HNF4α Regulates Claudin-7 Protein Expression during Intestinal Epithelial Differentiation
2015; Elsevier BV; Volume: 185; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2015.04.023
ISSN1525-2191
AutoresAttila E. Farkas, Roland S. Hilgarth, Christopher Capaldo, Christian Gerner‐Smidt, Doris R. Powell, Paula M. Vertino, Michael Koval, Charles A. Parkos, Asma Nusrat,
Tópico(s)Gut microbiota and health
ResumoThe intestinal epithelium is a dynamic barrier that maintains the distinct environments of intestinal tissue and lumen. Epithelial barrier function is defined principally by tight junctions, which, in turn, depend on the regulated expression of claudin family proteins. Claudins are expressed differentially during intestinal epithelial cell (IEC) differentiation. However, regulatory mechanisms governing claudin expression during epithelial differentiation are incompletely understood. We investigated the molecular mechanisms regulating claudin-7 during IEC differentiation. Claudin-7 expression is increased as epithelial cells differentiate along the intestinal crypt–luminal axis. By using model IECs we observed increased claudin-7 mRNA and nascent heteronuclear RNA levels during differentiation. A screen for potential regulators of the CLDN7 gene during IEC differentiation was performed using a transcription factor/DNA binding array, CLDN7 luciferase reporters, and in silico promoter analysis. We identified hepatocyte nuclear factor 4α as a regulatory factor that bound endogenous CLDN7 promoter in differentiating IECs and stimulated CLDN7 promoter activity. These findings support a role of hepatocyte nuclear factor 4α in controlling claudin-7 expression during IEC differentiation. The intestinal epithelium is a dynamic barrier that maintains the distinct environments of intestinal tissue and lumen. Epithelial barrier function is defined principally by tight junctions, which, in turn, depend on the regulated expression of claudin family proteins. Claudins are expressed differentially during intestinal epithelial cell (IEC) differentiation. However, regulatory mechanisms governing claudin expression during epithelial differentiation are incompletely understood. We investigated the molecular mechanisms regulating claudin-7 during IEC differentiation. Claudin-7 expression is increased as epithelial cells differentiate along the intestinal crypt–luminal axis. By using model IECs we observed increased claudin-7 mRNA and nascent heteronuclear RNA levels during differentiation. A screen for potential regulators of the CLDN7 gene during IEC differentiation was performed using a transcription factor/DNA binding array, CLDN7 luciferase reporters, and in silico promoter analysis. We identified hepatocyte nuclear factor 4α as a regulatory factor that bound endogenous CLDN7 promoter in differentiating IECs and stimulated CLDN7 promoter activity. These findings support a role of hepatocyte nuclear factor 4α in controlling claudin-7 expression during IEC differentiation. The intestinal epithelium constitutes a barrier that interfaces the distinct environment of the intestinal lumen and underlying tissue compartments. The intestinal epithelium is dynamic and actively is turned over as enterocytes proliferate in the crypt and migrate along the crypt–luminal axis, ultimately to be shed into the gut lumen. Epithelial barrier properties are achieved by intercellular junctions that include tight junctions (TJs), adherens junctions, and desmosomes.1Laukoetter M.G. Nava P. Nusrat A. Role of the intestinal barrier in inflammatory bowel disease.World J Gastroenterol. 