Hepatocyte growth factor (HGF) and Wnt signaling pathways have been shown to be important in embryogenesis and carcinogenesis. The aim of this study was to elucidate the mechanism of functional similarities observed in the two pathways. We used normal rat liver, primary hepatocyte cultures and a dominant-negative Met expression system to study the effect of HGF on Wnt pathway components. We demonstrate novel association of β-catenin and Met, a tyrosine kinase receptor of HGF, at the inner surface of the hepatocyte membrane. HGF induces dose-dependent nuclear translocation of β-catenin in primary hepatocyte cultures that is Wnt independent. The source of β-catenin for translocation in hepatocytes is the Met-β-catenin complex, which appears to be independent of the E-cadherin-β-catenin complex. To test the functionality of this association, we used a dominant-negative Met expression system that expresses only the extracellular and transmembrane regions of the β-subunit of Met. A loss of Met-β-catenin association resulted in abrogation of nuclear translocation of β-catenin upon HGF stimulation. This event is tyrosine phosphorylation dependent, and the association of Met and β-catenin is crucial for this event. We conclude that the HGF causes similar redistribution of β-catenin as Wnt-1 in the hepatocytes and that this effect is attributable to subcellular association of Met and β-catenin. The intracellular kinase domain of Met is essential for tyrosine phosphorylation and nuclear translocation of β-catenin. Part of the multifunctionality of HGF might be attributable to nuclear β-catenin and the resulting target gene expression.

Wnt signaling pathway has been shown to play diverse roles during embryogenesis and carcinogenesis (1, 2, 3). β-Catenin is an important component of this cascade, which is responsible for transactivating target genes after forming heterodimeric complexes with the T cell factor/lymphoid enhancement factor family upon its translocation to the nucleus (4, 5). We and others have shown previously that levels of β-catenin are tightly regulated with a minimal free monomeric form available inside a normal cell and during regulated growth (3, 6, 7). β-Catenin protein is bound to either the GSK3β/Axin/APC3 complex or E-cadherin inside a cell. Upon signaling by Wnt through its receptor frizzled and interactions with dishevelled, the GSK3β/Axin/APC/β-catenin complex undergoes dephosphorylation at specific serine and threonine residues (8, 9, 10, 11). This causes β-catenin dissociation from the complex, followed by its nuclear translocation. Several tumors have demonstrated mutations in APC and Axin proteins, resulting in stabilization of the β-catenin protein (2, 12). Also, mutations affecting serine/threonine phosphorylation sites in the β-catenin gene result in a stable protein that is resistant to ubiquitination, leading to enhanced cell proliferation and tumor formation (1, 13, 14, 15).

Previously, we have shown predominant β-catenin localization at the hepatocyte membrane in a normal adult rat liver with some cytoplasmic staining, suggesting a minimal association with GSK3β/Axin/APC complex (6). β-Catenin-E-cadherin association at the cell membrane is well recognized and has been shown to play a pivotal role in cell-cell adhesion. Tyrosine phosphorylation of β-catenin in tumors affects intercellular adhesion and promotes metastatic potential and local invasiveness of tumors (16, 17). Met, a tyrosine kinase receptor for HGF, has an established function in liver growth, development, and oncogenesis (18, 19, 20, 21, 22). In this report, we investigate the affect of Met activation in response to HGF on the Wnt pathway components with emphasis on membrane-associated β-catenin in hepatocytes.

HGF/scatter factor, a known mitogen, motogen, and morphogen for liver and other tissues, signals through membrane-associated Met, a tyrosine kinase receptor (18, 23, 24, 25). This pathway has been shown to be important during embryogenesis and tumorigenesis (19, 21). We wanted to analyze the mechanism of some of the functional coincidences seen in the Wnt and HGF signaling pathways. Although some earlier reports have shown association of Met and cadherin complexes with in tumors, no study is available on the mechanism of this association in normal or nontumor cells (26). Although few studies have shown tyrosine phosphorylation of β-catenin in response to HGF stimulation in tumor cells, very little is known about its fate and the mechanism of such event (27, 28).

In this report, we demonstrate and discuss the functional association of Met and β-catenin in normal rat liver. We also investigate the effect of HGF on the Wnt pathway components in primary hepatocyte cultures. Our results indicate the ability of HGF to induce Wnt-independent redistribution of β-catenin because of Met-β-catenin dissociation. To further our understanding of the molecular mechanism involved in this interaction, we used a dominant-negative system for HGF/Met signaling. We demonstrate the role of intact Met to tyrosine phosphorylate and translocate β-catenin to the nucleus after HGF stimulation. We discuss the importance of the interaction between these two independent signal transduction pathways, emphasizing the implications of elevated serum HGF levels observed in disease states including hepatocellular cancer.

Animals and Materials.

Male Fisher 344 rats were used for hepatocyte isolation and culture, and the experimentation was performed under the strict guidelines of the Institutional Animal Use and Care Committee at the University of Pittsburgh School of Medicine and the NIH.

Collagenase H for hepatocyte isolation was obtained from Boehringer Mannheim (Mannheim, Germany). Vitrogen (Celtrix Labs, Palo Alto, CA) was used for hepatocyte attachment to culture plates. General reagents were purchased from Sigma Chemical Co. (St. Louis, MO). HGF/SF (Δ5 variant) was kindly donated by Snow Brand Co. (Toshigi, Japan).

Hepatocyte Isolation and Culture.

Rat hepatocytes were isolated from at least three different animals by an adaptation of Seglen’s calcium two-step collagenase perfusion protocol as described previously from our laboratory (29, 30). Rat hepatocytes were plated at high density (1.5 × 106 cells/ml) after wet collagen coating (10% Vitrogen) for 1 h on 175-cm2 plates unless stated otherwise. Hepatocytes were allowed to attach for 2 h in basal hepatocyte growth medium (31). This was replaced by fresh hepatocyte growth medium with/without HGF at 12.5 ng/ml for 15 min (unless stated otherwise), and cells were used for protein isolation.

Preparation of Total Cell Lysates, Nuclear Extracts, and Differential Detergent Fractionation.

