Hepatocyte growth factor activates the AP-1 complex: a comparison between normal and transformed rat hepatocytes

Copyright 6 European Associatiorz for the Stud], o/‘ths Liw 1999

Journal of Hrpatology 1999; 30: 916925 Printed in Dmmark AN rights reserved Munksgaard Copenhagen

Journal of Hepatology ISSN 016X-8278

Hepatocyte growth factor activates the AP-1 complex: a comparison between normal and transformed rat hepatocytes Mohamed

Rahmani,

Farid Nadori,

Laboratoire de Biologie Celhdaire. INSERM

Anne-Marie Durand-Schneider, Dominique Bernuau

protein- 1 (AP- 1) is a transcription factor involved in the primary cellular response to growth factor stimuli (1). AP-1 is a dimer formed by one protein of the fos family (c-&s, FosB, Fra-1 and Fra-2) (2-5) and one protein of the jun family (c-Jun, JunB et JunD) (6-9). AP-1 interacts with a nucleotide sequence motif known as the phorbol 12-O tetradecanoylphorbol 13 acetate (TPA)-response element (TRE) (TGACYGTCA) located in the regulatory region of responsive genes, many of which are implicated in cell growth, differentiation and transformation. AP1 binds also, but with a lower affinity, to the CAMP responsive element (CRE) (10,ll). proteins possess CTIVATOR

Received 14 September:

916

Lardeux

and

U 327, Facultk de Mkdecine X, Bichat, Universitc’ Paris, Paris. France

Background/Aims: Stimulation of activator protein-l (AP-l), a FoslJun complex, is a key event in the cell response to growth factors. We have investigated whether hepatocyte growth factor (HGF) induces differential AP-1 responses in normal and transformed rat hepatocytes, the 7777 cells. Methods: Primocultures of isolated hepatocytes or 7777 cells were stimulated with HGF. Gene expression was evaluated by ribonuclease protection assay and Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay. Identification of the proteins bound to the probes was made by supershift assays with specific antibodies. Cells were electroporated with plasmids containing an AP-l-dependent chloramphenicol acetyl transferase (CAT) gene, and CAT activity was measured 24 h after treatment with medium alone or HGE Results: In both cell types, HGF triggered the same program of&n family mRNA activation, but distinct

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Bernard

FoslJun proteins accumulated in the nucleus. HGF increased DNA-binding activity to the phorbol 12-Otetradecanoate-13-acetate responsive element (TRE) in both cell types, but distinct TRE-binding proteins were recruited in the AP-1 dimers. HGF also increased consistently binding to a CAMP responsive element (CRE) in hepatocytes only. Finally, HGF triggered TRE- and CRE-dependent gene activations in hepatocytes but TRE-dependent gene activation alone in 7777 cells. Conclusions: HGF-induced AP-1 activation leads to the formation of distinct dimers with different functional capacities in normal and transformed hepatocytes. These data suggest the importance of qualitative abnormalities of the AP-1 complex for the establishment or maintainance of a transformed phenotype. Key words: AP-1; Transformation.

CRE; Fos; Hepatocytes;

Jun;

the ability to heterodimerize with Fos proteins and to homodimerize with themselves. Fos proteins, on the other hand, cannot form stable homodimers and thus cannot independently bind DNA (12-14). Since AP-1 dimers composed of different FoslJun proteins have been shown to exert distinct transcriptional activities (13-16) the composition of AP-1 dimers appears crucial for the cell biological response. In this connection, we recently reported that the composition of AP-1 dimers in normal and transformed rat hepatocytes differs under basal conditions, with the presence of JunD in normal hepatocytes, and both JunD and JunB, in transformed rat hepatocytes (17). We also observed that stimulation with epidermal growth factor (EGF) induced different programs of activation of thefos and jun genes, the recruitment of different proteins in the AP-1 complex, and different effects on the transactivation of TRE-dependent reporter genes, in untrans-

