Transforming Growth Factor-β (TGF-β) and EGF Promote Cord-like Structures That Indicate Terminal Differentiation of Fetal Hepatocytes in Primary Culture

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

242, 27–37 (1998)

EX984088

Transforming Growth Factor-b (TGF-b) and EGF Promote Cord-like Structures That Indicate Terminal Differentiation of Fetal Hepatocytes in Primary Culture ´ lvarez,‡ Ce´sar Roncero,* Sene´n Vilaro´,† Ara´nzazu Sa´nchez,* Roser Pagan,† Alberto M. A Manuel Benito,* and Isabel Fabregat*,1 * Departamento de Bioquı´mica y Biologı´a Molecular, Centro Mixto CSIC/UCM Facultad de Farmacia, and ‡Centro de Citometrı´a de Flujo y Microscopı´a Confocal UCM Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain; and †Departament de Biologia Cellular, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal, 645, 08028 Barcelona, Spain

INTRODUCTION When fetal hepatocytes were cultured in the presence of transforming growth factor-b (TGF-b1) and epidermal growth factor (EGF), some morphological changes were observed. Under these conditions, cells migrated, from typical clusters that hepatocytes adopt in culture, to form elongated, cord-like structures similar to the hepatic acinus organization. Immunocytochemical analysis of these cells revealed high levels of albumin and cytokeratin 18, phenotypic markers of parenchymal hepatocytes. Although some of the cells in the cord-like structures presented a cortical ring distribution of F-actin filaments, the cord also presented thick peripheral bundles and cells of the tips showed thin stress fibers oriented to the cell edges, typical of a migratory phenotype. In addition to these morphological effects, flow cytometric analysis of the cells revealed a larger size, granularity and intracellular lipid content (as a parameter related to liver metabolic function), in TGF-b 1 EGF-treated hepatocytes. Western blot analysis of the albumin levels revealed that both expression and secretion of albumin were increased in EGF 1 TGF-b-treated cells. Finally, all these changes were coincident with an enhancement in the DNA-binding activity for hepatocyte nuclear factors (HNF1, HNF3, and HNF4), as revealed in gel-shift experiments with nuclear extracts. We conclude that a cooperative action between TGF-b and EGF might modulate terminal maturation of fetal hepatocytes. © 1998 Academic Press Key Words: EGF; TGF-b; fetal hepatocytes; liver differentiation; liver plate formation.

The transforming growth factor-b (TGF-b) superfamily of cytokines comprises more than 25 related proteins from insects to man, with numerous biological activities, that include regulation of cell growth, differentiation, motility, organization, and death [for review, 1]. Normal proliferating liver in both regenerative growth and prenatal development contains increased TGF-b transcripts [2, 3]. TGF-b1 mediates inhibition of adult [4] and fetal [5] hepatocyte proliferation and it has been suggested that TGF-b may function as the effector of an inhibitory paracrine [2] or autocrine [6] loop that might regulate proliferation in fetal and regenerating liver. However, TGF-b may have functions in the liver other than the regulation of hepatic proliferation. Thus, TGF-b can regulate hepatic synthesis and secretion of a subset of acute-phase proteins, both directly and by modulating the effect of other cytokines [7, 8]. Moreover, targeted disruption of the TGF-b1 gene in mice results in an altered ultrastructural phenotype of hepatocytes [9] and a decrease in liver fatty acid binding protein [10], which is consistent with a role for TGF-b in normal development and regulation of physiological functions in hepatocytes. Fetal hepatocytes in primary culture are cells capable of carrying out both proliferation and differentiation processes simultaneously. We have shown that some growth factors, such as epidermal growth factor (EGF) or hepatocyte growth factor (HGF) are able to induce DNA replication in these cells [5, 11]. However, growth factors are also differentiation factors for fetal hepatocytes. In this way, EGF cooperates with some hormones, such as noradrenaline [12] or glucocorticoids [13], inducing the maximal expression of albumin and other liver-specific genes. TGF-b1 might regulate not only growth but also death and differentiation of fetal hepatocytes in primary culture. Thus, in these

1 To whom correspondence and reprint requests should be addressed at Dpto. de Bioquı´mica y Biologı´a Molecular, Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. Fax: 34-13941779. E-mail: [email protected].