2008; 14: 401-407Crossref PubMed Scopus (212) Google Scholar In addition to controlling epithelial polarity, proliferation, and differentiation, TJ proteins play an important role in the regulation of paracellular permeability.2Miyoshi J. Takai Y. Molecular perspective on tight-junction assembly and epithelial polarity.Adv Drug Deliv Rev. 2005; 57: 815-855Crossref PubMed Scopus (202) Google Scholar, 3Findley M.K. Koval M. Regulation and roles for claudin-family tight junction proteins.IUBMB Life. 2009; 61: 431-437Crossref PubMed Scopus (156) Google Scholar, 4Farkas A.E. Capaldo C.T. Nusrat A. Regulation of epithelial proliferation by tight junction proteins.Ann N Y Acad Sci. 2012; 1258: 115-124Crossref PubMed Scopus (40) Google Scholar Although all intestinal epithelial cells have TJs, the protein composition of these junctions changes during differentiation in the crypt–luminal axis. TJs comprise several transmembrane and associated scaffold proteins. The transmembrane proteins of the claudin family seal the intercellular space between epithelial or endothelial cells. Claudin proteins have four transmembrane domains, one intracellular loop, two extracellular loops, and both the N- and C-terminal domains are intracellular.5Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.J Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1708) Google Scholar Currently, 27 mammalian claudin genes have been described6Mineta K. Yamamoto Y. Yamazaki Y. Tanaka H. Tada Y. Saito K. Tamura A. Igarashi M. Endo T. Takeuchi K. Tsukita S. Predicted expansion of the claudin multigene family.FEBS Lett. 2011; 585: 606-612Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar and specific subsets of claudins determine the barrier properties of epithelia and endothelia in a tissue-specific manner.7Günzel D. Yu A.S.L. Claudins and the modulation of tight junction permeability.Physiol Rev. 2013; 93: 525-569Crossref PubMed Scopus (825) Google Scholar The expression of select claudins including claudin-2, -4, -7, -10, and -15 changes as intestinal epithelial cells (IECs) differentiate in the intestine and migrate along the crypt–luminal axis.8Fujita H. Chiba H. Yokozaki H. Sakai N. Sugimoto K. Wada T. Kojima T. Yamashita T. Sawada N. Differential expression and subcellular localization of claudin-7, −8, −12, −13, and −15 along the mouse intestine.J Histochem Cytochem. 2006; 54: 933-944Crossref PubMed Scopus (184) Google Scholar, 9Fujita H. Sugimoto K. Inatomi S. Maeda T. Osanai M. Uchiyama Y. Yamamoto Y. Wada T. Kojima T. Yokozaki H. Yamashita T. Kato S. Sawada N. Chiba H. Tight junction proteins claudin-2 and -12 are critical for vitamin D-dependent Ca2+ absorption between enterocytes.Mol Biol Cell. 2008; 19: 1912-1921Crossref PubMed Scopus (343) Google Scholar, 10Holmes J.L. Van Itallie C.M. 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In the colon, claudin-7 expression increases as the epithelial cells differentiate toward the luminal surface, resulting in a gradient with the highest expression at the luminal surface.8Fujita H. Chiba H. Yokozaki H. Sakai N. Sugimoto K. Wada T. Kojima T. Yamashita T. Sawada N. Differential expression and subcellular localization of claudin-7, −8, −12, −13, and −15 along the mouse intestine.J Histochem Cytochem. 2006; 54: 933-944Crossref PubMed Scopus (184) Google Scholar In addition to TJ localization, many claudin proteins such as claudin-7 also are distributed in the lateral membrane of IECs.8Fujita H. Chiba H. Yokozaki H. Sakai N. Sugimoto K. Wada T. Kojima T. Yamashita T. Sawada N. Differential expression and subcellular localization of claudin-7, −8, −12, −13, and −15 along the mouse intestine.J Histochem Cytochem. 