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• W2088136743 abstract "Stem cells of the gut epithelium constantly produce precursors that progressively undergo a succession of molecular changes resulting in growth arrest and commitment to a specific differentiation program. Few transcriptional repressors have been identified that maintain the normal intestinal epithelial cell (IEC) proliferation state. Herein, we show that the nuclear receptor co-repressor (NCoR1) is differentially expressed during the proliferation-to-differentiation IEC transition. Silencing of NCoR1 expression in proliferating cells of crypt origin resulted in a rapid growth arrest without associated cell death. A genechip profiling analysis identified several candidate genes to be up-regulated in NCoR1-deficient IEC. Pigment epithelium-derived factor (PEDF, also known as serpinf1), a suspected tumor suppressor gene that plays a key role in the inhibition of epithelial tissue growth, was significantly up-regulated in these cells. Chromatin immunoprecipitation experiments showed that the PEDF gene promoter was occupied by NCoR1 in proliferating epithelial cells. Multiple retinoid X receptor (RXR) heterodimers interacting sites of the PEDF promoter were confirmed to interact with RXR and retinoid acid receptor (RAR). Cotransfection assays showed that RXR and RAR were able to transactivate the PEDF promoter and that NCoR1 was repressing this effect. Finally, forced expression of PEDF in IEC resulted in a slower rate of proliferation. These observations suggest that NCoR1 expression is required to maintain IEC in a proliferative state and identify PEDF as a novel transcriptional target for NCoR1 repressive action. Stem cells of the gut epithelium constantly produce precursors that progressively undergo a succession of molecular changes resulting in growth arrest and commitment to a specific differentiation program. Few transcriptional repressors have been identified that maintain the normal intestinal epithelial cell (IEC) proliferation state. Herein, we show that the nuclear receptor co-repressor (NCoR1) is differentially expressed during the proliferation-to-differentiation IEC transition. Silencing of NCoR1 expression in proliferating cells of crypt origin resulted in a rapid growth arrest without associated cell death. A genechip profiling analysis identified several candidate genes to be up-regulated in NCoR1-deficient IEC. Pigment epithelium-derived factor (PEDF, also known as serpinf1), a suspected tumor suppressor gene that plays a key role in the inhibition of epithelial tissue growth, was significantly up-regulated in these cells. Chromatin immunoprecipitation experiments showed that the PEDF gene promoter was occupied by NCoR1 in proliferating epithelial cells. Multiple retinoid X receptor (RXR) heterodimers interacting sites of the PEDF promoter were confirmed to interact with RXR and retinoid acid receptor (RAR). Cotransfection assays showed that RXR and RAR were able to transactivate the PEDF promoter and that NCoR1 was repressing this effect. Finally, forced expression of PEDF in IEC resulted in a slower rate of proliferation. These observations suggest that NCoR1 expression is required to maintain IEC in a proliferative state and identify PEDF as a novel transcriptional target for NCoR1 repressive action. The intestinal epithelium consists of a cell monolayer organized in crypts and villi. This epithelium is under constant and rapid renewal, which is assured by constant division of the stem cells located at the base of the crypts (1Crosnier C. Stamataki D. Lewis J. Nat. Rev. Genet. 2006; 7: 349-359Crossref PubMed Scopus (571) Google Scholar). The descendant progenitor cells are progressively instructed to differentiate to exert their functional role during their journey along the villus compartment (2Menard D. Beaulieu J.F. Boudreau F. Perreault N. Rivard N. Vachon P.H. Unsicker K. Krieglstein K. Cell Signaling and Growth Factors in Development: From Molecules to Organogenesis. Wiley-VCH, 2006Google Scholar). The proliferation-to-differentiation transition of single progenitor cell is tightly regulated by morphogens, growth factors and hormones that impact on intracellular signaling pathways. Molecular alterations of specific components from these different classes of molecules are suspected to be important during the development of intestinal cancer (3van den Brink G.R. Offerhaus G.J. Cancer Cell. 2007; 11: 109-117Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In vivo studies have demonstrated the role of steroid hormones and ligands during intestinal epithelial development and homeostasis (4Henning S.J. Annu. Rev. Physiol. 