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0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

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cells TGF-b induces apoptosis [14] that is suppressed by the presence of EGF, but not by HGF [15]. Furthermore, TGF-b in combination with EGF or HGF maintains the expression of some liver specific genes, such as albumin and a-fetoprotein, above control values, inducing the expression of some hepatocyte transcription factors, such as HNF1 and HNF4 [15, 16]. The liver parenchyma derives from endodermal cells of the foregut, which after mesodermal induction differentiates into hepatocytes. In the rat, clear signs of terminal maturation, such as cell polarization and hepatic plate formation, are not seen until the end of gestation. It has been suggested that changes in adhesion receptors, actin assembly, and intermediate junction formation generate a shearing force by contraction that eventually mediates separation and polarization of the hepatocytes that, finally, are organized into oneto two-cell-thick hepatic plates characteristic of adult, quiescent liver [17]. Hepatocytes originate from intermediate, bipotential cells expressing hepatocyte and ductal markers [18]. At 19.5 days of development in the rat, cells of extrahepatic ducts no longer express a-fetoprotein or albumin [19], whereas the expression of both genes increases in hepatocytes. Furthermore, the expression of cytokeratins also differs between ductal cells and hepatocytes: adult hepatocytes contain only CK8 and CK18, while duct cells have CK7 and CK19 in addition to CK8 and CK18 [20]. The present knowledge of transcriptional specific regulation in the liver led to the identification of three families of liver-enriched transcription factors that participate in the restricted expression of liver genes in the adult hepatocytes [for review, 21]. These families include the variant homeodomain containing family of HNF1 (hepatocyte nuclear factor 1), the HNF3 (hepatocyte nuclear factor 3) wingex helix family, and members of the nuclear receptor superfamily, where the orphan receptor HNF4 (hepatocyte nuclear factor 4) is included. In this work, we show that when fetal hepatocytes are cultured with TGF-b and EGF, cells migrate to form cord-like structures similar to the hepatic acinus organization. EGF 1 TGF-b-treated cells also show high levels of albumin and cytokeratin 18, changes in F-actin distribution, a higher lipid content ,and notably enhanced DNA-binding activity for HNF1, HNF3, and HNF4 transcription factors. All these results agree with a role for TGF-b and EGF as modulators of terminal differentiation throughout fetal liver development. MATERIALS AND METHODS Materials. TGF-b (human recombinant) was from Calbiochem (La Jolla, CA) and EGF (human recombinant) was kindly provided by Serono Laboratories (Madrid, Spain). Collagenase was from Boehringer (Mannheim, Germany). Fetal and neonatal calf serum and culture media were from Imperial Laboratories (Hampshire,

UK). Bovine serum albumin (BSA), Azide, Nile red, and TRITCphalloidin were obtained from Sigma Chemical Co. (St. Louis, MO). FITC-conjugated sheep anti-mouse immunoglobulins, monoclonal anti-vimentin (clone V9), and monoclonal anti-cytokeratin 19 were obtained from Boehringer. TRITC-conjugated goat anti-rabbit immunoglobulins were obtained from Dako Corp. (Santa Barbara, CA). Polyclonal anti-cytokeratin 18 was kindly provided by Professor O. Bachs and Dr. R. Bastos [22]. Polyclonal anti-rat albumin was from Nordic Immunological Laboratories (U.S.A.). Immunofluorescence mounting medium was from ICN Biomedicals Inc. (Costa Mesa, CA). Cell isolation and culture. Hepatocytes from 20-day-old fetal Wistar rats were isolated as previously described by collagenase disruption [23]. The cells (about 2.5 3 106 cells/fetal liver) were plated on plastic noncoated dishes in arginine-free Medium 199, supplemented with ornithine (200 mM), fetal calf serum (10%), penicillin (120 mg/ml) and streptomycin (100 mg/ml). Cells were incubated in 5% CO2, at 37°C for 4 h, allowing cell attachment to plates. Medium was changed at that time and replaced by one of the same composition except that 10% fetal calf serum was changed by 2% newborn bovine serum. After 18 –20 h, the medium was again replaced for one of identical composition but in the absence of serum. Two hours later, growth factors were added. Light microscopy. After incubation of cells in the absence or presence of the different factors, they were washed twice with PBS and fixed in Bouin solution (71% picric acid/24% formaldehyde/5% acetic) at room temperature for 30 min. Fixative was then removed and cells were extensively rinsed with 70% ethanol and finally with PBS. Cells were visualized in a Nikon TMD 108 microscope and photographed using Kodak T-MAX 100 films. Confocal microscopy studies. For immunofluorescence detection of albumin and intermediate filaments (vimentin and cytokeratins), the cells were washed twice with PBS, fixed in methanol (220°C) for 2 min and processed for double immunofluorescence [24]. As primary antibodies we used monoclonal anti-vimentin, monoclonal anti-cytokeratin 19, polyclonal anti-albumin, and polyclonal anti-cytokeratin 18. To visualize the primary antibody, we used the following labeled secondary antibodies: FITC-conjugated sheep anti-mouse immunoglobulins and TRITC-conjugated goat anti-rabbit immunoglobulins. Double immunofluorescence assays were always performed by applying a mixture of mouse and rabbit primary antibodies, followed by extensive washing and incubation with a mixture of FITC-conjugated anti-mouse and TRITC-conjugated goat anti-rabbit. Primary antibodies were applied for 1 h at 37°C, followed by 4 3 5-min washes in PBS, a 45-min incubation with fluorescent conjugated antibodies, and four final washes of 5 min each in PBS. For detection of actin filaments, cells were washed with PBS and fixed in 3% paraformaldehyde for 30 min at room temperature. Following this period, after washing the cells with PBS, they were stained with TRITC-conjugated phalloidin that binds F-actin filaments. This probe was reconstituted in 60% methanol/40% DMSO at a concentration of 100 mg/ml and diluted 1/500 before using with PBS, 0.1% BSA, 0.01% azide, pH 7.2. It was applied for 1 h at 37°C in a humidified atmosphere in the dark, followed by extensive washing with PBS. For visualization, sections of the plates were coverslipped using immunofluorescence medium and examined in a Leica TCS 4D (Leica Lasertechnik GmbH, Heidelberg, Germany) confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope using the same conditions as described by Pagan et al. [25]. Final images were obtained by a color high resolution video printer Mitsubishi CP2000E. Western blot. To detect cytokeratin proteins, after washing the cells in PBS, they were incubated with detergent buffer (10 mM Pipes, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA) for 30 min at 4°C. The buffer extract was then decanted and the remaining insoluble cytoskeletal residues were scraped off and resuspended in the same buffer. The high-salt buffer-insoluble material obtained as a pellet after centrifugation (2500g, 30 min, 4°C) was

