Download Hepatocyte Growth Factor Induces Wnt

[CANCER RESEARCH 62, 2064 –2071, April 1, 2002]
Hepatocyte Growth Factor Induces Wnt-independent Nuclear Translocation of ␤Catenin after Met-␤-Catenin Dissociation in Hepatocytes1
Satdarshan P. S. Monga, Wendy M. Mars, Peter Pediaditakis, Aaron Bell, Karen Mulé, William C. Bowen,
Xue Wang, Reza Zarnegar, and George K. Michalopoulos2
Division of Cellular and Molecular Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261
ABSTRACT
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 ␤-catenin and Met, a tyrosine kinase receptor of HGF, at
the inner surface of the hepatocyte membrane. HGF induces dose-dependent nuclear translocation of ␤-catenin in primary hepatocyte cultures
that is Wnt independent. The source of ␤-catenin for translocation in
hepatocytes is the Met-␤-catenin complex, which appears to be independent of the E-cadherin-␤-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 ␤subunit of Met. A loss of Met-␤-catenin association resulted in abrogation
of nuclear translocation of ␤-catenin upon HGF stimulation. This event is
tyrosine phosphorylation dependent, and the association of Met and
␤-catenin is crucial for this event. We conclude that the HGF causes
similar redistribution of ␤-catenin as Wnt-1 in the hepatocytes and that
this effect is attributable to subcellular association of Met and ␤-catenin.
The intracellular kinase domain of Met is essential for tyrosine phosphorylation and nuclear translocation of ␤-catenin. Part of the multifunctionality of HGF might be attributable to nuclear ␤-catenin and the resulting
target gene expression.
INTRODUCTION
Wnt signaling pathway has been shown to play diverse roles
during embryogenesis and carcinogenesis (1–3). ␤-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 ␤-catenin are tightly regulated with a
minimal free monomeric form available inside a normal cell and
during regulated growth (3, 6, 7). ␤-Catenin protein is bound to
either the GSK3␤/Axin/APC3 complex or E-cadherin inside a cell.
Upon signaling by Wnt through its receptor frizzled and interactions with dishevelled, the GSK3␤/Axin/APC/␤-catenin complex
undergoes dephosphorylation at specific serine and threonine residues (8 –11). This causes ␤-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 ␤-catenin protein (2, 12). Also, mutations affecting
serine/threonine phosphorylation sites in the ␤-catenin gene result
in a stable protein that is resistant to ubiquitination, leading to
enhanced cell proliferation and tumor formation (1, 13–15).
Previously, we have shown predominant ␤-catenin localization at
the hepatocyte membrane in a normal adult rat liver with some
cytoplasmic staining, suggesting a minimal association with GSK3␤/
Axin/APC complex (6). ␤-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 ␤-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 –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 ␤-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, 23–25). 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 ␤-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 ␤-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 Wntindependent redistribution of ␤-catenin because of Met-␤-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 ␤-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.
MATERIALS AND METHODS
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 (⌬5 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
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Received 8/22/01; accepted 1/29/02.
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.).
2
To whom requests for reprints should be addressed, at Department of Pathology,
University of Pittsburgh, School of Medicine, S410 Biomedical Science Tower, 200
Lothrop Street, Pittsburgh, PA 15261. Phone: (412) 648-1040; Fax: (412) 648-9846;
E-mail: [email protected].
3
The abbreviations used are: GSK3␤, glycogen synthase kinase-3␤; APC, adenomatous polyposis coli gene product; HGF, hepatocyte growth factor; SF, scatter factor; HRP,
horseradish peroxidase; DN, dominant negative.
