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• W2065809107 abstract "Background & Aims: β-Catenin is the downstream effector of the Wnt signaling pathway and is involved in the process of colorectal carcinogenesis. However, it is still uncertain whether β-catenin exerts its oncogenic function solely by coactivating the target genes of T-cell factor-4 (TCF4). We previously reported that the β-catenin/TCF4 complex contains several classes of RNA-binding proteins and regulates the premessenger RNA splicing reaction, but the identity of the exact effector molecule downstream of the β-catenin/TCF4 complex has not been established. Methods: Using isotope-coded affinity tagging and mass spectrometry, we examined more than 4000 peptides derived from colorectal cancer cells and identified that splicing factor-1 (SF1) was one of the proteins whose expression is regulated by the β-catenin/TCF4 complex. Results: The expression of SF1 was found to be correlated with the differentiation status of intestinal epithelial cells and inversely correlated with tumorigenesis. Immunoprecipitation and immunofluorescence microscopy revealed that SF1 was a complex, and β-catenin-evoked gene transactivation and cell proliferation were negatively regulated by SF1 complementary DNA transfection. SF1 was essential for the induction of alternative splicing by the β-catenin/TCF4 complex, and SF1 complementary DNA transfection induced known cancer-related splice variants, such as Wnt-induced secreted protein-1v and fibroblast growth factor receptor-3-ATII. Conclusions: The β-catenin/TCF4 complex regulates the level of SF1 protein expression, and, conversely, SF1 interacts with the complex and regulates its gene transactivation and premessenger RNA splicing activities. Identification of the interaction may shed light on a novel aspect of the Wnt signaling pathway. Background & Aims: β-Catenin is the downstream effector of the Wnt signaling pathway and is involved in the process of colorectal carcinogenesis. However, it is still uncertain whether β-catenin exerts its oncogenic function solely by coactivating the target genes of T-cell factor-4 (TCF4). We previously reported that the β-catenin/TCF4 complex contains several classes of RNA-binding proteins and regulates the premessenger RNA splicing reaction, but the identity of the exact effector molecule downstream of the β-catenin/TCF4 complex has not been established. Methods: Using isotope-coded affinity tagging and mass spectrometry, we examined more than 4000 peptides derived from colorectal cancer cells and identified that splicing factor-1 (SF1) was one of the proteins whose expression is regulated by the β-catenin/TCF4 complex. Results: The expression of SF1 was found to be correlated with the differentiation status of intestinal epithelial cells and inversely correlated with tumorigenesis. Immunoprecipitation and immunofluorescence microscopy revealed that SF1 was a complex, and β-catenin-evoked gene transactivation and cell proliferation were negatively regulated by SF1 complementary DNA transfection. SF1 was essential for the induction of alternative splicing by the β-catenin/TCF4 complex, and SF1 complementary DNA transfection induced known cancer-related splice variants, such as Wnt-induced secreted protein-1v and fibroblast growth factor receptor-3-ATII. Conclusions: The β-catenin/TCF4 complex regulates the level of SF1 protein expression, and, conversely, SF1 interacts with the complex and regulates its gene transactivation and premessenger RNA splicing activities. Identification of the interaction may shed light on a novel aspect of the Wnt signaling pathway. The Wnt signaling pathway regulates cell fate, differentiation, proliferation, and death, and thus plays critical roles in embryonic development and carcinogenesis.1Peifer M. Polakis P. Wnt signaling in oncogenesis and embryogenesis—a look outside the nucleus.Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1137) Google Scholar, 2Moon R.T. Bowerman B. Boutros M. Perrimon N. The promise and perils of Wnt signaling through β-catenin.Science. 2002; 296: 1644-1646Crossref PubMed Scopus (878) Google Scholar, 3van Es J.H. Barker N. Clevers H. You Wnt some, you lose some: oncogenes in the Wnt signaling pathway.Curr Opin Genet Dev. 2003; 13: 28-33Crossref PubMed Scopus (211) Google Scholar, 4Nusse R. Wnt signaling in disease and in development.Cell Res. 2005; 15: 28-32Crossref PubMed Scopus (795) Google Scholar The Wnt signal is transmitted from the membrane receptors to the cytoplasmic multiprotein complex containing the adenomatous polyposis coli (APC) gene product, axin/axil, glycogen synthase kinase-3β (GSK-3β), and β-catenin.5Kikuchi A. Tumor formation by genetic mutations in the components of the Wnt signaling pathway.Cancer Sci. 2003; 94: 225-229Crossref PubMed Scopus (194) Google Scholar, 6Vogelstein B. Kinzler K.W. Cancer genes and the pathways they control.Nat Med. 2004; 10: 789-799Crossref PubMed Scopus (3286) Google Scholar This complex acts as a molecular chaperone that mediates the phosphorylation of β-catenin by GSK-3β, and the phosphorylated β-catenin protein is ubiquitinated and rapidly degraded.7Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. β-catenin is a target for the ubiquitin-proteasome pathway.EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2135) Google Scholar, 8Salomon D. Sacco P.A. Roy S.G. Simcha I. Johnson K.R. Wheelock M.J. Ben-Ze’ev A. Regulation of β-catenin levels and localization by overexpression of plakoglobin and inhibition of the ubiquitin-proteasome system.J Cell Biol. 1997; 139: 1325-1335Crossref PubMed Scopus (130) Google Scholar More than 80% of colorectal cancers carry mutations in the APC gene,9Kinzler K.W. Vogelstein B. Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4252) Google Scholar, 10Nakamura Y. Nishisho I. Kinzler K.W. Vogelstein B. Miyoshi Y. Miki Y. Ando H. Horii A. Nagase H. Mutations of the adenomatous polyposis coli gene in familial polyposis coli patients and sporadic colorectal tumors.Princess Takamatsu Symp. 1991; 22: 285-292PubMed Google Scholar and half of the remainder contain mutations in the β-catenin (CTNNB1) gene.11Sparks A.B. Morin P.J. Vogelstein B. Kinzler K.W. Mutational analysis of the APC/β-catenin/Tcf pathway in colorectal cancer.Cancer Res. 1998; 58: 1130-1134PubMed Google Scholar, 12Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Activation of β-catenin-Tcf signaling in colon cancer by mutations in β-catenin or APC.Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3480) Google Scholar These mutations cause dysfunction of the chaperone, which results in the accumulation of cytoplasmic β-catenin protein.13Munemitsu S. Albert I. Rubinfeld B. Polakis P. Deletion of an amino-terminal sequence stabilizes β-catenin in vivo and promotes hyperphosporylation of the adenomatous polyposis coli tumor suppressor protein.Mol Cell Biol. 1996; 16: 4088-4094Crossref PubMed Google Scholar The accumulated β-catenin is thought to cause colorectal carcinogenesis by forming a complex with T-cell factor-4 (TCF4), a member of the TCF/lymphoid enhancer factor (LEF) family, and by coactivating the target genes of TCF4.14Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google ScholarSeveral immediate transcriptional targets of TCF4 have been identified using complementary DNA (cDNA)/oligonucleotide microarrays,15Naishiro Y. Yamada T. Idogawa M. Honda K. Takada M. Kondo T. Imai K. Hirohashi S. Morphological and transcriptional responses of untransformed intestinal epithelial cells to an oncogenic β-catenin protein.Oncogene. 