1985; 47: 231-245Crossref PubMed Google Scholar). For example, the thyroid hormone exerts a positive effect on gut mucosal maturation (5Hodin R.A. Chamberlain S.M. Upton M.P. Gastroenterology. 1992; 103: 1529-1536Abstract Full Text PDF PubMed Scopus (46) Google Scholar) and enterocyte differentiation (6Malo M.S. Zhang W. Alkhoury F. Pushpakaran P. Abedrapo M.A. Mozumder M. Fleming E. Siddique A. Henderson J.W. Hodin R.A. Mol. Endocrinol. 2004; 18: 1941-1962Crossref PubMed Scopus (33) Google Scholar, 7Hodin R.A. Shei A. Morin M. Meng S. Surgery. 1996; 120: 138-143Abstract Full Text PDF PubMed Scopus (27) Google Scholar). Members of the nuclear hormone receptor superfamily are activated by metabolically transformed lipids that are absorbed by intestinal epithelial cells (IEC) 4The abbreviations used are:IECintestinal epithelial cellsAP-1activator protein-1β2-micβ2-microglobulinChIPchromatin immunoprecipitationeGFPenhanced green fluorescence proteinHDAChistone deacetylaseNCoR1nuclear receptor co-repressorPEDFpigment epithelium-derived factorPPARperoxisome proliferator-activated receptorsPBGDporphobilinogen deaminaseqRT-PCRquantitative reverse-transcription polymerase chain reactionRARretinoid acid receptorRXRretinoid X receptorSMRTsilencing mediator of retinoid and thyroid receptorsEMSAelectrophoretic mobility shift assay. before interacting with their receptors and associated gene targets (8Chawla A. Repa J.J. Evans R.M. Mangelsdorf D.J. Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1696) Google Scholar). Peroxisome proliferator-activated receptors (PPARs) are well-described examples of lipophilic ligand binding transcription factors that can influence intestinal epithelial proliferation, differentiation (9Varnat F. Heggeler B.B. Grisel P. Boucard N. Corthésy-Theulaz I. Wahli W. Desvergne B. Gastroenterology. 2006; 131: 538-553Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 10Gupta R.A. Sarraf P. Mueller E. Brockman J.A. Prusakiewicz J.J. Eng C. Willson T.M. DuBois R.N. J. Biol. Chem. 2003; 278: 22669-22677Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and inflammation (11Wu G.D. Gastroenterology. 2003; 124: 1538-1542Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). These nuclear receptors can repress gene transcription via the formation of a well defined co-repressor protein complex (12Lazar M.A. Nucl. Recept. Signal. 2003; 1: e001Crossref PubMed Google Scholar). One major player of that repression activity is the nuclear receptor co-repressor (NCoR1) that was originally identified for its potential to repress genes via physical interaction with the thyroid hormone receptor (13Hörlein A.J. Näär A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Söderström M. Glass C.K. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1714) Google Scholar). However, there is now growing evidence that NCoR1 can repress transcription through interaction with several other classes of transcriptional activators including activator protein (AP)-1 and NF-κB (14Perissi V. Aggarwal A. Glass C.K. Rose D.W. Rosenfeld M.G. Cell. 2004; 116: 511-526Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 15Lee S.K. Kim J.H. Lee Y.C. Cheong J. Lee J.W. J. Biol. Chem. 2000; 275: 12470-12474Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). One additional important class of nuclear receptors that are recruiting NCoR1 repressive action is the retinoid receptors (16Weston A.D. Blumberg B. Underhill T.M. J. Cell Biol. 2003; 161: 223-228Crossref PubMed Scopus (116) Google Scholar). These receptors are composed of either RAR-RXR heterodimers or RXR-RXR homodimers and can repress transcription in a ligand-independent manner. In absence of co-repressors, these receptors are involved in the differentiation of various epithelia (17Kubota H. Chiba H. Takakuwa Y. Osanai M. Tobioka H. Kohama G. Mori M. Sawada N. Exp. Cell Research. 