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Trans-repression of β-Catenin Activity by Nuclear Receptors

2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês

10.1074/jbc.m307154200

1083-351X

Salimuddin Shah, Andreas Hecht, Richard G. Pestell, Stephen W. Byers,

PI3K/AKT/mTOR signaling in cancer

The signaling/oncogenic activity of β-catenin can be repressed by the activation of nuclear receptors such as the vitamin A, vitamin D, and androgen receptors. Although these receptors directly interact with β-catenin and can sequester it away from its transcription factor partner T-cell factor, it is not known if this is the mechanism of trans-repression. Using several different promoter constructs and nuclear receptors and mammalian two-hybrid and mutation analyses we now show that interaction with the co-activator, p300, underlies the trans-repression of β-catenin signaling by nuclear receptors and their ligands. The signaling/oncogenic activity of β-catenin can be repressed by the activation of nuclear receptors such as the vitamin A, vitamin D, and androgen receptors. Although these receptors directly interact with β-catenin and can sequester it away from its transcription factor partner T-cell factor, it is not known if this is the mechanism of trans-repression. Using several different promoter constructs and nuclear receptors and mammalian two-hybrid and mutation analyses we now show that interaction with the co-activator, p300, underlies the trans-repression of β-catenin signaling by nuclear receptors and their ligands. By definition, the term chemoprevention refers to the treatment of individuals with premalignant lesions, individuals who are predisposed to the development of malignant lesions, and individuals early in the process of carcinogenesis, with the intention of preventing the later stages of carcinogenesis from developing (1Sporn M.B. Suh N. Nat. Rev. Cancer. 2002; 2: 537-543Crossref PubMed Scopus (329) Google Scholar). Although chemopreventative agents such as vitamins A and D are often effective in this context there is no de facto reason to assume that the same treatment would not be effective in more advanced stages of carcinogenesis. Nevertheless, this is often not the case because many advanced cancers in vivo and de-differentiated carcinoma cells in vitro are refractory to the anti-tumor, growth inhibitory activities of vitamins A and D. Thus, not only do more malignant tumors and cells have defects in the expression and function of several molecules characteristic of differentiated epithelial cells; they are also less responsive to differentiating agents. An obvious question arises from this realization; what are the molecular mechanisms that underlie resistance to vitamins A and D? In some cases, resistance is because of a defect in particular nuclear receptor isoforms. However, some cells, which are not growth-inhibited by vitamin A, express normal retinoic acid receptors (RARs) 1The abbreviations used are: RARretinoic acid (RA) receptorRARERAR elementVDRvitamin D receptorARandrogen receptorLEFlymphoid enhancer factorTCFT-cell factor9cRA9-cis-retinoic acidRXRretinoid X receptorAP-1activator protein-1SRFserum response factor.1The abbreviations used are: RARretinoic acid (RA) receptorRARERAR elementVDRvitamin D receptorARandrogen receptorLEFlymphoid enhancer factorTCFT-cell factor9cRA9-cis-retinoic acidRXRretinoid X receptorAP-1activator protein-1SRFserum response factor./RXRs and retain the ability to up-regulate retinoid-sensitive genes (2Waliszewski P. Waliszewska M.K. Gupta M. Milsom J.W. Hurst R.E. J. Surg. Oncol. 