2005; 24: 3141-3153Crossref PubMed Scopus (39) Google Scholar, 16van de Wetering M. Sancho E. Verweij C. de Lau W. Oving I. Hurlstone A. van der Horn K. Batlle E. Coudreuse D. Haramis A.P. Tjon-Pon-Fong M. Moerer P. van den Born M. Soete G. Pals S. Eilers M. Medema R. Clevers H. The β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1718) Google Scholar, 17Wong N.A. Pignatelli M. β-catenin—a linchpin in colorectal carcinogenesis?.Am J Pathol. 2002; 160: 389-401Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 18Lin Y.M. Ono K. Satoh S. Ishiguro H. Fujita M. Miwa N. Tanaka T. Tsunoda T. Yang K.C. Nakamura Y. Furukawa Y. Identification of AF17 as a downstream gene of the β-catenin/T-cell factor pathway and its involvement in colorectal carcinogenesis.Cancer Res. 2001; 61: 6345-6349PubMed Google Scholar, 19Yamada T. Takaoka A.S. Naishiro Y. Hayashi R. Maruyama K. Maesawa C. Ochiai A. Hirohashi S. Transactivation of the multidrug resistance 1 gene by T-cell factor 4/β-catenin complex in early colorectal carcinogenesis.Cancer Res. 2000; 60: 4761-4766PubMed Google Scholar but the entire protein network of β-catenin-mediated intestinal carcinogenesis seems complicated and has not yet been fully explained. In the present study, we used a liquid chromatography and mass spectrometry (LC-MS)-based quantitative proteomic approach20Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5540) Google Scholar to identify comprehensively a population of proteins whose expression is regulated by the β-catenin/TCF4 complex in colorectal cancer cells. As a result, we identified splicing factor-1 (SF1) as one of the proteins whose expression is negatively regulated by the β-catenin/TCF4 complex, and its expression was found to be directly correlated with the differentiation status of intestinal epithelial cells and inversely correlated with tumorigenesis. SF1 functioned as a component of the β-catenin/TCF4 complex and negatively regulated β-catenin-evoked gene transactivation and cell proliferation.We previously reported that the β-catenin/TCF4 complex contains several classes of RNA-binding proteins, including fusion (FUS) and heterogeneous nuclear ribonucleoproteins (hnRNPs), and regulates the pre-messenger RNA (mRNA) splicing reaction.21Sato S. Idogawa M. Honda K. Fujii G. Kawashima H. Takekuma K. Hoshika A. Hirohashi S. Yamada T. β-catenin interacts with the FUS proto-oncogene product and regulates pre-mRNA splicing.Gastroenterology. 2005; 129: 1225-1236Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar However, the exact effector molecule downstream of the β-catenin/TCF4 complex had not been identified. Here, we report finding that SF1 is essential for the induction of alternative splicing by the β-catenin/TCF4 complex. Human cancers express a large number of alternatively spliced transcripts.22Venables J.P. Aberrant and alternative splicing in cancer.Cancer Res. 2004; 64: 7647-7654Crossref PubMed Scopus (515) Google Scholar SF1 may play an important role in the process of carcinogenesis.Materials and MethodsCell LinesHuman embryonic kidney (HEK) cell line 293 and human colorectal cancer cell line DLD-1 were obtained from the Health Science Research Resources Bank (Osaka, Japan). Human cervical cancer cell line HeLa and simian kidney epithelial cell line Cos-7 were purchased from the Riken Cell Bank (Tsukuba, Japan). Human colorectal cancer cell line HCT-116 was purchased from