Adenomatous Polyposis Coli (APC)-independent Regulation of β-Catenin Degradation via a Retinoid X Receptor-mediated Pathway
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m304761200
ISSN1083-351X
AutoresJia‐Hao Xiao, Corine Ghosn, Cory Hinchman, Chad Forbes, Jenny W. Wang, Nonna Snider, Allison Cordrey, Yi Zhao, Roshantha A.S. Chandraratna,
Tópico(s)Ubiquitin and proteasome pathways
Resumoβ-catenin is a component of stable cell adherent complexes whereas its free form functions as a transcription factor that regulate genes involved in oncogenesis and metastasis. Free β-catenin is eliminated by two adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways regulated by glycogen synthase kinase 3β (GSK3β) or p53-inducible Siah-1. Dysregulation of β-catenin turnover consequent to mutations in critical genes of the APC-dependent pathways is implicated in cancers such as colorectal cancer. We have identified a novel retinoid X receptor (RXR)-mediated APC-independent pathway in the regulation of β-catenin. In this proteasomal pathway, RXR agonists induce degradation of β-catenin and RXRα and repress β-catenin-mediated transcription. In vivo, β-catenin interacts with RXRα in the absence of ligand, but RXR agonists enhanced the interaction. RXR agonist action was not impaired by GSK3β inhibitors or deletion of the GSK3β-targeted sequence from β-catenin. In APC- and p53-mutated colorectal cancer cells, RXR agonists still inactivated endogenous β-catenin via RXRα. Interestingly, deletion of the RXRα A/B region abolished ligand-induced β-catenin degradation but not RXRα-mediated transactivation. RXRα-mediated inactivation of oncogenic β-catenin paralleled a reduction in cell proliferation. These results suggest a potential role for RXR and its agonists in the regulation of β-catenin turnover and related biological events. β-catenin is a component of stable cell adherent complexes whereas its free form functions as a transcription factor that regulate genes involved in oncogenesis and metastasis. Free β-catenin is eliminated by two adenomatous polyposis coli (APC)-dependent proteasomal degradation pathways regulated by glycogen synthase kinase 3β (GSK3β) or p53-inducible Siah-1. Dysregulation of β-catenin turnover consequent to mutations in critical genes of the APC-dependent pathways is implicated in cancers such as colorectal cancer. We have identified a novel retinoid X receptor (RXR)-mediated APC-independent pathway in the regulation of β-catenin. In this proteasomal pathway, RXR agonists induce degradation of β-catenin and RXRα and repress β-catenin-mediated transcription. In vivo, β-catenin interacts with RXRα in the absence of ligand, but RXR agonists enhanced the interaction. RXR agonist action was not impaired by GSK3β inhibitors or deletion of the GSK3β-targeted sequence from β-catenin. In APC- and p53-mutated colorectal cancer cells, RXR agonists still inactivated endogenous β-catenin via RXRα. Interestingly, deletion of the RXRα A/B region abolished ligand-induced β-catenin degradation but not RXRα-mediated transactivation. RXRα-mediated inactivation of oncogenic β-catenin paralleled a reduction in cell proliferation. These results suggest a potential role for RXR and its agonists in the regulation of β-catenin turnover and related biological events. β-catenin is a key mediator in Wnt regulation of multiple cellular functions in embryogenesis and tumorigenesis (1Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1146) Google Scholar). In adult tissues, β-catenin is a component of stable cell adherent complexes whereas its free form functions as a co-activator for a family of transcription factors called T cell factor/lymphoid enhancer factor (TCF/LEF). 