Hepatocytes from the culture plates were washed in PBS, and total cell lysate was prepared in RIPA buffer (9.1 mm dibasic sodium phosphate, 1.7 mm monobasic sodium phosphate, 150 mm sodium chloride, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS, pH adjusted to 7.4) containing fresh protease and phosphatase inhibitor mixture (Sigma Chemical Co.; Ref. 6). Nuclear extracts were prepared in HEPES buffer (30). Briefly, cells were washed and harvested in PBS (80 g) and resuspended in 750 μl of hypotonic buffer [10 mm HEPES (pH 7.9), 10 mm NaH2PO4, 1.5 mm MgCl2, 0.5 mm spermidine, 1 mm NaF, 1% nonfat dry milk, and fresh protease and phosphatase inhibitor mixtures]. After incubation for 15 min at 4°C, cells were homogenized in a Dounce homogenizer (50–60 strokes). Released nuclei (5 min, 800 × g) were resuspended in hypertonic buffer [30 mm HEPES (pH 7.9), 25% glycerol, 450 mm NaCl, 12 mm MgCl2, and 0.3 mm Na2EDTA with fresh protease and phosphatase inhibitor mixture] for 45 min at 4°C. The supernatant (30 min; 30,000 × g) was subjected to dialysis for 2 h against the hypertonic buffer containing 150 mm NaCl.

Differential detergent fractionation has been described before (32). In short, a cytosolic enriched fraction of hepatocytes was isolated using ice-cold digitonin buffer [0.01% digitonin, 10 mm PIPES (pH 6.8), 300 mm sucrose, 100 mm NaCl, 3 mm MgCl2, and 5 mm EDTA] and centrifugation at 480 × g. Membrane-enriched fraction was isolated by subjecting the pellet from the above treatment to ice-cold Triton extraction buffer [0.5% Triton X-100, 10 mm PIPES (pH 7.4), 300 mm sucrose, 100 mm NaCl, 3 mm MgCl2, 3 mm EDTA] and centrifugation at 5000 × g (10 min). Nuclear enriched fraction was isolated by treatment of the above pellet in Tween 40/DOC extraction [1% Tween 40, 0.5% deoxycholate, PIPES 10 mm (pH 7.4), 10 mm NaCl, 1 mm MgCl2]. Supernatant after centrifugation 6780 × g is the nuclei-enriched fraction. All of the above buffers had appropriate protease and phosphatase inhibitor mixtures (Sigma Chemical Co.).

The concentration of the protein in the lysates was determined by bicinchoninic acid protein assay with BSA as a standard. Aliquots of the samples were stored at −80°C until use.

Gel Electrophoresis and Western Blotting.

All experiments were performed in triplicate, and the data shown were representative of all three sets of experiments. Fifty μg of protein from the extracts were resolved on ready gels ranging from 5 to 15%, depending on the molecular weight of the target protein, using the mini-PROTEAN 3 electrophoresis module assembly (Bio-Rad, Hercules, CA; Ref. 6). Proteins were subjected to overnight electrophoretic transfer at 30 V and 90 mA in transfer buffer [25 mm Tris (pH 8.3), 192 mm glycine, 20% methanol, and 0.025% SDS] to Immobilon-polyvinylidene difluoride membranes (Millipore, Bedford, MA) using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Blots were blocked with 5% nonfat dry instant milk in Tris-buffered saline-Tween 20 (5% milk Blotto) for 1 h and incubated with primary antibody in 5% milk Blotto for 2 h at room temperature or overnight at 4°C. This was followed by two washes for 10 min each in 1% milk Blotto and incubation with the HRP-conjugated secondary antibody in 1% milk Blotto for 1 h at room temperature. After four washes lasting 10 min each in Blotto, the blot was subjected to fresh SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) for 5 min, and the blot was visualized by autoradiography. Two 30-min washes at room temperature with IgG elution buffer (Pierce, Rockford, IL) were used for stripping the blots for reuse.

The blots were subjected to densitometric analysis after scanning the autoradiographs using NIH Image 1.58 software. The integrated absorbance obtained from this analysis was normalized to the actin levels. These values were plotted using KaleidaGraph software (Synergy software) to analyze quantitative changes.

Primary antibodies including anti β-catenin (mouse), anti-E-cadherin (rabbit), anti-GSK3β (mouse), and anti-APC (rabbit) were used at 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA). Anti-Wnt-1 and anti-T-cell factor 4 were used at 4 μg/ml (Upstate Biotechnology, Inc., Lake Placid, NY). The secondary antibodies including HRP-conjugated, antimouse and antirabbit were used at 1:75,000 (Chemicon, Temecula, CA).

Immunoprecipitation.

Four hundred μg of lysate in a 1-ml volume (in the presence of protease and phosphatase inhibitors) were precleared using appropriate control IgG (normal goat) together with 20 μl of protein A/G agarose for 30 min to 1 h at 4°C (Santa Cruz Biotechnology; Ref. 6). The supernatant obtained after centrifugation (1000 × g) at 4°C was incubated with 5 μl (10 μg) of agarose-conjugated, goat anti-β-catenin antibody (Santa Cruz Biotechnology) for 1 h or overnight at 4°C. Alternatively, the supernatant was incubated with 7 μl of anti-Met antibody (Santa Cruz Biotechnology) or 7 μl of anti-phosphotyrosine antibody PY20 (Transduction Labs) or anti-phosphoserine (Sigma Chemical Co.) for 1 h at 4°C using end-over-end rotation, followed by 20 μl of resuspended protein A/G agarose for 1 h or overnight at 4°C. The pellets were collected by centrifugation (1000 × g) and washed four times for 5 min each with RIPA buffer at 4°C. The pellets were resuspended in an equal volume of standard electrophoresis loading buffer with SDS and fresh β-mercaptoethanol and boiled for 5 min. Thirty μl of the samples were resolved on ready gels and transferred as described earlier. The antibodies used for blotting as well as HRP-conjugated secondary antibodies have been described elsewhere in this report. The blots were stripped and reprobed with the antibodies used for immunoprecipitation so that stoichiometric analysis could be performed.

Immunofluorescence Microscopy.

