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• W2019713047 abstract "The oncogenic protein β-catenin is overexpressed in many cancers, frequently accumulating in nuclei where it forms active complexes with lymphoid enhancer factor-1 (LEF-1)/T-cell transcription factors, inducing genes such as c-myc and cyclin D1. In normal cells, nuclear β-catenin levels are controlled by the adenomatous polyposis coli (APC) protein through nuclear export and cytoplasmic degradation. Transient expression of LEF-1 is known to increase nuclear β-catenin levels by an unknown mechanism. Here, we show that APC and LEF-1 compete for nuclear β-catenin with opposing consequences. APC can export nuclear β-catenin to the cytoplasm for degradation. In contrast, LEF-1 anchors β-catenin in the nucleus by blocking APC-mediated nuclear export. LEF-1 also prevented the APC/CRM1-independent nuclear export of β-catenin as revealed byin vitro assays. Importantly, LEF-1-bound β-catenin was protected from degradation by APC and axin in SW480 colon cancer cells. The ability of LEF-1 to trap β-catenin in the nucleus was down-regulated by histone deacetylase 1, and this correlated with a decrease in LEF1 transcription activity. Our findings identify LEF-1 as key regulator of β-catenin nuclear localization and stability and suggest that overexpression of LEF-1 in colon cancer and melanoma cells may contribute to the accumulation of oncogenic β-catenin in the nucleus. The oncogenic protein β-catenin is overexpressed in many cancers, frequently accumulating in nuclei where it forms active complexes with lymphoid enhancer factor-1 (LEF-1)/T-cell transcription factors, inducing genes such as c-myc and cyclin D1. In normal cells, nuclear β-catenin levels are controlled by the adenomatous polyposis coli (APC) protein through nuclear export and cytoplasmic degradation. Transient expression of LEF-1 is known to increase nuclear β-catenin levels by an unknown mechanism. Here, we show that APC and LEF-1 compete for nuclear β-catenin with opposing consequences. APC can export nuclear β-catenin to the cytoplasm for degradation. In contrast, LEF-1 anchors β-catenin in the nucleus by blocking APC-mediated nuclear export. LEF-1 also prevented the APC/CRM1-independent nuclear export of β-catenin as revealed byin vitro assays. Importantly, LEF-1-bound β-catenin was protected from degradation by APC and axin in SW480 colon cancer cells. The ability of LEF-1 to trap β-catenin in the nucleus was down-regulated by histone deacetylase 1, and this correlated with a decrease in LEF1 transcription activity. Our findings identify LEF-1 as key regulator of β-catenin nuclear localization and stability and suggest that overexpression of LEF-1 in colon cancer and melanoma cells may contribute to the accumulation of oncogenic β-catenin in the nucleus. lymphoid enhancer factor-1 adenomatous polyposis coli chromosome region maintenance 1 histone deacetylase 1 phosphate-buffered saline yellow fluorescent protein antibody T cell factor nuclear export signal β-Catenin accumulates to excessive levels in different cancers, including melanomas, colon cancer, breast cancer, and hepatocarcinomas (1Kinzler K.W. Vogelstein B. Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4286) Google Scholar, 2Morin P.J. Bioessays. 1999; 21: 1021-1030Crossref PubMed Scopus (817) Google Scholar, 3Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). Whether overexpressed in cancer (2Morin P.J. Bioessays. 1999; 21: 1021-1030Crossref PubMed Scopus (817) Google Scholar, 3Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar), in transfected cells (4Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2605) Google Scholar), or in transgenic mice (5Harada N. Tamai Y. Ishikawa T.-O. Sauer B. Takaku K. Oshima M. Taketo M.M. EMBO J. 1999; 18: 5931-5942Crossref PubMed Scopus (981) Google Scholar), the induced β-catenin accumulates throughout the cell with frequent concentration in the nucleus. β-Catenin is thought to be the key mediator of the Wnt signaling pathway (3Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar), and when overexpressed it can cause cell transformation (6Aoki M. Hecht A. Kruse U. Kemler R. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 139-144Crossref PubMed Scopus (155) Google Scholar). The oncogenic potential of β-catenin is mediated by its association with a class of related (but not identical) transcription factors that include lymphoid enhancer factor-1 (LEF-1),1 and the T cell factors including TCF-1, -3, and -4 (3Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). Of these, LEF-1 is of particular interest as it is overexpressed in colon cancer cell lines (7Porfiri E. Rubinfeld B. Albert I. Hovanes K. Waterman M. Polakis P. Oncogene. 1997; 15: 2833-2839Crossref PubMed Scopus (133) Google Scholar) and colon tumors (8Hovanes K., Li, T.W.H. Munguia J.E. Truong T. Milovanovic T. Marsh J.L. Holcombe R.F. Waterman M.L. Nat. Genet. 2001; 28: 53-57Crossref PubMed Google Scholar), and in metastatic melanoma cells (9Murakami T. Toda S. Fujimoto M. Ohtsuki M. Byers H.R. Etoh T. Nakagawa H. Biochem. Biophys. Res. Commun. 2001; 288: 8-15Crossref PubMed Scopus (66) Google Scholar), and is known to form nuclear β-catenin-LEF-1 complexes in vivo, which activate transcription of various transforming genes, including cyclin D1 (10Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3265) Google Scholar) and c-myc (11He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4092) Google Scholar). β-Catenin overexpression results from stabilizing cancer mutations within the β-catenin gene (see Ref. 2Morin P.J. Bioessays. 1999; 21: 1021-1030Crossref PubMed Scopus (817) Google Scholar) or in genes that regulate its degradation such as APC and axin (see Ref. 3Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). Recently, we and others showed that the nuclear build-up of β-catenin is normally prevented by a combination of nuclear export and cytoplasmic degradation (12Henderson B.R. Nat. Cell Biol. 2000; 2: 653-660Crossref PubMed Scopus (415) Google Scholar, 13Neufeld K.L. Zhang F. Cullen B. White R.L. EMBO Rep. 2000; 1: 519-523Crossref PubMed Scopus (144) Google Scholar, 14Rosin-Arbesfeld R. Townsley F. Bienz M. Nature. 2000; 406: 1009-1012Crossref PubMed Scopus (308) Google Scholar). β-Catenin can exit the nucleus by two distinct pathways: the CRM1 export pathway, which requires its association with the shuttling protein APC (12Henderson B.R. Nat. Cell Biol. 2000; 2: 653-660Crossref PubMed Scopus (415) Google Scholar, 13Neufeld K.L. Zhang F. Cullen B. White R.L. EMBO Rep. 2000; 1: 519-523Crossref PubMed Scopus (144) Google Scholar, 14Rosin-Arbesfeld R. Townsley F. Bienz M. Nature. 2000; 406: 1009-1012Crossref PubMed Scopus (308) Google Scholar), and an alternative CRM1-independent export route (15Wiechens N. Fagotto F. Curr. Biol. 2001; 11: 18-27Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 16Eleftheriou A. Yoshida M. Henderson B.R. J. Biol. Chem. 2001; 276: 25883-25888Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Despite the ability of β-catenin to efficiently exit the nucleus of SW480 colon cancer cells (16Eleftheriou A. Yoshida M. Henderson B.R. J. Biol. Chem. 2001; 276: 25883-25888Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), its subcellular distribution is biased toward the nucleus in those cells (16Eleftheriou A. Yoshida M. Henderson B.R. J. Biol. Chem. 2001; 276: 25883-25888Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 17Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (957) Google Scholar). Therefore, what factors influence nuclear retention of β-catenin? Despite the many nuclear proteins with which β-catenin associates (18Sharpe C. Lawrence N. Martinez Arias A. Bioessays. 2001; 23: 311-318Crossref PubMed Scopus (102) Google Scholar), β-catenin nuclear retention is most often correlated with binding to LEF-1 (4Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2605) Google Scholar, 7Porfiri E. Rubinfeld B. Albert I. Hovanes K. Waterman M. Polakis P. Oncogene. 