Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes

Lina Musallam1,2, Chantal Éthier1, Pierre S. Haddad1,2, and Marc Bilodeau1

1 Centre de Recherche, Centre Hospitalier de l'Université de Montréal-Hôpital Saint-Luc, and 2 Department of Pharmacology, Université de Montréal, Montreal, Quebec, Canada H2X 1P1


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The apoptotic Fas pathway is potentially involved in the pathogenesis of liver diseases. Growth factors, such as epidermal growth factor (EGF), can protect cells against apoptosis induced by a variety of stimuli, including Fas receptor stimulation. However, the underlying mechanisms of this protection have yet to be determined. We investigated the involvement of EGF receptor (EGFR) tyrosine kinase (TK) activity in the antiapoptotic effect of EGF on primary mouse hepatocyte cultures. Cells undergoing apoptosis after treatment with anti-Fas antibody were protected by EGF treatment. This protection was significantly but partially decreased when cells were treated with two specific inhibitors of the TK activity of EGFR. Evaluation of the efficacy of these compounds indicated that they were able to abolish EGFR autophosphorylation and postreceptor events such as activation of mitogen-activated protein kinases and the phosphatidylinositol 3'-kinase pathways as well as increases in Bcl-xL mRNA and protein levels. This leads us to postulate that EGF exerts its antiapoptotic action partially through the TK activity of EGFR. In addition, our results suggest that Bcl-xL protein upregulation caused by EGF is linked to the TK activity of its receptor.

apoptosis; Fas; Bcl-xL; receptor autophosphorylation; inhibitors of receptor tyrosine kinases


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PHENOMENON OF CELL DEATH by apoptosis is essential not only for the proper development of multiple systems and organs during embryogenesis (for example, through the elimination of self-reacting lymphocytes and the formation of digits) but also for the maintenance of adult tissue homeostasis (34, 54, 66). Given the widespread involvement of apoptosis, its potential role in several pathologies, including degenerative and autoimmune diseases, is not surprising (42, 66). The pathogenesis of several liver diseases, such as hepatocellular carcinoma and alcoholic liver disease, specifically implicates abnormal apoptotic regulation (20, 21, 27, 46, 49). In addition, several studies have reported a marked increase in apoptosis in viral infections such as hepatitis C (3) and B (59).

One of the apoptosis-inducing mechanisms that has been particularly linked to human liver diseases is the cell-surface death receptor Fas, because it is constitutively expressed on hepatocytes (10, 16, 21, 45). The Fas receptor induces apoptosis by activating intracellular effectors such as caspases (19, 31) and endonucleases (6, 12) and by recruiting proapoptotic proteins of the Bcl-2 family (32, 47). This family is divided into pro- (e.g., Bax, Bad, and Bik) and anti- (e.g., Bcl-2, Bcl-xL, and BAG-1) apoptotic proteins that respectively facilitate and inhibit apoptosis (51, 65).

It is well established that growth factors (GF), such as epidermal growth factor (EGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF), are capable of protecting various cell types against apoptosis induced by different apoptotic agents, although the underlying mechanisms of this effect remain poorly understood (22, 35, 44, 67). Several studies (2, 24, 35) have correlated the antiapoptotic effect of GF with increased expression of antiapoptotic Bcl-2-like proteins. However, it still remains unclear whether these proteins represent the major component of GF protection or whether other mechanisms exist.

GF signaling is mediated through enzymatic receptors that possess intrinsic tyrosine kinase (TK) activity. In response to the binding of their ligands, these receptors become oligomerized and then phosphorylated on specific tyrosine residues by their own catalytic activity (4, 30, 54). This phosphorylation leads to the activation of different intracellular pathways, such as mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3'-kinase (PI 3-K), which mediate the biological effects of GF (17, 39). For several GF, it has been demonstrated that both the oligomerization (15, 25, 28, 30) and the autophosphorylation (29, 38, 43) of their receptors are necessary for the transmission of their signal. On the other hand, it was reported (5) that mutation of the TK domain of c-met (the receptor of HGF) did not abrogate the antiapoptotic effect of HGF.

Therefore, we investigated the involvement of EGF receptor (EGFR) TK activity in the protection against Fas-induced apoptosis afforded by EGF to primary cultures of mouse hepatocytes. To achieve this aim, we used specific inhibitors of the TK activity of the EGFR, such as PD-168393 (18) and tyrphostin AG-1478 (37). We report that the antiapoptotic effect of EGF requires, at least in part, the function of EGFR catalytic activity. Moreover, this activity is responsible for the upregulation of Bcl-xL protein observed after EGF treatment.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

All animals received humane care according to the guidelines of the Canadian Council on Animal Care. Experimental protocols were approved by the institutional animal protection committee of Centre de recherche du CHUM-Hôpital Saint-Luc.