1997; 66: 156-167Crossref PubMed Scopus (9) Google Scholar). These data indicate that other molecules such as components of the cadherin/β-catenin-based signaling and adhesion system may also need for retinoic acid to exert its anti-proliferative effects (3Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar, 4Shah S. Pishvaian M.J. Easwaran V. Brown P.H. Byers S.W. J. Biol. Chem. 2002; 277: 25313-25322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar).Recently, several studies show that the signaling/oncogenic activity of β-catenin is trans-repressed by the activation of nuclear receptors such as RAR, vitamin D receptor (VDR), and androgen receptors (AR) (3Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar, 5Palmer H.G. Gonzalez-Sancho J.M. Espada J. Berciano M.T. Puig I. Baulida J. Quintanilla M. Cano A. de Herreros A.G. Lafarga M. Munoz A. J. Cell Biol. 2001; 154: 369-387Crossref PubMed Scopus (683) Google Scholar). In addition to its role in cell-cell adhesion β-catenin acts as a co-activator for the TCF/lymphoid enhancer factor (LEF) family of transcription factors. Although it is possible that all of these receptors directly interact with β-catenin and sequester it away from TCF, it is not known if this is the mechanism of trans-repression. Nuclear receptors also trans-repress AP-1, serum response factor (SRF), and NFκB activity by a mechanism likely involving interaction with a common pool of co-activators rather than a direct interaction with transcription factors (6Guberman A.S. Scassa M.E. Giono L.E. Varone C.L. Canepa E.T. J. Biol. Chem. 2003; 278: 2317-2326Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 7Zhou X.F. Shen X.Q. Shemshedini L. Mol. Endocrinol. 1999; 13: 276-285Crossref PubMed Scopus (96) Google Scholar, 8Kim S.W. Kim H.J. Jung D.J. Lee S.K. Kim Y.S. Kim J.H. Kim T.S. Lee J.W. Oncogene. 2001; 20: 6638-6642Crossref PubMed Scopus (12) Google Scholar). On the other hand, in contrast to AP-1, high levels of β-catenin markedly potentiate RARE, vitamin D receptor element, and androgen receptor element reporter activity as well as the expression of retinoid-sensitive genes, a phenomenon that is not likely to be a result of co-activator competition (3Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar, 5Palmer H.G. Gonzalez-Sancho J.M. Espada J. Berciano M.T. Puig I. Baulida J. Quintanilla M. Cano A. de Herreros A.G. Lafarga M. Munoz A. J. Cell Biol. 2001; 154: 369-387Crossref PubMed Scopus (683) Google Scholar, 9Truica C Byers S Gelmann E. Cancer Res. 2000; 60: 4709-4713PubMed Google Scholar). Using several different promoter constructs and nuclear receptors, mammalian two-hybrid analyses, and other biochemical methods we now show that interaction with the histone acetyl transferase, p300, underlies the trans-repression of β-catenin signaling by nuclear receptors. Taken together these data suggest that the failure of retinoic acid to inhibit the growth of some colon cancer cells is because of defects in trans-repression of β-catenin/TCF activity in addition to or instead of a failure to trans-activate retinoid-sensitive genes.EXPERIMENTAL PROCEDURESPlasmids—Vectors encoding either wild type or a degradation-resistant mutant of β-catenin (S37A) have been described previously (10Orford K. Crockett C. Jensen J.P. Weissman A.M. Byers S.W. J. Biol. Chem. 1997; 272: 24735-24738Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). Wild type and dominant negative c-Fos and c-Jun vectors were provided by Dr. Charles Vinson (11Olive M. Krylov D. Echlin D.R. Gardner K. Taparowsky E. Vinson C. J. Biol. Chem. 1997; 272: 18586-18594Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Wild type E1A and its mutant forms are described in Hecht et al. (12Hecht A. Vleminckx K. Stemmler M.P. Van Roy F. Kemler R. EMBO J. 2000; 19: 1839-1850Crossref PubMed Google Scholar). TCF reporter (TOPFLASH) and mutated TCF reporter (FOPFLASH) were provided by Marc van de Wetering (13van de Wetering M. Cavallo R. Dooijes D. van Beest M. van Es J. Louriero J. Ypma A. Hursh D. Jones T. Bejsovec A. Peifer M. Mortin M. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar). The mammalian two-hybrid system was used to assay protein-protein interactions in transfected cells. To construct the VP16/β-catenin fusion protein expression vector, full-length β-catenin from pcDNA-β-catenin-FLAG was cloned into pCMX-VP16 vector using 5′-GAGCTCGGATCCAGCCCGATCGGTACCT-3′ and 5′-ACTAGTGGATCCTTAGGCGTAGTCGGGGA-3′ as forward and reverse primers with BamHI sites at both ends. The PCR-amplified β-catenin fragment was cloned into pCMX-VP16 at the BamHI site. The orientation and reading frame of the resultant vector was confirmed by sequencing. To generate Gal4/β-catenin and Gal4/ARM-β-catenin expression vectors, we used PCR to amplify the full-length