Activation of AP-1 by HGF

formed vs transformed rat hepatocytes (17). These data suggested important differences in the functional activity of AP-1 in transformed hepatocytes and their normal counterpart, which could be a basis for the abnormal regulation of proliferation which is a characteristic of the transformed state. In order to investigate further the differences in AP1 function between normal and transformed cells, we analyzed in the present study AP-1 activation by hepatocyte growth factor (HGF). HGF, also known as scatter factor, is a disullide heterodimer consisting of one chain 01of 60 Kd and one chain @ of 34 Kd. The c-met protein, the HGF receptor, is a 190-Kd protein tyrosine kinase formed by two polypeptides chains of 50 and of 145 kd linked by disultide bonds. The signal transduction pathway utilized by HGF has not been completely clarified. Recent studies have shown that various molecules such as phosphatidyl inositol 3 kinase, phospholipase Cy, Grb2, YUS,mitogen-activated protein kinases and Janus kinases, are activated following binding of HGF to its receptor (18-23). The transmission of signals to target genes following HGF stimulation has not been completely characterized. Stimulation of signal transducers and activators of transcription factors by HGF has been reported in human hepatoma cells and hepatocytes (23). AP-1 activation by HGF has never been directly studied. Increased steady-state levels of c-j~n, junB and c-fos transcripts have been described after HGF stimulation of fetal and adult rat hepatocytes (24,25), and elevated levels of c-Fos and c-Jun proteins were reported in human hepatocytes stimulated by HGF (26). In a parallel study, we showed that HGF activates the AP-1 complex in transformed human hepatocytes, the HepG2 cell line (Malekkiani N, Rahmani M, Bernuau D, in preparation). One interesting issue, therefore, was to elucidate whether HGF activates the AP-1 complex in rat hepatocytes, and whether AP-1 stimulation of transformed hepatocytes, like EGF-induced AP-1 activation, triggers the recruitment of Fosl Jun proteins different from those in HGF-activated normal hepatocytes. In the present study, fos and jun gene activation at the mRNA and protein levels, DNAbinding activities and HGF-induced effect on the transcription of AP-1 dependent reporter genes were analyzed.

hepatocytes were purified by Percoll gradient centrifugation (29), and viability was found to be >85% by trypan blue exclusion. Hepatocytes were suspended in William’s E medium supplemented with 100 IU/ml penicillin, 10 &ml insulin, 100 ,ug/rnl streptomycin and 10% fetal bovine serum (FBS), and seeded at 3.54X lo4 cells/cm2 on collagen-coated Petri dishes. Cells were allowed to attach for 2 h at 37°C in a humitied atmosphere containing 5% COz. The medium was then changed and cells were cultured in the same medium without FBS for 24 h before HGF stimulation (10 @ml). Control hepatocytes were left untreated. Rat hepatoma 7777 cells were cultured in a mixture of Dulbecco’s modified essential medium (40%) and Ham’s F12 (60%) suppplemented with 100 IU/ml penicillin, 100 &ml streptomycin, 0.025 ptgiml fungizone and 5% FBS. The 7777 cells were seeded at 4X 104 cells/cm2 on Petri dishes cultures in 5% FBS containing medium for 18 h and deprived of serum 24 h before HGF stimulation (30 ngml). Ribonuclease protection assay

mRNA was quantified with complementary RNA probes and a ribonuclease protection assay, performed on cells directly solubihzed in 5 M guanidine thiocyanate containing 0.1 M EDTA, pH 7.0 (100 ,& lo6 cells), as previously described (30). The 32P-labeled antisense RNA probes were generated using T7 or T3 RNA polymerase (Promega) from linearized pBluescriptI1 SK+ (Stratagene, La Jolla, CA, USA) or pGEM-SZf(+) (Promega, Madison, WI, USA) cloning vectors containing the following cDNA fragments: a 343-bp Aat IZ/Sma I fragment from rat c-jun cDNA (31), a 491-bp Pst I/Nco I fragment from rat c-fos cDNA (32), a 212-bp Taq Z/Hint ZZ fragment from mouse junD cDNA, a 3 16-bp Sac Z/Taq I fragment from mouse junB cDNA, a 694-bp EcoRI/Pst Ifragment from mousejio.rB ( kind gifts from M. Yaniv, Paris), and a 851-bp Xba Z/Apa 2 fragment from rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA (33). Hybridization was carried out in the presence of an equal amount (20 pg) of unlabeled sp&ic sense mRNA as an internal standard for c-jun, junB and junD mRNA quantification. For c-jos and fosB mRNA’ quantification, cohybridization was performed with the GAPDH cRNA probe. The ribonuclease protection assay and polyacrylamide gel electrophoresis were performed as previously described (17,30). Quantitative analysis was performed from gels directly counted with the InstantImager (Packard, Groningen, The Netherlands). The ratio of the signals from protected fragments (intracellular mRNA/sense mRNA or GAPDH mRNA) was calculated and normalized for the DNA content in identical aliquots of hybridized lysates, measured by the method of Labarca & Paigen (34). Western blot analysis