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HGF INDUCES NUCLEAR TRANSLOCATION OF ␤-CATENIN
collagenase perfusion protocol as described previously from our laboratory secondary antibodies including HRP-conjugated, antimouse and antirabbit
(29, 30). Rat hepatocytes were plated at high density (1.5 ⫻ 106 cells/ml) after were used at 1:75,000 (Chemicon, Temecula, CA).
wet collagen coating (10% Vitrogen) for 1 h on 175-cm2 plates unless stated
Immunoprecipitation. Four hundred ␮g of lysate in a 1-ml volume (in the
otherwise. Hepatocytes were allowed to attach for 2 h in basal hepatocyte presence of protease and phosphatase inhibitors) were precleared using approgrowth medium (31). This was replaced by fresh hepatocyte growth medium priate control IgG (normal goat) together with 20 ␮l of protein A/G agarose for
with/without HGF at 12.5 ng/ml for 15 min (unless stated otherwise), and cells 30 min to 1 h at 4°C (Santa Cruz Biotechnology; Ref. 6). The supernatant
were used for protein isolation.
obtained after centrifugation (1000 ⫻ g) at 4°C was incubated with 5 ␮l (10
Preparation of Total Cell Lysates, Nuclear Extracts, and Differential ␮g) of agarose-conjugated, goat anti-␤-catenin antibody (Santa Cruz BiotechDetergent Fractionation. Hepatocytes from the culture plates were washed in nology) for 1 h or overnight at 4°C. Alternatively, the supernatant was
PBS, and total cell lysate was prepared in RIPA buffer (9.1 mM dibasic sodium
incubated with 7 ␮l of anti-Met antibody (Santa Cruz Biotechnology) or 7 ␮l
phosphate, 1.7 mM monobasic sodium phosphate, 150 mM sodium chloride,
of anti-phosphotyrosine antibody PY20 (Transduction Labs) or anti-phospho1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS, pH adjusted to 7.4)
serine (Sigma Chemical Co.) for 1 h at 4°C using end-over-end rotation,
containing fresh protease and phosphatase inhibitor mixture (Sigma Chemical
followed by 20 ␮l of resuspended protein A/G agarose for 1 h or overnight at
Co.; Ref. 6). Nuclear extracts were prepared in HEPES buffer (30). Briefly,
4°C.
The pellets were collected by centrifugation (1000 ⫻ g) and washed four
cells were washed and harvested in PBS (80 g) and resuspended in 750 ␮l of
hypotonic buffer [10 mM HEPES (pH 7.9), 10 mM NaH2PO4, 1.5 mM MgCl2, times for 5 min each with RIPA buffer at 4°C. The pellets were resuspended
0.5 mM spermidine, 1 mM NaF, 1% nonfat dry milk, and fresh protease and in an equal volume of standard electrophoresis loading buffer with SDS and
phosphatase inhibitor mixtures]. After incubation for 15 min at 4°C, cells were fresh ␤-mercaptoethanol and boiled for 5 min. Thirty ␮l of the samples were
homogenized in a Dounce homogenizer (50 – 60 strokes). Released nuclei (5 resolved on ready gels and transferred as described earlier. The antibodies used
min, 800 ⫻ g) were resuspended in hypertonic buffer [30 mM HEPES (pH 7.9), for blotting as well as HRP-conjugated secondary antibodies have been de25% glycerol, 450 mM NaCl, 12 mM MgCl2, and 0.3 mM Na2EDTA with fresh scribed elsewhere in this report. The blots were stripped and reprobed with the
protease and phosphatase inhibitor mixture] for 45 min at 4°C. The supernatant antibodies used for immunoprecipitation so that stoichiometric analysis could
(30 min; 30,000 ⫻ g) was subjected to dialysis for 2 h against the hypertonic be performed.
buffer containing 150 mM NaCl.
Immunofluorescence Microscopy. For the colocalization study, 4-␮m
Differential detergent fractionation has been described before (32). In short, liver cryosections were affixed to charged Superfrost/Plus slides (Fisher,
a cytosolic enriched fraction of hepatocytes was isolated using ice-cold digi- Pittsburgh, PA). The staining protocol has been described before (6).
tonin buffer [0.01% digitonin, 10 mM PIPES (pH 6.8), 300 mM sucrose, 100 Briefly, tissue was blocked in 20% nonimmune goat serum in PBG (PBS,
mM NaCl, 3 mM MgCl2, and 5 mM EDTA] and centrifugation at 480 ⫻ g.