1The abbreviations used are: TCF/LEF, T cell factor/lymphoid enhancer factor; APC, adenomatous polyposis coli; GSK3β, glycogen synthase kinase 3β; RAR, retinoic acid receptor; RXR, retinoid X receptor; TR, thyroid hormone receptor; AF-1/2, activation function 1 or 2; HRP, horseradish peroxidase; HEK293, human embryonic kidney 293 (cells); DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; DSP, dithiobis(succinimidyl propionate). Levels of free β-catenin are tightly regulated by two APC-dependent proteasomal degradation pathways, namely a GSK3β-regulated pathway involving the APC/Axin complex (2Polakis P. Curr. Biol. 2002; 12: R499—R501Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) and a p53-inducible pathway involving Siah-1 (3Liu J. Stevens J. Rote C.A. Yost H.J. Hu Y. Neufeld K.L. White R.L. Matsunami N. Mol. Cell. 2001; 7: 927-936Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 4Matsuzawa S.I. Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). In the GSK3β-regulated pathway, β-catenin associates with the APC/Axin complex and undergoes a two-step phosphorylation by casein kinase I (CKI) and GSK3β at serine/threonine residues within the first 50 N-terminal amino acids (2Polakis P. Curr. Biol. 2002; 12: R499—R501Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 5Schwarz-Romond T. Asbrand C. Bakkers J. Kuhl M. Schaeffer H.J. Huelsken J. Behrens J. Hammerschmidt M. Birchmeier W. Genes Dev. 2002; 16: 2073-2084Crossref PubMed Scopus (162) Google Scholar). β-catenin interacts with an ubiquitylation complex through the phosphorylated N terminus and undergoes proteasome-catalyzed degradation (6Polakis P. Cell. 2001; 105: 563-566Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Wnt inactivation of GSK3β leads to translocation of β-catenin to the nucleus, where it enables TCF/LEF to activate genes involved in embryogenesis and oncogenesis (1Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1146) Google Scholar, 7Bienz M. Clevers H. Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar). In the second pathway, p53-up-regulated Siah-1 interacts with the N-terminal region of APC, recruits an ubiquitylation complex to the N terminus of β-catenin, and targets it for proteasome-mediated degradation. Thus, both pathways require the intact N terminus of β-catenin. In cancers such as colorectal and hepatocellular cancers and melanoma, mutations in the key components of the two pathways, such as APC, p53, and Axin, or β-catenin itself, lead to dysregulation of β-catenin turnover and, consequently, high levels of nuclear β-catenin and abnormal activation of TCF/LEF-regulated genes that are involved in oncogenesis and metastasis (8Conacci-Sorrell M.E. Ben-Yedidia T. Shtutman M. Feinstein E. Einat P. Ben-Ze'ev A. Genes Dev. 2002; 16: 2058-2072Crossref PubMed Scopus (156) Google Scholar, 9Wong N.A. Pignatelli M. Am. J. Pathol. 2002; 160: 389-401Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar). Retinoids, which are natural and synthetic derivatives of vitamin A, regulate gene transcription through two families of nuclear receptors, i.e. retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (10Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2606) Google Scholar) and have significant anti-cancer effects (11Altucci L. Gronemeyer H. Nat. Rev Cancer. 2001; 1: 181-193Crossref PubMed Scopus (689) Google Scholar, 12Sun S.Y. Lotan R. Crit. Rev. Oncol. Hematol. 2002; 41: 41-55Crossref PubMed Scopus (297) Google Scholar). These receptors are ligand-dependent DNA binding transcription factors. Each receptor has an N-terminal A/B region that harbors the ligand-independent activation function-1 (AF-1), a central DNA-binding domain (the C region), and a C-terminal E region containing a ligand binding domain and a ligand-dependent activation function-2 (AF-2). RARs and RXRs bind to target genes as RAR-RXR heterodimers or RXR homodimers. In the absence of ligands, retinoid receptors are associated with co-repressors and repress gene transcription (13Westin S. Rosenfeld M.G. Glass C.K. Adv. Pharmacol. 2000; 47: 89-112Crossref PubMed Scopus (91) Google Scholar). Once associated with agonists, RARs and RXRs undergo conformational changes, recruit co-activators, and activate target gene transcription. Interestingly, instances of crosstalk between the Wnt/β-catenin- and retinoid-signaling pathways have been reported recently. For example, RAR was found to interact with β-catenin in vitro and inhibits β-catenin-mediated gene transcription in vivo (14Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar). Retinoic acid, an RAR agonist, was shown to synergize with Wnt signaling in the up-regulation of gene transcription (15Tice D.A. Szeto W. Soloviev I. Rubinfeld B. Fong S.E. Dugger D.L. Winer J. Williams P.M. Wieand D. Smith V. Schwall R.H. Pennica D. Polakis P. J. Biol. Chem. 2002; 277: 14329-14335Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 16Liu T. Lee Y.