Nuclear proteins prepared as below (30 pg) were fractionated by electrophoresis on a 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schull CCra-Labo, Ecquevilly, France). Membranes were incubated for 1 h in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST) and 5% nonfat dry milk to block nonspecific binding sites. The blots were then incubated for 1 h with a 1:1000 dilution of polyclonal rabbit anti-c-Jun, anti-JunB, anti-JunD, anti-c-Fos, anti-FosB, anti-Fral, or anti-&a2 (Santa-Cruz Biotechnology, Santa-Cruz, CA, USA) in PBST, at room temperature, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Biosys, Compiegne, France) diluted 1:lOOOin PBST for 1 h at room temperature. The blots were developed using an Enhanced Chemiluminescence Kit (Amersham) and exposed to Xray film for 5 s to 3 min for visualization. Electrophoretic mobility shift assay (EMSA)

Materials and Methods Cells Hepatocytes were obtained from adult male Sprague-Dawley rats (Charles River, Saint Aubin-les-Elbeuf, France) weighing 180-200 g. Animals were maintained on commercial chow (UAR, Villemoissonsur-Orge, France) and water ad libitum. Hepatocytes were isolated by collagenase perfusion (27), as modified by Balavoine et al. (28). The

Nuclear extracts were prepared by the method of Andrews & FalleL (35) with minor modifications. Cells were suspended in buffer A (10 mM Hepes/KOH, pH 7.9, 1.5 mM MgCls, 10 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 &ml leupeptin, 1 &ml aprotinin, 0.5 mM spermidine), and centrifuged at 500 g at 4°C for 30 s. The nuclear pellet was suspended in 20 mM Hepes/KOH, pH 7.9 containing 25 % glycerol, 420 mM NaCl, 1.5 mM MgCl2,

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0.2 mM ethylenediaminetetraacetic acid, 4 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 &ml leupeptin, 1 &ml aprotinin, and centrifuged at 18000 g at 4°C for 2 min. The supernatants were aliquoted and stored at -80°C. Protein concentration was determined with the BCA protein assay reagent (Pierce, Rockford, IL, USA). Single-stranded oligonucleotides corresponding to the TRE site (5’-TAA AGC ATG AGT CAG ACA CCT C-3’) and the CAMP responsive element (CRE) site (5’-AGA GAT TGC CTG ACG TCA CAG AGC TAG-3’) were synthesized (Genset, Paris, France) and end-labeled with T4 polynucleotide kinase in the presence of [Y~~P]ATP (5000 Wmmol, Amersham). The labeled oligonucleotides were annealed with corresponding unlabeled anti-sense synthetic oligonucleotides. Nuclear extracts (5.-20 pg) were incubated in binding buffer (20 mM Hepes, pH 7.9, 5 mM MgC12, 4 mM dithiothreitol, 20% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 2 mM levamisole, 0.1 pg/ml aprotinin, 0.1 &ml bestatin) containing 2 /cg poly(dI-dC) and 32P-labeled double-stranded probe (3~ lo4 cpm) for 20 min at 4°C. The reaction mixture was then loaded onto a 6% polyacrylamide gel in 0.09 M Tris borate. 2 mM EDTA, pH 8.0 buffer, and electrophoresed at 11 V/cm for 2 h at 20°C. The gels were dried and exposed to X-ray film for autoradiography. For supershift analyses, 2 ~1 of antibody specific to Jlrn family