For the colocalization study, 4-μm liver cryosections were affixed to charged Superfrost/Plus slides (Fisher, Pittsburgh, PA). The staining protocol has been described before (6). Briefly, tissue was blocked in 20% nonimmune goat serum in PBG (PBS, BSA, and glycine) buffer for 30 min at room temperature. Primary antibodies, including anti-β-catenin and anti-Met (Santa Cruz Biotechnology) at 1:50 dilution, were added to sections for 2 h at room temperature. After being washed, the fluorescently tagged secondary antibodies were applied to the sections for 1 h at room temperature. These antibodies were antimouse Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA) at a 1:3000 or antirabbit Alexa 488 (Molecular Probes, Eugene, OR) at a 1:500 dilution. After being washed, the nuclei were counterstained using 0.001% Hoechst dye (bis-benzimide). The tissue was coverslipped using gelvatol. For nuclear localization of β-catenin in primary hepatocyte cultures in response to HGF treatment, freshly isolated hepatocytes from three different animals were plated in six-well culture plates for 2 h, followed by addition of fresh medium without HGF and with HGF at 12.5 or 25 ng/ml for 15 min. Cells were fixed in methanol and stained for β-catenin as described above. Nuclei were counterstained by Sytox Green nucleic acid stain (Molecular Probes, Eugene, OR) at 100 nm in PBS for 30 s. Slides were viewed on a Nikon Eclipse epifluorescence microscope. Digital images were obtained on a Sony CCD camera using Optimas image acquisition software with a frame grabber board. Collages were prepared using Adobe Photoshop 5.0 software.

Construction of an Inducible DN-Met Expression System.

Generation of an inducible DN-Met expression system has been described recently (33). Briefly, plasmid pTet-On was transfected into Hepa1-6 cells. The cDNA for the truncated Met (DN-Met), which encodes the extracellular and transmembrane regions of the mouse Met protein (nucleotides −25 to +2906) was generated by PCR and cloned into the PCR 3.1 vector (InVitrogen, Carlsbad, CA), sequenced, and then subcloned into the pTRE plasmid containing the Tet-responsive promoter (Clontech, Palo Alto, CA). This recombinant plasmid, together with the pTK-Hyg plasmid, was cotransfected into the Hepa1-6 Tet cell line (clone 20) containing the pTet-on regulator plasmid. Positive clones were selected by adding hygromycin. Clone 20-312 was selected for further experimentation because it exhibited high expression of DN-Met after induction with doxycycline.

Cell Line Culture and Treatment.

Clone 20-312 cells (DN-Met Hepa1-6) culture has been described recently (33). Briefly, the cells were cultured in DMEM and serum starved for 24 h, followed by treatment with HGF for 30 min at 50 ng/ml. Induction of DN-Met was achieved by 1 mg/ml doxycycline for 48 h prior to studies. Total cell lysates were used to study association of β-catenin and Met as well as their tyrosine phosphorylation. Nuclear extracts were used to study differences in nuclear β-catenin levels.

HGF Induces Nuclear Translocation of β-Catenin in Primary Hepatocyte Cultures.

To determine whether HGF had any effect on the canonical Wnt signaling pathway components as well as some of the known target genes, we used normal hepatocytes isolated from rat liver. These isolated primary hepatocytes were cultured at high density for 2 h, followed by 15 min of HGF treatment. Proteins from these hepatocytes were isolated by differential detergent fractionation that enriches the lysate for membranes, cytoplasm, and nuclear components. Although this fractionation technique is not a very accurate enrichment method, this protocol allowed us to compare preliminary quantitative changes in the protein levels in response to HGF treatment. No obvious quantitative changes were seen in Wnt-1, GSK3β, T-cell factor 4, or cyclin D1 protein levels in the two groups (Fig. 1,A). An increase in β-catenin protein in the nuclei-enriched fraction was detectable in response to 15 min of treatment of HGF at 12.5 ng/ml (Fig. 1 A). There was also a corresponding increase in c-myc protein in the same compartment. There was a minimal decrease in Wnt-1 protein, if at all, in response to HGF treatment.

To confirm the nuclear redistribution of β-catenin protein in response to HGF treatment, we used traditional nuclear extracts (in HEPES buffer) from the HGF-treated and untreated hepatocytes. There was a significant increase (2–2.5-fold) in nuclear β-catenin protein (P < 0.05) in the presence of HGF at 12.5 ng/ml for 15 min in the hepatocyte cultures (Fig. 1,B). To determine whether there was any effect on the total β-catenin protein in response to HGF, we used whole cell lysates (in RIPA buffer) from the hepatocytes cultured under the two conditions. No significant increase was observed in the total β-catenin protein levels under these conditions (Fig. 1 C). However, we consistently observed a minimal increase in the total β-catenin protein levels that was statistically insignificant.

HGF Induces Nuclear Translocation of β-Catenin in a Dose-dependent Manner.

We analyzed β-catenin protein redistribution to the nuclei of cultured hepatocytes in response to increasing concentrations of HGF in the culture. HGF was added to the hepatocyte cultures at 12.5, 25, 50, and 100 ng/ml of culture medium. The nuclear isolates were tested for β-catenin levels in response to these increasing concentrations of HGF. Nuclear β-catenin appeared to increase in response to an increase in HGF concentration in the hepatocyte cultures (Fig. 2,A). After normalization, we confirmed an initial dose-dependent increase in nuclear β-catenin at 12.5 and 25 ng/ml of HGF with a peak 4-fold increase seen at 25 ng/ml (Fig. 2,B). This affect becomes blunted at higher HGF concentrations with a steady plateau observed in nuclear β-catenin levels at the 50–100 ng/ml HGF concentration. Thus, β-catenin translocation in response to increasing HGF concentration appears to follow first-order kinetics. We also used double immunofluorescence to reconfirm nuclear localization of β-catenin in response to the increasing HGF concentrations. Cy3-conjugated secondary antibody (red) detected β-catenin, and Sytox green was used as a nuclear counterstain. The overlay (yellow) was used to detect nuclear β-catenin. There was minimal nuclear β-catenin in most of the hepatocytes after 2 h of primary hepatocytes cultures without HGF (Fig. 2,C). There was a considerable increase in nuclear β-catenin in response to HGF treatment at 12.5 ng/ml, with a further elevation at 25 ng/ml (Fig. 2, D and E). An increase in nuclear β-catenin was indicated by increasing yellow color in the nuclei of the cultured hepatocytes in the presence of an increased dose of HGF. This also substantiates a dose-dependent redistribution of β-catenin in normal hepatocytes after HGF inclusion in the hepatocyte cultures.

