HGF upregulates and modifies subcellular distribution of proteins in colon cancer cell enterocytic differentiation

Stéphanie Kermorgant, Valérie Dessirier, Miguel J. M. Lewin, and Thérèse Lehy

Institut National de la Santé et de la Recherche Médicale Unité U 410, IFR Cellules Epithéliales, Faculté de Médecine Xavier-Bichat, 75870 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatocyte growth factor (HGF) and its receptor, c-Met, are involved in cell transformation. To study their role in intestinal cell differentiation, we used Caco-2 colon cancer cells, which differentiate spontaneously into enterocytes during culture. Cells grown continuously in the presence of HGF reached confluence more quickly than control cells. Markers of enterocytic differentiation, such as alkaline phosphatase and sucrase-isomaltase activities, adhesion molecules, and structural proteins such as E-cadherin, villin, and F-actin were upregulated by HGF throughout the 35 days of culture, and actin fibers were reorganized. HGF also stimulated expression and tyrosine phosphorylation of c-Met and Gab-1 as well as protein kinase C (PKC)-alpha expression. PKC-alpha has been shown to be involved in intestinal differentiation. We therefore investigated the possibility that increases in PKC-alpha protein levels were responsible for the HGF-promoted events. We did this by incubating cells with Gö-6976, an inhibitor of PKC-alpha and -beta 1, concomitantly with HGF. This inhibitor abolished the HGF-induced increase in villin levels before, but not after, confluence. Thus HGF accelerates Caco-2 cell differentiation and stimulates the metabolic and structural events accompanying this process. These HGF-promoted events may be mediated partly by Gab-1, and the effects of HGF on villin before confluence seem to involve PKC.

hepatocyte growth factor; c-Met; cell differentiation; protein kinase C; Gab-1


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HEPATOCYTE GROWTH FACTOR (HGF) was first isolated from the serum of hepatectomized rats. It circulates in large amounts associated with tumor progression in cancer patients, particularly those with gastrointestinal cancers (21, 51). HGF has pleiotropic biological actions (mitogenic, morphogenic, and motogenic) on various epithelial target cells (36, 56). Its receptor, c-Met, encoded by the c-met protooncogene, contains a 145-kDa transmembrane beta -chain with tyrosine kinase activity. The HGF-c-Met complex has been reported to have morphogenic effects during various processes, including embryogenesis (6, 33, 44, 47, 49, 52), in vitro malignant cell transformation during carcinogenesis (27, 34, 50), and cell differentiation, such as tubulogenesis of renal and mammary epithelial cells (7, 18, 48, 55). The mechanisms by which HGF-c-Met triggers morphogenesis involve the activation of several intracellular molecular pathways. Thus roles have recently been demonstrated for a functional Grb-2 site in the multifunctional docking site of c-Met (3, 17, 18), Grb-2-associated binding protein-1 (Gab-1) (3, 17, 55), and the transcription factor STAT-3 (7) in various cell types.

HGF and c-Met are present in normal human digestive tissues (28). Because they are strongly expressed in these tissues during the early stages of human fetal life, it has been suggested that they may be involved in morphogenesis of the digestive system (29, 31, 45, 54). However, little is known about the potential effect of HGF on the differentiation of gastrointestinal cells. One research group has carried out morphological studies and has reported that HGF promotes the formation in vitro of crypt-like structures by the human colon cancer cell line SW1222 (9). Caco-2 human colon carcinoma cells constitute another model for studying intestinal cell differentiation because these cells gradually undergo spontaneous enterocytic differentiation in culture after reaching confluence (40). It has been shown that insulin-like growth factor II may play a critical role in the differentiation of this cell line (46). HGF may also have an effect on this process.

Therefore, in this study we investigated, as a function of culture time, whether HGF was or was not able to activate various molecules involved in the acquisition by Caco-2 cells of the enterocytic phenotype. The molecules studied were alkaline phosphatase and sucrase-isomaltase, two brush border enzymes that are typical components of mature enterocytes, and proteins such as E-cadherin, an adhesion molecule present in adherens junctions of polarized epithelial cells, F-actin, and villin, a cytoskeleton molecule that participates in the organization and stabilization of the brush border F-actin bundle (12, 19). Recent studies have suggested that protein kinase C (PKC)-alpha appears to play an active role in inducing differentiation in Caco-2 cells (1, 43). This led us to investigate whether there is a relationship between PKC-alpha levels and the putative effects of HGF on Caco-2 cell differentiation. We demonstrated that HGF, in addition to promoting Caco-2 cell proliferation during growth, also upregulated, throughout the culture period, the metabolic and structural events associated with enterocytic-like differentiation. These effects were mediated, at least in part, by the Gab-1 and PKC pathways.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell line and culture reagents. Caco-2 cells, derived from a human colon carcinoma, passages 73-80, were cultured in 25-cm2 plastic flasks (Corning-Costar, Cambridge, MA) at 37°C in a humidified atmosphere containing 5% CO2. They were grown in DMEM (GIBCO BRL, Eragny, France) supplemented with 2 mM glutamine, 1% nonessential amino acids, 20% FCS (GIBCO BRL), 100 U/ml penicillin, and 100 µg/ml streptomycin in the presence or absence of 10 ng/ml HGF added to medium at each renewal. The medium was changed every 2 days, and confluence was reached 5-6 days after plating. Cells were harvested daily from day 2 to day 8 and then on days 10-11, 12, 15, 20, 25, 30, and 35 after plating.

