| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Molecular Biology and Genetics |
Division of Cellular and Molecular Pathology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Previously, we have shown predominant ß-catenin localization at the hepatocyte membrane in a normal adult rat liver with some cytoplasmic staining, suggesting a minimal association with GSK3ß/Axin/APC complex (6) . ß-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 ß-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 ß-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 ß-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 ß-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 ß-catenin because of Met-ß-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 ß-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 |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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 collagenase perfusion protocol as described previously from our laboratory (29
, 30)
. Rat hepatocytes were plated at high density (1.5 x 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 x 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 x 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 x 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 x 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 x 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 ß-catenin (mouse), anti-E-cadherin (rabbit), anti-GSK3ß (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 x g) at 4°C was incubated with 5 µl (10 µg) of agarose-conjugated, goat anti-ß-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 x 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 ß-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-ß-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 ß-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 ß-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 ß-catenin and Met as well as their tyrosine phosphorylation. Nuclear extracts were used to study differences in nuclear ß-catenin levels.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. An increase in ß-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 determine whether there was any effect on the total ß-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 ß-catenin protein levels under these conditions (Fig. 1C)
. However, we consistently observed a minimal increase in the total ß-catenin protein levels that was statistically insignificant.
HGF Induces Nuclear Translocation of ß-Catenin in a Dose-dependent Manner.
We analyzed ß-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 ß-catenin levels in response to these increasing concentrations of HGF. Nuclear ß-catenin appeared to increase in response to an increase in HGF concentration in the hepatocyte cultures (Fig. 2A)
. After normalization, we confirmed an initial dose-dependent increase in nuclear ß-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 ß-catenin levels at the 50–100 ng/ml HGF concentration. Thus, ß-catenin translocation in response to increasing HGF concentration appears to follow first-order kinetics. We also used double immunofluorescence to reconfirm nuclear localization of ß-catenin in response to the increasing HGF concentrations. Cy3-conjugated secondary antibody (red) detected ß-catenin, and Sytox green was used as a nuclear counterstain. The overlay (yellow) was used to detect nuclear ß-catenin. There was minimal nuclear ß-catenin in most of the hepatocytes after 2 h of primary hepatocytes cultures without HGF (Fig. 2C)
. There was a considerable increase in nuclear ß-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 ß-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 ß-catenin in normal hepatocytes after HGF inclusion in the hepatocyte cultures.
|
. After normalization, we conclude that the increase in nuclear ß-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 ß-catenin protein, and that a single dose of HGF is enough to sustain elevations in nuclear ß-catenin protein for an extended period of time.
HGF Does Not Affect E-Cadherin/ß-Catenin Complex or Serine/Threonine Phosphorylated ß-catenin Levels in Primary Hepatocyte Cultures.
We have shown previously that ß-catenin predominantly localizes at the membrane of the hepatocytes with some cytoplasmic distribution. E-cadherin-ß-catenin association at the membrane has also been well described previously. To determine the mechanism and source of nuclear mobilization of ß-catenin, we decided to study any changes in the E-cadherin-associated ß-catenin in response to HGF. Immunoprecipitation studies were used to assess changes in E-cadherin-ß-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-ß-catenin association in either culture condition (Fig. 3A)
. About 20% of ß-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-ß-catenin complex after HGF treatment (Fig. 3B)
. This indicates that the source of ß-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-ß-catenin as a complex from the hepatocyte membrane.
|
. There have been a few reports about redistribution of ß-catenin by HGF in tumor cells through transient inactivation of GSK3ß with resulting stabilization of ß-catenin protein (9)
. However, we detected only minimal levels of serine phosphorylated ß-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 GSK3ß-ß-catenin association. Coprecipitation studies using total cell lysates from the hepatocyte cultures detected a minimal association of endogenous ß-catenin and GSK3ß 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 ß-catenin in the normal cultured hepatocytes, and this effect may contribute, to a limited extent, in the promotion of ß-catenin translocation by imparting some stabilization to the protein (Fig. 1C)
.