EGF AND TGF-b ON TERMINAL LIVER DIFFERENTIATION washed briefly in PBS, pelleted once more (3000g, 10 min, 4°C) and resuspended in a lysis buffer (25 mM Hepes, 2.5 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, 5 mg/ml leupeptin). To detect intracellular albumin, cells were washed in PBS, scraped off, and lysed at 4°C in the same lysis buffer. All samples were sonicated 30 s at 1.5 mA and lysates were clarified by centrifugation at 12,000g for 10 min. Proteins were analyzed by SDS–PAGE and transferred by semidry transfer (Bio-Rad Labs, Richmond, CA) to Immobilon-P membranes (Millipore Corp., Bedford, MA). Membranes were blocked in TTBS (10 mM Tris/HCl, 150 mM NaCl, pH 7.5, 0.05% Tween 20) containing 5% non fat dried milk and incubated overnight in TTBS containing primary antibodies and 1% nonfat dried milk. Secondary antibodies were conjugated with peroxidase. The blot was developed with the ECL system (Amersham, Buckinghamshire, UK). Extracellular albumin was analyzed after recovering the culture medium from hepatocytes cultured in the absence or in the presence of factors and subsequent lyophilization of the medium. The lyophilized material was resuspended in PBS, and an identical aliquot of each sample was submitted to SDS–PAGE and analyzed as previously described for intracellular albumin and cytokeratin proteins. Flow cytometry studies. After incubation of the cells in the absence or in the presence of growth factors, they were washed with PBS and detached from dishes by addition of 0.25% trypsin, 0.02% EDTA. After 2–3 min, trypsinization was stopped with 10% fetal calf serum in culture medium. The lipids content per cell was analyzed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA) after staining cells with Nile red (10 mg/ml) for 10 min in the dark. Nile red was excited with 488 nm light of argon laser and emission recovery in PMT after selection through 575/24 BP filter. Nile red fluorescence increases in presence of polar lipids [26]. Mean fluorescence intensity (in arbitrary units) was recorded to statistical studies. Size and complexity (a parameter that measures surface integrity and organelle number, at the same time) of cells were quantified in arbitrary units for histograms of FSC (forward side scatter, size) and SSC (side scatter, complexity). Three experiments were performed in each case. Nuclear preparation and gel mobility shift assay. DNA-binding protein extracts from fetal hepatocytes were prepared as we previously described [14], starting with 5 3 106 cells. Cell suspension was resuspended in 10 mM Hepes–KOH, pH 7.9, at 4°C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM PMSF (Buffer A), allowed to swell on ice for 10 min, and then vortexed for 10 s. Samples were centrifuged at 14,000g for 10 s and the supernatant was discarded. The pellet was resuspended in cold Buffer C (20 mM Hepes–KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF, and 0.75 mg each of leupeptin, antipain, and aprotinin per milliliter) and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation at 14,000g for 2 min at 4°C and the supernatant fraction was stored at 280°C. Proteins were measured using the Bio-Rad protein reagent following the recommendations of the supplier. The gel mobility shift assay was performed essentially as described by Wen and Locker [27]. The following synthetic oligonucleotides were prepared using an oligonucleotide synthesizer (Pharmacia Biotech Inc.) as follows: HNF1, corresponding to proximal HNF1 motif, nucleotides 268 to 245 of a-fetoprotein promoter [28], tcgaACTGAAGGTTACTAGTTAACAGAC and tcgaGTCTGTTAACTAGTAACCTTCAGT; HNF3, corresponding to HNF3 site from 2111 to 285 of the transthyretin promoter [29], cGTTGACTAAGTCAATAATCAGAATCAG and aacGCTGATTCTGATTATTGAC-TTAGTC; and HNF4, corresponding to HNF4 site from 287 to 266 bp of the human apolipoprotein CIII (Apo CIII) [30], agctGCAGGTGACCTTTGCCCAGCGC and agctGCGCTGGGCAAAGGTCACCTGC. Finally, the double-stranded oligonucleotide used as AP-1 probe was composed of the sequence AGCTTGATGAGTCAGCCG and GATCCGGCTGACTCATCA. Labeling was performed by using Klenow polymerase and a-32P-labeled deoxyadenosine triphosphate. The

