Matrix metalloproteinase(s) mediate(s) NO-induced dissociation of ß-catenin from membrane bound E-cadherin and formation of nuclear ß-catenin/LEF-1 complex

Jay M. Mei2, Gregory L. Borchert1, Steven P. Donald and James M. Phang3

Metabolism and Cancer Susceptibility Section, Basic Research Laboratory, Center for Cancer Research, NCI-Frederick, MD and
1 Basic Research Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD 21702, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Modulation of the adenomatous polyposis coli (APC)-ß-catenin pathway by inflammatory mediators and extracellular matrix may be important in colon carcinogenesis. We have recently shown that nitric oxide (NO) induces the accumulation of cytosolic ß-catenin and subsequent formation of the nuclear ß-catenin/lymphocyte enhancing factor (LEF)-1 complex in conditionally immortalized young mouse colonic epithelial (YAMC) cells. In the present study, we explored the mechanism(s) through which NO exerts its effect on cytosolic ß-catenin accumulation and nuclear ß-catenin/LEF-1 complex formation. We found that NO-induced degradation of the membrane bound E-cadherin at tight junctions. Using an anti-E-cadherin antibody specific for its extracellular domain, we detected a 50kDa degradation fragment of E-cadherin (120 kDa) from the culture medium conditioned by YAMC cells exposed to the NO-releasing drug, NOR-1, for 4 and 24 h. As ß-catenin is normally bound to transmembrane E-cadherin and thus anchored to the cytoskeleton structure, the degradation of E-cadherin induced by NO may cause dissociation of ß-catenin from membrane bound E-cadherin. This was demonstrated by the detection of ß-catenin accumulation in the soluble cytosolic fractions in YAMC after exposure to NO-releasing drugs. Furthermore, the degradation of E-cadherin and the release of ß-catenin to cytosol were accompanied by the formation of nuclear ß-catenin/LEF-1 complex, demonstrating the dissociation of ß-catenin from E-cadherin may be responsible for the activation of ß-catenin/LEF-1 transcription complex. Co-treatment with NO donors and broad-spectrum matrix metalloproteinase (MMP) inhibitors TIMP-1 (100 ng/ml), GM6001 (10 µM) and GM1489 (10 µM) abolished the degradation of E-cadherin induced by NO as demonstrated by western blot analysis. These MMP inhibitors also blocked the cytosolic accumulation of ß-catenin and nuclear formation of ß-catenin/LEF-1 complex. The sum effect of MMP inhibitors demonstrated that NO-induced activation of MMP may cause the degradation of E-cadherin and the subsequent dissociation of ß-catenin, thereby contributing to the cytosolic accumulation of ß-catenin and nuclear formation of ß-catenin/LEF-1 complex.

Abbreviations: APC, adenomatous polyposis coli; EMSAs, electrophoretic mobility shift assays; LEF, lymphocyte enhancing factor; NO, nitric oxide; MMP, matrix metalloproteinases; Tcf, T-cell factor; YAMC, young adult mouse colon.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The role of the adenomatous polyposis coli (Apc) gene in colonic carcinogenesis and its link with the downstream transcription regulator ß-catenin/T-cell factor (Tcf)-lymphocyte enhancing factor (LEF) complexes have been well established (13). Although genes responsive to the ß-catenin/Tcf-LEF transcription pathway have been identified in part (4,5), the relationship of ß-catenin/Tcf-LEF to other transcriptional factors in regulating specific genes is complex (6,7). Nevertheless, an increase in cytoplasmic ß-catenin levels and subsequent ß-catenin/Tcf-LEF complex formation are believed to be important events in the early stages of colonic carcinogenesis (8,9). On the other hand, there is increasing evidence implicating nitric oxide (NO) in ulcerative colitis and Crohn's disease, conditions known to predispose patients to colon cancer. Overexpression of inducible NOS II has been frequently detected in colonic tissues from these patients (10) and NOS II mRNA and protein are overexpressed in colonic adenomas compared with normal tissues (11). Furthermore, NO has been implicated in the formation of intestinal polyposis in ApcMin mice (12).

To explore the connections involving Apc, ß-catenin and NO, we examined the formation of nuclear ß-catenin/LEF-1 DNA binding complex as well as the increase of cytoplasmic ß-catenin in response to NO-releasing drugs in non- transformed and non-tumorigenic murine colonic epithelial cells (13,14). These conditionally immortal cells are designated YAMC (young adult mouse colon). YAMC cells express the heat-labile SV40LT antigen that allows them to proliferate at 33°C. The restrictive temperature of 39°C causes SV40LT antigen instability, reversion to a non-transformed phenotype and cessation of proliferation (14). The temperature-sensitive SV40LT mutant antigen becomes inactivated and non- functional when cells are transferred to 39°C for 72 h before each experiment.