HGF/SF Receptor Met Associates to Endogenous ß-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 ß-catenin in response to HGF treatment in normal rat hepatocyte cultures, we began investigating any direct association of HGF pathway components with ß-catenin. It has been shown previously that ß-catenin phosphorylates at specific tyrosine residues, and this negatively influences cell-cell adhesion. We examined an association of endogenous ß-catenin to HGF receptor Met, a known receptor tyrosine kinase, in normal rat liver. We demonstrate the association of endogenous Met and ß-catenin in normal rat liver. Met and ß-catenin coprecipitate in unstimulated normal rat liver cell lysate (Fig. 4A)
. We also show Met-ß-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-ß-catenin association, revealed 
78% of Met to be associated to ß-catenin and 33% of ß-catenin to be associated to Met in the hepatocytes (Fig. 4C)
. Colocalization studies with double immunofluorescence using Met (green) and ß-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 ß-catenin in the hepatocytes.
|
. No comparable decrease in total ß-catenin protein was observed in the immunoprecipitate. Stoichiometric analysis depicts a significant decrease in Met-ß-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 ß-catenin.
Loss of ß-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 ß-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 ß-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 ß-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 ß-catenin-Met association.
|
. Similarly, we detected tyrosine-phosphorylated ß-catenin in the same lysates, indicating the ability of HGF to serially tyrosine phosphorylate Met and ß-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 ß-catenin by coprecipitation studies (Fig. 5B)
. This provides strong evidence of the role of HGF/Met signaling to induce tyrosine phosphorylation of ß-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 ß-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 ß-catenin levels. We detected an absence or a failure of increase in the nuclear ß-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. 5C)
. The nuclear levels of ß-catenin in the DN-Met-induced cells were comparable with the hepatocytes that were not treated with HGF (Fig. 1B)
. This difference in the nuclear ß-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-ß-catenin dissociation with the resulting nuclear translocation of ß-catenin.
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Using normal rat liver and primary hepatocytes cultures, we demonstrate a basal functional association between endogenous Met and ß-catenin. We provide further evidence that this association is significantly lost during HGF signaling, resulting in alteration in steady-state kinetics of the ß-catenin protein. Some studies have previously shown stabilization of ß-catenin protein by growth factors such as epidermal growth factor and HGF through their effect on GSK3ß (9 , 27 , 39 , 40) . We were unable to detect any significant changes in GSK3ß or serine/threonine phosphorylation of ß-catenin in response to HGF in the normal rat hepatocyte cultures. However, we consistently observed a minimal increase in the total ß-catenin protein that although it was statistically insignificant, it favored some stabilization. The above two factors might be acting in conjunction to induce ß-catenin nuclear translocation in response to HGF in hepatocytes.
Importantly, we found a novel association between HGF receptor Met and ß-catenin in normal hepatocytes. About 80% of Met is associated with ß-catenin, and about 30–40% of ß-catenin is associated with Met at the inner side of the hepatocyte membrane in normal rat liver. ß-Catenin is also associated with E-cadherin at the hepatocyte membrane, but stoichiometrically, this association is lower when compared with the Met-ß-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 ß-catenin complex might exist as a predominant complex and a functionally important pool of ß-catenin in hepatocytes.
Addition of HGF to the cultured hepatocytes induces Met tyrosine phosphorylation (Fig. 6)
. This in turn brings on phosphorylation of ß-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 ß-catenin favors dissociation of the Met-ß-catenin complex without affecting the E-cadherin-ß-catenin association that might be an independent pool at the membrane. These specific events along with a minimal ß-catenin stabilization (GSK3ß hypoactivity) result in nuclear translocation of ß-catenin upon HGF treatment in hepatocyte cultures. We cannot rule out dissociation of E-cadherin-ß-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 ß-catenin protein associating the Met-ß-catenin complex to the E-cadherin-ß-catenin complex is yet another possibility.