1997; 15: 2833-2839Crossref PubMed Scopus (133) Google Scholar, 19Huber O. Korn R. McLaughlin J. Ohsugi M. Herrman B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (784) Google Scholar, 20Simcha I. Shtutman M. Salomon D. Zhurinsky J. Sadot E. Geiger B. Ben-Ze'ev A. J. Cell Biol. 1998; 141: 1433-1448Crossref PubMed Scopus (238) Google Scholar, 21Hsu S.-C. Galceran J. Grosschedl R. Mol. Cell. Biol. 1998; 18: 4807-4818Crossref PubMed Scopus (339) Google Scholar, 22Prieve M.G. Waterman M.L. Mol. Cell. Biol. 1999; 19: 4503-4515Crossref PubMed Google Scholar). Some reports suggest that LEF-1 can compete directly with APC and with E-cadherin for the mutually exclusive binding to the armadillo repeat domain of β-cateninin vitro (23Orsulic S. Huber O. Aberle H. Arnold S. Kemler R. J. Cell Sci. 1999; 112: 1237-1245Crossref PubMed Google Scholar) or in vivo (20Simcha I. Shtutman M. Salomon D. Zhurinsky J. Sadot E. Geiger B. Ben-Ze'ev A. J. Cell Biol. 1998; 141: 1433-1448Crossref PubMed Scopus (238) Google Scholar, 23Orsulic S. Huber O. Aberle H. Arnold S. Kemler R. J. Cell Sci. 1999; 112: 1237-1245Crossref PubMed Google Scholar). On the other hand, Neufeld et al. (13Neufeld K.L. Zhang F. Cullen B. White R.L. EMBO Rep. 2000; 1: 519-523Crossref PubMed Scopus (144) Google Scholar) proposed that APC binding is dominant and that APC can displace nuclear β-catenin from LEF-1 complexes and subsequently export β-catenin to the cytoplasm. Given the well documented role of β-catenin-LEF-1 transcription complex activity in cell signaling and cancer (1Kinzler K.W. Vogelstein B. Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4286) Google Scholar, 2Morin P.J. Bioessays. 1999; 21: 1021-1030Crossref PubMed Scopus (817) Google Scholar, 3Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar), we have investigated the mechanism by which LEF-1 acts to retain β-catenin in the nucleus. We confirmed that LEF-1 and APC do compete for nuclear β-catenin; however, APC did not remove β-catenin once it was bound to LEF-1. In fact, LEF-1 effectively blocked APC-mediated nuclear export of β-catenin in vivo and blocked APC-independent export of β-catenin in vitro. Unexpectedly, LEF-1-bound β-catenin was highly protected in the nucleus from destruction by overexpressed APC and axin. We conclude that the β-catenin-LEF-1 interaction is not one-sided and that while β-catenin can activate transcriptionally silent LEF-1, and LEF-1 in turn functions to trap and stabilize β-catenin in the nucleus. NIH 3T3 mouse fibroblasts and SW480 colon carcinoma cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum and were confirmed free of mycoplasma. Leptomycin B was added to a final concentration of 6 ng/ml. MG132 (Calbiochem) was used at a final concentration of 20 μm. DNA transfection of cells (typically 1 μg of DNA/2 ml of medium) was performed with LipofectAMINE reagent as directed (Invitrogen), using cells at medium density seeded onto coverslips. The pAPC-YFP fusion vector is based on the pCMV-APC vector (25Smith K.J. Levy D.B. Maupin P. Pollard T.D. Vogelstein B. Kinzler K.W. Cancer Res. 1994; 54: 3672-3675PubMed Google Scholar) and contains yellow fluorescent protein (YFP) positioned at the C terminus of APC. We previously found that YFP was not properly expressed when inserted at the N terminus of APC, presumably due to folding constraints (data not shown). To construct pAPC-YFP, the pCMV-APC plasmid was first cut with AvrII at a unique C-terminal restriction site, and a modified PCR-amplified C-terminal fragment was then inserted to create the vector pCMV-APC(MCS). This plasmid was checked by DNA sequencing. The PCR primers used to amplify the APC C terminus were as follows: forward primer (FWDAVR, 5′-TTAATTACAACCCAAG-3′) and reverse primer (REVNOT1, 5′-CCAGTTCCTAGGTTATATCGATGCGGCCGCAACAGATGTCACAAGG-3′). The modified APC C terminus was engineered to incorporate the unique restriction sites NotI and ClaI just prior to the translation stop site, enabling the in-frame insertion of aNotI fragment containing the YFP cDNA. The resulting vector, pAPC-YFP, was confirmed by restriction mapping, and expression of both the APC and YFP domains was