We also wanted to determine the redistribution of β-catenin in response to HGF, as a function of time. Nuclear β-catenin levels were studied at 15, 30, 60, and 90 min after single HGF treatment at 12.5 ng/ml. Elevated β-catenin levels were detected as early as 15 min after HGF treatment, and this increase was maintained through 90 min (Fig. 2,F). After normalization, we conclude that the increase in nuclear β-catenin in response to 12.5 ng/ml of HGF in culture is constant after 15 min and is fairly maintained without any significant changes observed over the 90-min duration of culture (Fig. 2 G). This might imply that the factors responsible for nuclear translocation are still active after 90 min of treatment, thus maintaining continued elevations in nuclear β-catenin protein, and that a single dose of HGF is enough to sustain elevations in nuclear β-catenin protein for an extended period of time.

HGF Does Not Affect E-Cadherin/β-Catenin Complex or Serine/Threonine Phosphorylated β-catenin Levels in Primary Hepatocyte Cultures.

We have shown previously that β-catenin predominantly localizes at the membrane of the hepatocytes with some cytoplasmic distribution. E-cadherin-β-catenin association at the membrane has also been well described previously. To determine the mechanism and source of nuclear mobilization of β-catenin, we decided to study any changes in the E-cadherin-associated β-catenin in response to HGF. Immunoprecipitation studies were used to assess changes in E-cadherin-β-catenin association in response to HGF treatment at 12.5 ng/ml for 15 min in the primary hepatocyte cultures. No apparent change was detected in the E-cadherin-β-catenin association in either culture condition (Fig. 3,A). About 20% of β-catenin appeared to be associated to E-cadherin in normal cultured hepatocytes (data not shown). The stoichiometric analysis of this association depicts no modification in the endogenous E-cadherin-β-catenin complex after HGF treatment (Fig. 3 B). This indicates that the source of β-catenin translocating to the nucleus is very unlikely to be the E-cadherin-associated pool at the membrane. We cannot rule out release of E-cadherin-β-catenin as a complex from the hepatocyte membrane.

β-Catenin also associates to GSK3β in the cytoplasm. GSK3β phosphorylates β-catenin at specific serine and threonine residues to activate its degradation through ubiquitin-proteosome pathway. A decrease in serine-phosphorylated β-catenin would imply stabilization of the protein, resulting in increased levels in its total protein. We were unable to detect any changes in the serine phosphorylated β-catenin levels in the absence or presence of HGF during culture by coprecipitation studies (Fig. 3,C). There have been a few reports about redistribution of β-catenin by HGF in tumor cells through transient inactivation of GSK3β with resulting stabilization of β-catenin protein (9). However, we detected only minimal levels of serine phosphorylated β-catenin in the total cell lysates from hepatocytes that remained unchanged in presence of HGF. We also used immunoprecipitation studies to study the effect of HGF on GSK3β-β-catenin association. Coprecipitation studies using total cell lysates from the hepatocyte cultures detected a minimal association of endogenous β-catenin and GSK3β that remained unchanged upon HGF treatment (Fig. 3,D). Comparing our earlier results that showed consistent minimal stabilization of this protein after HGF treatment, it might be safe to presume that HGF might not be acting predominantly through the canonical Wnt pathway to influence nuclear translocation of β-catenin in the normal cultured hepatocytes, and this effect may contribute, to a limited extent, in the promotion of β-catenin translocation by imparting some stabilization to the protein (Fig. 1 C).

HGF/SF Receptor Met Associates to Endogenous β-Catenin in Normal Rat Liver, and This Complex Dissociates after HGF Treatment.

After ruling out some of the canonical Wnt pathway components to be significantly involved in the nuclear translocation of β-catenin in response to HGF treatment in normal rat hepatocyte cultures, we began investigating any direct association of HGF pathway components with β-catenin. It has been shown previously that β-catenin phosphorylates at specific tyrosine residues, and this negatively influences cell-cell adhesion. We examined an association of endogenous β-catenin to HGF receptor Met, a known receptor tyrosine kinase, in normal rat liver. We demonstrate the association of endogenous Met and β-catenin in normal rat liver. Met and β-catenin coprecipitate in unstimulated normal rat liver cell lysate (Fig. 4,A). We also show Met-β-catenin coprecipitation in lysates from hepatoma cell line (Hepa1-6) after 24-h culture (Fig. 4,B). The blots from the above studies, when subjected to densitometry for stoichiometric analysis of Met-β-catenin association, revealed ∼78% of Met to be associated to β-catenin and ∼33% of β-catenin to be associated to Met in the hepatocytes (Fig. 4,C). Colocalization studies with double immunofluorescence using Met (green) and β-catenin (red) antibodies demonstrate this association at the inner side of the hepatocyte membrane (yellow) in normal rat liver (Fig. 4 D). This study also demonstrates most of the Met to be associated with β-catenin in the hepatocytes.

Next, we determined whether this association underwent any change in the presence of HGF. Using total cell lysates from the hepatocytes cultured in absence or presence of HGF, an association of Met-β-catenin was investigated by coprecipitation studies. A decrease in association of Met to β-catenin occurred in response to HGF (Fig. 4,E). No comparable decrease in total β-catenin protein was observed in the immunoprecipitate. Stoichiometric analysis depicts a significant decrease in Met-β-catenin binding after HGF treatment (P < 0.05) that was apparent at our earliest time point of 15 min after HGF treatment at 12.5 ng/ml (Fig. 4 F). Thus, this complex undergoes dissociation in the presence of HGF with resulting nuclear translocation of β-catenin.

Loss of β-Catenin-Met Association in the DN-Met Expression System.

The purpose of this next study was to confirm the role of HGF/Met signaling in nuclear translocation of β-catenin and to provide further information on the mechanism. Functional inactivation of HGF signaling by DN receptor expression consisting of Met with deleted tyrosine kinase (intercellular) domain was used for this objective. An absence of this domain impairs Met dimerization, resulting in a failure of activation and hence the DN for HGF signaling. Total cell lysates from the Hepa1-6 cell line clone 20-312 cultured in presence of doxycycline for 48 h, which induced the DN-Met expression, was used to coprecipitate β-catenin and Met. Using an NH2-terminal antibody to Met for immunoprecipitation, we detected the higher species (Mr 140,000) representing the wild-type endogenous Met and the lower species (Mr 110,000) representing the truncated Met. None to minimal association of DN-Met and β-catenin was evident in the induced DN-Met-expressing cells (Fig. 5 A). The minimal association observed is apparently attributable to the endogenous wild-type Met in these cells. This demonstrates the requirement of an intact Met for optimal β-catenin-Met association.