2001; 263: 163-172Crossref PubMed Scopus (87) Google Scholar, 18Fisher G.J. Voorhees J.J. Faseb J. 1996; 10: 1002-1013Crossref PubMed Scopus (359) Google Scholar). intestinal epithelial cells activator protein-1 β2-microglobulin chromatin immunoprecipitation enhanced green fluorescence protein histone deacetylase nuclear receptor co-repressor pigment epithelium-derived factor peroxisome proliferator-activated receptors porphobilinogen deaminase quantitative reverse-transcription polymerase chain reaction retinoid acid receptor retinoid X receptor silencing mediator of retinoid and thyroid receptors electrophoretic mobility shift assay. Ncor1 gene deletion in mice resulted in an impairment of neural stem cell proliferation and spontaneous differentiation into astrocytes (19Hermanson O. Jepsen K. Rosenfeld M.G. Nature. 2002; 419: 934-939Crossref PubMed Scopus (263) Google Scholar). Other reports have recently suggested an antiproliferative role for NCoR1 in hepatocytes (20Feng X. Jiang Y. Meltzer P. Yen P.M. J. Biol. Chem. 2001; 276: 15066-15072Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and thyroid tumor cells (21Furuya F. Guigon C.J. Zhao L. Lu C. Hanover J.A. Cheng S.Y. Mol. Cell. Biol. 2007; 27: 6116-6126Crossref PubMed Scopus (32) Google Scholar). Thus, NCoR1 may influence cell proliferation by affecting different gene targets in specific cellular contexts. Because many NCoR1-interacting partners are crucial to the regulation of many IEC functions, we sought to evaluate the functional role of NCoR1 in this specific context. We provide here the evidence that NCoR1 nuclear expression is associated with proliferative and non-differentiated epithelial cells and that NCoR1 silencing by RNA interference causes cells to growth arrest. The pigment epithelial-derived factor (PEDF), a 50-kDa member of the serine protease inhibitor family, was further identified as a transcriptional target for NCoR1 repressive action during this process. Ectopic expression of PEDF in IEC reduced the proliferation rate, an observation that was consistent with the tumor suppressor properties of this regulator in epithelial tissues (22Doll J.A. Stellmach V.M. Bouck N.P. Bergh A.R. Lee C. Abramson L.P. Cornwell M.L. Pins M.R. Borensztajn J. Crawford S.E. Nat. Med. 2003; 9: 774-780Crossref PubMed Scopus (266) Google Scholar). Caco-2/15 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. IEC-6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum and 0.1 units/ml insulin. In both cases, the media were supplemented with 4.5g/liter d-glucose, 25 mm HEPES, 50 units/ml penicillin, and 50 μg/ml streptomycin. Cells were kept subconfluent for 5–10 passages. All cell lines were grown at 37 °C in 5% CO2. Cell proliferation assays were manually performed using a hemacytometer in the presence of 0.4% trypan blue to account for cell viability. Inhibition of proteasome activity was performed with supplementation of 50 μm MG132 into the cell culture medium (Sigma-Aldrich). Animal experimentation was approved by the Institutional Animal Research Review Committee in conformity with the Canadian Council on Animal Care. CD-1 wild-type mice obtained from Charles River Laboratories (Wilmington, MA) were sacrificed and the jejunum harvested, inverted onto polyethylene tubing, ligatured at both extremities, and washed with KRB buffer, pH 7.5, as described previously (23Boudreau F. Lussier C.R. Mongrain S. Darsigny M. Drouin J.L. Doyon G. Suh E.R. Beaulieu J.F. Rivard N. Perreault N. Faseb J. 2007; 21: 3853-3865Crossref PubMed Scopus (57) Google Scholar). Segments were then incubated under agitation in ice-cold isolation buffer (2.5 mm EDTA, 0.25 mm NaCl) for 2-min intervals. After each interval, cell suspensions were centrifuged at 400 × g for 5 min. Pellets were then washed with ice-cold KRB buffer and lysed for either nuclear protein or total RNA isolation (23Boudreau F. Lussier C.R. Mongrain S. Darsigny M. Drouin J.L. Doyon G. Suh E.R. Beaulieu J.F. Rivard N. Perreault N. Faseb J. 2007; 21: 3853-3865Crossref PubMed Scopus (57) Google Scholar). Total RNA was isolated from cultured cells as described previously (23Boudreau F. Lussier C.R. Mongrain S. Darsigny M. Drouin J.L. Doyon G. Suh E.R. Beaulieu