confirmed in transfected cells by immunostaining with the APC antibodies Ab7 (recognizes the N terminus, Oncogene Research) and C-20 (recognizes the C terminus, Santa Cruz Biotechnology). SW480 or NIH 3T3 cells were transfected with 0.5 μg of the luciferase reporter plasmids pGL3-OT or pGL3-OF (provided by Dr. B. Vogelstein), in addition to 0.5 μg of various APC plasmid DNAs, or 0.5/2.5 μg of histone deacetylase 1 cDNA (pcDNA3-HDAC1; kind gift of Dr. D. Ayer). The pGL3-OT promoter comprises three LEF-1/TCF binding sites, whereas pGL3-OF contains mutated inactive TCF sites and is a negative control (11He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4092) Google Scholar). Reporter gene activity was determined 48 h post-transfection with the luciferase assay system (Promega). Luciferase activity of APC-transfected samples was normalized for transfection efficiency by scoring for APC-positive SW480 cells in duplicate samples (immunostained with APC Ab7, Oncogene Research). Final luciferase activity values were arrived at by subtracting background (including the pGL3-OF value) and determining the mean ± S.D. from three experiments (in duplicate), where the pGL3-OT sample was set to 100%. Nuclear export of endogenous cellular β-catenin in SW480 cells was assessed using the digitonin permeabilized cell assay, as recently described in detail (16Eleftheriou A. Yoshida M. Henderson B.R. J. Biol. Chem. 2001; 276: 25883-25888Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Export reactions were performed at 30 °C for 30 min in transport buffer containing 50 mm Tris-HCl, pH 7.5, 5 mm magnesium acetate, 2 mm EGTA, 50 mm potassium acetate, 2 mm dithiothreitol, 50 μg/ml phenylmethylsulfonyl fluoride, 1 mm benzamidine, and 50 μg/ml leupeptin in the presence or absence of digitonin (50 μg/ml). All reactions included an energy-regenerating system comprising 1 mm ATP, 0.5 mm GTP, 4 units/ml of creatine kinase, and 10 mm creatine phosphate. Cells were immediately fixed and processed for immunostaining following the export reactions. Cells were grown on glass coverslips, fixed in 3% formalin/PBS for 20 min, followed by permeabilization with 0.2% Triton X-100/PBS for 10 min. Cells were preblocked in 3% bovine serum albumin/PBS for 30 min, incubated 50 min with primary antibody (diluted 1:70 in blocking solution), and washed three times with PBS. Primary antibodies used were as follows: β-catenin (monoclonal antibody C19220 from Transduction Laboratories and rabbit polyclonal antibody H-102 from Santa Cruz Biotechnology), FLAG-axin (anti-FLAG monoclonal antibody M2 from Sigma), HA-LEF-1 (rabbit polyclonal HA-probe Y-11 from Santa Cruz), and APC (monoclonal antibody Ab7 from Oncogene Research). Cells were then incubated with a secondary antibody (1:120 dilution of a fluorescein isothiocyanate- or Texas Red-conjugated anti-rabbit or anti-mouse antibody from Sigma) prior to mounting on slides with Vectashield (Vector Laboratories, Burlingame, CA) and fluorescence microscopy. Processing was at room temperature. Cells were analyzed and scored on an Olympus fluorescence microscope at ×400 magnification, and digital images were captured using an OptiScan confocal microscope at ×600 magnification. Quantification of fluorescence images was performed using the NIH Image software as described previously (12Henderson B.R. Nat. Cell Biol. 2000; 2: 653-660Crossref PubMed Scopus (415) Google Scholar). Cell fractionation and Western blot analysis were performed for transfected SW480 cells as described previously (24Galea M.A. Eleftheriou A. Henderson B.R. J. Biol. Chem. 2001; 276: 45833-45839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In mouse NIH 3T3 fibroblasts, cellular β-catenin is localized mainly at the plasma membrane (Fig.1 A). Prior to testing for regulation by LEF-1, we first confirmed that endogenous β-catenin is regulated by nuclear export and degradation in NIH 3T3 cells, by comparing the effects of drugs that block CRM1-specific nuclear export (leptomycin B, LMB) and proteasome-dependent degradation (MG132). We observed