-N. Malbon C.C. Wang H.-y. J. Biol. Chem. 2002; 277: 30887-30891Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Unlike the APC-dependent pathway, RAR signaling does not regulate β-catenin protein levels (14Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar). On the other hand, RXR agonists have been shown to cause degradation of RXRα and also its receptor heterodimerization partners, including RARs and TR (17Kopf E. Plassat J.L. Vivat V. de Thé H. Chambon P. Rochette-Egly C. J. Biol. Chem. 2000; 275: 33280-33288Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Osburn D.L. Shao G. Seidel H.M. Schulman I.G. Mol. Cell. Biol. 2001; 21: 4909-4918Crossref PubMed Scopus (43) Google Scholar). However, the biological consequences of such degradation phenomena have not been well understood. Here, we have investigated the role of RXR and its ligands in the regulation of β-catenin activity and identified a novel RXR-mediated APC-independent pathway. We show that RXR agonists reduce β-catenin-mediated activation of gene transcription and cell proliferation through a protein degradation mechanism. Retinoids—The RAR antagonist AGN194310 and the RXR agonist AGN194204 have been described previously (19Johnson A.T. Wang L. Standeven A.M. Escobar M. Chandraratna R.A.S. Bioorg Med. Chem. 1999; 7: 1321-1338Crossref PubMed Scopus (46) Google Scholar, 20Vuligonda V. Thacher S.M. Chandraratna R.A.S. J. Med. Chem. 2001; 44: 2298-2303Crossref PubMed Scopus (76) Google Scholar). The RXR-specific agonists AGN195362, AGN195456, AGN195741, AGN196060, and AGN196459 and the RXR-specific antagonist AGN195393 were synthesized at Allergan. Me2SO was used as a solvent for the compounds. Plasmids—TOPFLASH, which contains TCF/LEF binding sites placed in front of the TK-Luc reporter gene, was purchased from Upstate Biotechnology. The β-catenin expression vector, Gene Storm clone H-X87838 M in pcDNA3.1/GS, was purchased from Invitrogen. ΔNβ-catenin, a β-catenin mutant with an N-terminal deletion (amino acid residues 1–50), was made by PCR amplification from wild type β-catenin using the following pair of primers: 5′-AGG GAT CCA ACC ATG AAT CCT GAG GAA GAG-3′ and 5′-AGT CTA GAT TAC AGG TCA GTA TCA AAC CAG-3′. The resulting fragment was cloned into expression vector pcDNA3.1+ (Invitrogen Corp) between BamH1 and XbaI and confirmed by DNA sequencing. Finally, the N-terminal coding region with the deletion was released by digestion with MunI and XhoI and used to replace the 5′-end of wild type β-catenin in pcDNA3.1/GS. Human RXRα cDNA in a human keratinocyte cDNA library (21Nagpal S. Ghosn C. DiSepio D. Molina Y. Sutter M. Klein E.S. Chandraratna R.A.S. J. Biol. Chem. 1999; 274: 22563-22568Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) was identified in a yeast two-hybrid system using RARγ as a bait. The RXRα coding region was amplified from this clone by PCR using a pair of following primers: 5′-AG GAA TTC ATG GAC ACC AAA CAT TTC CTG CCG-3′ and 5′-AG CTG CAG CTA AGT CAT TTG GTG CGG CGG CTC-3′. The resulting fragment was subcloned into pEGFP-N2 (Clontech) between the EcoRI and PstI sites and then released by EcoRI and KpnI digestion. The released RXRα coding region was then cloned into a modified pCMV-FLAG vector (Sigma) containing the FLAG epitope DYKDDDDK. The RXRα deletion mutants (see Fig. 6A) were constructed by PCR amplification of hRXRα cDNA using primers as follows: 5′-AGG AAT TCT GCG CCA TCT GCG GGG ACC GC-3′ and 5′-AGG GTA CCC TAA GTC ATT TGG TGC GGC GCC TCC-3′ for RXRαCDE; 5′-AGG AAT TCA AGC GGG AAG CCG TGC AGG AGG AGC GG-3′ and 5′-AGG GTA CCC TAA GTC ATT TGG TGC GGC GCC TCC-3′ for RXRαDE; 5′-AGG AAT TCT CGC CGA ACG ACC CTG TCA CC-3′ and 5′-AGG GTA CCC TAA GTC ATT TGG TGC GGC GCC TCC-3′ for RXRαE; and 5′-AGG AAT TCA TGG ACA CCA AAC ATT TCC TGC CG-3′ and 5′-AGG GTA CCC TAG ATG AGC TTG AAG AAG AAG AG-3′ for RXRα ΔAF2. The resulting PCR fragments were cut by EcoR1 and KpnI and cloned into the pCMV-FLAG vector. For construction of RXRαΔC and RXRαΔCD, the EcoR1 fragment containing the A/B region of RXRα was obtained by PCR amplification with primers 5′-AGG AAT TCA TGG ACA CCA AAC ATT TCC TGC CG-3′ and 5′-AGG AAT TCG ATG TGC TTG GTG AAG GAA GCC-3′ and inserted into constructs RXRαDE and RXRαE at the EcoR1 site in front of the DE and E regions of RXRα, respectively. For making RXRαΔD, the EcoR1 fragment containing the ABC region of RXRα was prepared by PCR amplification using the following primers: 5′-AGG AAT TCA TGG ACA CCA AAC ATT TCC TGC CG-3′ and 5′-AGG AAT TCC ATG CCC ATG GCC AGG CAC TTC-3′ and inserted into the EcoR1 site in front of the E region in construct RXRαE. Antibodies—Native or horseradish peroxidase (HRP)-conjugated mouse monoclonal antibodies against the FLAG tag in RXRα, M2, and HRP-M2 were purchased from Sigma. Native or HRP-conjugated mouse monoclonal antibodies against the V5 tag in β-catenin, V5, and HRP-V5, respectively, were purchased from Invitrogen. Rabbit polyclonal antibodies against the N terminus of RXRα (D20), the C terminus of β-catenin (H102), poly(ADP-ribose) polymerase (PARP, H-250), GSK3β (H76), and a mouse monoclonal antibody against β-tubulin (D-10) were purchased from Santa Cruz Biotechnology. Cell Lines—HEK293, HeLa, CV1, and SW480 cells were purchased from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 10 μg/ml streptomycin at 37 °C in 5% CO2. To generate the cell lines CAT and mCAT that stably expresses β-catenin or ΔNβ-catenin, respectively, pGS-β-catenin or pGS-ΔNβ-catenin was transfected into HEK293 cells using LipofectAMINE. Twenty-four hours later, the cells were subjected to selection in the presence of zeocin (Invitrogen) at 400–500 μg/ml. The selection medium was changed every 3 days, and individual zeocin-resistant clones were isolated. Clones stably expressing β-catenin or ΔNβ-catenin were identified by Western blotting. To produce CATXα and mCATXα cell lines that stably express RXRα with wild type β-catenin or mutant ΔNβ-catenin, pGS-β-catenin and pGS-ΔNβ-catenin were transfected into cell line Xα that stably expresses FLAG-tagged RXRα. These cell lines were established as described above. Inhibitors—Proteasome inhibitors MG132 and MG262 were purchased from Biomol Research Laboratories and Calbiochem, respectively. Lysosome inhibitors, bafilomycin, leupeptin, E-64, and ammonium chloride were purchased from Sigma-Aldrich. Reporter Gene Assays—For measuring the TOPFLASH activity, cells were seeded at 50,000 cells per well in 24-well plates coated with poly-d-lysine (BD Biosciences). Twenty-four hours later, TOPFLASH and expression vectors were co-transfected into cells using FuGENE (Roche Applied Science) in DMEM containing 10% charcoal-treated FBS. To monitor transfection efficiency, either 15 ng of phRG-TK renilla or 100 ng of CMX-LacZ DNA were co-transfected. Five hours later, vehicle or retinoids were added. The cells were treated for 16 h before harvest. Luciferase activity was measured using the Dual luciferase reporter 100 assay system (Promega). Control Renilla activity was determined using the same kit. A second control, β-galactosidase activity, was measured by colorimetric assays. The reporter activity was normalized against either β-galactosidase or Renilla activity. Analysis of RXRα and its mutants in transactivation was performed as follows. 