A

Hepatocytes

(anti-c-&z, anti-JunB, anti-JunD), or Fos family members (anti cFos, anti-FosB, anti-Fral, anti-Fra2), and activating transcription factor (ATF)-2 and ATF-3 (Santa-Cruz Biotechnology, Santa-Cruz, CA, USA) were incubated with the nuclear extracts for 2 h at 4°C before addition of the 32P-labeled probe and electrophoresis. A rabbit polyclonal anti-JunB antiserum directed against the activation domain of &-zB protein (kindly provided by D. Lallemand, Paris) was also used in some experiments. Reporter plasmids The c-jun-TRE (a kind gift from T. Deng. University of California) contains the c-jun promoter from -79 to + 170, inserted 5’ to the luciferase (LUC) gene. The 2XCRE-TK-LUC plasmid (generously provided by J. Sobczak, Paris) contains 2 copies of the CRE from cyclin A inserted upstream of the thymidine kinase (TK) promoter in a TK-LUC reporter plasmid. Cell transfection and LUC assays Immediately after isolation, hepatocytes at a density of 20 million cells/O.8 ml PBS containing 5% FBS were electroporated (Gene Pulser, Bio-Rad Laboratories, Richmond, CA, USA) at 160 V and 960 FF, according to the method described by Le Cam (36). Subcon-

7777 cells

c-foe GAPDH (IS4 nt) fOS S (230 nt)

GAPDH (lS4 nt)

c-jun

mt.Sd (151 nt) ,“” Et (246 nt)

junB ht. Sd (195

nt)

juno (212 nt)

junD int. Sd (103 nt)

cl

0

0.5 1

2

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time of culture

6 24 (h)

i 0

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Fig. 1. Detection and quantification of fos and jun family transcripts by RNAse protection assay. (A) Representative autoradiograms after solution hybridization of cell lysates. GuSCN lysates from hepatocytes and 7777 cells were hybridized with specific cRNA probes and unlabeled sense mRNA as the internal standard (Int. Sd), or GAPDH cRNA probe (in the case of c-fos and fosB mRNA quanttjications). Specljk protectedfragments were separated by PAGE. The size of the synthesized cRNA probes is shown at right. (B) Q uantitative analysis of the mRNA levels in (0) hepatocytes and in (0) 7777 cells. Analyses were performedfrom gels directly counted with the InstantImager, and the signal ratio between protectedfragments (intracellular mRNA/sense mRNA or GAPDH mRNA) was calculated. When sense mRNA wus used us the internal standurd, the ratio was normalized for DNA content. The fold induction at the d@rent indicated times was determined by comparing the ratios to tfzose obtained without EGF treatment. The results are the mean of three independent experiments. 918

Activation of AP-1 by HGF Fig. 2. Western blot analyses of Jun and Fos nuclear proteins in hepatocytes and 7777 cells. Nuclear extracts (30 pg) from unstimulated cells or cells stimulated by HGFfor the indicated times were fractionated on a 10% SDSPAGEgel and transferred to nitrocellulose membranes. They were incubated with I:1000 dilutions of anti-Jun- or anti-Fos-spectfk antibodies, and then with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (1:lOOO dilution) as a secondary antibody. Antigen/antibody complexes were visualized by chemiluminescence.

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stimulation or not by HGF (30 &ml) for 24 h. For LUC assays, cells were washed in PBS, lysed for 15 min on ice with lysis buffer (25 mmolil Tris/HP04, pH 7.8, 8 mM MgClr, 1% Triton X 100, 1% bovine serum albumin, 15% glycerol, 1 mM EDTA and 1 mM DTT), centrifuged for 10 min at 13 000 rpm and stored at -20°C. LUC activity was determined with a luminometer (EGG Instruments, Evry, France), and was rapported to the amount of proteins in the nuclear extracts determined with the Biorad reagent (Biorad Laboratories, Mtinchen, Germany).

B

HEPATOCYTES 3omln

24h

--mqip

fluent 7777 cell cultures were trypsinized and electroporated at the same density as hepatocytes in PBS/Hepes 10 mM, pH 7.8 at 300 V and 960 #. Electroporation was performed in the presence of 50 pg reporter plasmid DNA and 50 pg sonicated salmon sperm DNA. After electroporation, the hepatocytes were plated in the presence of 10% FBS for 2 h, and deprived of serum for 18 h. They were then incubated in serum-free medium in the presence or absence of HGF (10 q/ml) for 24 h. After electroporation, 7777 cells were plated in medium with 5% FBS for 18 h, and deprived of serum for 8 h before