that a 16-h treatment with MG132 induced a stronger induction (9-fold) of nuclear β-catenin than did leptomycin B (4-fold), following immunostaining and confocal scanning microscopy (see images and fluorescence quantification in Fig.1 A). Thus, β-catenin accumulates in the nucleus of 3T3 cells following inhibition of the major nuclear export and degradation pathways. Previous studies reported an increase in nuclear β-catenin following the transient overexpression of LEF-1 (4Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2605) Google Scholar, 19Huber O. Korn R. McLaughlin J. Ohsugi M. Herrman B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (784) Google Scholar, 20Simcha I. Shtutman M. Salomon D. Zhurinsky J. Sadot E. Geiger B. Ben-Ze'ev A. J. Cell Biol. 1998; 141: 1433-1448Crossref PubMed Scopus (238) Google Scholar). We investigated this further and discovered that endogenous nuclear β-catenin (see confocal images in Fig. 1 B) increased in proportion to the amount of ectopically expressed LEF-1, as revealed by quantitative imaging of β-catenin/LEF-1 nuclear fluorescence and linear regression analysis (Fig. 1 B). LEF-1 had a similar effect on ectopically expressed β-catenin (data not shown), and unlike the previous study of Hsu et al. (21Hsu S.-C. Galceran J. Grosschedl R. Mol. Cell. Biol. 1998; 18: 4807-4818Crossref PubMed Scopus (339) Google Scholar), the positive effect of LEF-1 was not dependent on co-expression of wnt-1 protein. As a specificity control, overexpression of nuclear p53 had little effect on nuclear β-catenin (data not shown). We next tested whether co-expression of wild-type APC, or mutant APC-(1–1309) that binds but does not degrade β-catenin, caused a reduction in the effect of LEF-1. As shown in Fig. 1 C, both forms of APC did reduce the LEF-1-dependent induction of nuclear β-catenin (although somewhat modestly), suggesting that LEF-1 and APC can compete for nuclear β-catenin in vivo. This finding significantly extends earlier experiments that demonstrated an in vitrocompetition for β-catenin between LEF-1 and subfragments of APC (23Orsulic S. Huber O. Aberle H. Arnold S. Kemler R. J. Cell Sci. 1999; 112: 1237-1245Crossref PubMed Google Scholar). The overexpression of wild-type APC is known to inhibit β-catenin/LEF-1 transcriptional activity in APCmut/mut SW480 colon cancer cells (17Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (957) Google Scholar). This inhibitory action is likely to involve competition with LEF-1/TCF factors for binding to β-catenin. Interestingly, Neufeld et al. (13Neufeld K.L. Zhang F. Cullen B. White R.L. EMBO Rep. 2000; 1: 519-523Crossref PubMed Scopus (144) Google Scholar) reported that an export-defective form of APC could also disrupt β-catenin transcriptional activity. Our laboratory identified the same two N-terminal nuclear export signals (NESs; Ref. 12Henderson B.R. Nat. Cell Biol. 2000; 2: 653-660Crossref PubMed Scopus (415) Google Scholar) as did Neufeld and colleagues, and using a luciferase reporter assay we also found that APC isoforms containing mutations in NES1, or in both NES1 and NES2, effectively reduced β-catenin/LEF-1 transcription activity (see Fig. 2). Since these full-length APC NES mutants retain the β-catenin degradation sequences, however, we also tested a cancer mutant form of APC-(1–1309) which can bind and export nuclear β-catenin (12Henderson B.R. Nat. Cell Biol. 2000; 2: 653-660Crossref PubMed Scopus (415) Google Scholar), but cannot degrade it (17Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (957) Google Scholar). As shown in Fig. 2, APC-(1–1309) reduced promoter/luciferase activity by ∼50%, and this was unaffected by mutation of NES1, which is the dominant export signal in APC (24Galea M.A. Eleftheriou A. Henderson B.R. J. Biol. Chem. 2001; 276: 45833-45839Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). These results suggest that the ability of APC to inhibit β-catenin/LEF-1 transcription activity primarily involves nuclear sequestration of β-catenin, but is more efficient when APC retains the ability