and armadillo repeat regions of β-catenin using 5′-GAGCTCGGATCCACGCCCGATCGGTACCT-3′ and 5′-ACTAGTGGATCCTTAGGCGTAGTCGGGGA-3′ and 5′-ATCCCGGGAAACTATCAAGATGATGCAGAA-3′ and 5′-ATGGATCCTTGTGGCTTGTCCTCAGACATT-3′ forward and reverse primers, respectively. Both full-length β-catenin with the BamHI site and the ARM region of β-catenin with SmaI and BamHI sites were cloned downstream of Gal4 DNA binding sequences of pCMX-GAL4 vector (both pCMX VP16 and GAL4 vectors were kindly provided by Dr. Ronald Evans). To clone full-length RARα into pCMX-Gal4 and pCMX-VP16, a standard PCR procedure was followed using 5′-ACTGTCTGGATCCCATTCTGACT-3′ as forward and 5′-GCTTTAATAAGATCTGGATCCG-3′ as reverse primers. All the newly constructed vectors were sequenced using appropriate primers to confirm the reading frame. In addition, all vectors received from collaborators were sequenced before use in the experiments.Cell Culture, Treatments, and Antibodies—SKBR3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) plus 10% fetal bovine serum (FBS), SW480 cells were grown in DMEM with 5% FBS, and human embryonic kidney 293 cells were grown in DMEM with 10% horse serum. To study the effects of retinoic acid (RA) cells were treated with 1 μm 9-cis-RA or ethanol for 48 h. Several experiments were repeated using all-trans-retinoic acid with similar results. An anti-β-catenin monoclonal antibody (Transduction Labs) (1:1000), RARα (1:500) and RARβ (1:500) antibodies (Oncogene Research Products) were used for Western blotting. All antibodies were diluted in 5% skim milk in phosphate-buffered saline.Transient Transfection and Reporter Gene Assays—Cells were seeded in 12-well plates at 1 × 105 cells/well and transiently transfected using FuGENE 6 (Roche Applied Science). For luciferase assays cells were transfected with 50 ng of the expression vectors along with 100 ng of TCF reporter or control and mutated TCF reporter, and 10 ng of pCMV-Renilla luciferase (Promega). RA treatment was initiated 24 h post-transfection. Luciferase activity was monitored using the DUAL-luciferase assay system (Promega). The experimental reporter activity was controlled for transfection efficiency by comparison with the constitutively expressed Renilla luciferase. For mammalian two-hybrid analyses, Gal4-RARα and VP16-β-catenin were transiently transfected along with Renilla luciferase as an internal control.Western Blot Assays—The Bio-Rad DC protein assay kit was used to measure protein content in the samples. 25 μg of protein were separated on a 4-10% reducing polyacrylamide mini-gel (Novex), transferred onto nitrocellulose (Protran), and blocked overnight in 5% skim milk. The blot was then probed with an appropriate antibody followed by a secondary peroxidase-labeled antibody (KPL, Inc.), and the bands were visualized by enhanced chemiluminescence (Amersham Biosciences). The blots were then stripped at 50 °C for 30 min (stripping solution, 62.5 mm Tris, pH 7.5, 2% SDS, 1.7% (v/v) mercaptoethanol), washed two times in phosphate-buffered saline, and blocked in 5% milk before re-probing.RESULTSDifferential Response of β-Catenin/TCF and Retinoic Acid-responsive Promoters to Retinoic Acid in Colon Cancer Cells—In earlier studies we showed that β-catenin/TCF signaling activity could be inhibited by RA in Caco2 and HT29 colon cancer cells and in β-catenin-transfected SKBR3 and MCF-7 breast cancer cells (3Easwaran V. Pishvaian M. Salimuddin Byers S. Curr. Biol. 1999; 9: 1415-1418Abstract Full Text Full Text PDF PubMed Google Scholar). All these cell lines respond to retinoic acid by decreased proliferation and increased activation of retinoic acid-sensitive genes and RARE reporters (14Nicke B. Kaiser A. Wiedenmann B. Riecken E.O. Rosewicz S. Biochem. Biophys. Res. Commun. 1999; 261: 572-577Crossref PubMed Scopus (25) Google Scholar, 15O'Dwyer P.J. Ravikuma T.S. McCabe D.P. Steele Jr., G. J. Surg. Res. 1987; 43: 550-557Abstract Full Text PDF PubMed Scopus (24) Google Scholar). However, not all colon cancer cells respond to retinoic acid by decreasing growth even though RA-treatment continues to activate RARE reporters in these cells (2Waliszewski P. Waliszewska M.K. Gupta M. Milsom J.W. Hurst R.E. J. Surg. Oncol. 