Hepatocyte Isolation and Culture

Hepatocytes were isolated from the liver of fed male BALB/c mice (22-25 g) by using the two-step collagenase perfusion method described by Guguen et al. (23) with some modifications. After the induction of anesthesia with pentobarbital sodium (400 mg/kg ip), the peritoneal cavity was opened, and the liver was perfused in situ via the portal vein for 4 min at 37°C with calcium-free HEPES buffer and for 7 min with HEPES buffer containing 45 mg/100 ml collagenase D (Boehringer-Mannheim, Laval, QC, Canada) and 135 mg/100 ml CaCl2. The perfusion rate was set at 5 ml/min for both solutions. The cells were used only if cell viability, as determined by trypan blue exclusion, was >80%. The cells were seeded onto plastic petri dishes (26,000 cells/cm2) in Williams' medium E (GIBCO BRL, Toronto, ON, Canada) supplemented with 10% fetal bovine serum (GIBCO BRL) and allowed 90 min to attach. The serum-containing medium was then removed, and the cells were subjected to different culture conditions in serum-free medium. In control groups, cells were incubated with medium alone for the indicated time of the experiment. Apoptosis was induced in experimental groups with mouse anti-Fas Jo2 antibody (250 ng/ml, Pharmingen, Mississauga, CA). The antiapoptotic effect of EGF was studied by simultaneously incubating the cells with 50 ng/ml of EGF (Sigma, Oakville, ON, Canada) and the anti-Fas antibody.

Determination of PD-168393 and Tyrphostin AG-1478 Working Concentrations

The TK activity of EGFR was inactivated by the use of two specific inhibitors: PD-168393 (10 µM; Cedarlane Laboratories, Hornby, ON, Canada) and tyrphostin AG-1478 (25 µM; Sigma). Because PD-168393 is an irreversible inhibitor (18), its working concentration was determined by a dose-response curve in which cells were incubated with EGF alone or in the presence of different concentrations of this inhibitor for 10 min, then assayed for EGFR phosphorylation (see below). Total inhibition was obtained at 10 µM (data not shown).

On the other hand, tyrphostin AG-1478 is a reversible inhibitor. Consequently, its working concentration must ensure complete inhibition over long-term incubation. Therefore, in addition to the dose-response curve, we conducted time-course experiments in which cells were treated with EGF in the presence or absence of different concentrations of tyrphostin AG-1478 for 10 min, 1 h, 4 h, and 8 h. The dose-response curve of tyrphostin AG-1478 indicated that complete inhibition of EGFR autophosphorylation was obtained at 2.5 µM (data not shown). However, time-course experiments revealed that the inhibition produced by this concentration was not continuous over time (Fig. 1). Therefore, to obtain complete and continuous inhibition of the TK activity of EGFR over long periods of incubation, it was necessary to use 25 µM tyrphostin AG-1478. No evidence of cell toxicity was found at this concentration.


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Fig. 1.   Kinetics of epidermal growth factor receptor (EGFR) phosphorylation in mouse hepatocytes treated with EGF in the absence and presence of tyrphostin AG1478. Primary mouse hepatocyte cultures were incubated with EGF (50 ng/ml) alone or in the simultaneous presence of 2.5 or 25 µM tyrphostin AG-1478 for 10 min, 1 h, 4 h, and 8 h. The cells were then scraped off, lysed, and assayed for EGFR phosphorylation, as described in MATERIALS AND METHODS.

Morphological Determination of Apoptosis

After 24 h in culture, the medium was removed, and petri dishes were washed once with PBS. The cells were fixed with 3% paraformaldehyde solution (pH 7.4; Sigma) for 20 min at room temperature and then washed with PBS. To quantify apoptosis, hepatocyte nuclei were stained with Hoechst 33258 (250 ng/ml; Sigma) for 15 min, and petri dishes were then washed with distilled water and left to dry at room temperature in the dark. Hoechst 33258 fluorescence was visualized under a microscope (BX50F; Olympus Optical, Japan) equipped with ultraviolet epifluorescence using excitation and emission filters of 355 and 465 nm, respectively. When stained with this dye, normal hepatocyte nuclei appear homogenous and intact, as opposed to apoptotic nuclei, which are condensed, fragmented, and very bright (63). The percentage of apoptotic cells is expressed as the ratio of apoptotic nuclei vs. the total number of nuclei (normal + apoptotic). For each petri dish, 400 nuclei were evaluated.