BSA, and glycine) buffer for 30 min at room temperature. Primary antiMembrane-enriched fraction was isolated by subjecting the pellet from the bodies, including anti-␤-catenin and anti-Met (Santa Cruz Biotechnology)
above treatment to ice-cold Triton extraction buffer [0.5% Triton X-100, 10 at 1:50 dilution, were added to sections for 2 h at room temperature. After
mM PIPES (pH 7.4), 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 3 mM
being washed, the fluorescently tagged secondary antibodies were applied
EDTA] and centrifugation at 5000 ⫻ g (10 min). Nuclear enriched fraction was to the sections for 1 h at room temperature. These antibodies were antiisolated by treatment of the above pellet in Tween 40/DOC extraction [1%
mouse Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA) at a
Tween 40, 0.5% deoxycholate, PIPES 10 mM (pH 7.4), 10 mM NaCl, 1 mM
1:3000 or antirabbit Alexa 488 (Molecular Probes, Eugene, OR) at a 1:500
MgCl2]. Supernatant after centrifugation 6780 ⫻ g is the nuclei-enriched
dilution. After being washed, the nuclei were counterstained using 0.001%
fraction. All of the above buffers had appropriate protease and phosphatase
Hoechst dye (bis-benzimide). The tissue was coverslipped using gelvatol.
inhibitor mixtures (Sigma Chemical Co.).
The concentration of the protein in the lysates was determined by bicin- For nuclear localization of ␤-catenin in primary hepatocyte cultures in
response to HGF treatment, freshly isolated hepatocytes from three differchoninic acid protein assay with BSA as a standard. Aliquots of the samples
ent animals were plated in six-well culture plates for 2 h, followed by
were stored at ⫺80°C until use.
Gel Electrophoresis and Western Blotting. All experiments were per- addition of fresh medium without HGF and with HGF at 12.5 or 25 ng/ml
formed in triplicate, and the data shown were representative of all three sets of for 15 min. Cells were fixed in methanol and stained for ␤-catenin as
experiments. Fifty ␮g of protein from the extracts were resolved on ready gels described above. Nuclei were counterstained by Sytox Green nucleic acid
ranging from 5 to 15%, depending on the molecular weight of the target stain (Molecular Probes, Eugene, OR) at 100 nM in PBS for 30 s. Slides
protein, using the mini-PROTEAN 3 electrophoresis module assembly (Bio- were viewed on a Nikon Eclipse epifluorescence microscope. Digital
Rad, Hercules, CA; Ref. 6). Proteins were subjected to overnight electro- images were obtained on a Sony CCD camera using Optimas image
phoretic transfer at 30 V and 90 mA in transfer buffer [25 mM Tris (pH 8.3), acquisition software with a frame grabber board. Collages were prepared
192 mM glycine, 20% methanol, and 0.025% SDS] to Immobilon-polyvinyli- using Adobe Photoshop 5.0 software.
dene difluoride membranes (Millipore, Bedford, MA) using Mini Trans-Blot
Construction of an Inducible DN-Met Expression System. Generation
Electrophoretic Transfer Cell (Bio-Rad). Blots were blocked with 5% nonfat of an inducible DN-Met expression system has been described recently (33).