1997; 66: 156-167Crossref PubMed Scopus (9) Google Scholar). We wanted to investigate whether the failure of these cells to respond to RA by decreased proliferation was associated with loss of trans-repression of β-catenin signaling. We chose three colon cancer cell lines that had previously been shown to be only modestly responsive (SW480) or unresponsive (SW620, HCT116) to the growth inhibitory effects of RA (2Waliszewski P. Waliszewska M.K. Gupta M. Milsom J.W. Hurst R.E. J. Surg. Oncol. 1997; 66: 156-167Crossref PubMed Scopus (9) Google Scholar). Like Caco-2 and HT29 cells, SW480 and SW620 cells make large amounts of β-catenin protein as a result of a mutation in the adenomatous polyposis coli gene (APC); HCT116 cells have normal APC but an activating mutation of β-catenin. We first confirmed that all these cells did activate a RARE reporter after treatment with RA (Fig. 1A). SW480 and Caco-2 cells were somewhat more responsive than SW620 and HCT116 cells even though all four cell lines express similar levels of RARα and RARβ (Fig. 1, A and B, and see Sicinski and Weinberg (16Sicinski P. Weinberg R.A. J. Mammary Gland Biol. Neoplasia. 1997; 2: 335-342Crossref PubMed Scopus (56) Google Scholar)). β-Catenin signaling (TCF reporter activity) was also unaffected by RA treatment in HCT116 and SW620 cells; however, a small but consistent response was observed in SW480 cells (Fig. 1C). These data indicate that the reduced ability of these cells to stop cell growth in response to RA is associated with the failure of RA to trans-repress β-catenin signaling.The Ability of RAR/RA to Trans-repress β-Catenin Signaling Is Not Associated with Changes in β-Catenin or TCF4 Protein Levels—We showed earlier that trans-repression of β-catenin signaling by RA does not depend upon changes in the levels of β-catenin protein or on the phosphorylation status of the N-terminal serine residues important for targeting for ubiquitination (4Shah S. Pishvaian M.J. Easwaran V. Brown P.H. Byers S.W. J. Biol. Chem. 2002; 277: 25313-25322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Although Western analysis did not reveal any effect of RA on TCF4 levels (not shown), it is possible that RA might affect the activity or localization of TCF4 and indirectly regulate β-catenin signaling. In addition, dominant negative TCF4 stimulates the ability of β-catenin to potentiate the activity of RARE reporters (Fig. 1D) and vitamin D reporters (5Palmer H.G. Gonzalez-Sancho J.M. Espada J. Berciano M.T. Puig I. Baulida J. Quintanilla M. Cano A. de Herreros A.G. Lafarga M. Munoz A. J. Cell Biol. 2001; 154: 369-387Crossref PubMed Scopus (683) Google Scholar). In contrast, overexpression of TCF4 does not affect the ability of RAR or RA to trans-repress β-catenin signaling (Fig. 1E). These data show that in SW480 cells, the ability of RAR/RA to transrepress β-catenin signaling is not associated with changes in β-catenin or TCF4 protein levels.The Promoters of the β-Catenin-regulated Genes Cyclin D1 and siamois Are Also Regulated by RA in an RAR-dependent Manner—We next investigated the effects of RA and retinoid receptors on the activity of the β-catenin-sensitive cyclin D1 and siamois gene promoters. In a previous study we showed that RA-regulation of a cyclin D1 promoter fragment (-163 bp) was mediated through its TCF site in SW480 cells (4Shah S. Pishvaian M.J. Easwaran V. Brown P.H. Byers S.W. J. Biol. Chem. 2002; 277: 25313-25322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In the present study we used a much larger cyclin D1 promoter reporter (-1745 bp). We transfected SW480 cells with RARα and then treated them with RA. The expression of RARα alone significantly reduced the activity of the cyclin D1 and siamois promoters (Fig. 2) but did not affect the activity of cytomegalovirus-Renilla luciferase (not shown). It is interesting to note in Fig. 2 that the exogenous expression of RARα could decrease β-catenin signaling even in the absence of added RA. This is probably because of the small amount of endogenous RA present in serum, which is difficult to remove even after charcoal stripping. However, both promoters exhibited a dramatic increase in sensitivity to RA