Biochemical Determination of Cell Death

Alanine aminotransferase (ALT) is an enzyme normally present in the cytosol of hepatocytes. In response to cell damage (necrosis or late-stage apoptosis), ALT is released from the cells. Therefore, to determine cell death, we measured ALT levels released in the medium after 24 h in culture. To do this, the medium was collected to measure ALT activity. The adherent cells were then scraped off in ice-cold PBS. Both solutions were sonicated, then quantitative determination of ALT activity was performed by the Département de biochimie de l'Hôpital Saint-Luc with an automatic multianalyzer. ALT levels for each sample were calculated as the ratio of ALT present in the medium vs. the sum of the levels of ALT released in the medium and present in the cell homogenate.

Immunoblotting

Cell lysis. Based on the results of time-course experiments, it was determined that EGFR and MAPK phosphorylation assays should be performed after 1-h incubation. Akt phosphorylation peak, on the other hand, was at 5 min. The expression of Bcl-xL protein was evaluated after 24 h in culture. At the end of the experiment, the medium was removed and the cells were washed once with ice-cold PBS. Cells were then scraped off and pelleted by centrifugation. Cells were subsequently disrupted by sonication (Sonic & Materials, Danbury, CT) in the presence of lysis buffer (25 mM MOPS, pH 7.2, 60 mM beta -glycerophosphate, 15 mM 4-nitrophenylphosphate, 1 mM phenylphosphate, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 1 mM NaF, 15 mM EGTA, 15 mM MgCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, and 100 µM benzamidine). Protein concentration was determined according to the method of Bradford (7).

Western blot. Protein samples (75 µg) were separated by electrophoresis on 7% (EGFR and Akt) or 12% (MAPK and Bcl-xL) SDS-polyacrylamide gels (53) were then transferred electrophoretically overnight to Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham Pharmacia Biotechnology, Baie D'Urfée, QC, Canada). After transfer, equal protein loading was assessed by staining the membranes with Ponceau S (Sigma). Blots were probed with primary antibodies for 2 h and then with secondary antibodies for 1 h, both at room temperature with gentle agitation. To determine the levels of Akt, MAPK, and EGFR phosphorylation, blots were incubated with rabbit polyclonal anti-phospho Ser473 Akt (1:1,000; New England Biolabs, Mississauga, ON, Canada), mouse monoclonal anti-phospho ERK (1 µg/ml; E-4, Santa Cruz Biotechnology, Santa Cruz, CA), and anti-phosphotyrosine (1 µg/ml; PY20, Santa Cruz Biotechnology) antibodies, respectively. Bcl-xL protein expression was detected with mouse monoclonal anti-Bcl-x antibody (Transduction Laboratories, Lexington, KY) used at a concentration of 1.5 µg/ml. The activity of secondary anti-mouse IgG antibodies (1:2,000) coupled to alkaline phosphatase (Santa Cruz Biotechnology) was revealed by 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indoyl-phosphate reagents (Boehringer-Mannheim). Peroxidase-conjugated anti-rabbit IgG (1:5,000) activity was revealed using Renaissance Western blot chemiluminescence Reagent Plus (NEN Life Science Products, Boston, MA). Blots were then scanned, and bands were quantified by densitometry.

H3[32P]O4 Incorporation Into Mouse Hepatocytes in Culture

Cells were attached to 35-mm petri dishes (26,000 cells/cm2) for 90 min as described above. After cell attachment, the serum-containing medium was removed, and cells were incubated with phosphate-free MEM (GIBCO BRL) supplemented with sodium pyruvate (25 mg/l; Sigma) for 10 min at 37°C. H3[32P]O4 was then added (30 µCi/petri dish; ICN Biochemicals, Irvine, CA) for a 1-h incubation at 37°C. The medium was removed, and cells were washed once with Williams' medium E (37°C). The cells were incubated with 1 ml lysis buffer on ice for 30 min. Cerenkov radiation was quantitated in 200 µl of this homogenate using a beta -radiation counter. Proteins were precipitated from this homogenate by addition of 10% sulfosalicilic acid (1:1) and then resuspended in distilled water before protein concentration was determined according to the Bradford method (7).

RT-PCR

RNA preparation. Samples for RT-PCR were prepared from cell cultures treated with medium alone or EGF with or without PD-168393 for 6 h. Total RNA cellular content was isolated by TRIzol LS reagent (GIBCO BRL) according to the manufacturer's suggested protocol. Briefly, cells (26,000 cells/cm2) were homogenized with 2.25 ml TRIzol LS reagent. The aqueous phase containing total RNA was obtained by adding chloroform to the homogenate solution in 1:5 volume then separating by centrifugation at 12,000 g at 4°C. To precipitate RNA present in this phase, isopropanyl alcohol was added (1:1) and the resulting solution was incubated for 10 min at room temperature and then centrifuged at 12,000 g at 4°C. The pellet was washed once in 75% ethanol and resuspended in diethyl pyrocarbonate (DEPC)-treated water. RNA was then purified by RNase-free DNase I (0.5 U/µl; Roche Diagnostics, Laval, QC, Canada) treatment for 1 h at 37°C in a buffer containing RNase inhibitor (0.1 U/µl; Roche Diagnostics), 10 mM Tris (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2. The purified RNA was extracted by phenol-chloroform treatment and precipitated with 95% ethanol and 0.3 M sodium acetate. The pellet was washed once in 75% ethanol and resuspended in DEPC-treated water. RNA yield was determined by measuring absorbance (optical density, OD) of an aliquot of each sample in distilled water at 260 and 280 nm. RNA concentration was calculated as follows:
<FR><NU>OD<SUB><IT>260</IT></SUB><IT>−</IT>OD<SUB><IT>280</IT></SUB></NU><DE><IT>0.01</IT></DE></FR><IT>×</IT>dilution factor