Recent findings suggest that the association between ß-catenin and E-cadherin is vulnerable to enzymatic attack by matrix metalloproteinases (MMP) (15). MMP are a family of extracellular enzymes degrading matrix proteins, e.g. collagens and proteins involved in cell–cell interaction (16). These enzymes are linked to tumor cell invasion of the basement membrane, blood vessel penetration, angiogenesis and metastasis (17). It is noteworthy that NO has been implicated in all these activities as well (18), but what is most interesting is the finding that activation of MMPs may disrupt the association of ß-catenin with transmembrane E-cadherin by causing the degradation of the extracellular domain of E-cadherin, specifically, the proteolytic cleavage of the N-terminus extracellular domain of E-cadherin (19). This paper describes our studies showing that the effect of NO on the formation of ß-catenin/LEF-1 DNA complexes is dependent on the activity of MMP.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Reagents
Anti-ß-catenin and anti-mouse IgG HRP monoclonal antibodies were purchased from Transduction Laboratories (Lexington, KY). Anti-E-cadherin antibody was purchased from PanVera (Madison, WI). Anti-ß-catenin polyclonal antibody was from Sigma (St Louis, MO). Anti-actin monoclonal antibody was from Boehringer Mannheim (Indianapolis, IN). For cell culture, the following products were used and purchased from their respective sources: RPMI 1640 media and mouse IFN-{gamma} from Gibco-Invitrogen (Carlsbad, CA); neonatal calf serum from Gemini Bio-Products (Calabasas, CA), ITS+ from Collaborative Biomedical (Bedford, MA). NO donors, NOR-1 and SIN-1A, and MMP inhibitors were purchased from Calbiochem (San Diego, CA). All other chemicals and reagents were purchased from Sigma unless indicated otherwise.

Cell culture
Experiments were carried out using the conditionally immortalized murine colonic epithelial cells. All cells were grown on 75 cm2 culture flask coated with type I collagen (5 µg/cm2) in RPMI 1640 media supplemented with 5% neonatal calf serum, ITS+ (insulin 6.25 µg/ml, transferrin 6.25 µg/ml, selenious acid 6.25 ng/ml, linoleic acid 5.35 mg/ml and bovine serum albumin 1.25 mg/ml), 5 IU/ml of murine IFN-{gamma}, 100 000 IU/l penicillin and 100 mg/l streptomycin. They were cultured under transforming (permissive) conditions in a 33°C incubator with 5% CO2 plus all the aforementioned supplements in the media. All cells were then transferred, upon attaining confluency, into a 39°C incubator under non-transforming (non-permissive) conditions in serum-free and IFN-{gamma}-free media for 72 h before each experiment.

NOR-1 was dispersed in tissue culture medium to prepare a stock concentration of 10 mM and clumps were broken up by forceful repipetting and vigorous vortexing. After thorough mixing, an aliquot was diluted to the desired final concentration.

Western blotting
Briefly, cells were washed twice with cold PBS and harvested under either denaturing conditions by scraping in boiling 2x Laemmli sample buffer (Bio-Rad, Hercules, CA) or non-denaturing conditions by using a RIPA Buffer Set (Boehringer Mannheim, Indianapolis, IN). For total cell lysates under denaturing conditions, samples were heated at boiling temperature for an additional 5 min. Homogenates were then prepared in the Laemmli sample buffer by sonication (1 min each). After centrifugation at 2000 g for 5 min, the supernatants were used as the protein source. To make protein preparations that contain only soluble cytosolic fractions, cells were lysed in RIPA buffer under non-denaturing conditions at 4°C. After incubation on ice in a shaker for 15 min, the soluble supernatant was recovered by centrifugation at 100 000g for 30 min at 4°C. Harvested conditioned medium was concentrated using Centricon columns with a 3 kDa cut off. Approximately 10 ml were reduced to 100 µl by centrifugation at 2000 r.p.m.. Protein concentration was determined by the BCA method (Pierce, Rockford, IL). Electrophoresis samples were prepared by mixing the respective protein preparations with 2x Laemmli sample buffer. Samples were applied to precast Bio-Rad 7.5% Tris–HCl gels. After electrophoresis, proteins were transferred to nitrocellulose membranes using a semidry blotter (Bio-Rad). Blots were probed with primary antibody (1:500) followed by a secondary antimouse IgG antibody conjugated to horseradish peroxidase (1:1000). Detection was by the ECL method (Amersham, Arlington Heights, IL).