|
, STAT3, and others. Association of HGF receptor Met with ß-catenin provides strong evidence to explain the cross-talk and functional coincidences between the HGF/Met and Wnt/ß-catenin pathways. On the basis of our observations, we propose that ß-catenin is present as a third independent pool at the membrane of hepatocyte as a Met-ß-catenin complex, and this is an important regulator of free ß-catenin levels and signaling in hepatocytes. We have demonstrated the redistribution of ß-catenin in a hepatocyte and shown that the source of this ß-catenin is Met-ß-catenin complex. We have observed a minimal contribution from GSK3ß or E-cadherin-associated ß-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 ß-catenin levels in one cell type versus another. Tyrosine phosphorylation of ß-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 ß-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 ß-catenin. We demonstrate complete abrogation of nuclear translocation of ß-catenin in the DN-Met cells. We propose that HGF induces tyrosine phosphorylation-dependent nuclear translocation of ß-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 ß-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 |
|---|
|
FOOTNOTES |
|---|
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: michalopoulosgk{at}msx.upmc.edu
3 The abbreviations used are: GSK3ß, glycogen synthase kinase-3ß; APC, adenomatous polyposis coli gene product; HGF, hepatocyte growth factor; SF, scatter factor; HRP, horseradish peroxidase; DN, dominant negative.
Received 8/22/01. Accepted 1/29/02.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
in a chemically defined (HGM) medium. J. Cell Biol., 132: 1133-1149, 1996.This article has been cited by other articles:
|
|
 |
|
  S. Goldoni, A. Humphries, A. Nystrom, S. Sattar, R. T. Owens, D. J. McQuillan, K. Ireton, and R. V. Iozzo Decorin is a novel antagonistic ligand of the Met receptor J. Cell Biol., May 18, 2009; 185(4): 743 - 754. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Jean, A. Blanc, N. Prade-Houdellier, L. Ysebaert, H. Hernandez-Pigeon, T. Al Saati, M.-J. Haure, A.-M.-L. Coluccia, M. Charveron, E. Delabesse, et al. Epidermal Growth Factor Receptor/{beta}-Catenin/T-Cell Factor 4/Matrix Metalloproteinase 1: A New Pathway for Regulating Keratinocyte Invasiveness after UVA Irradiation Cancer Res., April 15, 2009; 69(8): 3291 - 3299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. David, A. Yeramian, M. Dunach, M. Llovera, C. Canti, A. G. de Herreros, J. X. Comella, and J. Herreros Signalling by neurotrophins and hepatocyte growth factor regulates axon morphogenesis by differential {beta}-catenin phosphorylation J. Cell Sci., August 15, 2008; 121(16): 2718 - 2730. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Litten and G. E. Tomlinson Liver Tumors in Children Oncologist, July 1, 2008; 13(7): 812 - 820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Marsh, S. Dickinson, G. W. Neill, J. F. Marshall, I. R. Hart, and G. J. Thomas {alpha}v{beta}6 Integrin Promotes the Invasion of Morphoeic Basal Cell Carcinoma through Stromal Modulation Cancer Res., May 1, 2008; 68(9): 3295 - 3303. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. S. Gujral, W. van Veelen, D. S. Richardson, S. M. Myers, J. A. Meens, D. S. Acton, M. Dunach, B. E. Elliott, J. W.M. Hoppener, and L. M. Mulligan A Novel RET Kinase-{beta}-Catenin Signaling Pathway Contributes to Tumorigenesis in Thyroid Carcinoma Cancer Res., March 1, 2008; 68(5): 1338 - 1346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ye, D. Hu, L. Tu, X. Zhou, F. Lu, B. Wen, W. Wu, Y. Lin, Z. Zhou, and J. Qu Involvement of PI3K/Akt Signaling Pathway in Hepatocyte Growth Factor-Induced Migration of Uveal Melanoma Cells Invest. Ophthalmol. Vis. Sci., February 1, 2008; 49(2): 497 - 504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. D. Tward, K. D. Jones, S. Yant, S. T. Cheung, S. T. Fan, X. Chen, M. A. Kay, R. Wang, and J. M. Bishop Distinct pathways of genomic progression to benign and malignant tumors of the liver PNAS, September 11, 2007; 104(37): 14771 - 14776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Apte, G. Zeng, M. D. Thompson, P. Muller, A. Micsenyi, B. Cieply, K. H. Kaestner, and S. P. S. Monga beta-Catenin is critical for early postnatal liver growth Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1578 - G1585. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Lutterbach, Q. Zeng, L. J. Davis, H. Hatch, G. Hang, N. E. Kohl, J. B. Gibbs, and B.-S. Pan Lung Cancer Cell Lines Harboring MET Gene Amplification Are Dependent on Met for Growth and Survival Cancer Res., March 1, 2007; 67(5): 2081 - 2088. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Anderson, R. Harrison, P. A. Atherfold, M. J. Campbell, S. J. Darnton, J. Obszynska, and J. A.Z Jankowski Met receptor signaling: a key effector in esophageal adenocarcinoma. Clin. Cancer Res., October 15, 2006; 12(20): 5936 - 5943. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Peruzzi, G. Athauda, and D. P. Bottaro The von Hippel-Lindau tumor suppressor gene product represses oncogenic beta-catenin signaling in renal carcinoma cells PNAS, September 26, 2006; 103(39): 14531 - 14536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Mazzone and P. M. Comoglio The Met pathway: master switch and drug target in cancer progression FASEB J, August 1, 2006; 20(10): 1611 - 1621. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Nakaigawa, M. Yao, M. Baba, S. Kato, T. Kishida, K. Hattori, Y. Nagashima, and Y. Kubota Inactivation of von Hippel-Lindau Gene Induces Constitutive Phosphorylation of MET Protein in Clear Cell Renal Carcinoma. Cancer Res., April 1, 2006; 66(7): 3699 - 3705. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-M. Xu, Y.-Q. Zhou, and M.-H. Wang Mechanisms of Cytoplasmic {beta}-Catenin Accumulation and Its Involvement in Tumorigenic Activities Mediated by Oncogenic Splicing Variant of the Receptor Originated from Nantes Tyrosine Kinase J. Biol. Chem., July 1, 2005; 280(26): 25087 - 25094. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Welm, S. Kim, B. E. Welm, and J. M. Bishop MET and MYC cooperate in mammary tumorigenesis PNAS, March 22, 2005; 102(12): 4324 - 4329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. M. Fischer, S. Giordano, P. M. Comoglio, and A. Ullrich Reactive Oxygen Species Mediate Met Receptor Transactivation by G Protein-coupled Receptors and the Epidermal Growth Factor Receptor in Human Carcinoma Cells J. Biol. Chem., July 9, 2004; 279(28): 28970 - 28978. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Slater, E. Koutsouki, C. L. Jackson, R. C. Bush, G. D. Angelini, A. C. Newby, and S. J. George R-Cadherin:{beta}-Catenin Complex and Its Association With Vascular Smooth Muscle Cell Proliferation Arterioscler Thromb Vasc Biol, July 1, 2004; 24(7): 1204 - 1210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. A. Graham and A. R. Asthagiri Epidermal Growth Factor-mediated T-cell Factor/Lymphoid Enhancer Factor Transcriptional Activity Is Essential but Not Sufficient for Cell Cycle Progression in Nontransformed Mammary Epithelial Cells J. Biol. Chem., May 28, 2004; 279(22): 23517 - 23524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Wadham, J. R Gamble, M. A Vadas, and Y. Khew-Goodall The Protein Tyrosine Phosphatase Pez Is a Major Phosphatase of Adherens Junctions and Dephosphorylates {beta}-Catenin Mol. Biol. Cell, June 1, 2003; 14(6): 2520 - 2529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Levy, C. Neuveut, C.-A. Renard, P. Charneau, S. Branchereau, F. Gauthier, J. T. Van Nhieu, D. Cherqui, A.-F. Petit-Bertron, D. Mathieu, et al. Transcriptional Activation of Interleukin-8 by beta -Catenin-Tcf4 J. Biol. Chem., October 25, 2002; 277(44): 42386 - 42393. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