RT-PCR. PCR primers were made against mouse Bcl-xL and 18S ribosomal protein sequences with oligonucleotide primers designed using the PRIMER program (Genetic Computer Group) and synthesized by the Sheldon Center (McGill University, Montreal, QC, Canada). The forward and reverse primers were as follows: for Bcl-xL, 5'-ATGGCAGCAGTGAAGCAAG-3' (forward) and 5'-GCAATCCGACTCACCAATACC-3' (reverse); and for 18S, 5'-TACCTGGTTGATCCTGCAGTA-3' (forward) and 5'-AATGGATCCTCGTTAAAGGATT-3' (reverse). RNA (1 µg) was mixed with 10 mM Tris (pH 8.3), 1 mM MgCl2, 50 mM KCl, 100 µg/ml BSA, 100 µM dNTPs, 1 µM primers, 100 U/ml RNase inhibitor, 125 U/ml avian myeloblastosis virus RT, 20 U/ml Taq polymerase, and 20 µCi/ml [alpha -32P]dCTP for a total reaction volume of 50 µl. RT reaction was conducted at 50°C for 15 min followed by 3 min at 95°C. PCR was conducted at 95°C for 30 s, 59°C for 45 s, and 72°C for 90 s. Bcl-xL and 18S were amplified for 23 and 15 cycles, respectively. These amplification conditions were determined by a kinetic study for each set of primers to ensure that PCR products remain proportional to initial gene expression template. After PCR reaction, samples were electrophoresed on 8% polyacrylamide gels and dried, and Cerenkov radiation in the excised band was quantitated using a beta -radiation counter.

Statistical Analysis

All data represent the values of at least three experiments, each from different cell isolation. Differences between groups were analyzed by one-way ANOVA for repeated measures (unless stated otherwise). P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EGF Protects Primary Mouse Hepatocyte Cultures Against Fas-Induced Apoptosis

Our control cultures displayed minimal levels of apoptosis (<1%), even though they remained without serum for 24 h, as depicted in Fig. 2. However, hepatocyte incubation with a monoclonal antibody specific for mouse Fas receptor (Jo2 clone) induced a significant increase in the level of apoptotic cells (21.9% ± 3.8; P < 0.001). The percentage of apoptotic cells was significantly lower when hepatocytes were simultaneously incubated with EGF and anti-Fas (5.0% ± 1.1; P < 0.001) than with anti-Fas alone, confirming that this GF is capable of protecting mouse hepatocytes against Fas-induced apoptosis, as previously reported by our laboratory (13).


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Fig. 2.   Antiapoptotic effect of EGF on primary mouse hepatocyte cultures subjected to Fas receptor stimulation. After attachment, mouse hepatocytes were treated with medium alone (untreated cells), EGF (50 ng/ml), anti-Fas (250 ng/ml), or anti-Fas and EGF simultaneously. After 24 h in culture, apoptosis was assessed by microscopy, as described in MATERIALS AND METHODS, using Hoechst 33258. Values are means ± SE from 7 experiments. *** P < 0.001.

EGR-R Catalytic Activity Effectively Inhibited by TK Inhibitors

As stated above, the TK activity of GF is important for signal transmission in various cell types. However, several lines of evidence suggest that the TK activity of GF receptors (GFR) may not be essential for all GF effects. To test the involvement of EGFR TK activity in the antiapoptotic effect of EGF, we assessed the content of EGFR in phosphotyrosine with and without EGF treatment in the absence and presence of two specific inhibitors of EGFR catalytic activity: PD-168393 and tyrphostin AG1478. We used anti-phosphotyrosine antibodies to detect the activated EGFR because GFR phosphorylates itself on tyrosine residues after ligand binding. The putative EGFR was detected at 170 kDa, as reported in the literature. EGFR content in phosphotyrosine after 1 h of EGF addition significantly increased compared with control untreated cultures [23.5 ± 3.8 vs. 3.25 ± 0.5 arbitrary units (AU), respectively; P < 0.001; Fig. 3], indicating increased TK activity. When cells were treated with EGF in the presence of PD-168393, EGFR displayed phosphotyrosine content (4 ± 1.2 AU) similar to that of EGFR in untreated cells (4 ± 1.1 AU; not significant; Fig. 3), indicating effective inhibition of EGFR TK activity by PD-168393. Similar results were obtained with tyrphostin AG-1478 (25 µM; data not shown). In addition, to ensure the absence of residual EGFR TK activity, we assessed H3[32P]O4 incorporation into mouse hepatocytes after 1 h treatment with EGF alone or in the presence of EGF and either inhibitor. As shown in Table 1, both inhibitors successfully prevented the EGF-induced increase in H3[32P]O4 incorporation (P < 0.01).


