver mass was delayed. Instead, extensive proliferation of oval cells occurred, and later, rounded areas of small strongly basophilic hepatocytes derived from the differentiating primitive oval cells were seen. These cells play an important role in restoring the original liver mass and functions after partial hepatectomy of livers from AAF-treated rats [17,18]. In the very early stage of differentiation, these small basophilic hepatocytes show a weak staining for OV-6, an oval cell-positive antibody (Figure 4). The distribution of the silver grains for TGF-a was uneven in the earliest foci; the preexisting acidophilic hepatocytes lacked or had very few silver grains. A sixfold increase in the expression of TGF-a was observed in the basophilic foci compared with the surrounding liver, which showed expression similar to the untreated adult liver (Figures 2 and 5). TGF-a Expression in Oval and Transitional Cells TGF-a has been demonstrated to be present in the epithelial components in several normal tissues, whereas cells derived from mesenchyma express a low level of TGF-a [25]. Oval cells are of endodermal origin, whereas perisinusoidal
Figure 3. Expression of TGF-a in 18-d embryonic liver. (A) antisense probe; (B) sense probe. Note the lack of silver grains in the hemopoietic cells. Only a few silver grains are present where the sense TGF-a probe was used. Bars = 50 pm.
stellate cells, which coproliferate with oval cells, are of mesodermal origin. By using serial sections, the dense areas of OV-6-positive oval cells and desmin-positive perisinusoidal stellate cells could be overlayered when detected with appropriate antibodies (data not shown). Treatment of the animals by the modified Solt-Farber protocol produced a prominent increase in the expression of liver TGF-a (Figure 6). Cells in the areas of oval-cell proliferationwere positive for TGF-a transcripts (Figure 7). The combination of in situ hybridization and immunocytochemistry using a desmin antibody revealedthe presence of silver grains both in oval cells and in perisinusoidal stellate cells (Figure 8). A high dose of AAF combined with partial hepatectomy causes focal-cell necrosis. This results in the formation of pseudoducts that become lined either with basophilicoval cells, as shown in figure 9, or with metaplasticallytransformed cells. These empty luminal spaces can also become filled with transitional cells (Figure lo), which are W-6 positive (data not shown). Both the cells lining the pseudoducts and the transitional cells proliferating inside the ducts demonstrated a high expression of TGF-a (Figure 111.
DISCUSSION Increasedexpression of TGF-a is strongly associated with neoplastic development. It is present at high levels in a variety of human tumors as well as in tumors experimentally induced in animals [8,9]. TGF-a is also expressed in
28
EVARTS EJAL.
Figure4. lntraductal proliferation of OV-6-positiveoval cells 2 wk after partial hepatectomy of an AAF-treated animal. Note
the slight positive staining of small hepatocytesfor OV-6 (arrow). Bar = 101 pm.
both chemically and virally transformed cell lines [I 0-1 21. This increased expression of TGF-a in neoplasia is commonly related to the increased growth that is observed in the tumor cell population. Our data demonstrated that TGF-a was expressed a t high levels both in prenatal and postnatal hepatocytes, and was also re-expressed, as has been shown by others [261, in hepatocytes after partial hepatectomy. It was also expressed a t high levels in replicating oval cells and perisinusoidalstellate cells, as well as in basophilic small hepatocytes. These findings support the notion that TGF-a is involved in growth regulation of both immature and fully differentiated hepatocytes. The presence of TGF-a in nonproliferating cells, such as brain cells, suggests that this growth factor might also have physiological functions unrelated to cell replication [27].Asignificant amount of TGF-a has been found in the embryonic brain and lung,
N
PH
AAF
nwfl l d after PH '
Figure 5. Dark-field illustration of a basophilicfocus 11 d after partial hepatectomy of an AAF-treated animal. (A) There is dense accumulation of silver grains in the basophilic area. Bar = 202 pm. (5) High magnification of TGF-a silver grains in the basophilicarea (surrounded by arrows). Bar = 50 prn.
TG F- (Y
- 4.5kb
GAPDH
- 1.4kb
Figure 6. Northern blot hybridizationwith 32P-labeledTGF-(U riboprobe of normal liver (N) and liver 24 h after partial hepatectomy (PH). Livers from five AAF-treated animals 11 d after partial hepatectomy (AAF) revealed marked expression of TGF-a. GAPDH was used as an internal standard.