Preparation of nuclear extracts and electrophoretic mobility shift assays (EMSAs)
Nuclear extracts were prepared from YAMC cells according to the method by Dignam et al. (20) with modifications (21). Briefly, cells were rinsed once with cold PBS followed by trypsinization. After centrifugation at 1000 g for 5 min, they were resuspended in five pellet volumes of hypotonic buffer containing 0.2 mM PMSF and 0.5 mM DTT. They were then chilled on ice for 10 min followed by lysis with a PT 3000 Polytron (Brinkmann, Littau, Switzerland) for 30 s and centrifuged at 4000 g for 15 min. The pellet was resuspended in 0.5 pellet volumes of low salt buffer. An equal volume of high salt buffer was added dropwise to the gently stirred suspension. The nuclear extracts were subjected to centrifugation at 16 000 g for 30 min followed by dialysis overnight. After dialysis, samples were spun at 100 000 g for 30 min. An aliquot of this nuclear preparation containing 5 µg protein was added to a 20 µl reaction mix containing 300 ng poly dI–dC; binding buffer (10 mM HEPES, pH 7.6; 60 µM KCl; 1 mM EDTA; 1 mM DTT; 12% glycerol); with or without double stranded mouse LEF-1 oligonucleotide (Gibco-BRL), CACCCTTTGAAGCTC with 5' overhang, as a specific competitor. Samples were incubated on ice for 10 min. Then LEF-1 oligonucleotide, radio-labeled using T4 kinase (Gibco-BRL) and [{gamma}-32P]ATP (NEN, Boston, MA), was added at 1.5–2x104 c.p.m. per reaction and incubated at room temperature for 30 min. DNA loading dye (Quality Biological, Gaithersburg, MD) was added to stop the reaction. Samples were run on a 4% polyacrylamide (37.5:1) (Protogel, National Diagnostics, Atlanta, GA) gel at 189 V for 2.5 h in 0.5x TBE running buffer. Gels were dried and exposed to XAR-5 film (Kodak). For super-shift studies, 3–5 µg nuclear lysate was mixed in a 20 µl reaction mixture as described for EMSA and incubated on ice for 10 min. Antibodies, 12 µg polyclonal anti-ß-catenin and 500 ng monoclonal anti-E-cadherin, or rabbit IgG, were then added to the respective reaction tubes. Reactions were incubated on ice for 15 min. 32P-Labeled murine LEF-1 oligonucleotide probe was added at 1.5–2x104 c.p.m./reaction and incubated at room temperature for 30 min.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Our recent studies on the activation of the ß-catenin/Tcf-LEF transcriptional factor by pro-inflammatory factors can be summarized. First, exogenously supplied NO from NO donors induce accumulation of ß-catenin in the cytosol of conditionally immortalized mouse colonic epithelial cells (21). Secondly, cytosolic accumulation of ß-catenin is due to the dissociation and translocation of membrane associated ß-catenin into the cytosol (21,22). Thirdly, accumulated cytosolic ß-catenin crosses the nuclear membrane and forms heterodimeric transcription complexes with LEF-1. The activated ß-catenin/ LEF-1 can be demonstrated by EMSAs (21,22).

The critical question is how NO induces the accumulation of ß-catenin in the cytosol. NO may affect the redox-sensitive tetrameric interactions among APC, GSK-3ß, axin and ß-catenin, thereby disrupting the ß-catenin degradation machinery. Certainly, truncation of the APC protein by the Min mutation or inhibition of GSK-3ß by the Wnt-1 pathway has been shown to stabilize ß-catenin in the cytosol. NO may also modulate the crossing of the nuclear membrane by ß-catenin. But based on our previous findings, the most probable site of action for NO is at the junction of ß-catenin and E-cadherin association. We propose that NO, most probably as peroxynitrite (the product of NO with reactive oxygen or released from NO donor) directly or indirectly disrupts the association of ß-catenin with transmembrane E-cadherin, increases cytosolic ß-catenin turnover and makes it available for formation of the heterodimeric transcriptional factor with Tcf/LEF.