to export/degrade β-catenin. Can LEF-1 block APC-mediated nuclear export of β-catenin? To address this question without the complication of β-catenin degradation, we again employed the APC-(1–1309) mutant (25Smith K.J. Levy D.B. Maupin P. Pollard T.D. Vogelstein B. Kinzler K.W. Cancer Res. 1994; 54: 3672-3675PubMed Google Scholar). We tested the ability of ectopic APC-(1–1309) to shift β-catenin from nucleus to cytoplasm in transfected SW480 cells, in the absence or presence of co-transfected LEF-1. As shown in the confocal images of Fig.3, transient expression of the mutant APC re-located β-catenin from nucleus to cytoplasm in 73% of transfected cells expressing cytoplasmic APC. However, the co-expression of LEF-1 caused nuclear retention of β-catenin in 85% of APC-transfected cells (see images and graphs in Fig. 3). The co-expression of ectopic APC and LEF-1 was very efficient, with >95% of APC-transfected cells also expressing recombinant LEF-1 (data not shown). The results demonstrate nuclear retention of β-catenin by co-expressed LEF-1 and indicate that APC is incapable of removing β-catenin from LEF-1 nuclear complexes. This contrasts with the recent prediction that APC might displace β-catenin bound to LEF-1 (13Neufeld K.L. Zhang F. Cullen B. White R.L. EMBO Rep. 2000; 1: 519-523Crossref PubMed Scopus (144) Google Scholar). Interestingly, LEF-1 did not affect APC localization, confirming that binding to β-catenin by LEF-1 and APC is mutually exclusive in vivo. The striking ability of LEF-1 to block nuclear export of β-catenin by APC-(1–1309) suggested that LEF-1 might also protect β-catenin from degradation by APC. To test this, we first transfected SW480 cells with plasmid encoding full-length untagged APC (pCMV-APC) and observed a total loss of β-catenin in ∼60% of transfected cells (see Fig. 4 A). This was accompanied by an overall 90% reduction in nuclear β-catenin fluorescence in APC-transfected cells (Fig. 4 C). When co-expressed with LEF-1, however, APC no longer exported and promoted degradation of β-catenin. In SW480 cells co-transfected with pHA-LEF-1 and pCMV-APC, nuclear β-catenin fluorescence levels decreased by only 5% in a randomly selected population of transfected cells (Fig. 4 C), although many transfected cells did show a reduction in cytoplasmic β-catenin (see images in Fig.4 A). To confirm these findings, we transiently expressed a full-length APC-YFP fusion vector in SW480 cells and stained these cells with a different β-catenin antibody (monoclonal Ab C19220) to that used above (rabbit polyclonal H-102). As expected, LEF-1 reduced the ability of APC-YFP to export and degrade cellular β-catenin (Fig.4 B). Quantitative imaging revealed that APC-YFP reduced β-catenin nuclear fluorescence by 92% in the absence of LEF-1, but only by 36% in the presence of LEF-1 (Fig. 4 C). Moreover, Western blot analysis of nuclear fractions prepared from SW480 cells untransfected or transfected with APC-YFP ± LEF-1 showed that LEF-1 reduced the disappearance of nuclear β-catenin caused by APC (Fig. 4 D). Collectively, these findings demonstrate for the first time that LEF-1 can inhibit APC-mediated degradation of β-catenin by protecting it in the nuclear compartment. The overexpression of axin, a key component in the cytoplasmic β-catenin degradation complex, is also known to induce β-catenin degradation when overexpressed in cells (26Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar), by a mechanism thought to require the APC/CRM1-independent nuclear export of β-catenin (15Wiechens N. Fagotto F. Curr. Biol. 2001; 11: 18-27Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar,16Eleftheriou A. Yoshida M. Henderson B.R. J. Biol. Chem. 2001; 276: 25883-25888Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). As we observed with APC, co-expression of axin with LEF-1 efficiently blocked the axin-dependent degradation of β-catenin in SW480 cells and caused β-catenin sequestration in the nucleus (Fig. 5). We recently showed that endogenous β-catenin can exit the nucleus of semipermeabilized SW480 