Absent Tyrosine Phosphorylation and Nuclear Translocation of β-Catenin in the DN, Met-expressing Cells in Response to HGF.

To further investigate the effect of HGF/Met signaling on β-catenin, we decided to study the ability of HGF to tyrosine phosphorylate β-catenin. We detected tyrosine-phosphorylated Met in the total cell lysates from the Hepa1-6 cells (expressing full-length Met) in response to HGF treatment (50 ng/ml for 30 min) by coprecipitation studies (Fig. 5,B). Similarly, we detected tyrosine-phosphorylated β-catenin in the same lysates, indicating the ability of HGF to serially tyrosine phosphorylate Met and β-catenin because of their close association (Fig. 5,B). Next, lysates from the Hepa1-6 cells (doxycycline treated) expressing truncated Met that underwent similar HGF treatment (50 ng/ml; 30 min) showed no detectable tyrosine-phosphorylated Met or β-catenin by coprecipitation studies (Fig. 5 B). This provides strong evidence of the role of HGF/Met signaling to induce tyrosine phosphorylation of β-catenin in response to elevated HGF levels.

Our final motive was to confirm the usefulness of this association by investigating the result of abrogation of tyrosine phosphorylation of β-catenin on its nuclear translocation in response to HGF treatment. Nuclear lysates from the HGF-treated (50 ng/ml; 30 min) Hepa1-6 cells (expressing full-length or truncated Met) were examined for β-catenin levels. We detected an absence or a failure of increase in the nuclear β-catenin levels in response to HGF in the Hepa1-6 cells expressing the DN-Met as compared with the cells expressing full-length Met (Fig. 5,C). The nuclear levels of β-catenin in the DN-Met-induced cells were comparable with the hepatocytes that were not treated with HGF (Fig. 1,B). This difference in the nuclear β-catenin levels in response to HGF in the uninduced and induced DN-Met cells was statistically significant (P < 0.05; Fig. 5 D). The above data strongly suggest the role of HGF/Met signaling in tyrosine phosphorylation-dependent Met-β-catenin dissociation with the resulting nuclear translocation of β-catenin.

β-Catenin is a pivotal component of the canonical Wnt pathway. It has been shown to be part of a transactivating complex with T-cell factor/lymphoid enhancement factor family members that induces several target genes in response to classical Wnt signal (5, 11). This event has been shown to follow stabilization of β-catenin protein by inactivation of the ubiquitin-proteosome pathway that comprises specific interactions with GSK3β, Axin, and APC, and this event is serine/threonine phosphorylation dependent (7, 9, 34, 35). Nuclear localization of this protein is primarily associated with induction or repression of several target genes, expressions depending on cell and tissue type (4, 14, 36, 37). Another major cellular component that β-catenin associates to is the adherens junction at the membrane of the cell, where it acts a linker between cadherins and actin cytoskeleton (38). It has been shown previously that β-catenin can become tyrosine phosphorylated, and this modification favors negative regulation of cell-cell adhesion by dissociation of the cadherin-catenin complex (16, 28). The fate of β-catenin upon tyrosine phosphorylation in response to certain growth factors is largely unknown. We demonstrate tyrosine phosphorylation-dependent nuclear translocation of β-catenin in response to hepatocyte growth factors in the normal hepatocytes. We thus identify a significant cross-talk between the Wnt and HGF pathways because of an direct interaction between endogenous Met and β-catenin.

Using normal rat liver and primary hepatocytes cultures, we demonstrate a basal functional association between endogenous Met and β-catenin. We provide further evidence that this association is significantly lost during HGF signaling, resulting in alteration in steady-state kinetics of the β-catenin protein. Some studies have previously shown stabilization of β-catenin protein by growth factors such as epidermal growth factor and HGF through their effect on GSK3β (9, 27, 39, 40). We were unable to detect any significant changes in GSK3β or serine/threonine phosphorylation of β-catenin in response to HGF in the normal rat hepatocyte cultures. However, we consistently observed a minimal increase in the total β-catenin protein that although it was statistically insignificant, it favored some stabilization. The above two factors might be acting in conjunction to induce β-catenin nuclear translocation in response to HGF in hepatocytes.

Importantly, we found a novel association between HGF receptor Met and β-catenin in normal hepatocytes. About 80% of Met is associated with β-catenin, and about 30–40% of β-catenin is associated with Met at the inner side of the hepatocyte membrane in normal rat liver. β-Catenin is also associated with E-cadherin at the hepatocyte membrane, but stoichiometrically, this association is lower when compared with the Met-β-catenin association. Although a few previous reports have demonstrated association of Met with cadherin complex in tumor cells, we were unable to detect any direct association of Met and E-cadherin in normal hepatocytes (26). Thus, we can conclude that the Met and β-catenin complex might exist as a predominant complex and a functionally important pool of β-catenin in hepatocytes.

Addition of HGF to the cultured hepatocytes induces Met tyrosine phosphorylation (Fig. 6). This in turn brings on phosphorylation of β-catenin at specific tyrosine residues because of a direct association between the two proteins. At this time, we cannot rule out an interplay of an intermediate adapter molecule like Gab-1 that might augment this association. Tyrosine phosphorylation of β-catenin favors dissociation of the Met-β-catenin complex without affecting the E-cadherin-β-catenin association that might be an independent pool at the membrane. These specific events along with a minimal β-catenin stabilization (GSK3β hypoactivity) result in nuclear translocation of β-catenin upon HGF treatment in hepatocyte cultures. We cannot rule out dissociation of E-cadherin-β-catenin as a complex from the Met at the hepatocyte membrane upon HGF stimulation. However, this process of coendocytosis is more likely to be associated in a recycling process of the membrane-associated proteins controlling their turnover, and nuclear translocation is an unlikely consequence of this process (41). Subcellular dimerization of β-catenin protein associating the Met-β-catenin complex to the E-cadherin-β-catenin complex is yet another possibility.