dry instant milk in Tris-buffered saline-Tween 20 (5% milk Blotto) for 1 h and Briefly, plasmid pTet-On was transfected into Hepa1-6 cells. The cDNA for
incubated with primary antibody in 5% milk Blotto for 2 h at room temperature
the truncated Met (DN-Met), which encodes the extracellular and transmemor overnight at 4°C. This was followed by two washes for 10 min each in 1% brane regions of the mouse Met protein (nucleotides ⫺25 to ⫹2906) was
milk Blotto and incubation with the HRP-conjugated secondary antibody in generated by PCR and cloned into the PCR 3.1 vector (InVitrogen, Carlsbad,
1% milk Blotto for 1 h at room temperature. After four washes lasting 10 min CA), sequenced, and then subcloned into the pTRE plasmid containing the
each in Blotto, the blot was subjected to fresh SuperSignal West Pico ChemiTet-responsive promoter (Clontech, Palo Alto, CA). This recombinant plasluminescent Substrate (Pierce, Rockford, IL) for 5 min, and the blot was
mid, together with the pTK-Hyg plasmid, was cotransfected into the Hepa1-6
visualized by autoradiography. Two 30-min washes at room temperature with
Tet cell line (clone 20) containing the pTet-on regulator plasmid. Positive
IgG elution buffer (Pierce, Rockford, IL) were used for stripping the blots for
clones were selected by adding hygromycin. Clone 20-312 was selected for
reuse.
The blots were subjected to densitometric analysis after scanning the auto- further experimentation because it exhibited high expression of DN-Met after
radiographs using NIH Image 1.58 software. The integrated absorbance ob- induction with doxycycline.
Cell Line Culture and Treatment. Clone 20-312 cells (DN-Met Hepa1-6)
tained from this analysis was normalized to the actin levels. These values were
plotted using KaleidaGraph software (Synergy software) to analyze quantita- 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
tive changes.
Primary antibodies including anti ␤-catenin (mouse), anti-E-cadherin (rab- min at 50 ng/ml. Induction of DN-Met was achieved by 1 mg/ml doxycycline
bit), anti-GSK3␤ (mouse), and anti-APC (rabbit) were used at 1:200 (Santa for 48 h prior to studies. Total cell lysates were used to study association of
Cruz Biotechnology, Santa Cruz, CA). Anti-Wnt-1 and anti-T-cell factor 4 ␤-catenin and Met as well as their tyrosine phosphorylation. Nuclear extracts
were used at 4 ␮g/ml (Upstate Biotechnology, Inc., Lake Placid, NY). The were used to study differences in nuclear ␤-catenin levels.
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HGF INDUCES NUCLEAR TRANSLOCATION OF ␤-CATENIN
RESULTS
HGF Induces Nuclear Translocation of ␤-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, GSK3␤,
T-cell factor 4, or cyclin D1 protein levels in the two groups (Fig. 1A).
An increase in ␤-catenin protein in the nuclei-enriched fraction was
detectable in response to 15 min of treatment of HGF at 12.5 ng/ml
(Fig. 1A). 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 ␤-catenin protein in re-
Fig. 1. HGF promotes nuclear translocation of ␤-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 ␤-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 ␤-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.
sponse 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 ␤-catenin
protein (P ⬍ 0.05) in the presence of HGF at 12.5 ng/ml for 15 min
in the hepatocyte cultures (Fig. 1B). To determine whether there was
any effect on the total ␤-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 ␤-catenin protein levels under these conditions (Fig. 1C). However, we consistently observed a minimal increase in the total ␤-catenin protein levels that was statistically insignificant.
HGF Induces Nuclear Translocation of ␤-Catenin in a Dosedependent Manner. We analyzed ␤-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 ␤-catenin levels in response to these increasing concentrations of HGF. Nuclear ␤-catenin appeared to increase in
response to an increase in HGF concentration in the hepatocyte
cultures (Fig. 2A). After normalization, we confirmed an initial dosedependent increase in nuclear ␤-catenin at 12.5 and 25 ng/ml of HGF
with a peak 4-fold increase seen at 25 ng/ml (Fig. 2B). This affect
becomes blunted at higher HGF concentrations with a steady plateau
observed in nuclear ␤-catenin levels at the 50 –100 ng/ml HGF concentration. Thus, ␤-catenin translocation in response to increasing
HGF concentration appears to follow first-order kinetics. We also
used double immunofluorescence to reconfirm nuclear localization of
␤-catenin in response to the increasing HGF concentrations. Cy3conjugated secondary antibody (red) detected ␤-catenin, and Sytox
green was used as a nuclear counterstain. The overlay (yellow) was
used to detect nuclear ␤-catenin. There was minimal nuclear ␤-catenin
in most of the hepatocytes after 2 h of primary hepatocytes cultures
without HGF (Fig. 2C). There was a considerable increase in nuclear
␤-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
␤-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 ␤-catenin in
normal hepatocytes after HGF inclusion in the hepatocyte cultures.