2006; 54: 933-944Crossref PubMed Scopus (184) Google Scholar We report a mechanism by which claudin-7 protein is regulated in differentiating IECs. A transcription factor (TF)/DNA binding array was used in combination with in silico analysis to screen for TFs that potentially control claudin-7 levels in differentiating intestinal epithelium. This screen identified hepatocyte nuclear factor 4α (HNF4α), PU.1, and Oct 2.1 as candidate TFs that bind CLDN7 promoter. Chromatin immunoprecipitation (ChIP) and promoter reporter assays showed that HNF-4α controls CLDN7 transcription in differentiating IECs. Thus, our study identified a novel direct regulation of the CLDN7 gene by HNF-4α during IEC differentiation. Caco-2 or HT29/B6 cells were grown in high-glucose (4.5 g/L) Dulbecco's modified Eagle's medium (Corning, Tewksbury, MA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 15 mmol/L HEPES, pH 7.4, 2 mmol/L l-glutamine, and 1% nonessential amino acids. Cells were seeded at high density (approximately 200,000/cm2) on cell culture–treated plates and harvested for Western blot or quantitative real-time PCR (qPCR) analysis after 1 to 12 days. Expended culture media was changed to fresh culture media daily. For luciferase reporter assays the cells were seeded at high density in 48-well, cell culture–treated plates, transfected the following day by luciferase reporter constructs, and harvested 1 to 5 days later. For the TF/DNA binding assay and ChIP analysis, Caco-2 cells were seeded at high density in 75 cm2 culture flasks and harvested after 2 or 12 days. Cells were harvested in ice-cold radioimmunoprecipitation assay [0.5% Triton X-100 (Fisher Scientific, Rockford, IL), 0.5% NP-40, 0.5% deoxycholic acid, 0.1% SDS, 150 mmol/L NaCl, 1 mmol/L EGTA (pH 8.0), 1 mmol/L EDTA, 0.2 mmol/L sodium orthovanadate, and 20 mmol/L Tris (pH 7.4)] buffer, and centrifuged at 10,000 × g for 20 minutes at 4°C to sediment cell debris. Supernatant protein concentration was determined using the bicinchoninic acid assay (Pierce/Thermo Fisher Scientific, Rockford, IL) and subjected to Western blot analysis. Primary antibodies (and dilutions) were claudin-7 (1:2000) and claudin-2 (1:250) polyclonal rabbit antibodies (Invitrogen, Life Technologies, Grand Island, NY), caudal type homeo box transcription factor 2 (Cdx2) (1:1000), p21 (1:1000), and HNF-4α (1:1000) monoclonal rabbit antibodies (CellSignaling, Danvers, MA), and mouse monoclonal glyceraldehyde-3-phosphate dehydrogenase (1:2000; Santa Cruz Biotechnology, Dallas, TX), mouse monoclonal α-tubulin (1:4000; Sigma, St. Louis, MO), or calnexin (1:5000; Sigma) as loading control. Cryostat sections of colon tissue mounted on glass microscope slides were fixed in methanol. Blocking of nonspecific binding was achieved by a 30-minute incubation in phosphate-buffered saline with 2% bovine serum albumin. Primary antibody incubation using rabbit polyclonal claudin-7, claudin-2, claudin-4, claudin-15 (1:200, 1:50, 1:200, 1:50, respectively; Invitrogen, Life Technologies) HNF-4α (1:100; CellSignaling) was performed in blocking solution overnight at 4°C. Secondary antibodies (Alexa Fluor-488–and Alexa Fluor-546–conjugated; Jackson ImmunoResearch Laboratories, West Grove, PA) were diluted 1:500 in blocking solution and incubated for 1 hour at room temperature, protected from light. Nuclei were stained with ToPro-3 iodide (Molecular Probes, Life Technologies), and coverslips were mounted in p-phenylene. Images were taken on an LSM 510 confocal microscope (Carl Zeiss Microscopy, Thornwood, NY), with software supplied by the vendor. PCR was performed using Apex Taq RED (Genesee, San Diego, CA) on a C1000 thermal cycler (Bio-Rad, Hercules, CA), 