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Fig. 3.   Effect of PD-168393 on EGFR phosphorylation in mouse hepatocytes treated with EGF. Hepatocytes were incubated for 1 h in medium alone or with EGF (50 ng/ml) in the absence or presence of PD-168393 (10 µM). They were then lysed and analyzed by Western blotting (7% polyacrylamide gel) using anti-phosphotyrosine antibodies. Values are means ± SE from 5 experiments. A representative blot is shown. NS, not significant. *** P < 0.001.


                              
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Table 1.   Effect of PD-168393 and tyrphostin AG-1478 on H3[32P]O4 incorporation into EGF-treated mouse hepatocytes

EGFR Downstream Signaling Blocked by TK Inhibitors

Cells treated with GF display increased activation of several intracellular signaling pathways (e.g., ERK pathway of MAPK family and PI 3-K pathway) and increased synthesis of growth and survival-promoting proteins (e.g., Bcl-xL). Therefore, to ascertain that EGFR signal was not transmitted via the TK activity of EGFR in the presence of TK inhibitors, we used Western blot analysis to measure 1) the phosphorylation (and therefore the activation) of two signal transduction pathways using phosphospecific antibodies against p42/p44 (ERK) and Akt (PI 3-K) proteins and 2) the level of expression of the antiapoptotic protein Bcl-xL. All of these experiments were performed in untreated cells and in cells treated either with or without EGF in the presence of the inhibitors.

Signal transduction pathways. Cell treatment with EGF increased basal phosphorylation levels of p42 (P < 0.05; Fig. 4A) and p44 (P < 0.05; data not shown) by twofold and induced the phosphorylation of Akt on Ser473 (P < 0.001; Fig. 4D). Cell treatment with PD-168393 (Fig. 4, A and D) or tyrphostin AG-1478 (data not shown) abolished EGF-induced increase in p42, p44, and Akt phosphorylations (P < 0.01), strongly suggesting that EGFR signal was not transmitted into the cell through the ERK or PI 3-K pathways in the presence of these inhibitors.



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Fig. 4.   Effect of PD-168393 on postreceptor events in mouse hepatocytes treated with EGF. Untreated and EGF-treated cells were incubated in the absence and presence of PD-168393 (10 µM). A: samples (n = 3) were incubated for 1 h with each condition then analyzed on 12% polyacrylamide gel. Membranes were blotted with anti-phospho mitogen-activated protein kinase antibodies. B: samples (n = 4), after 6 h incubation, were analyzed by RT-PCR for Bcl-xL gene using the ribosomal protein 18S as a control gene. Results are given as the ratio of Bcl-xL to 18S. C: samples (n = 4) were incubated for 24 h with each condition then analyzed on 12% polyacrylamide gel. Membranes were blotted with anti-mouse Bcl-xL antibodies. D: samples (n = 3) were incubated for 5 min with each condition then analyzed on 7% polyacrylamide gel. Membranes were blotted with anti-phospho Ser473 Akt antibodies. Values are means ± SE. Representative blots are shown. * P < 0.05; ** P < 0.01; *** P < 0.001.

Bcl-xL mRNA and protein expressions. Bcl-xL protein, a known homologue of the Bcl-2 antiapoptotic protein, is constitutively expressed in hepatocytes. As already observed in our laboratory (14), the level of Bcl-xL mRNA expression in untreated mouse hepatocytes (57.75 ± 5.4 AU) increased by 30% in EGF-treated cells (74.5 ± 7.6 AU; P < 0.01; Fig. 4B). As a consequence, Bcl-xL protein expression level was doubled after EGF treatment (17.1 ± 0.2 vs. 33.7 ± 5.9 AU for untreated vs. EGF-treated cultures, respectively; P < 0.05; Fig. 4C). PD-168393 slightly increased basal Bcl-xL protein cellular content in untreated cells, but this effect was not significant (22.4 ± 1.4 vs. 17.1 ± 0.2 AU for PD-168393-treated vs. control cells, respectively; not significant). Cell treatment with EGF in the presence of 10 µM PD-168393 totally blocked EGF-induced elevation in Bcl-xL mRNA compared with control conditions (57.75 ± 4.9 vs. 74.5 ± 7.6 AU, respectively; P < 0.01; Fig. 4B). This was confirmed by protein expression (19.4 ± 2.6 vs. 33.7 ± 5.9 AU, respectively; P < 0.05; Fig. 4C). Finally, treatment with this inhibitor prevented cell spreading, a characteristic morphological change of EGF-treated cells (Fig. 5).