cells independent of APC and the CRM1 export receptor (16Eleftheriou A. Yoshida M. Henderson B.R. J. Biol. Chem. 2001; 276: 25883-25888Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In this in vitro assay, cells are treated with the detergent digitonin, which permeabilizes the plasma membrane but leaves the nuclear envelope intact. The export reaction is unidirectional in that β-catenin can exit the nucleus, but is washed away before it re-enters the nucleus (see confocal images in Fig.6 A, top panel). We transfected SW480 cells with LEF-1 and performed the in vitro export reactions in transport buffer containing an energy-regenerating system (see “Materials and Methods”), in the absence or presence of digitonin (Fig. 6 A). Quantitative analysis of confocal images revealed that LEF-1 efficiently retained β-catenin in the nucleus during the in vitro export reaction (Fig. 6, A and B). Therefore, LEF-1 is able to block the general nuclear export of β-catenin. Interestingly, in this assay we also observed the presence of stable β-catenin/LEF-1 in the cytoplasm of some cells. This suggests that LEF-1 is also capable of stabilizing β-catenin in the cytoplasmic compartment, which is consistent with a recent study showing that a related protein, TCF-3, can stabilize β-catenin in the cytoplasm of Xenopusembryo cells (27Lee E. Salic A. Kirschner M.W. J. Cell Biol. 2001; 154: 983-993Crossref PubMed Scopus (128) Google Scholar). We next addressed possible mechanisms by which nuclear LEF-1-β-catenin complexes might be regulated in cells. Recently, histone deacetylase 1 (HDAC1) was found to interact with LEF-1 or with β-catenin (but apparently not both simultaneously) and caused transcriptional repression of a LEF-1-responsive promoter (28Billin A.N. Thirlwell H. Ayer D.E. Mol. Cell. Biol. 2000; 20: 6882-6890Crossref PubMed Scopus (193) Google Scholar). We postulated that HDAC1 might also interfere with the ability of LEF-1 to anchor β-catenin in the nucleus. To test this, we transfected NIH 3T3 cells with pHA-LEF-1 alone, or in the presence of a HDAC1 expression vector, and compared the level of β-catenin nuclear fluorescence in transfected cells (Fig. 7 A). As illustrated by confocal imaging and fluorescence quantification, co-expression of HDAC1 (2:1 ratio of DNA) reduced the LEF-1 induction of nuclear β-catenin by almost 40% (Fig. 7, A andB). Moreover, in the same cell line we confirmed that HDAC1 inhibited LEF-1 activation of a LEF-1 responsive promoter (Fig.7 C). HDAC1 had a similar inhibitory effect in cells co-transfected with both LEF-1 and β-catenin cDNAs (data not shown). We propose that HDAC1 disrupts the LEF-1-β-catenin complex, causing a decrease in nuclear β-catenin, which at least partly contributes to the reduced transcriptional activation of LEF-1-responsive genes. There is strong evidence linking the stabilization of β-catenin with its nuclear accumulation. For instance, nuclear levels of β-catenin are enhanced by its overexpression in transgenic mice (5Harada N. Tamai Y. Ishikawa T.-O. Sauer B. Takaku K. Oshima M. Taketo M.M. EMBO J. 1999; 18: 5931-5942Crossref PubMed Scopus (981) Google Scholar), by proteasome blockade (Ref. 20Simcha I. Shtutman M. Salomon D. Zhurinsky J. Sadot E. Geiger B. Ben-Ze'ev A. J. Cell Biol. 1998; 141: 1433-1448Crossref PubMed Scopus (238) Google Scholar and this study; Fig. 1 A), and by mutations in the β-catenin, APC, or axin genes (1Kinzler K.W. Vogelstein B. Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4286) Google Scholar, 2Morin P.J. Bioessays. 1999; 21: 1021-1030Crossref PubMed Scopus (817) Google Scholar, 3Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). In this study, we showed that LEF-1 can trap β-catenin in the nucleus and stabilize it against cytoplasmic destruction by APC-axin complexes, providing an additional mechanism by which cancer cells can elevate β-catenin and its transforming activity within the nucleus. Given that β-catenin can associate with several nuclear proteins including the chromatin remodelling factor BRG1 (29Barker N. Hurlstone A. Musisi H. Miles A. Bienz M. Clevers H. EMBO J. 2001; 20: 4935-4943Crossref PubMed Scopus (370) Google Scholar), the histone deacetylase HDAC1 (28Billin A.N. Thirlwell H. Ayer D.E. Mol. Cell. Biol. 2000; 20: 6882-6890Crossref PubMed Scopus (193) Google Scholar), and the transcription coactivator p300/CBP (30Sun Y. Kolligs F.T. Hottiger M.O. Mosavin R. Fearon E.R. Nabel G.