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Hepatocytes

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Fig. 3. TREbinding activities of hepatocyte and 7777 cell nuclear proteins detected by electrophoretic mobility shift assay (EMSA). (A) Equal amounts (1.5 ,ug) of nuclear extracts from hepatocytes either unstimulated or stimulated by HGFfor the indicated times in the absence or in the presence of cycloheximide (CHX) were incubated with a TRE probe labeled with [y-32P] ATP and electrophoresed in a 6%polyacrylamide gel. Specifi:city of the retarded complexes was assessed by coincubation with a 100X molar excess of cold TRE probe (competitor). (B) Equal amounts (15 ,ug) of nuclear extracts from 7777 cells either unstimulated or stimulated with HGF for the indicated times in the absence or in the presence of cycloheximide (CHX) were incubated with a 32P-labeled TRE probe and electrophoresed in a 6% polyacrylamide gel. Specificity of the binding was assessed by coincubation with a 100~ molar excess of cold TREprobe (competitor). 919

M. Rahmani et al.

Results Activation of the fos and jun genes at the mRNA level Detection and quantification of thefos and jun genes are illustrated in Fig. 1A and B. In hepatocytes stimulated by HGF, there was a rapid and transient accumulation of allfos and jun (except j,nD) mRNAs between 30 min and 1 h. The increase in C-$X andfosB mRNA was strong (30-50-fold), while c-jun and junB mRNAs were induced more weakly (between 2- and 3-fold). In 7777 cells, the profile offos and jun mRNA activation was very close to that in hepatocytes, but induction of c-fos andfosB mRNA was smaller (about 3-fold). As for hepatocytes, junD mRNA levels did not change significantly over the 24 h-period of study. Activation of the fos and jun genes at the protein level The nuclear concentrations of Fos and Jun proteins were assessed by Western blot analysis of nuclear extracts prepared 2 , 6 and 24 h after HGF stimulation (Fig. 2). In hepatocytes, the Fra-1 level was increased at 2 and 6 h, while c-Fos, FosB and Fra-2 levels did not change. Among the Jun-related proteins, JunB alone accumulated at 2 h, and it was still slightly elevated at 6 h. By 24 h, there was no difference in the level of any of the Fos- and Jun-related proteins between control and stimulated hepatocytes, except for a persistent elevation of the Fra-1 protein in HGF-treated hepatocytes. In 7777 cells, c-Fos, Fra-1 and to a lesser extent FosB levels, were increased at 2 h, and this induction persisted at 6 h only for Fra-1 and c-Fos. C-Jun and JunB proteins were also induced at 2 and 6 h. By 24 h, the levels of several Fos/Jun proteins (c-Fos, FosB, cJun, JunB) increased in untreated control 7777 cells compared to control cells, but none of the Fos- and Jun-related protein levels differed between control and stimulated 7777 cells, except for a slight decrease of c-Jun level in HGF-treated cells compared to control cells. TRE binding activity of nuclear proteins The binding of nuclear proteins to a TRE probe containing the consensus TRE sequence of the collagenase gene promoter was studied by EMSA. The same patterns of binding were obtained for HGF-stimulated hepatocytes (Fig. 3A) and 7777 cells (Fig. 3B). In both cases, the binding activity of nuclear proteins to the TRE probe was increased at 30 min, 2 and 6 h of HGF stimulation. By 24 h the level of TRE-binding activity in HGF-treated hepatocytes and 7777 cells had returned to the control level. Treatment with HGF in the presence of cycloheximide (20 pug/ml) induced different effects on the TRE binding in hepatocytes and 7777 cells. In hepatocytes, HGF-induced binding to the 920

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Antibody Control

2h

24h

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24h 4 Fig. 4. Identification of the Fos and Jun proteins in the TRE-binding compkves. Nuclear extracts (1.5 pg) from (A) hepatocytes or (B) 7777 cells, either unstimulated (control) or stimulated with HGF for the indicated times were incubated nith 2 ,ng spectfiic antibodies against Fos and Jun proteins, followed by incubation with a 32P-labeled TRE probe, and analysis by EMSA. Arrows point to the retarded bands and arrow heads indicate the supershtfted bands. The results illustrated are,from experiments with dfferent exposure times.