Growth factor, inhibitor, and antibodies. Human recombinant HGF was purchased from R&D Systems (Abingdon, UK). Gö-6976, a selective inhibitor of PKC-alpha and -beta 1, was purchased from Calbiochem (La Jolla, CA). The following antibodies were used: goat polyclonal antibody directed against recombinant human HGF (Sigma Chemical, St. Louis, MO); affinity-purified rabbit polyclonal anti-human c-Met intracellular domain (12 aa CT) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Gab-1 (Upstate Biological, Lake Placid, NY); anti-zonula occludens-1 (ZO-1) (Zymed Labs, San Francisco, CA); mouse monoclonal anti-phosphotyrosine (4G10) (UBI); anti-villin (ID2C3, gift from Louvard's group, Institut Curie-UMR, Paris, France, and Transduction Labs, Lexington, KY); anti-PKC-alpha and anti-E-cadherin intracellular domain (Transduction Labs); and anti-glyceraldehyde-3-phosphate dehydrogenase (Biodesign International, Kennebuck, ME). The secondary antibodies used for Western immunoblotting were peroxidase-labeled sheep (Amersham, Les Ulis, France) or goat (DAKO, Copenhagen, Denmark) anti-mouse IgG, monkey anti-rabbit IgG (Amersham), or rabbit anti-goat IgG (Biosys, Compiègne, France). The antibodies used for immunofluorescence experiments were Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes Europe, Leiden, Netherlands) or Texas red-conjugated affinity-purified donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA).

Tritiated thymidine incorporation. Cell proliferation was assessed at 4 days after plating. [3H]thymidine (0.1 µCi/ml) was added to cells cultured in the presence or absence of HGF 1 h before harvesting. Cells were washed twice with PBS and trypsinized. DNA was precipitated by incubation with 5% trichloroacetic acid at 4°C for 30 min. Precipitates were washed twice with 95% ethanol, then dissolved in 10% Triton. The amount of incorporated radioactivity was determined in a beta scintillation counter and expressed in cpm.

RNA extraction and RT-PCR. Total RNA was extracted from harvested Caco-2 cells with TRIzol reagent (GIBCO BRL), according to the manufacturer's protocol. The first-strand cDNA was synthesized from 1.6 µg of total RNA with murine RT and the first-strand cDNA synthesis kit from Pharmacia Biotech (Uppsala, Sweden). Oligonucleotide primers were synthesized by Genosys (Cambridge, UK). The following primer pairs were used: 1) For c-Met, the sense primer consisted of nucleotides 3932-3960 and the antisense primer consisted of nucleotides 4391-4416. The amplified product corresponded to the 484-bp sequence encoding the COOH terminal domain of the Met receptor (29). 2) For HGF, the sense primer consisted of nucleotides 979-1000 and the antisense primer consisted of nucleotides 1498-1518 (29). The reaction generated a 569-bp amplicon. 3) For Gab-1, the sense primer consisted of nucleotides 183-205 (5'-TGAAGCGTTATGCATGGAAGAGG-3') and the antisense primer consisted of nucleotides 554-573 (5'-GCCTGGGTGGATGGTGGTGC-3'). The reaction generated a 390-bp amplicon. The cDNA mixture was amplified by RT-PCR in a final volume of 50 µl, with 25 pmol of each primer. Thirty-five cycles were performed, consisting of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, and polymerization at 72°C for 2 min. The amplification was terminated by a final extension at 72°C for 10 min. The PCR products (10-µl samples) were subjected to electrophoresis in 1% agarose gels previously stained with ethidium bromide, and the bands were visualized under ultraviolet illumination. Amplified products were purified with the QIA Quick gel purification system (Quiagen, Courtaboeuf, France) and sequenced (Genome Express, Paris, France). For negative controls, the reaction was performed without RT, without polymerase, or by omitting specific primers.

Northern blot analysis. Total RNA (5 µg) was subjected to electrophoresis in 1% denaturing agarose gels, transferred to nylon membranes (Hybond-N+, Amersham) by capillary action, and ultraviolet crossed-linked using a Spectrolinker (Spectronis). After overnight prehybridization, the membranes were hybridized with probes at 42°C for 18 h in the presence of 40% formamide. Nylon membranes were washed under stringent conditions (0.1% SDS plus 2× SSC at room temperature for 10 min, 1× SSC at 42°C for 10 min, and 0.5× SSC at 42°C for 10 min), subjected to autoradiography by using Biomax MS Kodak films and intensifying screens, and left for 2 days at -80°C. The probe used for c-Met was the RT-PCR product obtained after purification.