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binding reaction mixture contained 0.5 ng of doubled-stranded oligonucleotide probe, 2 mg of poly(dI– dC) z poly(dI– dC) and 5 mg protein in Buffer C supplemented with 35 mM MgCl2. After a 20-min incubation at 4°C, the mixture was applied to a 6% polyacrylamide gel. Gels were run at 0.8 V/cm2 in 0.53 Tris– borate-EDTA running buffer for 2–3 h, at room temperature. The dried gel was then autoradiographed.

RESULTS

Changes in Morphology, Cell Organization and Cytoskeleton Distribution Induced by EGF and TGF-b in Fetal Hepatocytes in Primary Culture Hepatocytes from livers of 20-day-old fetuses were plated and maintained in the presence of serum for 24 h (10% fetal calf serum during the first 4 h, 2% newborn bovine serum the next 20 h). Then, cells were cultured in serum-free medium. Two hours later, EGF (20 ng/ml) or TGF-b (0.5 ng/ml) or both factors together were added to the culture medium. Since these cells respond to EGF increasing DNA synthesis and division [5], 24 h later the number of cells was higher in the EGF-treated dishes (Fig. 1). When both EGF and TGF-b were present, TGF-b blocked proliferation induced by EGF [16] and the cell number did not increase (Fig. 1). The attached cells in control (no addition), EGF- or TGF-b-treated dishes were organized in typical clusters of polygonal cells (Fig. 1). Some apparent morphological changes and different organization of the clusters were observed by phase contrast analysis on different fields on the dish after addition of these factors. In control (serum-free medium without any addition) and in TGF-b-supplemented cultures, small clusters of hepatocytes were observed. Some fibroblast like cells were found in empty spaces of the culture dish. In EGF- supplemented cultures, hepatocyte number increased and clusters were larger than under the other conditions. However, the most striking change in morphology and organization of the cells was observed when both factors, EGF and TGF-b, were added simultaneously to the culture medium. Regardless, little clusters of epithelial cells were also observed under this condition; hepatocytes were organized in elongated, cord-like structures (Fig. 1). These structures were often formed by a row of hepatocytes of one or two cells of thickness. A dose–response analysis for both EGF and TGF-b revealed that the concentrations necessary for these morphological changes were identical to those required for the mitogenic/survival effect in the case of EGF [15] and the growth inhibitory effect in the case of TGF-b [16] (data not shown). To characterize phenotypically the cells in these structures, immunolocalization studies were performed by using markers that distinguish hepatocytes from bile duct cells in adult rat liver, such as albumin, CK18, and CK19. Since we have previously reported

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FIG. 1. Phase contrast microscopy of fetal hepatocytes cultured in the presence of EGF and/or TGF-b. Fetal hepatocytes, isolated and plated as described under Materials and Methods, were maintained in the presence of serum for 24 h. Then, the medium was replaced with serum-free medium. Two hours later, some dishes were supplemented with EGF (20 ng/ml) or TGF-b (0.5 ng/ml) or both factors together (EGF 1 TGF-b) or without any external growth factor (control). Twenty-four hours later, cultures were analyzed by phase contrast microscopy and micrographs were obtained. Note the cord-like organization of the cells treated with EGF 1 TGF-b (bottom right). The panels are representative from more than 10 different cultures. Bar, 50 mm.

that hepatocytes from neonatal rat livers in culture may present coexpression of vimentin and cytokeratin proteins (as a consequence of an epithelial–mesenchyma transition, [24, 31]), we decided to also analyze the presence of vimentin in these fetal hepatocyte cultures. Results comparing control (nontreated) and EGF 1 TGF-b-treated cells are presented in Fig. 2. Double immunofluorescence performed with antibodies against albumin and vimentin revealed that all the cells present in these cord-like structures contained high levels of albumin. Vimentin-positive cells were of fibroblastic appearance and were mainly located in the spaces between clusters, these cells being completely absent in the cord-like structures. Double immunofluorescence performed with anti-CK18 and anti-CK19 showed that in control cells we could find double CK18/ CK19-positive cells. In contrast, cells present in these morphological structures, induced by the combination

of EGF and TGF-b, were only CK18 positive. All these results indicate that the phenotype of the cells organized in these cord-like structures are differentiated hepatocytes. We wanted to quantify the CK18/19 contents after incubation of the fetal hepatocytes in the presence of each one of the factors separately. As it is shown in Fig. 3, Western blot analysis of the CK18 levels revealed that EGF- and EGF 1 TGF-b-treated hepatocytes presented higher levels of this protein, whereas TGF-b treatment did not modify the expression of this protein. A similar response was observed for CK19 expression, although in this case the changes in the protein levels were lower. Thus, EGF alone is able to modify the expression of intermediate filaments in fetal hepatocytes in primary culture. Actin assembly and intermediate junction formation may cause plate formation in the liver [17]. Accordingly, we decided to study the effect of EGF