the American Type Culture Collection (Rockville, MD).Plasmid Construction and Establishment of DLD1 Tet-off TCF4BΔN30AU1-tagged TCF4B cDNA (nucleotides 398–2138, accession number Y11306 ) was subcloned into pTRE2-pur (BD Biosciences, San Jose, CA) to create pTRE2-TCF4BΔN30. DLD1 cells were double transfected sequentially with regulatory pTet-OFF (BD Biosciences) and responsive pTRE2-TCF4BΔN30 or pTRE2-pur control plasmid.The human SF1 expression construct (pcDNA-3.1-His-SF1) was kindly provided by Dr. M. A. Garcia-Blanco (Duke University Medical Center).23Goldstrohm A.C. Albrecht T.R. Sune C. Bedford M.T. Garcia-Blanco M.A. The transcription elongation factor CA150 interacts with RNA polymerase II and the pre-mRNA splicing factor SF1.Mol Cell Biol. 2001; 21: 7617-7628Crossref PubMed Scopus (102) Google Scholar Full-length human β-catenin cDNA and its truncated form (lacking a 134-amino acid sequence in its NH2-terminus15Naishiro Y. Yamada T. Idogawa M. Honda K. Takada M. Kondo T. Imai K. Hirohashi S. Morphological and transcriptional responses of untransformed intestinal epithelial cells to an oncogenic β-catenin protein.Oncogene. 2005; 24: 3141-3153Crossref PubMed Scopus (39) Google Scholar) were subcloned into pFLAG-CMV4 (Sigma-Aldrich, St Louis, MO) to prepare pFLAG-β-catenin and pFLAG-β-cateninΔN134, respectively. Human TCF4E cDNA lacking a 30-amino acid β-catenin-binding site in its NH2-terminus14Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar was subcloned into pFLAG-CMV4 to prepare pFLAG-TCF4EΔN30. The composition of all constructs described in this study was confirmed by restriction endonuclease digestion and sequencing.Isotope-Coded Affinity Tagging AnalysisDLD1 Tet-off TCF4BΔN30 cells were cultured for 7 days in the absence or presence of 0.1 μg/mL doxycycline (Dox). The cells were washed with ice-cold PBS, allowed to swell for 15 minutes in hypotonic buffer (50 mmol/L Tris-HCl, pH 7.4, 5 mmol/L MgCl2, 5 mmol/L CaCl2) supplemented with protease inhibitor cocktail (Boehringer Mannheim, Indianapolis, IN), and then centrifuged for 60 minutes at 105,000g to separate the soluble fraction (fraction 1). The pellet was resuspended in lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 5 mmol/L MgCl2, 5 mmol/L CaCl2, and protease inhibitor cocktail) and homogenized. The cell homogenates were fractionated into fractions 2–5 by sequential differential centrifugation (see Supplementary Figure S1 online at www.gastrojournal.org), and each fraction was dissolved in dissolving buffer (50 mmol/L Tris-HCl, pH 8.3, 5 mmol/L EDTA, 0.5% SDS, 1% octyl glucoside, 8 mol/L urea). After adjusting the concentration of urea to 5 mol/L, 300-μg protein samples were reduced with 2 mmol/L tris (2-carboxyethyl)phosphine (TCEP) for 30 minutes at 37°C and differentially labeled with the isotopically light (12C0) or heavy (13C9) acid-cleavable isotope-coded affinity tagging (ICAT) reagent (Applied Biosystems, Foster City, CA). The 12C0- and 13C9-labelled samples were combined, digested with modified trypsin (Promega, Madison, WI), fractionated via cation-exchange chromatography, and purified by avidin-affinity chromatography.The ICAT-labeled peptides were concentrated and desalted on a 500-μm ID × 1 mm HiQ sil C18-3 trapping column (KYA Technologies, Tokyo, Japan) before loading onto a 150-μm ID × 5-cm C18W-3 separation column (KYA Technologies). Peptides were then fractionated with an acetonitrile gradient (0%–80%, 200 nL/minute for 3 hours) and analyzed with a Q-Star Pulsar-i mass spectrometer equipped with a nanospray ionization source (Applied Biosystems). Data were processed with ProICAT software (Applied Biosystems).AntibodiesAnti-78-kilodalton