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Fig. 5.   Morphology of EGF-treated mouse hepatocytes in the presence and absence of PD-168393. Primary mouse hepatocyte cultures were incubated with medium (untreated) or EGF (50 ng/ml) in the presence or absence of 10 µM PD-168393. After 24 h in culture, EGF-treated hepatocytes were spread, and their cell surface increased compared with untreated cells. Hepatocytes treated with EGF in the presence of PD-168393 were spheroid and resembled control cells with or without PD-168393. Magnification, ×100.

Together, these results establish the efficacy of PD-168393 and tyrphostin AG-1478 as inhibitors of EGFR TK activity and its downstream signaling events. Therefore, these compounds represent adequate tools to study the implication of the catalytic activity of EGFR in the antiapoptotic effect of EGF.

Inhibition of EGFR TK Activity Diminishes Antiapoptotic Effect of EGF

The level of apoptosis in anti-Fas plus EGF-treated cultures was significantly increased by PD-168393 addition as depicted in Fig. 6A (13.4% ± 3.4 vs. 44.8% ± 1.0 for control vs. PD-168393, respectively; P < 0.05). Interestingly, however, addition of this inhibitor did not fully abolish EGF protective capacity against Fas-induced apoptosis (P < 0.01). This phenomenon was also observed when we measured the level of ALT released in the medium. Indeed, PD-168393 addition significantly increased ALT levels in the medium of anti-Fas plus EGF-treated cultures (84.2% ± 5.2) compared with control anti-Fas plus EGF cultures (52.1% ± 9.3; P < 0.01; Fig. 6B). Nonetheless, ALT levels in the medium of cultures treated with anti-Fas and EGF in the presence of PD-168393 remained significantly lower than in cultures treated with anti-Fas alone (P < 0.05). Similarly, EGF provided partial protection to mouse hepatocytes against Fas-induced apoptosis in the presence of tyrphostin AG-1478 (Fig. 6C). It is important to point out that treatment of primary mouse hepatocyte cultures with PD-168393 or tyrphostin AG-1478 did not significantly affect the level of apoptosis in untreated or anti-Fas-treated cultures (data not shown).



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Fig. 6.   Effect of PD-168393 and tyrphostin AG-1478 on the antiapoptotic response of EGF in mouse hepatocytes. Hepatocytes were incubated with anti-Fas (250 ng/ml) alone or with EGF (50 ng/ml) in the absence or presence of PD-168393 [10 µM; A and B; n = 3] or tyrphostin AG-1478 [25 µM; C; n = 7] for 24 h. Results are presented as the following ratio: %apoptotic cells (anti-Fas + EGF)/%apoptotic cells (anti-Fas) × 100. At the end of the experiment, cell death was assessed by morphological criteria, using Hoechst 33258 (A and C) or by biochemical analysis by quantitative determination of alanine aminotransferase (ALT) activity released in the medium (B). Differences between groups were analyzed by paired t-test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Together, these observations strongly suggest that, in spite of the potency and efficacy of the inhibitors we established above, the presence of either inhibitor caused only a partial decrease in the ability of EGF to protect mouse hepatocytes against Fas-induced apoptosis.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GF (such as EGF, HGF, and NGF) are essential for several cell functions, including proliferation, differentiation, and survival (1). Therefore, several studies have focused on the identification and characterization of the intracellular effectors involved in the execution of GF biological responses in various cell types. Hence, a general model regarding the mode of action of GF was elaborated (26, 68): the interaction of GF with receptors induces the oligomerization of the latter and, consequently, activates the TK domain of the same receptor. This domain was shown to be responsible for the phosphorylation of the receptor itself on specific tyrosine residues, which then serve as docking sites for Src homology 2-containing molecules such as Grb2 and PI 3-K. These molecules lead to the activation of several intracellular pathways (such as MAPK, phospholipase C-gamma , and PI 3-K), which consequently mediate the diverse biological responses of GF (17, 39, 40, 50, 62). Thus, according to this hypothesis, the TK activity of GFR plays a central role in the transmission of the GF signal.