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FIG. 2. Immunofluorescence detection of albumin, vimentin, and cytokeratin 18 and 19 of fetal hepatocytes cultured in presence of EGF and TGF-b. Fetal rat liver cells were obtained and treated with EGF and TGF-b (EGF 1 TGF-b) or without (control) as in Fig. 1. Twenty-four hours later, cells were fixed and double immunostained with rabbit anti-albumin and mouse anti-vimentin (ALB-VIM) or rabbit anticytokeratin 18 and mouse anti-cytokeratin 19 (CK19-CK18). Fluorescent secondary antibodies were TRITC-conjugated goat anti-rabbit immunoglobulins (in red, to visualize albumin and CK 18) and FITC-conjugated goat anti-mouse immunoglobulins (in green, to visualize vimentin and CK 19). A three-dimensional projection of six horizontal sections (xy, 0.31 mm; step size, 0.25 mm) was obtained by a confocal microscope. Panels show an overlay of the fluorescence signal from the two channels (red and green). The yellow indicates colocalization between FITC and TRITC fluorescence. Bar, 30 mm.

and TGF-b on actin filament distribution. Laser confocal microscopy observations allowed us to visualize the distribution of F-actin in cells stained with rhodamine–phalloidin (Fig. 4). In nontreated cells two distribution patterns of F-actin could be observed: hepatocytes in the middle of the clusters presented a ring-like F-actin distribution, which is typical of ep-

ithelial cells that organize actin bundles in areas of cell to cell contact. In contrast, cells at the periphery of the clusters (Fig. 4, arrowheads) did not present these cortical bundles but showed some stress fibers oriented to the cell margins. Ring-like, cortical actin bundles were more pronounced in EGF- supplemented cultures, suggesting that EGF stimulated

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FIG. 3. Western blot analysis for cytokeratins 18 (CK 18) and 19 (CK 19) in fetal hepatocytes cultured in presence of EGF and/or TGF-b. Fetal hepatocytes were cultured in the absence (C) or presence of 20 ng/ml EGF (E), 0.5 ng/ml TGF-b (T), or the combination of both factors (E 1 T) and processed for the extraction of the cytoskeleton after 24 h of treatment. 10 mg extract was submitted to SDS– polyacrylamide gel electrophoresis and Western blotting using a polyclonal antibody against CK 18 and a monoclonal antibody against CK 19, as described under Materials and Methods. A representative blot of each protein is shown (top) with the densitometric analysis corresponding to the mean 6 SEM of three independent experiments (bottom). In this last case, results are expressed as the percentage of the control value.

intercellular adhesion. The thin-stress fiber formation appeared only in the lamellipodial projections, without apparent differences with respect to the control pattern. In contrast, in TGF-b-supplemented cultures, the hepatocytes presented abundant stress fibers organization of the F-actin, which were oriented to the cell edges. The ring-like cortical F-actin distribution was not observed. When both EGF and TGF-b were present, the F-actin filaments in the hepatocytes of the cord-like structures showed a different distribution. Some cells showed a cortical ring of actin bundles, suggestive that they are in cell to cell contact; however, most of the cells of the tips in the cords showed thin stress fibers oriented to the cell edges. The cord also presented thick peripheral bundles. All together seemed to indicate that the elongated structures induced by TGF-b and EGF have the capacity to migrate. Fetal Hepatocytes Incubated in the Presence of EGF 1 TGF-b Presented Larger Size, Increased Granularity, and Enhanced Lipid and Albumin Content To address if the morphological changes induced by EGF and TGF-b could be also coincident with a higher maturation degree of the fetal hepatocytes, we performed a flow cytometric analysis of the cell size and granularity. Increased cytoplasmic granularity is related to maturation of the endoplasmic reticulum, Golgi apparatus, lysosomes, and mitochondria [32]. The results are presented in Fig. 5. Fetal hepatocytes incubated with EGF 1 TGF-b showed more cells with