25 μL reactions were run using the following parameters: 3 minutes of initial denaturation at 95°C, followed by 30 cycles of 95°C denaturation, 60°C annealing, and 72°C extension for 30 seconds each. Real-time PCR was performed on a MyIQ real-time PCR detection system (Bio-Rad), using Maxima SYBR Green (Fisher Scientific, Pittsburgh, PA) according to the kit protocol. Briefly, 10 minutes of 90°C initial denaturation followed by 40 cycles of 15 seconds of 95°C denaturation, 30 seconds of 62°C annealing, and 30 seconds of 72°C extension with the detection of the SYBR green signal set to each extension step. For first-strand synthesis, RNA was purified using TRI Reagent (Life Technologies) according to the manufacturer's protocol. First-strand synthesis was performed on a C1000 thermal cycler (Bio-Rad) with RevertAid (Fisher Scientific) according to the manufacturer's protocol, using 5 μg total RNA and oligo dT primers. The primer pairs that were used are listed in Tables 1 and 2.Table 1Primers Used in PCR or qPCR Reactions to Detect Claudin-7 mRNA and hnRNAPrimer nameSequenceNotesForward 35′-TGCAGGCCACTCGAGCCCTA-3′Claudin-7 mRNA, forward on exonForward hn25′-GAACACGGCGCCGGACAGAA-3′Claudin-7 hnRNA, forward on exon–intron boundaryReverse 35′-GGCCTTTGTCCGGCACCCTG-3′Reverse primer for both forward aboveβ-actin forward5′-AGTCCATCACGATGCCAGT-3′β-actin reverse5′-TGACCCAGATCATGTTTGAGA-3′ Open table in a new tab Table 2Primers for the Detection of CLDN7 Genomic DNA from ChIPPrimer nameSequenceNotesCLDN7HNF4 forward 25′-AGTGAGACCCGGCCTCTAAG-3′ChIP, first HNF-4α siteCLDN7HNF4 reverse 25′-GACCTCAGGTGATCCCAACC-3′CLDN7HNF4 forward 35′-ATCGACCAGGGTAGGAGACA-3′ChIP, second HNF-4α siteCLDN7HNF4 reverse 35′-AAGCCCCTTCCTTTCTTCCC-3′CLDN7-H1-H2 forward 15′-CAGGAAGCTCCAAAGCAGGA-3′CTL1, negative controlCLDN7-H1-H2 reverse 15′-TCTGTCACGCCTCACTGTTC-3′CLDN7-H2 forward5′-AGCCAGAACAATGATGACACGTGGCTGTAA-3′CTL2, negative controlCLDN7-H2 reverse5′-TCACTGCTCCCAGCCATGAACTGTTTGTTA-3′PU1 CLDN7 3 reverse5′-CCTTCCTCCTTCATCTTTGGTCATCTC-3′ChIP, PU.1 sitePU1 CLDN7 3 forward5′-CTCTGCACTCCAGCCTGGGTAAC-3′POU2F2 CLDN7 1 forward5′-GTTAGGAGCCTTGATGCCGGAG-3′ChIP, Oct2 sitePOU2F2 CLDN7 1 reverse5′-CTTAGCGTGGCTCCCCAAC-3′ Open table in a new tab TF DNA binding activity was assayed in nuclear lysates from 2- and 12-day-old confluent monolayers of Caco-2 cells using a Combo Protein/DNA Array (MA1215; Panomics/Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. Briefly, the nuclear lysates were incubated with a mixture of 345 biotinylated oligonucleotide probes representing unique TF binding (TFB) motifs. The protein/DNA complexes were bound to a spin column and unbound oligonucleotides were removed by elution with a wash buffer. The bound oligonucleotides subsequently were eluted from the column, denatured, and hybridized to a nylon membrane printed with oligonucleotides complementary to the probes. The membranes carrying the biotinylated probes were incubated with streptavidin–horseradish peroxidase and visualized by chemiluminescence on X-ray film. The films were scanned and subsequently processed using ImageJ version 1.46d (NIH, Bethesda, MD; http://imagej.nih.gov/ij),12Abramoff M.D. Magalhaes P.J. Ram S.J. Image processing with ImageJ.Biophotonics Int. 2004; 11: 36-42Google Scholar with the Protein Array Analyser plugin (available at image.bio.methods.free.fr/ImageJ/?Protein-Array-Analyzer-for-ImageJ.html, last accessed April 8, 2015).13Carpentier G, Henault E: Protein array analyzer for ImageJ. Proceedings of the ImageJ User and Developer Conference, Centre de Recherche Public Henri Tudor, Ed., 2010, 238–240Google Scholar Signal intensity values from