J.F. Rivard N. Perreault N. Faseb J. 2007; 21: 3853-3865Crossref PubMed Scopus (57) Google Scholar). Reverse transcription reactions were carried out at 42 °C for 1 h in the presence of 1 μg of RNA, 40 milliunits of poly-oligo(dT)12–18 (Amersham Biosciences, Baie d'Urfé, QC) and 40 units of reverse transcriptase (Roche Molecular Biochemicals). PCR reactions were performed in a total volume of 25 μl with 1 μl of the RT reaction, 1 unit of TaqDNA polymerase (New England Biolabs, Pickering, ON), and 100 ng of each specific oligonucleotide. Real-time PCR was performed using a Lightcycler apparatus (Roche Molecular Biochemicals) as described previously (23Boudreau F. Lussier C.R. Mongrain S. Darsigny M. Drouin J.L. Doyon G. Suh E.R. Beaulieu J.F. Rivard N. Perreault N. Faseb J. 2007; 21: 3853-3865Crossref PubMed Scopus (57) Google Scholar). Experiments were run and analyzed using the Lightcycler software 4.0 (relative quantification monocolor) according to the manufacturer's instructions (Roche Molecular Biochemicals). Double-stranded DNA amplification during PCR was monitored using SYBR Green I (QuantiTect SYBR Green PCR Kit; Qiagen, Valencia, CA) and PCR amplification programs designed as described in the QuantiTect SYBR Green PCR Handbook (Qiagen). A serial dilution of a calibrator sample was used for the standard curve for each gene analyzed, which was then used to correct for the differences in the efficiency of the PCR. Primers sequences are available upon request. Cell protein fractionation (cytosol, membranes and organelles, nucleus, and cytoskeleton) was performed with the ProteoExtract subcellular proteome extraction kit according to the manufacturer's instructions (Calbiochem, EMD Biosciences, San Diego, CA). 5 μg of protein extract was analyzed by 3–8% Tris acetate NuPAGE (Invitrogen, Burlington, ON) and transferred onto a polyvinylidene difluoride membrane (Roche Molecular Biochemicals). Western blot was performed as described previously (23Boudreau F. Lussier C.R. Mongrain S. Darsigny M. Drouin J.L. Doyon G. Suh E.R. Beaulieu J.F. Rivard N. Perreault N. Faseb J. 2007; 21: 3853-3865Crossref PubMed Scopus (57) Google Scholar). NCoR1 and SMRTe antibodies were purchased from Millipore (Upstate). Actin, histone H1, and PPARγ antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Three different sets of shRNA oligonucleotides were designed for rat NCoR1 targeting according to Ambion guidelines (technical bulletin 506) using the siRNA sequences tacttgccttacttcttca (1), aaaccaaagctgatcaaca (2), or gcagaactacttaggaact (3) with ttcaagaga as the loop sequence (see Fig. 3A). The oligonucleotide-annealed products were subcloned into pLenti6-U6 (23Boudreau F. Lussier C.R. Mongrain S. Darsigny M. Drouin J.L. Doyon G. Suh E.R. Beaulieu J.F. Rivard N. Perreault N. Faseb J. 2007; 21: 3853-3865Crossref PubMed Scopus (57) Google Scholar) between the BamHI and XhoI sites, giving rise to pLenti6-shNCoR. Lentiviruses were produced and used for cell infection according to Invitrogen recommendations (ViraPower lentiviral expression system instruction manual). The cDNA of rat PEDF was PCR-amplified from IEC-6 total RNA and subcloned into BamH1 and EcoRI restriction sites of the retroviral vector pBabe-puro. The HEK 293T cell line was used for transfection with Lipofectamine 2000 (Invitrogen) and both the retroviral DNA construct and helper amphotropic DNA as described previously (24Gheorghiu I. Deschênes C. Blais M. Boudreau F. Rivard N. Asselin C. J. Biol. Chem. 2001; 276: 44331-44337Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Subconfluent IEC-6 cells were infected with either an empty vector (control) or PEDF recombinant viruses in the presence of 2 μg/ml of polybrene (Sigma-Aldrich). Two days after infection, cells were selected with 2 μg/ml puromycin (Sigma-Aldrich). Probes for the microarray analysis were generated from isolated RNA obtained 2 days after RNAi-dependent shutdown of NcoR1 expression. Affymetrix GeneChip® Rat Genome 230 2.0 arrays were screened with the generated probes via the microarray platform of McGill University and Génome Québec Innovation Center as described previously (25Novak J.P. Sladek R. Hudson T.J. Genomics. 