a larger size and/or a higher granularity, both parameters related to a higher maturation of the hepatocytes [33]. These results were confirmed by microscopy (not shown results). We could calculate changes in the sizes of the cells that varied from about 245 mm2 in nontreated cells up to 290 mm2 in EGF 1 TGF-b treated hepatocytes. These changes were not observed when hepatocytes were incubated only in the presence of EGF or TGF-b (data not shown). Adult liver plays an essential role in the metabolism of lipids and lipoproteins. For this, lipid synthesis and secretion have been previously related to hepatic-differentiated function [34, 35]. We decided to analyze the intracellular lipid content by staining cells with Nile red. FACScan flow cytometric analysis revealed that, after 24 h, EGF 1 TGF-b-treated hepatocytes presented a significantly higher fluorescence, i.e., an increased intracellular lipid content, with respect to control (nontreated) cells (Fig. 5), that was not observed with each one of the factors separately (data not shown). One of the specialized functions of the liver is the production and secretion of albumin. Western blot analysis of the intracellular content of albumin revealed that when fetal hepatocytes were incubated in the presence of EGF and TGF-b, a significant increase in the albumin levels was observed (Fig. 6). When we analyzed the extracellular albumin levels, they were also increased in EGF 1 TGF-b-treated cells (Fig. 6). Cycloheximide completely inhibited the cell response to the factors in terms of both synthesis and release of albumin. The use of cycloheximide has been previously used by other authors to demonstrate that the albumin is synthesized by the cells and is not an artifact of culture, such as protein being released from dead cells or carried over from fetal serum at the time of plating [36]. All these results together suggest that, in addition to the morphological changes described above, EGF 1 TGF-b treatment also induces a functional maturation of fetal hepatocytes. Fetal Hepatocytes Incubated in the Presence of EGF 1 TGF-b Present Enhanced DNA-Binding Activity for Some Liver-Enriched Transcription Factors The maturation processes showed in previous sections in fetal hepatocytes in culture with EGF and TGF-b must be a reflection of changes in the level of liver-enriched transcription factors activity related to differentiation of hepatocytes, such as HNF-1, HNF-3, and HNF-4. To analyze this, after different times of treatment, fetal hepatocyte nuclear extracts were performed, and DNA-binding activity for the different factors was evaluated in gel-shift experiments with the specific oligonucleotide. Results are

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FIG. 4. Actin filament organization of fetal hepatocytes cultured in the presence of EGF and/or TGF-b. Fetal rat liver cells were processed as indicated in Fig. 1. Twenty-four hours after treatment with or without growth factors (control, EGF, TGF-b, and EGF 1 TGF-b) cells were fixed and actin filaments were stained with rhodamine-conjugated phalloidin. A three-dimensional projection of six horizontal sections (xy, 0.31 mm; step size, 0.25 mm) was obtained by confocal microscope. Arrows point to ring-like cortical actin filaments. Arrowheads indicate stress fibers. The right arrowhead in the EGF 1 TGF-b panel is also pointing to a peripheral bundle. Bar, 10 mm.

presented in Fig. 7. As can be observed, HNF1 and HNF4 DNA-binding activities were almost absent in the fetal hepatocytes at the beginning of the culture. Cells treated in the presence of both EGF and TGF-b showed an increase in their DNA-binding activity, being maximal after 48 h (Fig. 7). Nuclear extracts from cells at the beginning of the culture showed a relevant HNF3 DNA-binding activity that clearly decreased in control and EGF- and TGF-b-treated cells. However, hepatocytes incubated for 48 h in the presence of both EGF and TGF-b always showed the maximal HNF3 DNA-binding activity. To compare with a liver nonspecific transcription factor, we evaluated the DNA-binding activity of AP-1. Results shown in Fig. 7 indicate that the activity of this factor decreased during culture in the absence of serum, although, as it could be expected, it was always higher when EGF was present (Fig. 5). These results suggest that the cells incubated with both EGF and TGF-b also present a significant activation of the nuclear machinery implied in the transcription of liver-specific genes.

DISCUSSION

The processes of terminal differentiation are generally associated with growth arrest of differentiating cells and the increase in the expression of specialized functions. Thus, a growth inhibitor, such as TGF-b, might be potentially involved in the terminal maturation of the fetal hepatocyte. The formation of parenchymal organs during embryonic development involves a complex series of morphogenetic processes that ultimately result in the construction of the organized multicellular structure. During early embryogenesis, in poorly differentiated carcinomas and in proliferating liver parenchyma during regeneration, hepatocytes are either completely or partially nonpolarized. Perinatally, during the postreplicative phase of regeneration and in neoplastic nodules, hepatocytes assemble around lumena to form acinar, duct-like structures. Finally, the parenchyma is organized into one-cellthick hepatic plates characteristic of adult, quiescent liver [for review, 17]. Much is known about the factors that control this process.

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FIG. 5. Effect of EGF 1 TGF-b on cell size, granularity, and intracellular lipid content by FACScan analysis. (Right) Biparametric analysis of fetal rat hepatocytes presented as SSC (side scatter), a measure of cytoplasmic complexity or granularity vs size (forward light scatter). (A) Control, nontreated cells; (B) Cells incubated for 24 h in the presence of 20 ng/ml EGF and 0.5 ng/ml TGF-b. Arrows show the peak for small (on the left) and large (on the right) size. The oval circle marks the area for cells with the higher size and complexity. (Left) Quantitative analysis of the mean for fetal hepatocyte size, granularity, and intracellular lipid content in cells in the absence (C) or in the presence for 24 h of EGF 1 TGF-b (E 1 T). To quantify the intracellular lipid content, cells were preincubated for 20 –30 min with Nile red, as described under Materials and Methods, before FACScan analysis. The mean 6 SEM from three independent experiments is shown.