glucose-regulated protein (clone 40), antinucleoporin p62 (clone 63), and anti-β-catenin (clone 14) mouse monoclonal antibodies were purchased from BD Transduction Laboratories (Palo Alto, CA). Antialcohol dehydrogenase rabbit (sc-22750), antilaminin receptor rabbit (sc-20979), anti-SF1 goat (sc-21157), anti-β-catenin goat (sc-1496), anti-FUS goat (sc-8531), anti-Wnt-induced secreted protein-1 (WISP1) rabbit (sc-25441), and anti-WISP2 goat (sc-8867) polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antialdolase goat polyclonal antibody (100–1141) was obtained from Rockland (Gilbertsville, PA). Antihemeoxygenase-2 rabbit polyclonal antibody (OSA-200) was purchased from Stressgen (British Columbia, Canada). Anti-Bcl-2 mouse monoclonal antibody (clone 124) was obtained from Dako (Glostrup, Denmark). Antivesicle-associated membrane protein (VAMP)-associated protein A rabbit polyclonal antibody (AB5741) was purchased from Chemicon (Temecula, CA). Anti-β-actin mouse monoclonal antibody (AC-74) and anti-SF1 goat polyclonal antibody (S1945) were obtained from Sigma-Aldrich. Anti-TCF4 (6H5-3) and anti-TCF-3/4 (6F12-3) monoclonal antibodies were purchased from Upstate (Charlottesville, VA). Anti-poly (ADP-ribose) polymerase (PARP) rabbit polyclonal antibody (No. 9542) was purchased from Cell Signaling (Boston, MA). Anti-SF1 (AB/SF20) rabbit polyclonal antibody was purchased from CeMines (Golden, CO). Anti-FUS rabbit polyclonal antibody was generously provided by Dr. K. Shimizu (National Cancer Center Research Institute, Tokyo, Japan).ImmunoprecipitationNuclear extracts were prepared with the CelLytic nuclear extraction kit (Sigma-Aldrich). The extracts were incubated at 4°C overnight with anti-β-catenin monoclonal antibody, anti-SF1 goat polyclonal antibody, anti-FUS rabbit polyclonal antibody, anti-TCF4 monoclonal antibody, normal mouse IgG, normal goat IgG, or normal rabbit IgG and precipitated with Dynabeads protein G (Dynal Biotech, Oslo, Norway). The immunoprecipitated proteins were released by boiling in SDS loading buffer for 5 minutes and fractionated by SDS-PAGE.Immunoblot AnalysisProtein samples were fractionated by SDS-PAGE and blotted onto Immobilon-P membranes (Millipore, Billerica, MA). After incubation with the primary antibodies at 4°C overnight, the blots were detected with the relevant horseradish peroxidase-conjugated anti-mouse, anti-rabbit, anti-goat, or anti-rat IgG antibody and ECL Western blotting detection reagents (Amersham Biosciences, Amersham, United Kingdom).Tissue SamplesMale Min mice (C57BL/6J-ApcMin/+) were obtained from the Jackson Laboratory (Bar Harbor, ME). Eleven colorectal cancer cases were selected from the surgical pathology archive panel of the National Cancer Center Hospital (Tokyo, Japan). Paraffin-embedded intestinal tissue was stained by the avidin-biotin complex method. Protein samples were extracted from the small intestine and polyp tissues of a Min mouse as described previously.15Naishiro Y. Yamada T. Idogawa M. Honda K. Takada M. Kondo T. Imai K. Hirohashi S. Morphological and transcriptional responses of untransformed intestinal epithelial cells to an oncogenic β-catenin protein.Oncogene. 