after transfection with RARα. In the presence of exogenous RARα, cyclin D1 reporter activity was reduced to background levels by RA treatment, and the siamois promoter only became retinoid-sensitive after transfection with RARα. As was the case with the cyclin D1 promoter, the ability of the siamois reporter to be trans-repressed by RA/RAR depended on the presence of its TCF sites (Fig 2D). These data confirm that the trans-repressive effects of RA on β-catenin signaling are dependent upon RAR and are mediated through TCF binding elements.Fig. 2RA effects on β-catenin/TCF-responsive promoter activity.A and B, SW480 cells were transiently transfected with wt RARα, RARα-Δ403, siamois promoter-luc, or TCF site-mutated siamois promoter constructs. Cells were harvested 24 h after RA treatment, and luciferase activity was measured. C and D, SW480 cells were transiently transfected with wild type RARα, RARα-Δ403, cyclin D1 promoter-luc (-1745-bp CD1), or TCF site-mutated cyclin D1 promoter-luc promoter constructs. Cells were harvested 24 h after RA treatment, and luciferase activity was measured.View Large Image Figure ViewerDownload (PPT)The Ability of RAR and Other Nuclear Steroid Receptors to Trans-repress β-Catenin Signaling Depends on Their Co-activator Interacting Regions—Ligand-activated vitamin D and androgen receptors can also trans-repress β-catenin signaling (5Palmer H.G. Gonzalez-Sancho J.M. Espada J. Berciano M.T. Puig I. Baulida J. Quintanilla M. Cano A. de Herreros A.G. Lafarga M. Munoz A. J. Cell Biol. 2001; 154: 369-387Crossref PubMed Scopus (683) Google Scholar, 17Song L.N. Herrell R. Byers S. Shah S. Wilson E.M. Gelmann E.P. Mol. Cell. Biol. 2003; 23: 1674-1687Crossref PubMed Scopus (143) Google Scholar). Like RAR these receptors directly interact with β-catenin and can sequester it away from TCF, but it is not known if this is the mechanism of trans-repression. Nuclear receptors also trans-repress AP-1, SRF, and NFκB activity by a mechanism likely involving competition for a common pool of co-activators rather than or as well as a direct interaction with Fos or Jun (18Benkoussa M. Brand C. Delmotte M.H. Formstecher P. Lefebvre P. Mol. Cell. Biol. 2002; 22: 4522-4534Crossref PubMed Scopus (99) Google Scholar, 19Lee S.K. Jung S.Y. Kim Y.S. Na S.Y. Lee Y.C. Lee J.W. Mol. Endocrinol. 2001; 15: 241-254Crossref PubMed Scopus (44) Google Scholar). In view of these observations we examined whether nuclear receptor trans-repression of β-catenin signaling involves structures that are required for co-activator recruitment. In most nuclear receptors, full transcriptional activity depends upon regions at their N-(AF-1 domain, A/B) and C-terminal (AF-2) domains. Deletion of either domain has a profound effect on the ability of the RAR, VDR, and AR to activate target genes (20Slagsvold T. Kraus I. Bentzen T. Palvimo J. Saatcioglu F. Mol. Endocrinol. 2000; 14: 1603-1617Crossref PubMed Scopus (38) Google Scholar, 21Liu Y.Y. Nguyen C. Peleg S. Mol. Endocrinol. 2000; 14: 1776-1786Crossref PubMed Scopus (39) Google Scholar, 22Bommer M. Benecke A. Gronemeyer H. Rochette-Egly C. J. Biol. Chem. 2002; 277: 37961-37966Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We transfected cells with RARα, RARαΔA/B, RARαΔF-2, and RARΔ403, a naturally occurring oncogenic mutant missing 60 amino acids at the C terminus (Fig. 3A). Unlike RARα, expression of RARα, RARαΔA/B, RARαΔF-2, and RARΔ403 did not reduce β-catenin signaling measured by TCF reporter activity (Fig. 3B). In addition none of these RAR mutants supported the trans-repressive effects of RA; rather, when compared with RAR, all mutants hyperactivated β-catenin signaling (Fig. 3B). Similar results were obtained using the β-catenin-responsive cyclin D1 and siamois reporters (Fig. 2 A and C). These data indicate that the ability of RARα to inhibit β-catenin signaling is associated with trans-activation in general and with the regions that recruit co-activators such as members of the steroid receptor coactivator (SRC) family and p300 (23van Wely K.H. Molijn A.C. Buijs A. Meester-Smoor M.A. Aarnoudse A.J. Hellemons A. den Besten P. Grosveld G.C. Zwarthoff E.C. Oncogene. 