Nevertheless, there have been some conflicting reports regarding the importance of this activity and of the subsequent receptor autophosphorylation in the mediation of some of the biological functions of GF. Several studies (29, 38, 43, 68) have indeed reported that the TK activity of GFR is required for the transmission of biological responses such as proliferation and gene transcription. On the other hand, evidence has accumulated to show that some GF effects may occur independently of the TK activity of their receptors. For instance, Schreiber et al. (55) have reported that cross-linking of cell-bound monoclonal EGFR antibodies resulted in the clustering of the receptor and the stimulation of DNA synthesis without activation of the TK domain of this receptor (55). Therefore, the aim of the present study was to determine the importance of EGFR TK activity in the antiapoptotic effect of EGF.

We first examined the effect of EGF on normal and Fas-stimulated mouse hepatocytes in primary cultures. EGF addition to Fas-treated cultures protected >70% of mouse hepatocytes susceptible to Fas-induced apoptosis. This protection occurred in parallel with increased EGFR phosphotyrosine content as well as MAPK and Akt phosphorylation. In addition, EGF treatment increased the cellular content of Bcl-xL mRNA and protein, suggesting the implication of this protein in the antiapoptotic effect of EGF.

It is important to point out that Fas receptor stimulation had no influence on either baseline levels or EGF-induced levels of EGFR, p42/p44, and Akt phosphorylation (data not shown). In addition, Fas-induced apoptosis in primary mouse hepatocytes cultures was shown by our laboratory (14) to occur without any modulation of Bcl-xL protein expression, because Fas receptor stimulation had no significant effect on Bcl-xL mRNA and protein levels.

The involvement of EGFR TK activity in the antiapoptotic effect of EGF was then tested using two inhibitors of this activity: PD-168393 and tyrphostin AG1478. As previously reported (18, 52), these compounds selectively inhibit the TK activity of the EGFR by competing for its substrate subsite. Consistent with this fact, the findings of the present study demonstrate that these inhibitors did not affect the basal state of any of our tested parameters. Indeed, PD-168393 and tyrphostin AG-1478 addition to the culture medium of untreated cultures had no significant effect on either overall H3 32PO4 incorporation into the cells (see Table 1) or basal MAPK and Akt phosphorylation levels (Fig. 4, A and D, respectively). In addition, the level of apoptosis in untreated and Fas-treated cultures was not significantly affected when these inhibitors were present (data not shown). Therefore, these results clearly indicate that PD-168393 and tyrphostin AG-1478 have no direct effect on other kinase activities in mouse hepatocytes. Consequently, these compounds represent adequate tools to selectively inhibit the TK activity of EGFR. In addition, these compounds permitted us to utilize normal mouse hepatocytes instead of cell lines transfected with mutated receptors and thus allowed us to test conditions close to physiological settings.

In addition to being selective, these inhibitors are efficient. Indeed, cell treatment with either compound completely abolished EGF-induced 1) EGFR autophosphorylation, 2) p42/p44 (ERK) phosphorylation, 3) Akt (PI 3-K) phosphorylation, 4) Bcl-xL protein expression, and 5) H3 32PO4 incorporation into cells. Their effect was observed irrespective of the presence of proapoptotic Fas receptor stimulation (data not shown). Therefore, these data strongly demonstrate that, in our experimental conditions, no residual TK activity (and consequently no TK-dependent EGFR signal) could be detected in the presence of these inhibitors.

However, in spite of these observations, we were surprised to find that the EGF-induced antiapoptotic effect, even though significantly reduced by both TK inhibitors, was not completely abolished. Indeed, we found that the level of apoptotic cells was higher in anti-Fas plus EGF-treated cultures in the presence of PD-168393 (by at least 35%) and tyrphostin AG-1478 (by 50%) compared with control Fas plus EGF-treated cultures. This level, however, never reached that observed in Fas-treated cultures, indicating that some of the EGF protective signal still passes through into the cell in the presence of these inhibitors. Therefore, our results strongly suggest that part of this protection is not related to EGFR TK activity.

Several studies (8, 48) have shown that TK activity is important for the antiapoptotic effect of GF. For instance, it was demonstrated (48) that an insulin-like growth factor-I (IGF-I) receptor lacking a functional ATP binding site provided no protection from apoptosis. In addition, it was reported (8) that tyrphostin AG-1478 was able to negate the antiapoptotic action of EGF in a human bladder carcinoma cell line: transformed cells that express high levels of EGFR. Here we report that the EGF antiapoptotic signal requires, at least in part, the catalytic activity of EGFR to be able to modulate certain cell death pathways. This modulation would probably be mediated through the well-established TK-dependent signaling cascades such as MAPK and PI 3-K, which either induce the activation of survival-promoting proteins (i.e., Bcl-xL protein) or the downregulation (i.e., Bid and Blk; Ref. 14) or inactivation of proapoptotic proteins [i.e., PI 3-K-mediated suppression of caspase-9 activation (33, 64) or Bad protein inactivation by phosphorylation after GF treatment (9, 69)].