2005; 24: 3141-3153Crossref PubMed Scopus (39) Google Scholar Animal experiments were reviewed and approved by the Ethics Committee of the National Cancer Center Research Institute (Tokyo, Japan).Immunofluorescence CytochemistryCells cultured on glass coverslips (Asahi Technoglass Corp., Tokyo, Japan) were fixed with 4% paraformaldehyde at room temperature for 10 minutes and permeabilized with 0.2% Triton X-100. After blocking with 10% normal swine serum (Vector Laboratory, Inc., Burlingame, CA), the cells were incubated with anti-β-catenin mouse monoclonal and anti-SF1 goat polyclonal antibodies at 4°C overnight. After incubation with Alexa Fluor-594 anti-mouse IgG and Alexa fluor-488 anti-goat IgG antibodies (Invitrogen, Carlsbad, CA), the specimens were examined with a laser scanning microscope (LSM5 PASCAL, Carl Zeiss, Jena, Germany).24Honda K. Yamada T. Hayashida Y. Idogawa M. Sato S. Hasegawa F. Ino Y. Ono M. Hirohashi S. Actinin-4 increases cell motility and promotes lymph node metastasis of colorectal cancer.Gastroenterology. 2005; 128: 51-62Abstract Full Text Full Text PDF PubMed Scopus (168) Google ScholarLuciferase Reporter AssayA pair of luciferase reporter constructs, TOP-FLASH and FOP-FLASH (Upstate), was used to evaluate TCF/LEF transcriptional activity. Cells were transiently transfected in triplicate with one of the luciferase reporters and phRL-TK (Promega) by using the Lipofectamine 2000 reagent (Invitrogen). Luciferase activity was measured with the Dual-luciferase Reporter Assay system (Promega).Colony Formation AssayHEK293 cells were transfected with pcDNA3.1-His-SF1 and/or pFLAG-β-cateninΔN134 or appropriate empty plasmids by using the Lipofectamine 2000 reagent, and, 24 hours later, 750 μg/mL G418 (Geneticin; Invitrogen) was added to the culture medium. Cells were stained with Giemsa stain solution (Wako, Osaka, Japan) after 8 days of selection. A soft-agar colony formation assay was performed as described previously.25Naishiro Y. Yamada T. Takaoka A.S. Hayashi R. Hasegawa F. Imai K. Hirohashi S. Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of β-catenin/T-cell factor 4-mediated gene transactivation.Cancer Res. 2001; 61: 2751-2758PubMed Google ScholarIn Vivo Splicing Analysis and RNA InterferenceCells were cotransfected with 0.25 μg pCS3-MT-E1A, which carries the adenovirus E1A minigene26Hallier M. Lerga A. Barnache S. Tavitian A. Moreau-Gachelin F. The transcription factor Spi-1/PU.1 interacts with the potential splicing factor TLS.J Biol Chem. 1998; 273: 4838-4842Crossref PubMed Scopus (118) Google Scholar (kindly provided by Dr. F. Moreau-Gachelin, Institut Curie, Paris, France, and obtained through Dr. N. Ohkura, National Cancer Center Research Institute), and pFLAG-CMV4 TCF4EΔN30, pcDNA3.1-His-SF1, or the various small interfering RNAs (siRNA) (siGENOME duplex, D-012662; Non-Specific Control Duplex X, VI) (Dharmacon, Lafayette, CO) by using Lipofectamine 2000 reagent, and, 48 hours later, the total RNA was extracted and analyzed by reverse transcription (RT)-polymerase chain reaction (PCR) with a pair of primers, 5′-GAGCTTGGGCGACCTCA-3′ (RR67) and 5′-TCTAGACACAGGTGATGTCG-3′ (E1A2). Quantification was performed with a LAS-3000 scanner and Science Lab 2003 software (Fujifilm, Tokyo, Japan).Conventional RT-PCR AnalysisTotal RNA was prepared from cell lines with the RNeasy Mini Kit (Qiagen, Valencia, CA). DNase-I-treated total RNA was random primed and reverse transcribed using SuperScript reverse transcriptase (Invitrogen). The following PCR primers were used: for SF1, 5′-CCTTCGGGAAGACGATAACA-3′ and 5′-TTCAGCCATGAGGGACAAAT-3′; for estrogen receptor (ER) β, 5′-CGCTAGAACACACCTTACCTG-3′ and 5′-CTGTGACCAGAGGGTACAT-3′; for WISP1, 5′-CGAGGTACGCAATAGGAGTGTGT-3′ and 5′-CCCTGCCTTAATGAGTGTATGGA-3′; for WISP2, 5′-TTTCTGGCCTTGTCTCTTCC-3′ and 5′-GTGTGTGTAGGCAGGGAGTG-3′; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-AAGGCTGAGAACGGGAAGCTTGTCATCAAT-3′ and 5′-TTCCCGTCTAGCTCAGGGATGACCTTGCCC-3′; for fibroblast growth factor receptor (FGFR) 3, 5′-CGCACCGGCCCATCCTG-3′ and 5′-GCGTCACAGCCGCCACCACC-3′. The PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining.Real-Time RT-PCR