the array analysis were recorded in Supplemental Table S1, and fold changes of TF DNA binding activity are shown in Supplemental Table S2. A 3072-bp fragment upstream of the human CLDN7 translation start site (−3072 to −1 bp, where 0 is the A in the start ATG) was cloned into a pGL4.10 luciferase reporter vector [expresses firefly (Photinus pyralis) luciferase; Promega, Madison, WI] at the KpnI and NcoI sites. Introducing the NcoI recognition sequence during cloning resulted in an extra cytidine before the start ATG, which was removed via site-directed mutagenesis using the Quickchange Site Directed Mutagenesis kit following the manufacturer's instructions (Agilent Technologies, Santa Clara, CA). This construct expresses luciferase driven by the wild-type CLDN7 promoter and was designated pGL4C7. The cloned fragment includes the transcription start site (at −1150 bp) and the 5′ untranslated region of CLDN7 because regulatory elements often reside in this region.14Bianchi M. Crinelli R. Giacomini E. Carloni E. Magnani M. A potent enhancer element in the 5′-UTR intron is crucial for transcriptional regulation of the human ubiquitin C gene.Gene. 2009; 448: 88-101Crossref PubMed Scopus (54) Google Scholar, 15Vostrov A. Taheny M. Izkhakov N. Quitschke W. A nuclear factor-binding domain in the 5'-untranslated region of the amyloid precursor protein promoter: implications for the regulation of gene expression.BMC Res Notes. 2010; 3: 4Crossref PubMed Scopus (11) Google Scholar Initially, two promoter truncation constructs were generated spanning −1409 to −1 bp (pGL4C7Δ1) and −494 to −1 bp (pGL4C7Δ2) by restriction endonuclease digestion using NcoI + KpnI and SmaI + KpnI, respectively. To map the promoter activity between pGL4C7Δ1 and pGL4C7Δ2, we generated four 200-bp to 800-bp deletion promoter constructs of pGL4C7Δ1 (pGL4C7Δ1.1-4) by a PCR-based method. To create the pGL4C7Δ1.1-4 constructs, primers were generated every 200 bp downstream from the NcoI site, with an additional KpnI site added artificially to the 5′ end. The opposite primer was the same for each PCR reaction at the ApaI site in the beginning of the luc2 gene. The resulting PCR products were ligated into the pGL4.10 vector at the KpnI and ApaI restriction sites. Restriction endonucleases were purchased from New England Biolabs (Ipswich, MA). Primer pairs used for cloning are listed in Table 3.Table 3Primers Used to Create CLDN7/Luciferase ReportersPrimer nameSequenceNotesCloning primers for the initial CLDN7 construct CLDN7 forward 25′-ATGGTACCAAGGAAAGTCAGTTGCGGTAG-3′Cloning primer CLDN7 reverse5′-GTCACATGTTCCGCCCTCAGAAAACAG-3′Cloning primer CLDN7 mut forward5′-GTGTTTTCTGAGGGCGGAAATGGAAGCTTAATGTTTTTGGCATCTTCCATTC-3′Quickchange primer CLDN7 mut reverse5′-GAATGGAAGATGCCAAAAACATTAAGCTTCCATTTCCGCCCTCAGAAAACAC-3′Quickchange primerCloning primers for deletion mutant CLDN7 luciferase constructs in the NcoI-SmaI region of the promoter Luc2 ApaI reverse V5′-GGCGCTGGGCCCTTCTTAAT-3′Cloning primer at the ApaI site in the luc2 gene Cl7 trunc forward 15′-ATATGGTACCTAGCCCTCACCCTGCTC-3′Cloning primer 200 after the NcoI site Cl7 trunc forward 25′-ATATGGTACCTGTAGCAGAGCCAGAGAAC-3′Cloning primer 400 after the NcoI site Cl7 trunc forward 3b5′-ATATGGTACCAGGCGCACCTGTTGGGAA-3′Cloning primer 625 after the NcoI site Cl7 trunc forward 45′-ATATGGTACCTGGGCAAGGAGGGGTGG-3′Cloning primer 801 after the NcoI siteArtificial KpnI (ATATGGTA) site is marked in bold at 5′ end.mut, mutation. Open table in a new tab Artificial KpnI (ATATGGTA) site is marked in bold at 5′ end. mut, mutation. Mutations of the individual HNF-4α sites in the pGL4C7 construct were generated using "megaprimer" mutagenesis.16Barik