3.5 × 103 CV-1 cells per well of a 96-well opaque plate (Falcon) were transiently transfected using LipofectAMINE with the reporter plasmid CRBPII-TK-Luc together with 0.04 μg of RXRα mutants. Five hours later, DMEM containing 10% charcoal-treated FBS and retinoids were added to the wells. Cells were grown for a further 18 h and lysed for determination of luciferase activity. Protein Expression and Western Blotting Analysis—Cells were seeded at 2 × 106 per plate into 100-mm dishes in DMEM containing 10% FBS and, on the next day, transfected with 1–4 μg of various cDNA or parental expression vectors (8 μg of DNA in total) using LipofectAMINE (Invitrogen). Five hours later, the cells were fed with fresh DMEM medium containing 10% charcoal-treated FBS and vehicle or retinoids. After the retinoid treatment, the total cell lysates were prepared using a buffer containing 1% Nonidet P-40, 30 mm Tris-HCl (pH 7.4), 0.5 mm EDTA (pH 8.0), 150 mm NaCl, 10% glycerol, 1 mm sodium orthovanadate, 40 mm NaF, 0.5 mm phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (Merck). The cell lysates were homogenized by passing through a QIAshredder (Qiagen) and cleared from insoluble materials by centrifugation at 12,000 rpm in a bench top Eppendorf centrifuge. Protein concentrations were determined using the Bradford kit (Bio-Rad). Proteins were resolved on 4–12% SDS-PAGE and transferred to either nitrocellulose or polyvinylidene difluoride membranes. The membranes were blocked with 10% nonfat milk in phosphate-buffered saline (PBS, Invitrogen) containing 0.1% Tween 20 (PBST). The membranes were incubated with primary antibodies at room temperature for 2 h or at 4 °C overnight. After the removal of unbound antibodies, membranes were incubated with HRP-conjugated secondary antibodies for1hat room temperature and washed five times with PBST. The antibody-associated protein bands were revealed using the ECL plus system (Amersham Pharmacia Biotech). Pulse-Chase Analysis—Cells in growth phase were washed with RPMI 1640 medium without cysteine, methionine, and glutamine (Cell-gro), starved in the same medium for 60 min, and pulse-labeled using 200 μCi of 35S Promix (Amersham Biosciences) for 1 h. Then, the cells were washed with RPMI 1640 three times and chased with the medium supplemented with cysteine (50 μg/ml), methionine (15 μg/ml), and glutamine (2 mm) in the presence or absence of AGN194204. Total cell extracts were prepared as described above. RXRα and β-catenin were immunoprecipitated using the M2 and V5 antibodies, respectively. The immunoprecipitated proteins were resolved on 4–12% SDS-PAGE. Gels were stained with Coomassie Blue and treated with the reagent Amplify (Amersham Biosciences) for 15 min, vacuum-dried, and subjected to autoradiography. In Vivo Protein Crosslinking and Immunoprecipitation—HEK293 cells at ∼80% confluence in 150-mm poly-d-lysine-coated plates (BD Biosciences) were transfected with expression vectors for β-catenin (8 μg), ΔN-β-catenin (8 μg), and RXRα (4 μg) and cultured overnight in DMEM containing high glucose and 10% activated charcoal-treated fetal bovine serum. The cells were treated with vehicle or 1 μm AGN194204 for 20 min in the same medium and then with 1 mm dithiobis(succinimidyl propionate) (DSP; Pierce), a reversible crosslinking reagent, in phosphate-buffered saline for 15 min. The reaction was quenched for 15 min by Tris-HCl buffer (pH 7.5) at 20 mm. The cells were lysed in ice-chilled radioimmune precipitation assay buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in phosphate-buffered saline) containing a mixture of protease inhibitors (Sigma) and homogenized by QIAshredder (Qiagen). The extracts (1.5 mg per immunoprecipitation) were incubated overnight with specific antibodies and protein G-agarose beads with constant shaking at 4 °C. Immunoprecipitated materials were washed with the ice-cold radioimmune precipitation assay buffer and dissolved in SDS-PAGE loading dye containing β-mercaptoethanol by heating at 100 °C for 5 min. This procedure frees the DSP-crosslinked molecules pulled down by the antibodies. Proteins were resolved on 4–12% SDS-polyacrylamide gels followed by Western blotting. Cell Proliferation Assay—Cell proliferation assays were performed in 96-well microtiter plates. HEK293, mCAT (HEK293-derived cells stably expressing ΔNβ-catenin), Xα (HEK293-derived cells stably expressing RXRα), and mCATXα cells were seeded at 200–400 cells/well in regular growth medium. The next day, vehicle or retinoids were added. Cell proliferation was measured after 6 days of treatment using a cell proliferation kit purchased from Chemicon International. RXR Agonists Inactivate