Western immunoblotting. Harvested cells were either homogenized in PBS with protease inhibitors and sonicated or were lysed by incubation with lysis buffer at 4°C (29) for 15 min. Protein concentration was determined by a colorimetric BCA protein assay (Pierce, Rockford, IL). Then proteins were solubilized in boiling Laemmli buffer, separated by SDS-7.5% PAGE with equal amounts (5 or 10 µg) loaded in each lane, and transferred to nitrocellulose sheets. The equivalence of loading across the gel lanes was checked by Ponceau red staining. Blots were probed with antibodies directed against the various proteins, at appropriate dilutions, and then with the corresponding secondary antibodies (diluted 1:1,000 to 1:10,000). Immune complexes were detected by enhanced chemiluminescence (Amersham, Paris, France). Blots were then quantified by densitometric analysis with the NIH Image 1.61/ppc program. Western blots were also performed on particulate and soluble fractions prepared from the PBS homogenates by centrifugation at 100,000 g for 60 min at 4°C. The pellets were resuspended in PBS by sonication.

c-Met immunoprecipitation and detection of tyrosine-phosphorylated proteins. Assays were performed 11 and 20 days after plating, with Caco-2 cell lysates prepared as for Western immunoblotting. Bead-conjugated sheep anti-rabbit IgG (Dynabead; Dynal, Compiègne, France) (25 µl) was incubated with 1.5 µg of c-Met antibody for 45 min. The beads were then washed twice with PBS-BSA and incubated with 100 µg of each lysate for 2 h, with stirring. Bound proteins were washed three times with PBS-BSA, solubilized in boiling Laemmli buffer, and separated by SDS-PAGE. Immunoblotting was then performed with 1 µg/ml anti-phosphotyrosine antibody or, to check receptor immunoprecipitation, with anti-c-Met antibody.

Immunofluorescence staining. Confocal microscopy. Cells were grown on glass coverslips (CML, Angers, France) 4, 8, or 20 days after plating and were fixed in 2% paraformaldehyde in PBS containing CaCl2 and MgCl2 for 10 min. Free aldehydes were quenched with 50 mM NH4Cl in PBS for 10 min. To detect villin, E-cadherin, and PKC-alpha , it was necessary to permeabilize the cells in 0.1% Triton X-100 in PBS-BSA for 15 min. Cells were then incubated with primary antibodies directed against villin, E-cadherin, or ZO-1 (5 µg/ml) for 1-2 h at room temperature, rinsed, and incubated with secondary antibodies (5 µg/ml) for 45 min. For actin localization, cells were incubated with 10 U/ml rhodamine-phalloidin (Molecular Probes) for 20 min at 4°C. Cells were examined with a Leica microscope equipped for transmission fluorescence or a Leica TCS-4D confocal laser-scanning microscope (Leica, Heidelberg, Germany) with the appropriate excitation and emission filters. For confocal microscopy, seven to nine horizontal scans corresponding to a 75 × 75-µm optical field were analyzed per cell.

Alkaline phosphatase and sucrase-isomaltase activities. At 4, 11, 20, and 35 days after plating, alkaline phosphatase activity was measured in cell lysates at 26°C by following p-nitrophenol phosphate hydrolysis with a commercially available kit (Sigma). Sucrase-isomaltase activity was determined as described by Messer and Dahlqvist (35). Results are expressed in milliunits per milligram protein.

Statistical analysis. Experiments were performed at least in triplicate, and data are expressed as means ± SE. We used the nonparametric Kruskall Wallis test for comparisons involving more than two groups. Differences between two groups were then evaluated with the Mann-Whitney U-test. The level of statistical significance was set at P = 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of HGF and c-Met in Caco-2 cells as a function of culture time. Caco-2 cells undergo spontaneous differentiation on the formation of confluent monolayers, exhibiting several of the morphological and functional characteristics of mature enterocytes. We investigated whether HGF and c-Met were expressed and the way in which this expression changed during the course of differentiation by studying Caco-2 cells at various times before and after confluence was reached. Neither HGF mRNA nor HGF protein was detected in the Caco-2 cells tested, whatever the passage and culture time considered, whereas HGF mRNA was detected in human fetal liver tissue used as a positive control (29) (Fig. 1A). Human fetal liver tissue was obtained after voluntary abortions from the Gynecology and Obstetrics Department of Bichat-Claude Bernard Hospital and were used in accordance with the requirements of the Human Research Committee of this hospital (29).