AnalysisThe TaqMan universal PCR master mix and predesigned TaqMan Gene Expression probe and primer sets were purchased from Applied Biosystems. Amplification data measured as an increase in reporter fluorescence were collected in real time with the PRISM 7000 Sequence Detection system (Applied Biosystems). The mRNA expression level of SF1 relative to β-actin was calculated by the comparative threshold cycle (CT) method.DNA SequencingAutomated sequencing was performed using the ABI PRISM Big dye terminator cycle sequencing kit (Applied Biosystems) and a Genetic Analyzer 3100 (Applied Biosystems).ChemicalsDox (Sigma-Aldrich), a derivative of tetracycline, was dissolved in deionized water to a stock concentration of 1 mg/mL Dox and added to culture medium to a final concentration of 0.1 μ/mL. Sodium butyrate was obtained from Wako.ResultsIdentification of Proteins Whose Expression Is Regulated by the β-Catenin/TCF4 ComplexTCF/LEF lacking the N-terminal β-catenin-binding site suppresses transcriptional activity in a dominant negative manner.14Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC−/− colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2911) Google Scholar By using a strict tetracycline-regulation system,27Gossen M. Freundlieb S. Bender G. Muller G. Hillen W. Bujard H. Transcriptional activation by tetracyclines in mammalian cells.Science. 1995; 268: 1766-1769Crossref PubMed Scopus (2020) Google Scholar we established a pair of transfectants: DLD1 Tet-off TCF4BΔN30 and DLD1 Tet-off control. DLD1 Tet-off TCF4BΔN30 was capable of inducing TCF4B lacking the NH2-terminal 30 amino acids (TCF4BΔN30), and DLD1 Tet-off control served as a mock transfectant. Induction of TCF4BΔN30 protein and suppression of the TCF/LEF transcriptional activity in DLD1 Tet-off TCF4BΔN30 cells cultured in the absence of Dox were confirmed by immunoblotting (Figure 1A) and reporter luciferase assay (Figure 1B). The formation of piled-up foci25Naishiro Y. Yamada T. Takaoka A.S. Hayashi R. Hasegawa F. Imai K. Hirohashi S. Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of β-catenin/T-cell factor 4-mediated gene transactivation.Cancer Res. 2001; 61: 2751-2758PubMed Google Scholar (Figure 1C) and colonies in soft agar25Naishiro Y. Yamada T. Takaoka A.S. Hayashi R. Hasegawa F. Imai K. Hirohashi S. Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of β-catenin/T-cell factor 4-mediated gene transactivation.Cancer Res. 2001; 61: 2751-2758PubMed Google Scholar (Figure 1D and 1E) was significantly suppressed by the induction of TCF4BΔN30. DLD1 Tet-off TCF4BΔN30 cells and DLD1 Tet-off control cells were cultured for 7 days in the presence and absence of Dox. Whole protein extracts were fractionated into F1 to F5 by stepwise centrifugation (see Supplementary Figure S1 online at www.gastrojournal.org), and the overall protein content was confirmed to be equal by Bradford protein assay (data not shown) and immunoblotting with antibodies against organelle proteins representative of each fraction (see Supplementary Figure S2 online at www.gastrojournal.org).We used ICAT-MS28Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4321) Google Scholar to search for proteins differentially expressed in DLD1 Tet-off TCF4BΔN30 after induction of dominant negative TCF4 (Figure 2A and 2B). Whole proteins are enzymatically digested into a large array of small peptide fragments having uniform physical and chemical characteristics. The use of nano (nL/minute)-level flow rate high-performance liquid chromatography (HPLC) coupled with small reverse-phase columns significantly increased the sensitivity of electrospray ionization (ESI)-MS. Among the 4115 and 4441 