In this work, we show that TGF-b and EGF promote changes in the morphology and organization of fetal hepatocytes in primary culture that appear organized in elongated, cord-like structures. We propose that these morphological changes may be related to liverplate formation based on the next observations: (i) The

FIG. 6. Analysis of the intracellular and extracellular albumin levels after incubation of the fetal hepatocytes in presence of EGF and/or TGF-b. Cells were cultured for 24 h in the absence (C) or in the presence of 20 ng/ml EGF, 0.5 ng/ml TGF-b, or the combination of both factors (EGF 1 TGF-b), with or without 0.5 mg/ml cycloheximide (CHX). After this period, medium (for detection of extracellular albumin) or cells (for detection of intracellular albumin) were collected and processed for Western blot analysis, as described under Materials and Methods. A representative blot of three independent experiments is shown.

cells are organized in one- to two-cell-thick structures (Fig. 1); (ii) immunocytochemical characterization of the cells in these structures shows that they express the characteristic markers of parenchymal hepatocytes, i.e., high levels of albumin and cytokeratin 18 (Fig. 2); and (iii) although some of the cells present in these elongated structures showed a cortical ring of F-actin bundles (Fig. 4, arrows), resembling the cytoskeleton organization observed in the adult liver [37], the cord also presents thick peripherical bundles and cells of the tips in the cords show thin stress fibers oriented to the cell edges (Fig. 4, arrowheads). The observation that alterations in actin microfilament assembly occur in local regions of the leading edge of migrating cells has led to the concept that actin polymerization events act as a driving force for cell extension during locomotion. In accordance with this, the cord-like structures, induced by TGF-b and EGF, could be dynamically multicellular structures, composed by differentiated hepatocytes that have the capacity to migrate. The morphological changes observed in EGF 1 TGF-b treated hepatocytes are coincident with an enhancement in cell size, granularity, and intracellular lipid content (Fig. 5). It has been previously described that with maturation, cell granularity (a flow cytomet-

EGF AND TGF-b ON TERMINAL LIVER DIFFERENTIATION

FIG. 7. Analysis of DNA-binding activity of liver transcription factors in fetal hepatocytes: regulatory role of EGF and/or TGF-b. Cells were collected before (to) or after 48 h in the absence (C) or in the presence of: 20 ng/ml EGF (E), 0.5 ng/ml TGF-b (T), or both (E 1 T) and nuclear extracts were prepared as described under Materials and Methods. Five micrograms of protein was incubated for 20 min at 4°C with 0.5 ng of 32P-labeled doubled-stranded oligonucleotide containing the specific responsive elements for HNF1, HNF3, HNF4, and AP-1. After this time, the mixture was electrophoresed on a 6% polyacrylamide gel under nondenaturing conditions. (Left) The autoradiograph of a representative experiment out of three is shown. (Right) Relative DNA-binding activity has been calculated after densitometric analysis of the bands and is expressed as percentage (%) with respect to the EGF 1 TGF-b treatment. The mean 6 SEM of three independent experiments is shown.

ric parameter termed 90° light scatter, or side scatter, composed primarily for light reflected by internal structures or membrane undulations) and autofluorescence exponentially increased from fetal liver to suckling and adult liver [33]. The rates of lipogenesis in the liver are low in the last 2 days of gestation in the rat and just after birth [38]. However, fetal hepatocytes in primary culture may respond to hormones that induce the metabolic maturation of the liver [23] or growth factors that are also differentiating factors, such as hepatocyte growth factor [34], increasing the rate of lipogenesis. In accordance with this, the increase in size, granularity, and intracellular lipids observed in EGF 1 TGF-b-treated hepatocytes indicate that these factors might induce a metabolic maturation of these cells. The majority of the proteins for the specialized functions of the adult liver, including production of plasma proteins and regulation of carbohydrate, lipid, and urea metabolism, are produced exclusively by hepato-