TRE probe was not modified by cycloheximide at 30 min, but it was reduced by this treatment after 2 (data not shown) and 6 h of HGF stimulation (Fig. 3A). By contrast, cycloheximide did not reduce the HGFinduced TRE-binding activity of nuclear extracts from 7777 cells at 30 min and 6 h (Fig. 3B). To gain insight into the composition of AP-1 dimers binding to the TRE probe, antibodies against the Fos

Activation of AP-1 by HGF

and Jun proteins were added to the reaction mixture in the binding assay With nuclear extracts from unstimulated hepatocytes (Fig. 4A), anti-JunD antibody induced an intense super-shifted band, while weak retarded bands were detected with the anti-c-Jun and anti-Fral antibodies. None of the other antibodies disrupted or retarded the AP-1 complex. With nuclear extracts from hepatocytes stimulated by HGF for 2, 6 (data not shown) and 24 h, the intensity of the retarded bands obtained with the anti-JunD, anti-c-Jun and anti-Fra-1 antibodies was stronger than in unstimulated cells (Fig. 4A). In 7777 cells (Fig. 4B), supershift analysis of nuclear extracts prepared from control cells showed that anti-JunD and anti-JunB induced a supershifted and a decreased band, respectively, while antic-Jun antibody did not modify the TRE-binding. Using the anti-&s antibodies, only anti-Fra-1 induced a weak supershift. Analysis of nuclear extracts from 7777 cells stimulated with HGF for 2 (Fig, 4B) and 6 h (data not shown) showed a weak supershifted band with the anti-c&s antibody, besides the presence of JunD, JunB and I;ral. By 24 h of stimulation, the patterns of supershift in HGF-treated and control 7777 cells were comparable.

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CRE-binding activity of nuclear proteins

Since the AP-1 complex is also able to bind to CRE sequences (9,l l), we also analyzed the binding activity to a CRE probe of nuclear proteins from hepatocytes (Fig. 5A) and 7777 cells (Fig. 5B). Two distinct retarded bands of unequal intensity were visible with extracts from both cells, with a strong faster migrating band and a faint slower migrating band. Several minor fast migrating bands were also visible in extracts from 7777 cells. Specificity of these bands was assessed by their complete disappearance in the presence of a 1OOXmolar excess of unlabeled CRE probe. Extracts from HGF-stimulated hepatocytes showed an increased intensity of the main retarded band after 30 min, 2 h and 6 h, which was partially inhibited by treatment with cycloheximide at 30 min but not at 6 h. Binding of nuclear extracts from HGF-stimulated 7777 cells to the CRE probe was very weakly increased at 30 min and 2 h, and returned to control level by 6 h. Supershift analyses with nuclear extracts from control (data not shown) and 2 h-stimulated hepatocytes showed that the anti-c-Jun and anti-JunD antibodies induced partial disruption of the 2 bands. None of the anti-Fos antibodies modified the binding to the CRE probe (Fig. 6A). Since the main proteins binding to CRE sites are proteins of the ATF/CRE binding (ATF/ CREB) family, we also used anti-ATF2 and anti-ATF3 antibodies in the supershift assays. Only the anti-ATF3

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Fig. 5. CRE-binding activities of hepatocytes and 7777 cells detected by EMSA. (A) Equal amounts (15 pg) of nuclear extracts from hepatocytes either non-stimulated or stimulated with HGFfor the indicated times in the absence or in the presence of cycloheximide (CHX) were incubated with a 32P-labeled CREprobe and electrophoresed in a 6%polyacrylamide gel. Specificity of the binding was assessed by coincubation with a 100X molar excess of cold CREprobe (competitor). (B) Equal amounts (15 ,ug) of nuclear extracts from 7777 cells non-stimulated or stimulated with HGFfor the indicated times in the absence or in the presence of cycloheximide (CHX) were incubated with a “Plabeled CRE probe and electrophoresed in a 6% polyacrylamide gel. Spectfkity of the binding was assessed by coincubation with a 100X molar excess of cold CRE probe (competitor).