We also wanted to determine the redistribution of ␤-catenin in
response to HGF, as a function of time. Nuclear ␤-catenin levels were
studied at 15, 30, 60, and 90 min after single HGF treatment at 12.5
ng/ml. Elevated ␤-catenin levels were detected as early as 15 min after
HGF treatment, and this increase was maintained through 90 min (Fig.
2F). After normalization, we conclude that the increase in nuclear
␤-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. 2G). This might
imply that the factors responsible for nuclear translocation are still
active after 90 min of treatment, thus maintaining continued elevations in nuclear ␤-catenin protein, and that a single dose of HGF is
enough to sustain elevations in nuclear ␤-catenin protein for an
extended period of time.
HGF Does Not Affect E-Cadherin/␤-Catenin Complex or
Serine/Threonine Phosphorylated ␤-catenin Levels in Primary
Hepatocyte Cultures. We have shown previously that ␤-catenin
predominantly localizes at the membrane of the hepatocytes with
some cytoplasmic distribution. E-cadherin-␤-catenin association at
the membrane has also been well described previously. To determine
the mechanism and source of nuclear mobilization of ␤-catenin, we
decided to study any changes in the E-cadherin-associated ␤-catenin
in response to HGF. Immunoprecipitation studies were used to assess
changes in E-cadherin-␤-catenin association in response to HGF
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HGF INDUCES NUCLEAR TRANSLOCATION OF ␤-CATENIN
Fig. 2. HGF induces a dose-dependent increase in
the nuclear translocation of ␤-catenin. A, a representative Western blot shows an increase in nuclear ␤-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 ␤-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 ␤-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 ␤-catenin (red)
and counterstained with Sytox green. Nuclear localization of ␤-catenin was confirmed by overlay (yellow). In
the absence of HGF, most of the hepatocytes showed
minimal nuclear ␤-catenin. D, in the presence of HGF
at 12.5 ng/ml of HGF, there was a substantial increase
in nuclear ␤-catenin as shown. E, there was a further
increase in nuclear ␤-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 ␤-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 ␤-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.
treatment at 12.5 ng/ml for 15 min in the primary hepatocyte cultures.
No apparent change was detected in the E-cadherin-␤-catenin association in either culture condition (Fig. 3A). About 20% of ␤-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-␤-catenin complex after HGF treatment (Fig. 3B). This indicates that the source of
␤-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-␤-catenin as a complex from the hepatocyte
membrane.
␤-Catenin also associates to GSK3␤ in the cytoplasm. GSK3␤
phosphorylates ␤-catenin at specific serine and threonine residues to
activate its degradation through ubiquitin-proteosome pathway. A
decrease in serine-phosphorylated ␤-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
␤-catenin levels in the absence or presence of HGF during culture by
coprecipitation studies (Fig. 3C). There have been a few reports about
redistribution of ␤-catenin by HGF in tumor cells through transient
inactivation of GSK3␤ with resulting stabilization of ␤-catenin protein (9). However, we detected only minimal levels of serine phosphorylated ␤-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 GSK3␤-␤-catenin
association. Coprecipitation studies using total cell lysates from the
hepatocyte cultures detected a minimal association of endogenous
␤-catenin and GSK3␤ that remained unchanged upon HGF treatment
(Fig. 3D). 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 ␤-catenin
in the normal cultured hepatocytes, and this effect may contribute, to
a limited extent, in the promotion of ␤-catenin translocation by
imparting some stabilization to the protein (Fig. 1C).