2002; 79: 104-113Crossref PubMed Scopus (164) Google Scholar). To test for changes in signal intensity, compiled data (RMA analysis) were screened using the software available on the microarray platform website. Genes were then filtered for up- or down-regulation of expression of a minimum of 2-fold and a minimal magnitude change of 200 fluorescence units between control and NCoR1 knockdown cells. Chromatin immunoprecipitation assays (ChIP) were performed using the protocol from the ChIP assay kit (Upstate, VA). Subconfluent and confluent Caco-2/15 cells were incubated with 1% formaldehyde for 10 min at 37 °C for DNA cross-link. Chromatin was immunoprecipitated with rabbit IgG (Santa Cruz Biotechnology) or with NCoR1 antibody (catalog 06–892; Millipore, Upstate, VA). One percent of the lysate was kept to control the amount of DNA used for immunoprecipitation (Input). Immunoprecipitated DNA was purified and diluted 1:20 before PCR amplification. The following sequences were used for PEDF promoter amplification; −878 up: 5′-gaggcaggagaacctcttga-3′; −529 down: 5′-cgagaccccgtctcaaaaa-3′; −411 up: 5′-gccaacacacctgggtaattt; and −81 down: 5′-cacacatttgcacaccttcc-3′. Electrophoretic mobility shift assays (EMSA) were performed essentially as described previously (26Boudreau F. Rings E.H. Swain G.P. Sinclair A.M. Suh E.R. Silberg D.G. Scheuermann R.H. Traber P.G. Mol. Cell. Biol. 2002; 22: 5467-5478Crossref PubMed Scopus (28) Google Scholar, 27Laniel M.A. Béliveau A. Guérin S.L. Methods Mol. Biol. 2001; 148: 13-30PubMed Google Scholar) with some modifications. The reactions were performed in a volume of 24 μl of binding buffer D (10 mm Hepes pH 7.9, 10% glycerol, 0.1 mm EDTA, and 0.25 mm phenylmethylsulfonyl fluoride) containing 4 μg of nuclear protein extracts from 293T cells transfected or not with RXRα (Open Biosystems, MHS1010-98051588) and RARα (Open Biosystems, MHS1010-97228051) expression vectors, 50 mm KCl, 50 ng of poly(dI-dC), and 25,000 cpm of 32P-labeled DNA probes for 10 min. For the supershift analysis, 600 ng of RXRα (D20SC-553) or RARα (C20SC-551) antibodies (Santa Cruz Biotechnology) were added, and the binding reactions were pursued for 10 min at room temperature. Retarded complexes were then separated on a 5% polyacrylamide gel at 4 °C for 4 h, dried for 1 h at 80 °C, and exposed overnight on a Molecular Imager FX screen (Bio-Rad). The running buffer used was a Tris-glycine 0.5× buffer (0.2 m glycine, 0.025 m Tris, and 1 mm EDTA). The DNA probes consisted of double-stranded oligonucleotides of 8 potential RXR binding sites within the promoter region of the human PEDF gene (Fig. 6). The human PEDF gene promoter was amplified by PCR from purified genomic DNA isolated from human fetal colon. The primers used from the amplification included positions −995 to +1 relatively to the transcriptional initiation site. The PCR product was subcloned in the pGL3basic luciferase reporter vector (Promega). Integrity of the subcloned PCR product was confirmed by sequence analysis. 293T cells were transfected by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Cells at 50% confluence wre incubated with 0.2 μg of luciferase reporter, 0.1 μg of RARα, and/or RXRα expression vectors, 0.2 μg of NCoR1 expression vector, 0.018 μg of the pRL SV40 Renilla luciferase vector (Promega), and a constant total DNA amount of 0.8 μg per transfected well in the presence of 2 μl of Lipofectamine 2000/100 μl of OptiMEM. The medium was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum after an incubation of 4 h. The luciferase and Renilla activities were determined 48 h after the transfection using the dual luciferase assay kit (Promega Biotech). Each experiment was repeated three times with five replicates. All data were expressed as mean ± S.E. Groups were compared using the Student's t test (GraphPad Prism 4, GraphPad Software, San Diego). Statistical significance was defined as p < .05. To investigate the profile of NCoR1 expression during epithelial cell differentiation, populations of epithelial cells from the mouse small intestinal mucosa were progressively isolated along the villus-to-crypt axis using a modified Weiser procedure (23Boudreau F. Lussier C.R. Mongrain S. Darsigny M. Drouin J.L. Doyon G. Suh E.R. Beaulieu J.F. Rivard N. Perreault N. Faseb J. 2007; 21: 3853-3865Crossref