2003; 22: 699-709Crossref PubMed Scopus (66) Google Scholar). In addition, this explanation is also consistent with hyper-activation of β-catenin signaling by mutants of RARα. It is known that the all RARα mutants used in this study are able to hetero-dimerize with RXR, which means when RARα mutants are overexpressed they can sequester much of RXR in the cell and can deprive other nuclear receptors from the formation of dimers with the RXR. The impairment of nuclear receptor activation, due to a limited amount of RXR, could free up components of transcriptional machinery, which in turn might be available to augment the β-catenin signaling. Other experiments performed using the VDR and AR also showed trans-repression of β-catenin signaling (Fig. 3, C and D). In these experiments the full-length receptor trans-repressed β-catenin signaling; however, the deletion of AF2 from VDR and A/B from AR completely impaired the trans-repression ability of these receptors. Taken together these data strongly indicate that the ability of nuclear receptors to bind co-activators and/or co-repressors is necessary for their effects on β-catenin signaling.Fig. 3Effects of nuclear receptor transactivation domains on β-catenin signaling in SW480 cells.A, schematic representation of the RARα constructs. WT, wild type. B, 50 ng of the indicated RARα constructs were transfected either with or without RA (10-6m). TCF reporter activity was measured as described above. C, control. C, SW480 cells were transiently transfected with VDR or AB domain-deleted VDR constructs along with TCF reporter. Luciferase activity was measured 24 h after vitamin D3 treatment. D, SW480 cells were transiently transfected with AR or AB domain-deleted AR constructs along with TCF reporter. Luciferase activity was measured 24 h after treatment with the androgen receptor ligand, R1881.View Large Image Figure ViewerDownload (PPT)RA-mediated Trans-repression of the Cyclin D1 Promoter Is Mediated via Changes in β-Catenin/TCF, Not AP-1, in Colon Cancer Cells—We showed recently that the trans-repressive effects of RA on a short fragment of the cyclin D1 promoter (-163-bp CD1) expressed in SW480 colon cancer cells required the presence of a TCF binding element (4Shah S. Pishvaian M.J. Easwaran V. Brown P.H. Byers S.W. J. Biol. Chem. 2002; 277: 25313-25322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, RA trans-repression of the same promoter expressed in SKBR3 breast cancer cells was independent of the TCF site. This promoter fragment contains adjacent AP-1 and TCF sites, and we reasoned that RA-mediated trans-repression of its activity was mediated by β-catenin/TCF in colon cancer cells, which have high levels of β-catenin signaling activity, and by the AP-1 site in SKBR3 cells, which have high AP-1 activity (4Shah S. Pishvaian M.J. Easwaran V. Brown P.H. Byers S.W. J. Biol. Chem. 2002; 277: 25313-25322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Consistent with this, the basal activity of -163-bp CD1 was also markedly inhibited by mutation of the TCF site in SW480 cells but not in SKBR3 cells. To study this further we used another cyclin D1 promoter (-1745-bp CD1), which contains many potential regulatory elements including several AP-1 sites. The basal activity of -1745-bp CD1 is less dependent on the TCF site (Fig. 4). Expression of a dominant negative form of fos (to inhibit AP-1) actually increased the basal activity of -1745-bp CD1 but markedly reduced the basal activity of -1745-bp CD1ΔTCF. This experiment indicates that in the absence of TCF binding, the activity of the cyclin D1 promoter is regulated by AP-1. Nevertheless, like -163-bp D1, the ability of RA and RAR to trans-repress the activity of -1745-bp CD1 in SW480 cells depends on the TCF site.Fig. 4Role of AP-1 in RA-mediated trans-repression of cyclin D1 reporter.A, SW480 cells were transiently transfected with RARα, and dominant negative AP-1 (dnAP-1), and CD1-luciferase reporter activity was measure in the absence (-) and in the presence (+) of RA. B, same as in A except CD1-luciferase with its TCF site mutated was used as reporter.View Large Image Figure ViewerDownload (PPT)RA Trans-repression of β-Catenin Signaling Can Occur on a Heterologous Promoter