HGF/SF has diverse functional roles including morphogenesis, mitogenesis, and motogenesis (18). It has been shown to function through several components including phosphatidylinositol 3-kinase, phospholipase Cγ, STAT3, and others. Association of HGF receptor Met with β-catenin provides strong evidence to explain the cross-talk and functional coincidences between the HGF/Met and Wnt/β-catenin pathways. On the basis of our observations, we propose that β-catenin is present as a third independent pool at the membrane of hepatocyte as a Met-β-catenin complex, and this is an important regulator of free β-catenin levels and signaling in hepatocytes. We have demonstrated the redistribution of β-catenin in a hepatocyte and shown that the source of this β-catenin is Met-β-catenin complex. We have observed a minimal contribution from GSK3β or E-cadherin-associated β-catenin toward its nuclear levels, after HGF stimulation in the hepatocyte cultures. This substantiates cell and tissue-specific differences in protein-protein interactions observed in the Wnt pathway, where a different molecule may be playing a more important role in regulating β-catenin levels in one cell type versus another.

Tyrosine phosphorylation of β-catenin has been shown to be important for cell-cell adhesion (16). However, we report nuclear localization of this protein upon HGF stimulation that supports its role in regulating gene expression after its tyrosine phosphorylation. We have satisfactorily shown in this report that tyrosine phosphorylation of β-catenin favors its dissociation from Met in response to HGF. Using the DN-Met expression system, we demonstrate the importance of an intact tyrosine kinase domain of Met to tyrosine-phosphorylate β-catenin. We demonstrate complete abrogation of nuclear translocation of β-catenin in the DN-Met cells. We propose that HGF induces tyrosine phosphorylation-dependent nuclear translocation of β-catenin that might be Wnt independent in hepatocyte cultures. This might be an important factor that regulates target gene expression, especially in hepatocytes. This might be significant clinically because increased serum HGF levels have been reported previously in several liver disease states including advanced hepatic cirrhosis, hepatocellular cancers, or acute liver failure (42, 43, 44). We are investigating the in vivo significance of elevated HGF and its effect on the canonical Wnt pathway. Our previous study on analysis of canonical Wnt pathway during liver regeneration provides some in vivo correlation to this novel observation (6). Heightened nuclear translocation of β-catenin was observed at 5 min during liver regeneration that corresponds to an early peak of tyrosine phosphorylation of Met after partial hepatectomy (45). More in vivo studies are under way to directly investigate this correlation. This might provide insight into the molecular basis of hepatic tumorigenesis in an unexplainable subset of hepatocellular cancers and progression of other disease states associated with elevated HGF levels.

Fig. 1.

HGF promotes nuclear translocation of β-catenin in primary hepatocyte cultures. A, differential detergent fractionation technique was used to enrich the lysate for cytoplasm, membranes, and nuclei from hepatocytes (+/− HGF). Western blot analysis depicts increased levels of β-catenin and c-myc protein in the nuclei-enriched fraction and minor increase in cytoplasmic cyclin D1 levels. There was a minor decrease in Wnt-1 protein levels in the presence of HGF. No other significant changes were observed. B, nuclear extracts were prepared in HEPES for the same two conditions. A representative Western blot shows significant increase (P < 0.05) in nuclear β-catenin levels in the presence of HGF. Bars, SE. w/, with; w/o, without. C, total cell lysate was prepared in RIPA buffer for the same two conditions. A representative blot demonstrates minimal although consistent increase that was statistically insignificant (P > 0.05) after HGF treatment. Bars, SE. w/, with; w/o, without.

Fig. 1.

HGF promotes nuclear translocation of β-catenin in primary hepatocyte cultures. A, differential detergent fractionation technique was used to enrich the lysate for cytoplasm, membranes, and nuclei from hepatocytes (+/− HGF). Western blot analysis depicts increased levels of β-catenin and c-myc protein in the nuclei-enriched fraction and minor increase in cytoplasmic cyclin D1 levels. There was a minor decrease in Wnt-1 protein levels in the presence of HGF. No other significant changes were observed. B, nuclear extracts were prepared in HEPES for the same two conditions. A representative Western blot shows significant increase (P < 0.05) in nuclear β-catenin levels in the presence of HGF. Bars, SE. w/, with; w/o, without. C, total cell lysate was prepared in RIPA buffer for the same two conditions. A representative blot demonstrates minimal although consistent increase that was statistically insignificant (P > 0.05) after HGF treatment. Bars, SE. w/, with; w/o, without.

Close modal
Fig. 2.

HGF induces a dose-dependent increase in the nuclear translocation of β-catenin. A, a representative Western blot shows an increase in nuclear β-catenin protein with increasing doses of HGF. A substantial increase was evident at 12.5 and 25 ng/ml. The same blot was stripped and probed for β-actin and demonstrates equal loading. B, the representative Western blot was subjected to densitometric analysis and shows an almost 3-fold increase in quantity of nuclear β-catenin protein levels after HGF treatment at 12.5 ng/ml. There was a further elevation in its levels at 25 ng/ml (4-fold increase). No further increase is observed with any more increase in the dose of HGF and the affect plateaus. C, cultured cells were stained for β-catenin (red) and counterstained with Sytox green. Nuclear localization of β-catenin was confirmed by overlay (yellow). In the absence of HGF, most of the hepatocytes showed minimal nuclear β-catenin. D, in the presence of HGF at 12.5 ng/ml of HGF, there was a substantial increase in nuclear β-catenin as shown. E, there was a further increase in nuclear β-catenin in cultured hepatocytes in the presence of 25 ng/ml of HGF as shown. F, a representative Western blot analysis shows an increase in nuclear β-catenin after 15 min of HGF treatment at 12.5 ng/ml. This increase was maintained throughout the duration of the experiment for 90 min after single treatment. The bottom panel confirms equal protein loading by comparable β-actin levels confirmed by Western blot. G, densitometric analysis of the Western blot reconfirms an initial increase at 15 min. This increase is ∼3-fold and maintained through 90 min. Fifteen min was the earliest time point we used for the experiment.

Fig. 2.