PubMed Scopus (57) Google Scholar) (Fig. 1A). Total RNA was isolated from these epithelial cell populations, and NCoR1 gene transcript levels were assessed by real-time RT-PCR analyses. No significant change in NCoR1 mRNA expression was detected along the villus-to-crypt axis (Fig. 1B) in contrast to the induction of the sucrase-isomaltase gene transcript in the villus epithelial cell fractions, a specific marker of intestinal epithelial cell differentiation (Fig. 1C). Because it has been previously reported that NCoR1 regulation of expression could be dependent on post-transcriptional mechanisms in other systems (28Zhang J. Guenther M.G. Carthew R.W. Lazar M.A. Genes Dev. 1998; 12: 1775-1780Crossref PubMed Scopus (190) Google Scholar), we next evaluated the NCoR1 protein profile of expression in intestinal epithelial cell fractions that were progressively harvested from the villus-to-crypt axis. NCoR1 protein was detectable in mouse crypt IEC fractions and disappeared in the most upper villus-associated epithelial fractions (Fig. 1D). The decrease of NCoR1 protein expression correlated with the detection of faster migrating dephosphorylated forms of the Rb protein that are normally associated with non-cycling G1-arrested cells (29Houde M. Laprise P. Jean D. Blais M. Asselin C. Rivard N. J. Biol. Chem. 2001; 276: 21885-21894Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and the induction of the PPARγ isoform, an inhibitor of IEC proliferation (9Varnat F. Heggeler B.B. Grisel P. Boucard N. Corthésy-Theulaz I. Wahli W. Desvergne B. Gastroenterology. 2006; 131: 538-553Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 10Gupta R.A. Sarraf P. Mueller E. Brockman J.A. Prusakiewicz J.J. Eng C. Willson T.M. DuBois R.N. J. Biol. Chem. 2003; 278: 22669-22677Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) (Fig. 1D). We next undertook to explore the pattern of NCoR1 expression during the proliferation-to-differentiation transition of pure intestinal epithelial cells in culture. The Caco-2/15 cell line that spontaneously differentiates into enterocytes upon reaching confluence was chosen because it has been extensively used as a model of IEC differentiation (30Boudreau F. Zhu Y. Traber P.G. J. Biol. Chem. 2001; 276: 32122-32128Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Vachon P.H. Beaulieu J.F. Gastroenterology. 1992; 103: 414-423Abstract Full Text PDF PubMed Scopus (187) Google Scholar). Again, no significant change in NCoR1 gene transcript expression was detected during the proliferation-to-differentiation transition of Caco-2/15 cells in culture (Fig. 2A). The sucrase-isomaltase gene transcript was strongly increased during this process (Fig. 2B) as has extensively been reported in the past (30Boudreau F. Zhu Y. Traber P.G. J. Biol. Chem. 2001; 276: 32122-32128Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 31Vachon P.H. Beaulieu J.F. Gastroenterology. 1992; 103: 414-423Abstract Full Text PDF PubMed Scopus (187) Google Scholar). We then investigated the NCoR1 protein profile during cell growth arrest and differentiation. Nuclear and cytosolic protein fractions were harvested at different Caco-2/15 cell confluence stages and subjected to Western blot analysis. Nuclear NCoR1 protein levels significantly decreased during the proliferation-to-differentiation transition of Caco-2/15 cells in culture, a pattern that was not observed for cytosolic NCoR1 protein levels (Fig. 2, C and D). In contrast, PPARγ protein was strongly induced in postconfluent-differentiated cells (Fig. 2C). Taken together, these observations indicate that NCoR1 protein expression was associated with proliferating, non-differentiated IEC.FIGURE 2.NCoR1 protein is down-modulated during the proliferation-to-differentiation transition of Caco-2/15 cells in culture. Total RNA from Caco-2/15 cells at different stages of confluence was used to monitor mRNA levels of the NCoR (A) and sucrase-isomaltase (B) genes (n = 3). C, subcellular fractions of Caco-2/15 cells were harvested at different stages of confluence. Western blots were performed with NCoR1, histone H1, and α-tubulin antibodies. Total extracts of Caco-2/15 cells were subjected to Western blot with PPARγ antibody. Specific detection of actin was done to control