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cytes and their synthesis is regulated primarily at the level of transcription initiation [39]. Although a number of liver-enriched transcription factors are not exclusive of the mature hepatocytes, expression of HNF4 is first observed when the progenitors differentiate morphologically and functionally into hepatocytes [40]. We show here that fetal hepatocytes in EGF 1 TGFb-treated dishes showed an enhancement in albumin production and secretion, that it is dependent on protein synthesis (Fig. 6), and that it correlates with an increase in the DNA-binding activity of HNF1, HNF3, and HNF4 transcription factors (Fig. 7). Taken together, these results led us to propose TGF-b and EGF as important factors responsible for the terminal maturation of fetal hepatocytes. This agrees with the findings that targeted disruption of the TGF-b1 gene in mice results in an altered phenotype of adult hepatocytes [9] and a decrease in liver fatty acid binding protein [10], suggesting a role for TGF-b in regulation of physiological functions and terminal differentiation of hepatocytes. Previous results obtained in our laboratory had shown that growth factors are also differentiation factors for fetal hepatocytes. Thus, EGF may act with some hormones, such as noradrenaline [5] or glucocorticoids [13], inducing the maximal expression of albumin and other liver-specific genes. Recently, we found that EGF or HGF in the presence of TGF-b maintains the liver specific gene expression of fetal hepatocytes [16]. However, among all the combinations described above, only EGF 1 TGF-b has been able to induce significant morphological changes related to liver-plate formation in fetal hepatocytes. Although it is the first time that a role for EGF and TGF-b in final morphogenic and functional differentiation of the liver is proposed, there is some previous evidence implicating growth factors in tubulogenic or branching morphogenetic activities in epithelial cells. Thus, Barros et al. [41] have described the induction of branching tubular structures in some kidney epithelial cell lines, when cultured not only with HGF, that has well-known morphogen properties, but also with TGF-a or EGF. Furthermore, it is well established that TGF-b1 and other members of this superfamily promote chondreogenesis and osteogenesis in the rat [42, 43]. The neural crest and hematopoietic systems appear to use the different members of the TGF-b superfamily in different ways to generate cellular diversity and differentiation [44, 45]. Finally, TGF-b also promotes actin cytoskeleton reorganization and migratory phenotype in epithelial tracheal cells in primary culture [46]. In the case of the liver, it is well documented that HGF, EGF, and TGF-a are not only mitogenic, but also motogenic for a wide variety of liver cells in vitro [47, 48]. In the case of neonatal hepatocytes, we have recently reported that EGF is not motogen by itself [49], and this seems to

36

´ NCHEZ ET AL. SA

also be the case for fetal hepatocytes. However, in the presence of both EGF and TGF-b, Fig. 3 clearly shows the appearance of dynamic structures reflecting motility of the fetal hepatocytes. In agreement with this, Stolz and Michalopoulos [50] have also recently reported a synergism between EGF (not HGF) and TGF-b in promoting hepatocyte motility in vitro. Block et al. [51] have recently described that if adult mature parenchymal hepatocytes enter into clonal growth under the influence of mitogens, they lose expression of hepatocyte specific genes, acquire expression of markers characteristic of bile duct epithelium (CK19), and assume a very simplified morphologic phenotype. These hepatocyte cultures can be induced to form acinar/ductular structures in the presence of HGF and an adequate extracellular matrix. Extracellular matrix is the main responsible for the actin cytoskeleton organization in cultured rat hepatocytes [52]. There is now clear evidence that a change in hepatocyte phenotype, polarity, proliferation, and function can be mediated through interaction with the surrounding matrix [17, 53]. TGF-b is one of the most powerful factors that induces extracellular matrix protein expression [54]. We have recently described that TGF-b increases fibronectin mRNA levels in fetal hepatocytes [16]. An attractive working hypothesis would be that an induction in the expression of fibronectin (or other extracellular matrix proteins?) by TGF-b might be responsible for the assembly of hepatocytes to form the plate structures. The requirement of two signals, EGF and TGF-b, indicates that this process is even more complex and, in addition to the extracellular matrix, other intracellular events related to EGF and/or TGF-b signal transduction might be implicated. Thus, EGF by itself induces changes in the expression of CKs, increasing the CK18/19 ratio (Fig. 3), whereas TGF-b seems to be the main factor responsible for the changes in F-actin organization (Fig. 4). Furthermore, although there are some lines of evidence suggesting that only arginine-synthesizing cells, i.e., hepatocytes, would be capable of cell division in cultures in arginine-deficient medium [55], the existence of some fibroblastic-like, vimentin-positive cells in our cultures (Fig. 1) could indicate that these cells might also participate in the production of collagen and other matrix components. Further work will be necessary to address that complexity, and primary cultures of fetal hepatocytes might be a good model to look at both cellular and molecular levels. We are most grateful to Susanna Castel, from Serveis CientificoTe`cnics of the University of Barcelona, for her expert assistance with the confocal microscopy; Drs. Oriol Bachs and Ricard Bastos for their kind gift of polyclonal anti-CK45; Dr. J. L. Danan for his stimulating comments and for providing us the oligonucleotides used in the gel mobility shift assays; and Dr. H. L. Leffert for helpful discussion of the results presented in this paper. This work was supported by

grants from the Fondo de Investigaciones Sanitarias from Ministerio de Sanidad, Spain (Grants 95/1530 to M.B. and 96/2099 to S.V.), and the Comisio´n Interministerial de Ciencia y Tecnologı´a, Spain (Grant PB94-1548 to S.V.). A.S. is recipient of a fellowship from Comunidad de Madrid, Spain.

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