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Fig. 1.   Hepatocyte growth factor (HGF) and c-Met expression during the differentiation of Caco-2 cells. Representative examples of at least 4 independent experiments, here on passage 75, are shown. Cells were harvested at various times after plating. They reached confluence on day 5 after plating (indicated by the *). A: time course of HGF and c-Met mRNA levels by RT-PCR. HGF mRNA was not detected at any time but was detected in human fetal liver (L). B: expression of c-Met mRNA by Northern blots. The amount of 2 bands (the majority of RNA at 8 kb and another band at 5.9 kb) increased after confluence from day 8 after plating, reaching a plateau and then decreasing from day 25 after plating, when the differentiation was well established. The 28S ribosomal RNA did not vary. C: detection of c-Met and GAPDH proteins on Western blots. GAPDH levels did not vary during differentiation, unlike levels of the standard c-Met beta -chain, at 145 or at 85 kDa.

c-Met transcripts were detected by RT-PCR in these cells at all culture times tested (Fig. 1A). Analysis of Northern blots indicated that the amount of c-Met mRNA increased after confluence, from day 8 after plating, reaching a plateau and then decreasing from day 25 onwards. The expression of the standard 145-kDa c-Met beta -chain protein, detected on immunoblots, increased during cell growth, reached a plateau from day 8 after plating, and then decreased from day 20 onward. Another band, 85 kDa in size, was detected only after confluence, and the amount of protein corresponding to this band increased from day 15 after plating onward (Fig. 1, B and C).

Human recombinant HGF stimulates cell proliferation and the activity/expression of certain differentiation markers during Caco-2 cell differentiation. First of all, we investigated the effect of HGF (10 ng/ml added to the culture medium each time the medium was replaced) on Caco-2 cell proliferation. Four days after plating, the level of radiolabeled thymidine incorporation was 40.9 ± 11.4% (n = 3, not significant) higher for cells incubated with this dose of HGF than for controls. We also found, in ~20 different experiments, that cells cultured in the presence of HGF reached confluence ~1/2-1 day earlier than did control cells. This growth-promoting effect was HGF specific because it was totally abolished by an anti-HGF antibody added at a concentration of 30 µg/ml, whereas this antibody had no effect on control cell proliferation (data not shown).

In a second experiment, we examined the behavior of a number of markers of differentiation as a function of culture time and, afterward, investigated whether HGF modified the behavior of these markers. We checked for spontaneous Caco-2 cell differentiation by providing evidence of developing brush border alkaline phosphatase and sucrase-isomaltase activities in postconfluent cells (Fig. 2A). Mean alkaline phosphatase activity was 31-49% higher and mean sucrase-isomaltase activity was 39-104% higher, depending on the time in culture, in cells continuously cultured in the presence of HGF than in control cells (P value at least <0.05) (Fig. 2B). Concerning adhesion and cytoskeleton molecules, analysis of immunoblots revealed that control cells showed a gradual increase in levels of E-cadherin and villin proteins with time in culture after confluence (Fig. 2C, top). Compared with control cells, HGF markedly stimulated the expression of E-cadherin and villin proteins at all time points examined (Fig. 2C, bottom). The largest increase was observed on day 4 after plating with, in the experiment shown in Fig. 2C, an increase by a factor of 8 for villin and of 7 for E-cadherin, whereas no increase in the levels of these proteins was seen between days 4 and 11 after plating in cells cultured in the absence of HGF. In an additional experiment, it was shown again that the stimulating effect noted on these different variables was HGF specific because it was abolished by adding the anti-HGF antibody, which had no effect when added in the absence of HGF (Fig. 2D).


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Fig. 2.   A: development in control Caco-2 cells of alkaline phosphatase (ALP) and sucrase-isomaltase (SI) activities as determined in cell lysates. Representative example of at least 4 experiments for ALP and 2 experiments for SI are shown. *P < 0.05, **P < 0.02, and ***P < 0.001 vs. day 4 after plating. B: stimulating effects of HGF (10 ng/ml added to the culture medium each time the medium was replaced) on ALP activity (a; mean of 4 separate experiments, with assays performed in triplicate for each experiment) and SI activity (b; 2 experiments) expressed as percentage of control value. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. the corresponding time points in control cells. C: HGF effect on villin and E-cadherin protein levels, as shown by Western blots. Representative examples, here on passage 80, of at least 4 independent experiments are shown. X is the factor of increase for each protein relative to its level in control cells cultured without HGF on the same day. Note the increases by a factor of 8 for villin (a) and of 7 for E-cadherin (b) in the presence of HGF before confluence on day 4 after plating and the lack of increase in these protein levels in control cells between days 4 and 11 after plating. HGF had no effect on GAPDH levels (c). D: effect of neutralizing anti-HGF antibody in the presence or absence of HGF on day 4 after plating (experiment performed in triplicate). The antibody abolished the stimulation by HGF of ALP (a) and SI activities (b) as well as villin (c) and E-cadherin expression (d). *P < 0.02 and **P < 0.01 vs. control values. E: immunoblots showing the translocation of villin from the soluble to the particulate fraction after confluence on day 15 after plating.

HGF affects the subcellular redistribution of adhesion and structural proteins during Caco-2 cell differentiation. The upregulation of E-cadherin and villin proteins by HGF was confirmed by the results of immunofluorescence studies carried out 4 (Fig. 3), 8, and 20 days (Fig. 4) after plating. We found that signals for these two proteins and for F-actin were stronger in Caco-2 cells grown in the presence of HGF, whereas the signal at the cell membrane for ZO-1, a tight junction protein, was less intense and expressed in fewer cells (Figs. 3 and 4).