for protein loading. D, nuclear NCoR1 protein levels were quantified and calibrated with the histone H1 protein level (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The functional relationship between NCoR1 protein expression and maintenance of cellular proliferation was next investigated. The non-transformed IEC-6 cell line was further used because it has been described to phenotypically correspond to intestinal epithelial crypt cells (32Quaroni A. Wands J. Trelstad R.L. Isselbacher K.J. J. Cell Biol. 1979; 80: 248-265Crossref PubMed Scopus (680) Google Scholar) and has been extensively used as a model to study normal and non-cancerous IEC proliferation. We sought to directly neutralize NCoR1 expression by an RNA interference approach. In general, IEC-6 cell transfection with an enhanced green fluorescence protein (eGFP) expression vector resulted in poor efficiency of detection. However, stable infection of a lentivirus-eGFP into IEC-6/Cdx2 cells consistently resulted in over 90% of positive eGFP cells among the cell population. We thus generated lentiviral constructs that contained shRNA sequences under the control of a U6 promoter that were predicted to target the rat Ncor1 mRNA (Fig. 3A). Three independent shRNA NCoR1 lentiviruses were tested to down-regulate NCoR1 synthesis. A Western blot was performed with total extracts obtained from short term infected IEC-6 cell populations. The NCoR1 protein level was efficiently decreased in IEC-6 cells infected with the shRNA lentivirus 3 as opposed to cells infected with an eGFP lentivirus, an irrelevant shRNA lentivirus and two other NCoR1-specific shRNA constructs (Fig. 3B). The silencing mediator of retinoid and thyroid receptors (SMRT), a functionally related family member of NCoR1 (33Hu X. Lazar M.A. Trends Endocrinol. Metab. 2000; 11: 6-10Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), was not influenced under these specific conditions. The specific shRNA/NCoR3 construct was further utilized to efficiently interfere with NCoR1 expression. Stable IEC-6 cell populations were first generated with either NCoR1 shRNA or control shRNA lentiviruses. Long term antibiotic resistance selection of these recombinant cells consistently resulted in poor cell recovery of the NCoR1 shRNA populations. In addition, these cells progressively overcame the loss of NCoR1 protein expression (data not shown). A short term strategy was thus chosen to investigate whether the loss of NCoR1 could impact on cell proliferation and/or cell death. IEC-6 cells were infected with either control or shRNA/NCoR#3 lentiviruses and distributed into 6-well tissue culture plates. An inhibition of cell growth was rapidly observed for shRNA/NCoR3 cells that persisted for several days when compared with control infected cells (Fig. 4A). Western blot confirmed that NCoR1 expression was reduced in these cell populations during the kinetics (Fig. 4B). No significant increase in cell death was observed between control and NCoR1 knockdown cells as determined by the trypan blue exclusion test or by detection of the cytoplasmic specific 89-kDa cleaved form (34Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3804) Google Scholar) of polyadenosine diphosphate-ribose polymerase (PARP) (Fig. 4C).FIGURE 4.Knockdown of NCoR1 reduces IEC-6 cell proliferation. A, IEC-6 cells were infected with shNCoR 3 (NCoR) and control (ctl) shRNA lentiviruses, and cells were subsequently counted at different days (representative of three independent experiments). Protein extracts were isolated at several days during the kinetics and subjected to Western blot analysis with the" @default.
• W2088136743 created "2016-06-24" @default.
• W2088136743 creator A5020028874 @default.
• W2088136743 creator A5027821252 @default.
• W2088136743 creator A5029335213 @default.
• W2088136743 creator A5039422874 @default.
• W2088136743 creator A5040380731 @default.
• W2088136743 date "2009-09-01" @default.
• W2088136743 modified "2023-09-27" @default.
• W2088136743 title "Nuclear Receptor Co-repressor Is Required to Maintain Proliferation of Normal Intestinal Epithelial Cells in Culture and Down-modulates the Expression of Pigment Epithelium-derived Factor" @default.
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