TGF-a, HEPATIC PROLIFERATION,AND DlFFERENTlATlON
Figure 7. (A) Small cells composed of both oval cells and It0 cells in the oval-cell area are both positive for TGF-a. (6)Only a few silver grains are present where the sense probe for TGF-a was used. Bars = 50 bm.
Figure 8. A combination of in situ hybridization for TGF-a and immunohistochemistry for desmin-positive perisinusoidal stellate cells revealed the presence of TGF-a transcripts both in
29
and this level remained constant, whereas in the liver, the highest concentration was observed by embryonic day 20 and then progressively decreased after birth [7].Our data agrees with these findings, demonstrating high levels of transcripts for TGF-a in both prenatal and postnatal liver. Among the first replicating cell populationsthat appear after activation of the hepatic stem cell compartment are the oval cells and perisinusoidal stellate cells [I 71. Stellate cells may control, to a significant extent, the fate of oval cells by both paracrine means and the disposition of extracellular matrix. It has been established that stellate cells are the main source of TGF-p, and their production of extracellular matrix proteins can lead to fibrosis [28].These cells may, therefore, create a microenvironment that either promotes or inhibits oval-cell differentiation. They can effectively isolate the ductular oval cells by synthesizing extracellular matrix proteins, which then leads to the formation of adenofibrosis, or they can synthesize matrix proteins that are important for the further differentiation of the oval cells. In this study, we demonstrated that both oval cells and perisinusoidal stellate cells produced TGF-a transcripts. This indicates that TGF-a may play an important part in the proliferation of both mesenchymal cells and primitive hepatic epithelial cells and that TGF-a possibly contributes to their migration into the liver acini. In vitro studies have demonstrated that both TGF-a and extracellular matrix components are required for kidney tubulogenesis [29]. Therefore, it is conceivable that these factors are also required for the architecturalarrangementof both embyronic and adultliver. High levels Of TGF-a were also observed in the intraductally proliferating oval
desmin-positive (arrows) and -negative cells. Note the presence of large pseudoducts lined with oval cells. Bar = 50 pm.
30
EVARTS ETAL.
Figure 9. Hematoxylin-and-eosinstaining of a liver 13 d after partial hepatectomy. Short white arrows point t o dying hepatocytes. The long white arrow points t o intestinal metaplasia, while the short black arrow points to a pseudoduct. The long black arrow points t o a small basophilic hepatocyte, and the open arrows point t o a transitional cell. Bar = 33 pm.
Figure 1 1 . (A) Expression o f TGF-a in proliferating intraductal OV-6-positive cells. (B) Corresponding duct probed with sense probe. Bars = 33 km.
Figure 10. lntraductal proliferation of oval cells. Black arrows point t o surrounding perisinusoidal stellate cells. Hematoxylinand-eosin staining 13 d after partial hepatectomy o f an AAFtreated animal. Bar = 33 km.
cells (Figure 1 l ) , which also remained positivefor the ovalcell antibody. Oval cells lining the pseudoducts that are derived from the spaces left by the dying hepatocytes were also strongly positive for TGF-a (Figure 8). Prolonged expression of TGF-a in transgenic mice leads to the hypertrophy of several organs, including the liver, and also results in neoplastic transformation 113-1 51. It is not known, however, whether TGF-a expression in the preneoplastic foci is elevated throughout their progression to hepatocellular carcinoma or whether this expression is restricted to the rapidly proliferating hepatocytes. In the hamster oral epithelium, TGF-a is abundantly expressed in the basal layer and increases in the hyperplastic lesions produced by carcinogen administration. However, transformation to carcinoma is not associated with further elevation of TGF-a [30]. Even though TGF-a is thought to play an important role in cellular proliferation and transformation, its role in the normal cellular physiology is poorly understood. However, its presence in preimplantation blastocysts [31] and in nonproliferating brain cells 1271seems to indicate other important functions for this polypeptide growth factor. Other liver-specificgrowth factors are involvedwith TGF-a
in the proliferation of the hepatocytes after partial hepatectomy. However, their role in the proliferation and differentiation of embryonic hepatocytes is entirely unknown. Our study suggests a prominent role for this cell-membrane protein during liver development and may also indicate other important functions in addition to its mitogenic action. Received June 1 1, 1991; revisedAugust 5,1991 ; accepted August 8, 1991.
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TGF-a, HEPATIC PROLIFERATION,AND DIFFERENTIATION
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