HGF/SF Receptor Met Associates to Endogenous ␤-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
␤-catenin in response to HGF treatment in normal rat hepatocyte
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HGF INDUCES NUCLEAR TRANSLOCATION OF ␤-CATENIN
Fig. 3. Affect of HGF on ␤-catenin-E-cadherin complex, ␤-catenin-GSK3␤ complex,
and serine-phosphorylated ␤-catenin. A, a representative immunoprecipitation study
shown suggests no change in association of ␤-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 ␤-catenin and probed for E-cadherin. The blot was stripped and reprobed
for ␤-catenin for stochiometric analysis. IP, immunoprecipitation. B, after normalization
for ␤-catenin levels, the E-cadherin Western blots were subjected to densitometric
analysis. HGF does not influence the stoichiometry of ␤-catenin-E-cadherin association in
hepatocyte cultures at 12.5 ng/ml. C, representative blot shows minimal change in
serine-phosphorylated ␤-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 ␤-catenin. IP, immunoprecipitation. D, a representative blot depicts no obvious changes in the ␤-catenin-GSK3␤ association after HGF
treatment in hepatocyte cultures. Total cell lysates were subjected to immunoprecipitation
by anti-␤-catenin. IP, immunoprecipitation.
cultures, we began investigating any direct association of HGF pathway components with ␤-catenin. It has been shown previously that
␤-catenin phosphorylates at specific tyrosine residues, and this negatively influences cell-cell adhesion. We examined an association of
endogenous ␤-catenin to HGF receptor Met, a known receptor tyrosine kinase, in normal rat liver. We demonstrate the association of
endogenous Met and ␤-catenin in normal rat liver. Met and ␤-catenin
coprecipitate in unstimulated normal rat liver cell lysate (Fig. 4A). We
also show Met-␤-catenin coprecipitation in lysates from hepatoma
cell line (Hepa1-6) after 24-h culture (Fig. 4B). The blots from the
above studies, when subjected to densitometry for stoichiometric
analysis of Met-␤-catenin association, revealed ⬃78% of Met to be
associated to ␤-catenin and ⬃33% of ␤-catenin to be associated to
Met in the hepatocytes (Fig. 4C). Colocalization studies with double
immunofluorescence using Met (green) and ␤-catenin (red) antibodies demonstrate this association at the inner side of the hepatocyte
membrane (yellow) in normal rat liver (Fig. 4D). This study also
demonstrates most of the Met to be associated with ␤-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-␤-catenin was investigated by coprecipitation studies. A decrease
in association of Met to ␤-catenin occurred in response to HGF (Fig.
4E). No comparable decrease in total ␤-catenin protein was observed
in the immunoprecipitate. Stoichiometric analysis depicts a significant
decrease in Met-␤-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. 4F). Thus, this complex undergoes
dissociation in the presence of HGF with resulting nuclear translocation of ␤-catenin.
Loss of ␤-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 ␤-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 ␤-catenin and Met. Using an NH2terminal 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 ␤-catenin was
evident in the induced DN-Met-expressing cells (Fig. 5A). 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 ␤-catenin-Met association.
Absent Tyrosine Phosphorylation and Nuclear Translocation of
␤-Catenin in the DN, Met-expressing Cells in Response to HGF.