β-Catenin-mediated Transcription via Endogenous and Transfected RXRs—We investigated the effect of RXR-specific agonists on β-catenin-mediated TCF/LEF transcriptional activity, a surrogate marker for the oncogenic activity of β-catenin, using the TOPFLASH reporter gene, which contains TCF/LEF binding sites (22Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2950) Google Scholar, 23Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar). In HEK293 cells, the significant reporter activity produced by endogenous β-catenin was reduced by AGN194204, an RXR-specific agonist (20Vuligonda V. Thacher S.M. Chandraratna R.A.S. J. Med. Chem. 2001; 44: 2298-2303Crossref PubMed Scopus (76) Google Scholar), in the absence (∼50%) or presence (∼70%) of transfected RXRα (Fig. 1, a and b). The significantly increased reporter activity obtained with β-catenin transfection was still very effectively (∼80%) reduced by AGN194204 treatment in the presence of cotransfected RXRα (Fig 1, a and b). Similar inhibition was observed in CATXα cells that were stably transfected with both β-catenin and RXRα, whereas the AGN194204 effect was less pronounced in CAT cells that were stably transfected with only β-catenin (Fig. 1c). RXR Agonists Induce β-Catenin Degradation via Endogenous and Overexpressed RXRs—Because APC-dependent protein degradation pathways regulate β-catenin-mediated transcription (6Polakis P. Cell. 2001; 105: 563-566Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 22Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2950) Google Scholar, 23Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar) and RXR agonists induce degradation of RXR and associated receptor partners (17Kopf E. Plassat J.L. Vivat V. de Thé H. Chambon P. Rochette-Egly C. J. Biol. Chem. 2000; 275: 33280-33288Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Osburn D.L. Shao G. Seidel H.M. Schulman I.G. Mol. Cell. Biol. 2001; 21: 4909-4918Crossref PubMed Scopus (43) Google Scholar), we sought to determine whether AGN194204 inhibited TOPFLASH reporter activity by reducing β-catenin protein levels. HEK293 cells were treated with vehicle or the RXR agonist, and total cell lysates were analyzed by immunoprecipitation and Western blotting. As shown in Fig. 2a, AGN194204 decreased endogenous β-catenin (∼50%) and RXRα levels in HEK293 cells in the absence of transfected RXRα. AGN194204 further reduced endogenous β-catenin (∼80%) in the presence of transfected RXRα. In HEK293 cells transfected with β-catenin alone, AGN194204 had no effect on β-catenin because of the low levels of endogenous RXRα relative to transfected β-catenin (Fig. 2b). However, it dramatically reduced β-catenin protein levels concurrent with RXRα protein levels in cells transfected with both RXRα and β-catenin. Similar AGN194204 effects were obtained in stably transfected HEK293 cells (CATXα; Fig. 2c) or transiently transfected CV1, HeLa, and SW480 cells (Fig. 2e), indicating the ubiquitous nature of this phenomenon. The AGN194204 effects on reducing β-catenin protein levels were time- (Fig. 2c) and dose-dependent (Fig. 2f), and the efficiency of the reduction depended on RXR protein levels (Fig. 2, a and d). AGN194204 readily caused a significant decrease of β-catenin at a dose as low as 1 nm (Fig. 2f), reflecting its high affinity for RXRα. Several different RXR agonists, including AGN195362, AGN195456, AGN195741, AGN196060, AGN196459, and 9-cis retinoic acid, similarly reduced β-catenin protein levels (Fig. 2g, and data not shown). An RXR-specific antagonist, AGN195393 (24Dussault I. Beard R. Lin M. Hollister K. Chen J. Xiao J.H. Chandraratna R. Forman B.M. J. Biol. Chem. 2003; 278: 7027-7033Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), dose-dependently inhibited the AGN194204 effects on β-catenin and RXR protein levels (Fig. 2f). An RAR agonist, TTNPB, or an RAR antagonist, AGN194310, showed no effects (Fig. 2g). Pulse-chase analysis was performed to ascertain whether the RXR agonist effect occurs at the level of protein degradation. AGN194204 accelerated degradation of both 35S-labeled RXRα and β-catenin (Fig. 2h). In the presence of