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Fig. 3.   Expression and distribution of adhesion molecules and structural proteins in cells cultured in the presence (B) or absence (A) of HGF before confluence 4 days after plating. Cells were grown on glass coverslips. They were fixed in 2% paraformaldehyde, permeabilized or not permeabilized with 0.1% Triton, immunostained for E-cadherin (an adherens junction protein), villin, and zonula occludens (ZO)-1 (a tight-junction protein), and observed by transmission fluorescence microscopy. Bar = 15 µm. F-actin was observed after staining with rhodamine-phalloidin by confocal laser-scanning microscopy. Bar = 10 µm. In cells grown in the presence of HGF, a stronger signal was detected for E-cadherin, villin, and F-actin, with the start of reorganization of F-actin bundles. Conversely, the ZO-1 signal at the plasma membrane was weaker and detected in fewer cells incubated in the presence of HGF than in its absence.



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Fig. 4.   Expression and localization of adhesion and structural proteins in cells cultured in the presence or absence of HGF after confluence, on days 8 (A) and 20 (B) after plating. Transmission fluorescence microscopy (a, b, k, l) or confocal laser microscopy (c-j, m, n). Methods were as described in Fig. 3. A stronger signal for E-cadherin and villin was still observed in cells grown in the presence of HGF than in control cells, although the difference in intensity was less than on day 4 after plating. For villin, c and d are composite pictures corresponding to the projection of all horizontal scans, and e and f to that of all vertical scans. Note that villin, which was located in the cytoplasm before confluence, was mostly associated with cell borders after confluence and then with the apical surface and lateral membranes of differentiated cells on day 20 after plating. For F-actin, g-j are composite pictures of the two basal horizontal scans, m and n, of the two apical horizontal scans. On day 8 after plating, in the presence of HGF, F-actin staining was stronger and more tightly associated with the cell membranes. On day 20 after plating, there was less F-actin in the basal part of HGF-treated cells (j) than in control cells (i), with F-actin instead detected mostly at the apical surface (n vs. m). Bar = 15 µm.

It was also shown that villin, which was localized in the cytoplasm in undifferentiated cells (Fig. 3), mainly with a perinuclear distribution as revealed by confocal microscopy, was translocated to cell membranes once enterocytic differentiation began (Fig. 4a). Its typical distribution, at the apical surface of differentiated cells, was observed 20 days after plating (Fig. 4, d and f). This translocation was also evident on immunoblots because villin was mostly present in the soluble fraction before and just after confluence but was detected, on the contrary, in the particulate fraction by day 15 after plating (Fig. 2E). In addition, in the presence of HGF, F-actin bundles began to be redistributed toward cell membranes from day 4 after plating onward (Fig. 3) and, after confluence, changes in F-actin expression mirrored those of villin, with apical reorganization of its distribution observed 20 days after plating (compare Fig. 4, i and m with j and n).

Overall, findings indicated that HGF was clearly involved not only in stimulating cell growth but also in modifying enzyme activities and the expression and distribution of proteins accompanying the process of enterocytic differentiation.

HGF stimulates the expression and tyrosine phosphorylation of c-Met in the course of Caco-2 cell differentiation. We wondered whether the continuous presence of HGF in the culture medium of Caco-2 cells also affected the expression and functioning of c-Met in these cells. We found that c-Met protein expression was actually stimulated under these conditions. In the representative example depicted in Fig. 5A, c-Met protein levels were 1.5-3.2 times higher in cells incubated in the presence of HGF. The anti-c-Met antibody was used to immunoprecipitate c-Met from the cell lysate, and the resulting precipitate was subjected to Western blot analysis with an anti-phosphotyrosine antibody. Figure 5B shows the profile of tyrosine phosphorylation of the c-Met 145-kDa band at days 11 and 20 after plating. We found that tyrosine phosphorylation was stimulated by HGF. Another protein, with a molecular mass of 115 kDa, was immunoprecipitated with c-Met and showed a similar profile of tyrosine phosphorylation.


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Fig. 5.   A: Stimulating effects of HGF on c-Met protein levels as a function of culture time. Representative example of Western blots are shown (here at passage 80). X is the factor of increase relative to control cells without HGF on the same day. B: effect of HGF on the tyrosine phosphorylation of c-Met in postconfluent cells on day 11 and day 20. Lysates were immunoprecipitated with anti-c-Met antibody and then subjected to immunoblotting with 4G10 anti-phosphotyrosine antibody. HGF clearly stimulated phosphorylation of the 145-kDa band of c-Met but also that of another band at 115-kDa, which coimmunoprecipitated with c-Met and which was probably Gab-1. C: time course of Gab-1 mRNA levels by RT-PCR during the differentiation of Caco-2 cells. Cells reached confluence on day 5 after plating (indicated by *). D: stimulation of Gab-1 protein expression by the continuous presence of HGF added in the culture medium (here at passage 80). X is the factor of increase in protein levels relative to control cells cultured in the absence of HGF on the same day. E: successive c-Met and Gab-1 immunoblottings after c-Met immunoprecipitation.