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Fig. 6. Identijikation of the Fos and Jun proteins in the CRE-binding complexesfiom cells. Nuclear extracts from 2 h were incubated with 2 Fos, Jun and ATF proteins, 32P-labeled CRE probe and

(A) hepatocytes or (B) 7777 cells stimulated with HGF for ,ug spectjk antibodies against followed by incubation with a analysis by EMSA.

antibody decreased the intensity of the faster migrating band (Fig. 6A). Analysis of the composition of the CRE-binding dimers in cell extracts from unstimulated (data not shown) or HGF-stimulated 7777 cells showed partial disruption of the upper band with the antiJunD antibody, while the anti-ATF2 and anti-ATF3 antibodies did not modify the CRE-binding (Fig. 6B). Analysis of TRE- and CRE-dependent reporter gene activation We next compared the functional consequences of HGF-induced AP-1 stimulation on TRE- (Fig. 7A) and CRE-dependent (Fig. 7B) transactivation in hepatocytes and 7777 cells. In hepatocytes transiently transfected with the c-&n-TRE-LUC or the 2XCRELUC plasmid, and stimulated with HGF (10 ng/ml) for 24 h, LUC expression was increased 1.5-fold and 2-fold, respectively. In cells transfected with the control TK-LUC plasmid and stimulated with HGF, no significant variation in LUC expression was noted (data not shown). In 7777 cells transiently transfected with the c-jun-TRE-LUC construct LUC expression was increased 1.6-fold. By contrast, HGF treatment (30 ng/ ml) of 7777 cells transfected with the 2XCRE-LUC plasmid did not induce significant variations in LUC expression. 922

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Fig. 7. Transcriptional activation of (A) c-jun-TRE-LUC and (B) 2X-CRE-LUC, in hepatocytes (open bars) and 7777 cells (hatched bars). Cells were transientl_v transfected b-y electroporation with the reporter constructs, as described in Materiuls and Methods, and left untreated (C) or stimulated by) HGF (IO ng/ml ,for hepatocytes, and 30 ng/mljor 7777 cells) ,for 24 h. At the end of the stimulation period, LUC activity was assa,ved and normalizecl,for protein content of cellular extracts. The results are the mean? SEM of 3 to 8 independent experiments.

Discussion In this study we have shown that HGF treatment of normal and transformed rat hepatocytes stimulates AP-1 activity. This result is in agreement with parallel data obtained in our laboratory demonstrating HGFinduced AP-I activation in transformed human hepatocarcinoma cells, the HepG2 cell line (Malekkiani N, Rahmani M, Bernuau D et al., in preparation). We also show that the HGF-induced AP-1 activation in untransformed and transformed rat hepatocytes dif-

Activation of AP-I by HGF

fered at several levels: the induction of the Fos and Jun genes mainly at the protein level, the composition and DNA-binding specificity of the AP-1 dimers (data summarized in Table l), and the transactivating functions of the AP-1 complex. The pattern of activation of the jun family genes at the mRNA level was very close in HGF-stimulated hepatocytes and 7777 cells, with an immediate and transient elevation of c-jun and junB transcripts, but no modification ofjunD mRNA levels. However, activation of the fos and jun genes differed at the protein level. Increased nuclear concentrations of FEZ-1 were noted in both cell types, but the increase was more sustained in hepatocytes. Conversely, the increases in nuclear JunB levels persisted longer in 7777 cells than in hepatocytes. Moreover, despite the fact that the inductions of c-fos and fosB mRNA were much more important in hepatocytes than in 7777 cells, c-Fos and FosB proteins accumulated in 7777 cells only, suggesting a blockade of translation or a high instability of Fos proteins in normal but not in transformed hepatocytes. Increased stability of Fos and Jun proteins is a well-recognized abnormality in vivo in virus-induced mouse osteosarcomas and chicken fibrosarcomas (1). HGF is known to transduce its signal via the rasl ruflmitogen activated protein kinases and the signal transducer and activator of transcription/Janus activated kinase signaling pathways (14,20,21,37), which can phosphorylate Fos and Jun proteins, thus stimulating their transcription (14,38,39). These kinases may also play a role on FodJun protein stability by reducing

TABLE 1 Summary of the changes in fos/jun mRNA and protein levels and in AP-1 composition induced by HGF in normal hepatocytes and 7777 cells Normal hepatocytes

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mRNA (increase)

c-jun junB c-fos

c-jun junB c-fos

Nuclear proteins (increase)

JunB Fra-1

c-Jun JunB c-Fos Fra-1 (Fos B)