peptides detected and sequenced in 2 independent ICAT-MS experiments (experiment 1 and experiment 2), 148 (57 up-regulated and 91 down-regulated) and 151 (62 up-regulated and 89 down-regulated) peptides, respectively, were found to be differentially expressed more than 2-fold (see Supplementary Table S1 online at www.gastrojournal.org). Tandem mass spectrometry (MS/MS) (Figure 2B) and a database search revealed the 148 and 151 peptides to be derived from 81 (31 up-regulated and 50 down-regulated) and 72 (30 up-regulated and 42 down-regulated) individual proteins, respectively, with 66 overlapping (see Supplementary Table S1 online at www.gastrojournal.org). Based on the functional annotation provided by the Celera Discovery System (CDS) (http://www.celeradiscoverysystem.com/index.cfm), all 87 (= 81 + 72 − 66) proteins compiled from the 2 experiments were classified into 14 (16%) nucleic acid-binding proteins, 7 (8%) oxidoreductases, 7 (8%) select regulatory molecules, 6 (7%) cytoskeletal proteins, and others (see Supplementary Figure S3 online at www.gastrojournal.org). The known subcellular localization of proteins identified by ICAT-MS was generally consistent with results of the organelle fractionation (see Supplementary Table S2 online at www.gastrojournal.org). We previously reported that 22 out of >2000 protein spots displayed by 2-dimensional difference gel electrophoresis were found to be up- or down-regulated after induction of stabilized β-catenin (β-cateninΔN89),29Seike M. Kondo T. Mori Y. Gemma A. Kudoh S. Sakamoto M. Yamada T. Hirohashi S. Proteomic analysis of intestinal epithelial cells expressing stabilized β-catenin.Cancer Res. 2003; 63: 4641-4647PubMed Google Scholar and 9 of the 22 proteins were found to be involved in redox and cytoskeletal regulation.29Seike M. Kondo T. Mori Y. Gemma A. Kudoh S. Sakamoto M. Yamada T. Hirohashi S. Proteomic analysis of intestinal epithelial cells expressing stabilized β-catenin.Cancer Res. 2003; 63: 4641-4647PubMed Google Scholar Consistent with those findings, the majority of proteins identified in the current study fell into 2 categories: redox-regulatory proteins and cytoskeleton-associated proteins. Differential expression of representative proteins identified by ICAT-MS was confirmed by immunoblotting with available antibodies (Figure 2C). Supplementary Table S2 (see Supplementary Table S2 online at www.gastrojournal.org) lists the proteins identified in this study.Figure 2Identification of downstream proteins of the β-catenin/TCF4 complex. DLD1 Tet-off TCF4BΔN30 cells were cultured in the presence or absence of Dox for 7 days. The whole protein extracts were fractionated by a differential centrifugation technique as described in Supplementary Figure 1 (see Supplementary Figure 1 online at www.gastrojournal.org), labeled differentially with the 12C0 (Dox [+]) or heavy 13C9 (Dox [−]) ICAT reagent, and analyzed by nanoflow LC-MS (A) and MS/MS (B). cps, count(s) per second; amu, atomic mass unit(s). (A) MS spectrum showing the light (12C0)- and heavy (13C9)-labeled peptides of SF1 protein. The r" @default.
• W2065809107 created "2016-06-24" @default.
• W2065809107 creator A5005311940 @default.
• W2065809107 creator A5006409268 @default.
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• W2065809107 creator A5026500116 @default.
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• W2065809107 date "2007-03-01" @default.
• W2065809107 modified "2023-10-14" @default.
• W2065809107 title "Involvement of Splicing Factor-1 in β-Catenin/T-Cell Factor-4-Mediated Gene Transactivation and Pre-mRNA Splicing" @default.
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