As discussed previously, MMP-mediated dissociation of E-cadherin and ß-catenin was an attractive hypothesis. To test this hypothesis, we first examined whether NO does indeed cause E-cadherin degradation, and if it does, whether the degradation can be blocked by MMP inhibitors. After NOR-1 treatment for 4 and 24 h, we collected the conditioned culture medium and concentrated the solutes using centricon columns. Using an anti-E-cadherin antibody that specifically recognizes the N-terminal extracellular domain, we found that NO stimulated the degradation of E-cadherin, as demonstrated by western blot (Figure 1Go). Full-length E-cadherin is 120 kDa, whereas we detected a cleavage fragment of E-cadherin consistent with the extracellular domain. Importantly, the accumulation of this cleavage product was blocked or decreased by three broad-spectrum MMP inhibitors, TIMP-1, GM 6001 and GM 1489 (Figure 1Go).



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Fig. 1. After cells were treated with NOR-1 (5 µM) for 4 and 24 h, the conditioned culture media were collected and concentrated ~100-fold using Centricon columns with a 3 kDa cut off. As demonstrated in this western blot using an anti-E-cadherin antibody, which specifically recognizes the N-terminal extracellular domain, a 50 kDa degradation fragment of E-cadherin extracellular domain was detected. The full-length E-cadherin is 120 kDa. This degradation was blocked or reduced by three broad-spectrum MMP inhibitors, TIMP-1 (100 ng/ml), GM6001 (10 µM) and GM1489 (10 µM).

 
We know from our previous studies that cells treated with NO donors accumulate free ß-catenin in the cytosol (21,22). In the current experiments, treatment with NO resulted in similar accumulation of free cytosolic ß-catenin at 30 and 120 min (Figure 2Go). However, when treated concomitantly with MMP inhibitors, the levels of ß-catenin were markedly decreased. This finding suggested that the NO-induced ß-catenin accumulation was mediated by MMP, since others have shown that proteolytic degradation of the extracellular domain of E-cadherin led to release of ß-catenin from its binding to the intracellular domain of E-cadherin.



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Fig. 2. The accumulation of free ß-catenin in the cytosol at 30 and 120 min in response to NOR-1 (5 µM) treatment or SIN-1A (5 µM) treatment was significantly decreased by MMP inhibitors, TIMP-1 (100 ng/ml), GM6001 (10 µM) and GM1489 (10 µM).

 
The inhibition of MMP activity markedly decreased the NO-induced ß-catenin accumulation in the cytosol. Therefore, we tested whether it would also affect the NO-induced formation of nuclear ß-catenin/LEF-1 complex. As reported previously, using EMSA, we showed that NO treatment increased the formation of ß-catenin/LEF-1:DNA complexes which were supershifted by anti-ß-catenin antibodies (Figure 3Go). Consistent with the aforementioned studies, inhibitors of MMP markedly decreased the formation of the complex.



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Fig. 3. As demonstrated in this experiment using gel-shift assays, NO-induced ß-catenin/LEF-1 complex formation was abolished by all three MMP inhibitors, TIMP-1 (100 ng/ml), GM6001 (10 µM) and GM1489 (10 µM).

 
These results clearly implicate MMP in the NO induction of the ß-catenin/Tcf-LEF complex. We are actively pursuing the mechanisms through which NO interacts with MMP. There are at least two possible ways for NO to increase the degradation of E-cadherin by certain MMP. MMP-7 (a.k.a. matrilysin) is an attractive target as it is known to degrade E-cadherin (15). Furthermore, MMP-7 can be regulated by ß-catenin (6) and the effect of NO would thus activate a positive feedback loop. Another possibility is MMP-2, which is released into the medium when cells were treated with NO donors (23). We are testing these possibilities using substrate specific zymography methods (24). There is also another possibility as suggested by some studies (24), that oxidative modifications of extracellular matrix proteins, e.g. the extracellular domain of E-cadherin, can change their sensitivity and susceptibility of these proteins to MMP degradation. In other words, NO may alter the substrates for MMP so that they are more readily degraded.

An important implication of this study is that the pro-inflammatory signaling effects of NO on the APC/ß-catenin pathway involve MMP and matrix proteins. It is tempting to speculate that this regulatory loop may contribute to the demonstrated effects of NO on the interaction of chronic inflammation with tumorigenesis, tissue invasion and metastases.


    Notes
 
2 Present address: Johnson & Johnson Pharmaceutical and Development, L.L.C., Welsh and McKean Roads, Spring House, PA 19477, USA Back

3 To whom correspondence should be addressed Email: phang{at}mail.ncifcrf.gov Back


    Acknowledgments
 
This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received July 9, 2002; revised August 27, 2002; accepted September 5, 2002.