HGF stimulates the expression and tyrosine phosphorylation of Gab-1 in the course of Caco-2 cell differentiation. We thought that the protein coimmunoprecipitated with c-Met was Gab-1, a Grb2-associated multisubstrate docking protein that plays an important role in mediating the c-Met morphogenic signal (3, 17, 55). To determine whether Caco-2 cells express Gab-1, we carried out RT-PCR with specific primers and Western blotting with an anti-Gab-1 antibody at various time points in the course of Caco-2 cell culture. We detected Gab-1 mRNA (Fig. 5C) and Gab-1 protein (Fig. 5D, top) in control Caco-2 cells. The levels of this protein were low before confluence. We investigated whether the tyrosine-phosphorylated 115-kDa band that coimmunoprecipitated with c-Met corresponded to Gab-1 by reprobing membranes with anti-c-Met antibody and then with anti-Gab-1 antibody. We found that the 115-kDa band did indeed correspond to Gab-1 (Fig. 5E). Finally, HGF increased Gab-1 protein levels by 20-100% (Fig. 5D, bottom).

HGF stimulates PKC-alpha expression: role of PKC in the spontaneous or HGF-promoted enterocytic differentiation of Caco-2 cells. It has been reported that changes in PKC-alpha expression are involved in the regulation of Caco-2 cell differentiation (1, 43). We therefore investigated whether HGF stimulated the synthesis of PKC-alpha protein in Caco-2 cells. PKC-alpha protein levels, studied by Western blotting as a function of spontaneous cell differentiation, were significantly higher in cells from day 11 to 35 after plating than in cells 3-5 days after plating, a profile similar to that previously described (1) (Fig. 6A, top). HGF clearly increased the level of this protein, throughout culture, as shown by Western blotting and immunofluorescence (Fig. 6, A, bottom, and B).


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Fig. 6.   Effect of HGF on protein kinase C (PKC)-alpha protein levels during the differentiation of Caco-2 cells. A: Western blots. Two different experiments from passage 74 are shown. X is the factor of increase in protein levels relative to control cells on the same day. B: immunofluorescence on day 20 after plating. The signal for PKC-alpha was more intense in cells cultured in the presence of HGF than in control cells and seemed to be associated with membranes.

In an attempt to elucidate whether PKC-alpha was involved in HGF-promoted Caco-2 cell differentiation, we cultured Caco-2 cells in the presence or absence of HGF and Gö-6976, a specific inhibitor of the conventional isoforms PKC-alpha and -beta 1. This inhibitor was used at a concentration of 10-6 M, as in other studies with Caco-2 cells (10). Under these conditions, Caco-2 cells were harvested 4, 11, or 14 days after plating. We assessed the activity of alkaline phosphatase and sucrase-isomaltase and analyzed villin and E-cadherin protein levels in cell lysates. The inhibition of PKC-alpha and -beta 1 by Gö-6976 differentially modulated these variables, with quite opposite effects on pre- and postconfluent cells.

On day 4 of culture, Gö-6976 was found to stimulate alkaline phosphatase and sucrase-isomaltase activities in control and HGF-treated Caco-2 cells (P < 0.01 and P < 0.001, respectively) (Fig. 7A). In particular, it potentiated the effect of HGF on sucrase-isomaltase activity. It also tended to increase villin and E-cadherin protein levels in control cells, as shown by immunoblots and densitometric analysis (Fig. 7, B and C). In contrast, it totally abolished the increase in villin levels induced by HGF but seemed to have no marked effect on the HGF-induced increase in E-cadherin levels.


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Fig. 7.   Effect of Gö-6976 (an inhibitor of PKC-alpha and -beta 1, used at a concentration of 10-6 M) on ALP and SI activities as well as villin and cadherin expression in control and HGF-treated cells: A-B: day 4 after plating. C-D: day 11 or 14 after plating. A: on day 4 after plating, Gö-6976 stimulated ALP and SI activities in control and HGF-treated cells. Gö-6976 and HGF had a synergetic effect on SI activity. *P < 0.01; **P < 0.001. B: representative examples of immunoblots showing villin and E-cadherin expression levels. Gö-6976 tended to increase villin expression levels in control Caco-2 cells but totally inhibited the HGF-induced increase in villin levels. This was confirmed by densitometric analysis of immunoblots (NIH Image 1.61/ppc), each value corresponding to the mean from 3 independent experiments. Gö-6976 increased E-cadherin expression levels in control cells but did not affect the increase induced by HGF; this was also confirmed by densitometric analysis of 2 experiments performed in triplicate. C: after confluence, Gö-6976 inhibited alkaline phosphatase activity in control and HGF-treated cells, *P < 0.02; **P < 0.001. D: for villin, the immunoblots used were from experiments performed in plastic flasks (series 1) and Transwell chambers (series 2). On day 11 after plating, Gö-6976 enhanced villin expression levels in control cells but had no effect on villin levels in HGF-treated cells. This was confirmed by densitometric analysis of the 2 immunoblots.