HGF induces a dose-dependent increase in the nuclear translocation of β-catenin. A, a representative Western blot shows an increase in nuclear β-catenin protein with increasing doses of HGF. A substantial increase was evident at 12.5 and 25 ng/ml. The same blot was stripped and probed for β-actin and demonstrates equal loading. B, the representative Western blot was subjected to densitometric analysis and shows an almost 3-fold increase in quantity of nuclear β-catenin protein levels after HGF treatment at 12.5 ng/ml. There was a further elevation in its levels at 25 ng/ml (4-fold increase). No further increase is observed with any more increase in the dose of HGF and the affect plateaus. C, cultured cells were stained for β-catenin (red) and counterstained with Sytox green. Nuclear localization of β-catenin was confirmed by overlay (yellow). In the absence of HGF, most of the hepatocytes showed minimal nuclear β-catenin. D, in the presence of HGF at 12.5 ng/ml of HGF, there was a substantial increase in nuclear β-catenin as shown. E, there was a further increase in nuclear β-catenin in cultured hepatocytes in the presence of 25 ng/ml of HGF as shown. F, a representative Western blot analysis shows an increase in nuclear β-catenin after 15 min of HGF treatment at 12.5 ng/ml. This increase was maintained throughout the duration of the experiment for 90 min after single treatment. The bottom panel confirms equal protein loading by comparable β-actin levels confirmed by Western blot. G, densitometric analysis of the Western blot reconfirms an initial increase at 15 min. This increase is ∼3-fold and maintained through 90 min. Fifteen min was the earliest time point we used for the experiment.

Close modal
Fig. 3.

Affect of HGF on β-catenin-E-cadherin complex, β-catenin-GSK3β complex, and serine-phosphorylated β-catenin. A, a representative immunoprecipitation study shown suggests no change in association of β-catenin-E-cadherin in the presence of HGF. The hepatocytes were cultured for 2 h, and one batch was treated with HGF at 12.5 ng/ml versus no HGF for 15 min. Total cell lysates from the above conditions were immunoprecipitated by β-catenin and probed for E-cadherin. The blot was stripped and reprobed for β-catenin for stochiometric analysis. IP, immunoprecipitation. B, after normalization for β-catenin levels, the E-cadherin Western blots were subjected to densitometric analysis. HGF does not influence the stoichiometry of β-catenin-E-cadherin association in hepatocyte cultures at 12.5 ng/ml. C, representative blot shows minimal change in serine-phosphorylated β-catenin after HGF treatment of the hepatocytes in culture. Phosphoserine antibody was used for immunoprecipitation, and the products were analyzed by Western blot using antibody against β-catenin. IP, immunoprecipitation. D, a representative blot depicts no obvious changes in the β-catenin-GSK3β association after HGF treatment in hepatocyte cultures. Total cell lysates were subjected to immunoprecipitation by anti-β-catenin. IP, immunoprecipitation.

Fig. 3.

Affect of HGF on β-catenin-E-cadherin complex, β-catenin-GSK3β complex, and serine-phosphorylated β-catenin. A, a representative immunoprecipitation study shown suggests no change in association of β-catenin-E-cadherin in the presence of HGF. The hepatocytes were cultured for 2 h, and one batch was treated with HGF at 12.5 ng/ml versus no HGF for 15 min. Total cell lysates from the above conditions were immunoprecipitated by β-catenin and probed for E-cadherin. The blot was stripped and reprobed for β-catenin for stochiometric analysis. IP, immunoprecipitation. B, after normalization for β-catenin levels, the E-cadherin Western blots were subjected to densitometric analysis. HGF does not influence the stoichiometry of β-catenin-E-cadherin association in hepatocyte cultures at 12.5 ng/ml. C, representative blot shows minimal change in serine-phosphorylated β-catenin after HGF treatment of the hepatocytes in culture. Phosphoserine antibody was used for immunoprecipitation, and the products were analyzed by Western blot using antibody against β-catenin. IP, immunoprecipitation. D, a representative blot depicts no obvious changes in the β-catenin-GSK3β association after HGF treatment in hepatocyte cultures. Total cell lysates were subjected to immunoprecipitation by anti-β-catenin. IP, immunoprecipitation.

Close modal
Fig. 4.

Endogenous Met-β-catenin association and effect of HGF on this complex. A, endogenous Met-β-catenin association is shown in a representative immunoprecipitation study. Immunoprecipitation was carried out in total cell lysate from normal rat liver by anti-Met and β-catenin antibodies. The Western blots show coprecipitation of endogenous Met and β-catenin in unstimulated normal rat liver. B, coprecipitation studies using hepatoma cells also demonstrate similar association of Met-β-catenin. IP, immunoprecipitation. C, stoichiometric analysis of blots from the coprecipitation studies on Met-β-catenin in normal rat liver as well as hepatoma cells demonstrates at least 78% of total Met to be associated with β-catenin (shaded area) and ∼33% of total β-catenin (shaded) to be associated with Met in these cells. D, double immunofluorescence showed colocalization (yellow) of Met (green) and β-catenin (red) at the inner side of the hepatocyte membrane. The nuclei were counterstained with Hoechst dye (blue). Arrowheads, colocalization. E, HGF promotes dissociation of Met-β-catenin complex, as can be seen in the representative coprecipitation studies. There are decreases in association of β-catenin and Met in the presence of 12.5 ng/ml of HGF in hepatocyte cultures. The total β-catenin in immunoprecipitate is comparable, as can be seen in the lower panel. IP, immunoprecipitation. F, after normalization for β-catenin levels, the Western blots were subjected to densitometric analysis. The bar graph depicts a significant (P < 0.05) decrease in association of Met and β-catenin in the presence of 12.5 ng/ml of HGF in the hepatocyte cultures.

Fig. 4.

Endogenous Met-β-catenin association and effect of HGF on this complex. A, endogenous Met-β-catenin association is shown in a representative immunoprecipitation study. Immunoprecipitation was carried out in total cell lysate from normal rat liver by anti-Met and β-catenin antibodies. The Western blots show coprecipitation of endogenous Met and β-catenin in unstimulated normal rat liver. B, coprecipitation studies using hepatoma cells also demonstrate similar association of Met-β-catenin. IP, immunoprecipitation. C, stoichiometric analysis of blots from the coprecipitation studies on Met-β-catenin in normal rat liver as well as hepatoma cells demonstrates at least 78% of total Met to be associated with β-catenin (shaded area) and ∼33% of total β-catenin (shaded) to be associated with Met in these cells. D, double immunofluorescence showed colocalization (yellow) of Met (green) and β-catenin (red) at the inner side of the hepatocyte membrane. The nuclei were counterstained with Hoechst dye (blue). Arrowheads, colocalization. E, HGF promotes dissociation of Met-β-catenin complex, as can be seen in the representative coprecipitation studies. There are decreases in association of β-catenin and Met in the presence of 12.5 ng/ml of HGF in hepatocyte cultures. The total β-catenin in immunoprecipitate is comparable, as can be seen in the lower panel. IP, immunoprecipitation. F, after normalization for β-catenin levels, the Western blots were subjected to densitometric analysis. The bar graph depicts a significant (P < 0.05) decrease in association of Met and β-catenin in the presence of 12.5 ng/ml of HGF in the hepatocyte cultures.