Composition of TRE-binding dimers

c-Jun JunD Fra-1

JunB JunD c-Fos Fra-1

Composition of CRE-binding dimers

c-Jun JunD ATF-3

JunD

Parentheses indicate that the increase is weak.

ubiquitin-dependent degradation mechanisms (40,41). We hypothesize that differential activation of these protein kinases and distinct effects on the Fos and Jun proteins occur in normal and transformed hepatocytes upon HGF stimulation. Altogether, these results strongly suggest that the abnormal regulation of the fos and&n genes in transformed hepatocytes is mainly due to abnormal translational and/or post-translational mechanisms. Differences in AP-1 regulation were also reflected by the activation of different FoslJun-containing AP-1 dimers in the 2 cell types (Table 1). Under basal conditions the AP-1 complexes of hepatocytes and 7777 cells are predominantly composed of JunlJun homodimers (this study) and (17). We show that HGF stimulation induced a modification of the constitution of the AP-1 subunits, with the recruitment of Fra-1 in hepatocytes while c-Fos and Fra-1 were recruited in 7777 cells, resulting in both cases in the formation of distinct JunlFos heterodimers. It could be argued that the formation of different dimers might artificially result from the use of different culture systems, with distinct proliferative rates and different cell-to-cell contacts. However, we observed that the basal composition of the AP-1 dimers remained the same in control hepatocytes left untreated for 2 h or for 24 h of culture, a time when a confluent monolayer is formed and numerous cell contacts are established. In the same way, the basal composition of the AP-1 dimers in control untreated 7777 cells did not vary over the 24-h period of culture, despite a doubling of the cell number. Therefore, the recruitment of different Fos proteins in the AP-1 dimers of normal and transformed hepatocytes appears to be clearly related to HGF stimulation. Variations in the composition of AP-1 dimers in transformed cells and their normal counterpart have been previously described in other cell types such as adipocytes and fibroblasts (42,43). Other important differences in the AP-1 function of normal and transformed hepatocytes were observed at the levels of DNA-binding specificity and activity induced by HGE Increased binding of nuclear proteins from HGF-treated cells to the main AP-1 consensus DNA sequence, the TRE site, occurred rapidly after HGF treatment, and persisted for 6 h. However, ongoing protein synthesis was required for this activation in hepatocytes but not in 7777 cells. These data again point to a high stability of Fos and Jun proteins in transformed hepatocytes. Furthermore, nuclear proteins from HGF-treated hepatocytes, but not from 7777 cells, exhibited increased binding to a CRE probe, another potential target for AP-1 binding (10,ll). It is also interesting to note that, as for the TRE-binding 923

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complexes, the composition of the CRE-binding complexes of HGF-stimulated cells differed in the 2 cell types, with the presence of c-.Jun, JunD and ATF3 (or LRF-1) in hepatocytes, whereas JunD alone was identified in the CRE-binding i complexes of 7777 cells (Table 1). Since AP-1 composition can selectively regulate gene transcription, it is likely that activation of distinct target genes occurs in transformed hepatocytes and in their normal counterpart upon HGF stimulation. Finally, we have shown differences in the transactivating capacities of the AP-1 complex. In hepatocytes, activation of both TRE- and CRE-dependent genes was induced by HGF, whereas in 7777 cells TREdependent gene activation alone was stimulated, consistent with the inconspicuous effect of HGF on AP-1 binding to the CRE site in these cells. Our observations might be relevant to in vivo events. It has been shown that normal and preneoplastic hepatocytes, but not hepatocarcinoma‘cells, proliferate in response to HGF in rat in viva (44). These variable responses might be due, at least in part, to abnormalities of the AP-1 complex, although a direct demonstration that differences exist in the composition of the AP-1 dimers of normal and transformed hepatocytes in viva is still lacking, Increased AP-1 activity has been raised as an important mechanism of cell transformation (4548). Our study emphasizes that differences in the constitution of AP-1 dimers both under basal conditions and after growth factor stimulation stand also as an important characteristic of transformed cells. Whether such abnormalities are a cause or a consequence of the transformation process remains to be established.

Acknowledgements We thank A.F Bringuier for help with Western blot analyses. This work was partially supported by a grant from La Ligue contre le Cancer.

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