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12613-12618Crossref PubMed Scopus (107) Google Scholar,31Takemaru K.-I. Moon R.T. J. Cell Biol. 2000; 149: 249-254Crossref PubMed Scopus (407) Google Scholar), it is likely that additional factors will contribute to its nuclear retention in mammalian cells. Indeed, we observed that co-expression of HDAC1 diminished the ability of LEF-1 to sequester β-catenin in the nucleus and to stimulate transcription of a LEF-1 responsive promoter (Fig. 7). Therefore, the modulation of β-catenin nuclear transport or retention by protein co-factors may reflect a common cellular mechanism for regulating its oncogenic transcription function. This is supported by the finding that the XenopusTCF-3 protein can block nuclear export of β-catenin in microinjectedXenopus oocytes (15Wiechens N. Fagotto F. Curr. Biol. 2001; 11: 18-27Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The LEF-1 gene is under positive feedback control by β-catenin-LEF-1-TCF transcription complexes (8Hovanes K., Li, T.W.H. Munguia J.E. Truong T. Milovanovic T. Marsh J.L. Holcombe R.F. Waterman M.L. Nat. Genet. 2001; 28: 53-57Crossref PubMed Google Scholar), providing one explanation for the elevated expression of LEF-1 in colon tumor cell lines (7Porfiri E. Rubinfeld B. Albert I. Hovanes K. Waterman M. Polakis P. Oncogene. 1997; 15: 2833-2839Crossref PubMed Scopus (133) Google Scholar), colon tumors (8Hovanes K., Li, T.W.H. Munguia J.E. Truong T. Milovanovic T. Marsh J.L. Holcombe R.F. Waterman M.L. Nat. Genet. 2001; 28: 53-57Crossref PubMed Google Scholar), and malignant melanoma cells (9Murakami T. Toda S. Fujimoto M. Ohtsuki M. Byers H.R. Etoh T. Nakagawa H. Biochem. Biophys. Res. Commun. 2001; 288: 8-15Crossref PubMed Scopus (66) Google Scholar). LEF-1 forms active complexes with β-catenin in vivo in colon cancer cells (7Porfiri E. Rubinfeld B. Albert I. Hovanes K. Waterman M. Polakis P. Oncogene. 1997; 15: 2833-2839Crossref PubMed Scopus (133) Google Scholar). We propose that enhanced LEF-1 levels provide an alternative mechanism for stabilizing β-catenin in cells. This may be of particular relevance in cancers where elevated nuclear β-catenin does not correlate with known gene mutations (32Rimm D.L. Caca K., Hu, G. Harrison F.B. Fearon E.R. Am. J. Pathol. 1999; 154: 325-329Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 33Omholt K. Platz A. Ringborg U. Hansson J. Int. J. Cancer. 2001; 92: 839-842Crossref PubMed Scopus (83) Google Scholar). The ability of LEF-1 to stabilize β-catenin is highly consistent with a positive feedback model that favors accumulation of transcriptionally active and oncogenic β-catenin/LEF-1 complexes. In future experiments it will be of interest to identify additional factors and signaling pathways that modulate the stability/dissociation of LEF-1-β-catenin complexes. The ability to regulate the nuclear activity or nuclear exclusion of β-catenin has implications for potential anticancer therapies. We thank Drs. Bert Vogelstein and Kenneth Kinzler for pCMV-APC vector, Dr. Trevor Dale for FLAG-tagged Axin plasmid, Dr. J. Behrens for the HA-tagged LEF-1 plasmid, Dr. Donald Ayer for the pcDNA3-HDAC1 plasmid, and Dr. M. Yoshida for leptomycin B. We also thank Ken Kinzler, Francois Fagotto, and Paul Polakis for helpful discussions and sharing of information. We acknowledge the generous technical help and advice of Dr. Helen Rizos and Mary Sartor." @default.
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• W2019713047 title "Lymphoid Enhancer Factor-1 Blocks Adenomatous Polyposis Coli-mediated Nuclear Export and Degradation of β-Catenin" @default.
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