On day 11 as on day 14 after plating, Gö-6976 was found to decrease alkaline phosphatase activity in control and HGF-treated Caco-2 cells (P < 0.02 and P < 0.001, respectively), as shown in Fig. 7C. Gö-6976 also increased villin expression in control Caco-2 cells, whereas it had no clear effect on the increase in villin levels due to HGF (Fig. 7D). Similar results were obtained in an additional experiment in which the Caco-2 cells were cultured on porous polycarbonate Transwell filters with double compartments containing medium supplemented with HGF (Fig. 7D) to improve the level of contact of the basal parts of these cells with HGF. These results were confirmed by densitometry.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Consistent with recent observations (22), we found that Caco-2 cells did not express HGF but its receptor alone. The highest levels of c-Met (mRNA and protein) occurred 8-15 days after plating. HGF is known to stimulate the proliferation of most epithelial cells in vitro. In this study, we found that 10 ng/ml HGF exerted only a moderate mitogenic effect on Caco-2 cells, consistent with the results of other studies in which Caco-2 cells were incubated with the same concentration of HGF (22). This led to a modest shortening of the time required to reach confluence. The differentiation process therefore began earlier in the presence of HGF, and this may partly account for the large increase in expression of E-cadherin and villin proteins noted on immunoblots 4 days after plating. However, at that time, the magnitude of increase observed in the amounts of these two proteins in cells incubated with HGF compared with controls was much greater than that observed between cells before and after confluence in spontaneous Caco-2 cell differentiation. This provides evidence for two simultaneous HGF-mediated events: the acceleration of differentiation as described above and true upregulation of protein levels in each cell. This upregulation was confirmed by immunofluorescence studies showing higher levels of E-cadherin, villin, and actin in cells cultured with HGF. The subcellular distribution of structural proteins was also altered, particularly the reorganization of actin fibers and their association with cell membranes, which began sooner in HGF-treated cells than in controls. This effect of HGF was maintained throughout the culture period, and HGF was also found to affect alkaline phosphatase and sucrase-isomaltase activities.

To our knowledge, the potential promoting effect of HGF on the differentiation of gastrointestinal cells has been investigated in only three works. Thus HGF has been shown to induce H+-K+-ATPase expression in rabbit stomach (57). A second study reported that human SW1222 colon cancer cells cultured in the presence of HGF formed crypt-like structures and displayed a polarized phenotype with a brush border. However, this report mentioned the lack of effect of HGF on Caco-2 cells (9). The experimental procedures differed from those described here: the molecules that might be affected during cell differentiation were not investigated and cells were grown in three-dimensional collagen matrices, whereas the cells in this study were cultured directly on plastic. In a third study, HGF (20 ng/ml) increased the proliferation rate of T84 human intestinal epithelial cells but did not induce the three-dimensional organization of these cells within collagen gels (25). In the current study, although the spontaneous differentiation of Caco-2 cells may be affected by the basal levels of growth factors provided by FCS, our findings nonetheless clearly demonstrate that exogenous HGF is indeed able to have an effect on the enterocytic differentiation of Caco-2 cells. Because HGF is secreted in part by mesenchyme tissue in vivo, the physiological differentiation of epithelial crypt cells into enterocytes may be influenced by HGF secretion from the neighboring mesenchyme. It has been suggested that HGF and c-Met signals are exchanged between mesenchyme and epithelia during various types of organogenesis, including intestinal morphogenesis (20, 22, 29, 39, 40, 47).

In HGF-treated cells, the higher levels observed for E-cadherin, an adherens junction molecule present in large amounts in polarized cells, are consistent with a similar increase observed in the presence of HGF in Madin-Darby canine kidney (MDCK) epithelial cells (2) and with that accompanying the induction by HGF of differentiation markers in rat hepatocytes (30). The observed lower levels of ZO-1 protein expression at the plasma membrane of HGF-treated Caco-2 cells is also consistent with observations made by others in MDCK cells after HGF treatment (23). However, our results conflict with those of Nusrat et al. (38), who reported similar distributions and staining intensities for E-cadherin and ZO-1 in control and HGF-exposed monolayers of T84 cells. The experimental conditions differed in this previous study and in the study reported here. Such differences may account for these discrepancies. Nusrat et al. cultured cell monolayers on collagen-coated supports and exposed them to 200-500 ng/ml HGF for 48 h, whereas we cultured Caco-2 cells on plastic for 35 days in the continuous presence of 10 ng/ml HGF. Moreover, we report for the first time a link between HGF and villin, a cytoskeleton protein of the brush border (12, 19). Interestingly, as differentiation of Caco-2 cells progressed, we observed the translocation of villin from the cytoplasm to plasma membranes and then to the apical region of differentiated cells. Although villin has been detected in the leading edges of lamellipodia-like extensions of migrating cells, suggesting that it is involved in cell motility (37), such a role may be excluded here as the effect of HGF was studied in postconfluent cells that were not migrating and were developing intercellular junctions. Similarly, ezrin, another cytoskeleton protein of the brush border, has been shown to be involved in the morphogenic action of HGF on a kidney-derived epithelial cell line (14).