and Depends on the N- and C-terminal Regulatory Domains of β-Catenin—The preceding experiments indicate that the ability of nuclear receptors to trans-repress β-catenin signaling depends upon their N- and C-terminal co-activator recruitment domains. If interaction with a common co-activator is involved in the mechanism of trans-repression one would expect that the co-activator recruitment N- and/or C-terminal domains of β-catenin would also be necessary for trans-repression to occur. Other studies have demonstrated that β-catenin can activate heterologous promoters such as Gal-4 if it is fused to the appropriate DNA binding domain. However no studies have investigated whether the trans-repressive effects of nuclear receptor ligands can occur in this situation. To test this we made various β-catenin-Gal-4 fusion constructs and expressed them in 293 cells (Fig. 5). We first confirmed that 293 cells made RARs and were responsive to both the trans-repressor and trans-activation functions of RA in the context of TCF- and RARE-reporter activity (Fig. 5A). 293 cells make significant amounts of RARα and RARβ (Fig. 5B). Wild type, N-terminal-deleted, C-terminal-deleted and N- and C-terminal-deleted β-catenin were cloned in a Gal4 expression vector, and wild type LEF and ΔNLEF, which cannot bind β-catenin, were cloned in a VP16 expression vector (Fig. 5C). The ability of each of the β-catenin-Gal4 constructs to bind either VP16-LEF or VP16-ΔNLEF and activate the Gal4-promoter is demonstrated in Fig. 5D. We next tested the affects of RA/RAR on the ability of the various β-catenin-Gal4 fusion proteins to activate the Gal4 promoter (Fig. 6). The trans-repressive effects of RA/RAR on wild type β-catenin-Gal4 activation of the Gal-4 promoter was similar to that observed with TCF reporter (cf.Fig. 1C with Fig. 6A). Similarly, RARΔAF-2 hyperactivated β-catenin-Gal4 activation of the Gal-4 promoter and completely prevented RA-mediated trans-repression. These data show that RA/RAR effects on β-catenin signaling activity can be exerted on a heterologous promoter and confirm that neither TCF nor TCF binding elements are required for the trans-repressive effects of RA. Deletion of the N-terminal of β-catenin-Gal4 only reduced basal activity of the Gal4 promoter by ∼50% but completely abrogated response to RA/RAR (Fig. 6A). In contrast to β-catenin-Gal4, RARΔAF-2 did not hyperactivate N-terminal-deleted β-catenin-Gal4. Deletion of the C-terminal of β-catenin-Gal4 reduced basal activity of the Gal4 promoter by ∼80%, but this activity could be further reduced by the addition of RA/RAR (Fig. 6B). Although the degree of trans-repression was small (20-50%), it was consistently observed. In contrast to ΔN-β-catenin-Gal4, RARΔAF-2 potentiated the activity of ΔC-β-catenin-Gal4. Deletion of both the N and C termini of β-catenin-Gal4 (ARM-β-catenin Gal4) also reduced basal activity of the Gal4 promoter by ∼80% compared with wild type β-catenin. Although the basal activity of ARM-β-catenin-Gal4 was similar to the ΔC-β-catenin-Gal4, it was not trans-repressed by RA/RAR and was not hyperactivated by RARΔAF-2. Taken together these data show that although both N- and C-terminal regulatory domains of β-catenin-Gal4 are required, for full trans-activation of the Gal4 promoter and for the trans-repressive effects of RA/RAR, the N-terminal region is absolutely essential.Fig. 5Mammalian two-hybrid assays for protein-protein interactions.A, to perform mammalian two hybrid assay in 293 cells, we measured TCF and RARE reporter activity. Cells were transfected with TCF or RARE reporter plasmids along with Renilla luciferase as an internal control. Reporter activity was measured at 24 h after RA treatment. B, Western blot of RARα and RARβ in 293 cells. C, schematic representation of β-catenin and Gal4-DNA binding domain. D, to confirm the activity of various Gal4-β-catenin constructs, the assay was performed using VP16-LEF and VP16-LEF missing the β-catenin binding site. 293 cells were transfected with the indicated plasmids, and Gal4 reporter activity was measured after 24 h.View Large Image Figure ViewerDownload (PPT)Fig. 6The role of N- and C-terminal regulatory domains of β-catenin.A,