cycloheximide and the absence of AGN194204, β-catenin is readily subjected to degradation by the active APC-pathways in HEK293 cells (compare lane 3 to lane 1 in Fig. 2i) as expected. However, cycloheximide did not block the AGN194204-induced degradation of β-catenin and RXRα (comparing lane 4 to lane 3 in Fig. 2i), indicating that induction of transcriptional activity is not required for this effect. Together, these data indicate that RXR agonists reduce β-catenin protein levels by an RXR-mediated protein degradation pathway, which is independent of the RXR-mediated gene transcription activation pathway. In cells where the Siah- and GSK3β-regulated APC pathways are impaired by mutations or GSK3β is inhibited by Wnt signaling, levels of β-catenin are increased in the cytoplasmic compartment, and, ultimately, β-catenin is translocated to the nucleus where it transactivates the TCF/LEF-targeted genes. Our transactivation data indicated that nuclear β-catenin-related transcriptional activity was reduced by RXR agonists. We further examined whether inhibition of β-catenin-mediated gene transcription by AGN194204 is due to a reduction of β-catenin protein levels in the nucleus with an analysis of nuclear and cytosolic fractions of CATXα cells. Poly(ADP-ribose) polymerase and β-tubulin were used as nuclear and cytoplasmic markers, respectively, for monitoring the efficiency of separation of the two fractions. Decreases in β-catenin protein levels as a result of AGN194204 treatment were observed in both nuclear and cytosolic compartments (Fig. 2, j and k). The APC-dependent degradation of β-catenin and the agonist-dependent degradation of RXR proceed by proteasomal pathways (6Polakis P. Cell. 2001; 105: 563-566Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 17Kopf E. Plassat J.L. Vivat V. de Thé H. Chambon P. Rochette-Egly C. J. Biol. Chem. 2000; 275: 33280-33288Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Osburn D.L. Shao G. Seidel H.M. Schulman I.G. Mol. Cell. Biol. 2001; 21: 4909-4918Crossref PubMed Scopus (43) Google Scholar). To determine whether the RXR agonist-induced degradation of β-catenin involves this pathway, we treated cells with the proteasome inhibitors MG262 and MG132. As shown in Fig. 2, l and m, these two inhibitors dose-dependently blocked AGN 194204-induced degradation of both β-catenin and RXRα. However, lysosomal inhibitors such as bafilomycin, E64, NH4Cl, and leupeptin had no effect (data not shown). These data indicate that RXR agonist-induced degradation of β-catenin also proceeds by a proteasomal pathway. The RXR-regulated β-Catenin Degradation Pathway Is Independent of the p53/Siah-1- and GSK3β-regulated APC Pathways—To determine whether the GSK3β- or p53/Siah-regulated APC pathways are involved in the RXR agonist effects, a β-catenin mutant (ΔNβ-catenin) with a deletion of the N-terminal sequence (50 amino acids) that is targeted by the two APC-dependent pathways was prepared (6Polakis P. Cell. 2001; 105: 563-566Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar). Although this mutant, which is resistant to APC-mediated degradation pathways, showed higher TOPFLASH reporter gene activity than wild-type β-catenin, its increased activity was very effectively inhibited by AGN194204 in the presence of RXRα (Fig. 1d). High levels of TOPFLASH reporter activity associated with elevated β-catenin levels have been reported in SW480 colorectal cancer cells wherein both APC and p53 genes contain loss-of-function mutations (22Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2950) Google Scholar, 23Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar, 25Abarzua P. LoSardo J.E. Gubler M.L. Neri A. Cancer Res. 1995; 55: 3490-3494PubMed Google Scholar). However, AGN194204 effectively inhibited reporter activity in SW480 cells containing cotransfected RXRα (Fig. 1e), which is consistent with the observed decrease in endogenous β-catenin levels in these cells (Fig. 2a). Similarly, whereas LiCl, a GSK3β inhibitor that is known to elevate free β-catenin levels (26Playford M.P. Bicknell D.
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