We showed that, in Caco-2 cells, HGF is able to stimulate the protein expression and phosphorylation of its own receptor. These findings are consistent with previous data. Indeed, HGF has been shown to stimulate the c-Met expression (mRNA and/or protein) in a lung tumor cell line (8) and in the human tumoral hepatocyte line Hep G2 (16). A large dose of HGF (200 ng/ml) has also been found to induce the tyrosine phosphorylation of c-Met in intestinal T84 cells (38). Moreover, in the current study, we found that Gab-1, one of the molecules involved in intracellular transduction of the morphogenic HGF signal, was present in Caco-2 cells. We also showed that Gab-1 protein levels increased with cell differentiation and that HGF stimulated the expression and phosphorylation of this protein. These findings strongly suggest that HGF activates the enterocytic differentiation of Caco-2 cells, at least partly via a pathway involving Gab-1.

PKC is known to be involved in the control of cell proliferation (5, 42, 43), and a number of studies have investigated the potential roles of the various PKC isoforms in intestinal cell differentiation (4, 11, 13, 24, 26, 32, 53). Higher levels of PKC-alpha expression have been found to be associated with the tumorigenic progression of Caco-2 cells (15). However, more recent studies have reported the converse, since Caco-2 cell enterocytic differentiation has been shown to be associated with an increase in PKC-alpha expression/activity (1), whereas the transfection of Caco-2 cells with an antisense PKC-alpha construct resulted in a lower level of differentiation and a more aggressive transformed phenotype (43). In the present study, we confirmed that PKC-alpha expression levels increased during cell differentiation and found that HGF stimulated PKC-alpha expression by 1.5- to 6.5-fold. This suggested that increases in PKC-alpha expression might be responsible for the HGF-promoted events during enterocytic differentiation. We therefore investigated the effects of a specific inhibitor of PKC-alpha and -beta 1, Gö-6976, in the presence or absence of HGF. The results obtained with this inhibitor indirectly indicated that, during the growth phase, the PKC-alpha and/or -beta 1 isoform inhibit(s) the development of alkaline phosphatase and sucrase-isomaltase activities in control and HGF-treated cells. Gö-6976 clearly inhibited, at that time, the HGF-induced increase in villin levels, suggesting that the increase in PKC-alpha expression levels in Caco-2 cells cultured with HGF does participate in the large increase in villin levels detected in these cells just before confluence but was not involved in the increase in E-cadherin levels. On the contrary, our findings indicated, during the postconfluent phase: 1) a stimulating effect of PKC on alkaline phosphatase activity, whether cells were cultured in the presence or absence of HGF and 2) that one or both PKC isoforms downregulate(s) villin expression in Caco-2 cells undergoing spontaneous differentiation. They also suggested that, in HGF-treated cells, the increase in PKC-alpha expression levels merely accompanies and does not actively contribute to the increase in villin levels. Abraham et al. (1) reported that total PKC-alpha protein levels increase with Caco-2 cell differentiation, whereas PKC-beta 1 levels do not change. The two PKC isoforms may have different functions. It is therefore possible that the opposite results obtained with the PKC inhibitor during pre- and postconfluent periods are due to differences in the relative amounts of these isozymes during these two periods. Further studies are required to confirm this.

In conclusion, our data provide evidence that HGF accelerates the enterocytic differentiation process in Caco-2 cells and markedly stimulates the cellular metabolic and structural events implicated in this process. These effects of HGF are probably, at least in part, mediated by Gab-1. At least for villin protein production, the effects of HGF during the preconfluent period involved PKC, probably PKC-alpha . In addition, during spontaneous Caco-2 cell differentiation, an inhibitor of PKC-alpha /-beta 1 modulated villin production and alkaline phosphatase activity differently as a function of time in culture.


    ACKNOWLEDGEMENTS

We thank Dr. Sylvie Robine for generously providing us with the anti-alpha -villin antibody and Caroline Vignes-Colombeix for confocal microscopy.


    FOOTNOTES

This work was supported by the Institut de la Santé et de la Recherche Médicale and by Institut de Recherche des Maladies de l'Appareil Digestif (grant to S. Kermorgant). S. Kermorgant was a recipient of grants from the Ministère de l'Enseignement Supérieur et de la Recherche and the Association pour la Recherche sur le Cancer.

Address for reprint requests and other correspondence: T. Lehy, INSERM U 410, Faculté de Médecine Xavier-Bichat, 16, rue Henri Huchard, F-75870 Paris Cedex 18, France (E-mail: tlehy{at}bichat.inserm.fr).

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 14 November 2000; accepted in final form 1 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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