To further investigate the effect of HGF/Met signaling on ␤-catenin,
we decided to study the ability of HGF to tyrosine phosphorylate
␤-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. 5B). Similarly, we detected tyrosine-phosphorylated
␤-catenin in the same lysates, indicating the ability of HGF to serially
tyrosine phosphorylate Met and ␤-catenin because of their close
association (Fig. 5B). 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 ␤-catenin by coprecipitation studies (Fig. 5B). This
provides strong evidence of the role of HGF/Met signaling to induce
tyrosine phosphorylation of ␤-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 ␤-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
␤-catenin levels. We detected an absence or a failure of increase in the
nuclear ␤-catenin levels in response to HGF in the Hepa1-6 cells
expressing the DN-Met as compared with the cells expressing fulllength Met (Fig. 5C). The nuclear levels of ␤-catenin in the DN-Metinduced cells were comparable with the hepatocytes that were not
treated with HGF (Fig. 1B). This difference in the nuclear ␤-catenin
levels in response to HGF in the uninduced and induced DN-Met cells
was statistically significant (P ⬍ 0.05; Fig. 5D). The above data
strongly suggest the role of HGF/Met signaling in tyrosine phosphorylation-dependent Met-␤-catenin dissociation with the resulting nuclear translocation of ␤-catenin.
DISCUSSION
␤-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 ␤-catenin protein by
inactivation of the ubiquitin-proteosome pathway that comprises spe-
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HGF INDUCES NUCLEAR TRANSLOCATION OF ␤-CATENIN
Fig. 4. Endogenous Met-␤-catenin association and effect
of HGF on this complex. A, endogenous Met-␤-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 ␤-catenin antibodies.
The Western blots show coprecipitation of endogenous Met
and ␤-catenin in unstimulated normal rat liver. B, coprecipitation studies using hepatoma cells also demonstrate similar
association of Met-␤-catenin. IP, immunoprecipitation. C,
stoichiometric analysis of blots from the coprecipitation studies on Met-␤-catenin in normal rat liver as well as hepatoma
cells demonstrates at least 78% of total Met to be associated
with ␤-catenin (shaded area) and ⬃33% of total ␤-catenin
(shaded) to be associated with Met in these cells. D, double
immunofluorescence showed colocalization (yellow) of Met
(green) and ␤-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-␤-catenin complex, as can be seen in the
representative coprecipitation studies. There are decreases in
association of ␤-catenin and Met in the presence of 12.5
ng/ml of HGF in hepatocyte cultures. The total ␤-catenin in
immunoprecipitate is comparable, as can be seen in the lower
panel. IP, immunoprecipitation. F, after normalization for
␤-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 ␤-catenin in
the presence of 12.5 ng/ml of HGF in the hepatocyte cultures.
cific interactions with GSK3␤, 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
␤-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 ␤-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 ␤-catenin upon tyrosine phosphorylation in
response to certain growth factors is largely unknown. We demonstrate tyrosine phosphorylation-dependent nuclear translocation of
␤-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 ␤-catenin.
Using normal rat liver and primary hepatocytes cultures, we demonstrate a basal functional association between endogenous Met and
␤-catenin. We provide further evidence that this association is significantly lost during HGF signaling, resulting in alteration in steadystate kinetics of the ␤-catenin protein. Some studies have previously
shown stabilization of ␤-catenin protein by growth factors such as
epidermal growth factor and HGF through their effect on GSK3␤ (9,
27, 39, 40). We were unable to detect any significant changes in
GSK3␤ or serine/threonine phosphorylation of ␤-catenin in response
to HGF in the normal rat hepatocyte cultures. However, we consistently observed a minimal increase in the total ␤-catenin protein that
although it was statistically insignificant, it favored some stabilization. The above two factors might be acting in conjunction to induce
␤-catenin nuclear translocation in response to HGF in hepatocytes.
Importantly, we found a novel association between HGF receptor
Met and ␤-catenin in normal hepatocytes. About 80% of Met is
associated with ␤-catenin, and about 30 – 40% of ␤-catenin is associated with Met at the inner side of the hepatocyte membrane in normal
rat liver. ␤-Catenin is also associated with E-cadherin at the hepatocyte membrane, but stoichiometrically, this association is lower when
compared with the Met-␤-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 ␤-catenin complex might exist as a predominant complex and a functionally important pool of ␤-catenin in
hepatocytes.