Close modal
Fig. 5.

Intact intracellular (tyrosine kinase) domain of Met is required for HGF-induced nuclear translocation of β-catenin. A, both species of Met, the Mr 140,000 wild-type endogenous (β-subunit) as well as the Mr 110,000 DN-truncated Met, were detected after induction by doxycycline treatment, as seen in the upper right panel. Coprecipitation and Western blots were done using NH2-terminal anti-Met and anti-β-catenin antibodies. No or a minimal Met-β-catenin association was observed in the DN cells. IP, immunoprecipitation. B, total cell lysates from DN Hepa1-6 cells (+/− doxycycline) cells were immunoprecipitated for tyrosine-phosphorylated proteins using anti-phosphotyrosine antibody (PY20) after 30 min of HGF treatment at 50 g/ml. The Western blots were analyzed for Met and β-catenin. Left panel, tyrosine-phosphorylated Met and β-catenin in response to HGF treatment in the uninduced DN cells. There was no detectable tyrosine-phosphorylated Met or β-catenin in the induced DN-Met-expressing cells (after doxycycline treatment) in response to HGF. IP, immunoprecipitation. C, a representative blot shown here depicts failure of nuclear β-catenin levels to increase in the presence of HGF in the Hepa1-6 cells expressing DN-Met as compared with the wild-type Met (uninduced versus induced DN-Met). This nuclear β-catenin in the induced DN cells was comparable with the nuclear β-catenin in the unstimulated hepatocyte cultures. D, densitometric analysis demonstrates the difference between the nuclear β-catenin in the two conditions to be statistically significant (P < 0.05). After normalizing for loading, we observed a 50% less nuclear β-catenin in response to HGF in the DN Hepa1-6 cells that was comparable with the normal nuclear β-catenin levels in the non-HGF-treated hepatocytes. Bars, SD.

Fig. 5.

Intact intracellular (tyrosine kinase) domain of Met is required for HGF-induced nuclear translocation of β-catenin. A, both species of Met, the Mr 140,000 wild-type endogenous (β-subunit) as well as the Mr 110,000 DN-truncated Met, were detected after induction by doxycycline treatment, as seen in the upper right panel. Coprecipitation and Western blots were done using NH2-terminal anti-Met and anti-β-catenin antibodies. No or a minimal Met-β-catenin association was observed in the DN cells. IP, immunoprecipitation. B, total cell lysates from DN Hepa1-6 cells (+/− doxycycline) cells were immunoprecipitated for tyrosine-phosphorylated proteins using anti-phosphotyrosine antibody (PY20) after 30 min of HGF treatment at 50 g/ml. The Western blots were analyzed for Met and β-catenin. Left panel, tyrosine-phosphorylated Met and β-catenin in response to HGF treatment in the uninduced DN cells. There was no detectable tyrosine-phosphorylated Met or β-catenin in the induced DN-Met-expressing cells (after doxycycline treatment) in response to HGF. IP, immunoprecipitation. C, a representative blot shown here depicts failure of nuclear β-catenin levels to increase in the presence of HGF in the Hepa1-6 cells expressing DN-Met as compared with the wild-type Met (uninduced versus induced DN-Met). This nuclear β-catenin in the induced DN cells was comparable with the nuclear β-catenin in the unstimulated hepatocyte cultures. D, densitometric analysis demonstrates the difference between the nuclear β-catenin in the two conditions to be statistically significant (P < 0.05). After normalizing for loading, we observed a 50% less nuclear β-catenin in response to HGF in the DN Hepa1-6 cells that was comparable with the normal nuclear β-catenin levels in the non-HGF-treated hepatocytes. Bars, SD.

Close modal
Fig. 6.

Proposed model of nuclear translocation of β-catenin in response to HGF signaling. HGF induces tyrosine phosphorylation of Met that in turn phosphorylates β-catenin at tyrosine residues. This causes dissociation of β-catenin from Met at the membrane (A). E-cadherin-β-catenin appears to be an independent pool at the membrane of the hepatocyte, although we cannot rule out the possibility of dimerization of β-catenin (B). HGF might induce dissociation from Met of the β-catenin-E-cadherin complex itself, with resulting nuclear translocation of β-catenin (C). Minimal cytoplasmic stabilization of β-catenin after its release from the hepatocyte membrane might be through GSK3β hypoactivation, promoting nuclear translocation of β-catenin.

Fig. 6.

Proposed model of nuclear translocation of β-catenin in response to HGF signaling. HGF induces tyrosine phosphorylation of Met that in turn phosphorylates β-catenin at tyrosine residues. This causes dissociation of β-catenin from Met at the membrane (A). E-cadherin-β-catenin appears to be an independent pool at the membrane of the hepatocyte, although we cannot rule out the possibility of dimerization of β-catenin (B). HGF might induce dissociation from Met of the β-catenin-E-cadherin complex itself, with resulting nuclear translocation of β-catenin (C). Minimal cytoplasmic stabilization of β-catenin after its release from the hepatocyte membrane might be through GSK3β hypoactivation, promoting nuclear translocation of β-catenin.

Close modal

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

1

This work was supported by NIH Grants CA30241 and CA35373 (to G. K. M.) and Pathology Postdoctoral Research Grant (to S. P. S. M.).

3

The abbreviations used are: GSK3β, glycogen synthase kinase-3β; APC, adenomatous polyposis coli gene product; HGF, hepatocyte growth factor; SF, scatter factor; HRP, horseradish peroxidase; DN, dominant negative.

We thank Dr. Donna Beer Stolz and Mark Ross for assistance with fluorescence microscopy. We also acknowledge strong technical assistance provided by Kari Nejak in differential detergent fractionation, Western blots, and immunoprecipitation studies.

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