However, contrary to the above-cited studies (8, 48), we found that part of the EGF antiapoptotic response persisted independently of the TK activity of the receptor. As mentioned earlier, there are precedents to antiapoptotic signal transduction being independent from the catalytic activity of GFR. Indeed, Bardelli et al. (5) have reported that mutation of HGF receptor in its catalytic domain did not affect the HGF protective effect. More interestingly, partial involvement of the TK domain in the antiapoptotic effect of a GF was previously reported: Dews et al. (11) have demonstrated that mutation of the IGF-I receptor in its TK domain resulted in reduction of the antiapoptotic action of IGF-I without completely abolishing it. Here we report similar observations for EGFR in primary mouse hepatocyte cultures.

To date, these TK-independent pathways have not been identified. However, in an attempt to understand these processes, it is important to remember that after GF binding to their receptors, another important initial event occurs, i.e., receptor oligomerization. This phenomenon of receptor association and subsequent conformational changes may favor interactions between certain intracellular proteins in a manner that would link the oligomerized receptor to certain effectors of the antiapoptotic machinery. Bardelli et al. (5) have indeed proposed that the antiapoptotic protein BAG-1, a cytoplasmic protein of the Bcl-2 family expressed in many cell types including hepatocytes (5, 14, 60), may play a role in these interactions. Bardelli et al. (5) has shown that BAG-1 is capable of interacting with the cytoplasmic domain of GFR through its COOH terminus independent of the GFR phosphorylation state. At the same time, the BAG-1 NH2 terminus (which contains a ubiquitin-like domain) has been implicated in the mediation of protein-protein interactions (61). It is known that only full BAG-1 protein is capable of transmitting the antiapoptotic signal of certain GF (61). Hence, with such functional and structural properties, BAG-1 (and other BAG-1-like proteins) may link GFR with intracellular antiapoptotic effectors without the need for GFR autophosphorylation.

Another interesting point is raised by our observations. We showed that Bcl-xL protein is upregulated by EGF treatment only when TK activity of its receptor is functional and that this increase was also observed in Fas plus EGF-treated cells (14). Expression of Bcl-2-like proteins by gene manipulation (overexpression) has been shown by many studies (2, 56, 57) to be sufficient to protect cells against apoptosis induced by different stimuli. For instance, it was reported (36) that overexpression of the Bcl-2 transgene was able to protect mouse hepatocytes against apoptosis induced by Fas receptor stimulation. However, our observations suggest that it might not be the sole antiapoptotic pathway activated by the EGF signal. Indeed, Bcl-xL levels did not increase when hepatocytes were treated with EGFR TK inhibitors, yet part of the EGF protection persisted. It has also been found that overexpression of Bcl-2 and Bcl-xL proteins did not abrogate Fas/tumor necrosis factor-alpha -induced apoptosis but protected the same cell types against radiation- (58) or glucocorticoid (41)-induced apoptosis. Therefore, protection against apoptosis can occur without the involvement of Bcl-2-like proteins. In the present study, we focused on the protective effect of EGF against Fas-induced apoptosis in primary mouse hepatocyte cultures. Therefore, our results lead us to postulate that when Bcl-xL protein is increased in physiological settings in mouse hepatocytes, it represents one of several key constituents of the intracellular antiapoptotic machinery.

In conclusion, our studies clearly showed that the protection afforded by EGF against Fas-induced apoptosis is mediated, at least in part, through intracellular mechanisms that depend on EGFR TK activity (receptor autophosphorylation, MAPK and PI 3-K activation, and Bcl-xL protein increase). Nevertheless, our results strongly suggest that part of the EGFR signal might be transmitted into the cell independent of these mechanisms.


    ACKNOWLEDGEMENTS

We thank André Claude for expertise in photography and Christian Demers for valuable technical advice. In addition, we also thank Ovid Da Silva (Bureau d'aide à la recherche du Centre de recherche du CHUM) for manuscript editing.


    FOOTNOTES

This study was sponsored by a grant from the Canadian Liver Foundation. L. Musallam received a joint graduate studentship from the Fonds de la Recherche en Santé du Québec (FRSQ) and Fonds pour la Formation de Chercheurs et d'Aide à la Recherche. M. Bilodeau and P. S. Haddad are FRSQ research scholars.

Address for reprint requests and other correspondence: M. Bilodeau, Centre de Recherche du CHUM-Hôpital Saint-Luc, 264, Blvd. René-Lévesque est, Montréal, Québec, Canada H2X 1P1 (E-mail: Marc.Bilodeau{at}umontreal.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 October 2000; accepted in final form 12 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 280(6):G1360-G1369
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