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HGF INDUCES NUCLEAR TRANSLOCATION OF ␤-CATENIN
Fig. 5. Intact intracellular (tyrosine kinase) domain of Met is required for HGF-induced
nuclear translocation of ␤-catenin. A, both species of Met, the Mr 140,000 wild-type
endogenous (␤-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-␤-catenin antibodies. No
or a minimal Met-␤-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 ␤-catenin. Left panel, tyrosine-phosphorylated Met and ␤-catenin in response
to HGF treatment in the uninduced DN cells. There was no detectable tyrosine-phosphorylated Met or ␤-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 ␤-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 ␤-catenin in the induced DN cells was comparable with
the nuclear ␤-catenin in the unstimulated hepatocyte cultures. D, densitometric analysis
demonstrates the difference between the nuclear ␤-catenin in the two conditions to be
statistically significant (P ⬍ 0.05). After normalizing for loading, we observed a 50% less
nuclear ␤-catenin in response to HGF in the DN Hepa1-6 cells that was comparable with
the normal nuclear ␤-catenin levels in the non-HGF-treated hepatocytes. Bars, SD.
Addition of HGF to the cultured hepatocytes induces Met tyrosine
phosphorylation (Fig. 6). This in turn brings on phosphorylation of
␤-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 ␤-catenin favors dissociation of the Met-␤-catenin complex without affecting the Ecadherin-␤-catenin association that might be an independent pool at
the membrane. These specific events along with a minimal ␤-catenin
stabilization (GSK3␤ hypoactivity) result in nuclear translocation of
␤-catenin upon HGF treatment in hepatocyte cultures. We cannot rule
out dissociation of E-cadherin-␤-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 ␤-catenin protein associating
the Met-␤-catenin complex to the E-cadherin-␤-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 C␥, STAT3, and others. Association of HGF receptor
Met with ␤-catenin provides strong evidence to explain the cross-talk
and functional coincidences between the HGF/Met and Wnt/␤-catenin
pathways. On the basis of our observations, we propose that ␤-catenin
is present as a third independent pool at the membrane of hepatocyte
as a Met-␤-catenin complex, and this is an important regulator of free
␤-catenin levels and signaling in hepatocytes. We have demonstrated
the redistribution of ␤-catenin in a hepatocyte and shown that the
source of this ␤-catenin is Met-␤-catenin complex. We have observed
a minimal contribution from GSK3␤ or E-cadherin-associated ␤-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
␤-catenin levels in one cell type versus another.
Tyrosine phosphorylation of ␤-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
␤-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
␤-catenin. We demonstrate complete abrogation of nuclear translocation of ␤-catenin in the DN-Met cells. We propose that HGF induces
tyrosine phosphorylation-dependent nuclear translocation of ␤-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– 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
Fig. 6. Proposed model of nuclear translocation of ␤-catenin in response to HGF
signaling. HGF induces tyrosine phosphorylation of Met that in turn phosphorylates
␤-catenin at tyrosine residues. This causes dissociation of ␤-catenin from Met at the
membrane (A). E-cadherin-␤-catenin appears to be an independent pool at the membrane
of the hepatocyte, although we cannot rule out the possibility of dimerization of ␤-catenin
(B). HGF might induce dissociation from Met of the ␤-catenin-E-cadherin complex itself,
with resulting nuclear translocation of ␤-catenin (C). Minimal cytoplasmic stabilization of
␤-catenin after its release from the hepatocyte membrane might be through GSK3␤
hypoactivation, promoting nuclear translocation of ␤-catenin.
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HGF INDUCES NUCLEAR TRANSLOCATION OF ␤-CATENIN
during liver regeneration provides some in vivo correlation to this
novel observation (6). Heightened nuclear translocation of ␤-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.
ACKNOWLEDGMENTS
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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Hepatocyte Growth Factor Induces Wnt-independent Nuclear
Translocation of β-Catenin after Met-β-Catenin Dissociation in
Hepatocytes
Satdarshan P. S. Monga, Wendy M. Mars, Peter Pediaditakis, et al.
Cancer Res 2002;62:2064-2071.
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