S. Site-directed mutagenesis in vitro by megaprimer PCR.Methods Mol Biol. 1996; 57: 203-215PubMed Google Scholar The primer pairs that were used are listed in Table 4. All PCR reactions were performed using Pfu Ultra (Agilent Technologies) according to the manufacturer's instructions. Ten microliters of Qiaquick (Qiagen, Valencia, CA) gel extraction purified megaprimer was used in the second PCR with a standard concentration of reverse primer. The resulting mutations containing PCR products then were digested by SphI/EcorI or AvrII/PpmuI, resulting in 246-bp or 871-bp fragments, respectively. Digested fragments were gel-purified and ligated into the appropriate gel-purified, wild-type, claudin-7 promoter. pGL4C7 Mut1 and pGL4C7 Mut2 constructs were generated containing mutated HNF-4α binding sites at −2519 bp or −856 bp upstream from the start ATG, respectively. The pGL4C7 Mut1+2 promoter construct was generated using standard cloning techniques from the pGL4C7 Mut1 and pGL4C7 Mut2 promoter constructs.Table 4Primers for Megaprimer Mutagenesis of the CLDN7 PromoterPrimer nameSequenceUseCLDN7 SphI5′-ATGCGCATGCTTTGAGCCCAGG-3′With CLDN7 Mut1, produces Mut 1 MegaCLDN7 Mut15′-CACCTCGACCTCCCAAAGTGGTCGGATTACAGGCGTGAG-3′With CLDN7 SphI produces Mut 1 MegaCLDN7 EcorI5′-CCTCAGCCTCCCAAGTAGCT-3′With Mut 1 MegaCLDN7 forward 15′-CTCGGCCGGCTTTAGGTCCCAGTGGTTTC-3′With CLDN7 Mut2, produces Mut2 MegaCLDN7 Mut25′-TCCCCGCCGGGTTCTGTCCAAAGTGGTCTCTCCTCGGCCCTGCTT-3′With CLDN7 F1, produces Mut 2 MegaCLDN7 reverse 15′-GTCCCCGAAGGCCAGGCGGT-3′ Open table in a new tab Cells were transfected with 0.2 μg/well pGL4C7 vector [or equimolar amounts of pGL4C7Δ1.1-4 or pGL4.10 (empty vector) based on their respective size in base pairs] and 0.04 μg/well pRL-TK (expressing Renilla luciferase; Promega) as a control for transfection efficiency. Samples were generated in triplicate in 48-well tissue culture plates, each well was transfected at the same time because older cultures are resistant to transfection. Luciferase activity was measured and reporter activity was determined using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Putative TF binding motifs were identified on the 3072-bp CLDN7 promoter sequence, which also was used to create luciferase reporters. The in silico analysis was performed using the PROMO web service (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3, last accessed April 8, 2015) with default settings for Homo sapiens chromosome 17, RCh38.p2 Primary Assembly, Sequence ID: ref NC_000017.11, range: 7265114 to 7262044 (Supplemental Table S3). We mapped putative TFB motifs on the pGL4C7Δ constructs for further analysis (Supplemental Table S4). PROMO uses the TRANSFAC 8.3 database to construct specific binding site weight matrices for TFB site prediction.17Messeguer X. Escudero R. Farré D. Núñez O. Martínez J. Albà M.M. PROMO: detection of known transcription regulatory elements using species-tailored searches.Bioinformatics. 2002; 18: 333-334Crossref PubMed Scopus (946) Google Scholar, 18Farré D. Roset R. Huerta M. Adsuara J.E. Roselló L. Albà M.M. Messeguer X. Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN.Nucleic Acids Res. 2003; 31: 3651-3653Crossref PubMed Scopus (753) Google Scholar ChIP was performed using the EZ-ChIP or Magna-ChIP kits (EMD Millipore, Billerica, MA) according to the manufacturer's recommendations. Confluent monolayers of Caco-2 cells were fixed with fresh 0.5% formaldehyde, quenched with glycine, and collected in ice-cold, phosphate-buffered saline containing protease inhibitors. The cells then were lysed in ChIP lysis buffer and the chromatin was sheared with a Branson 450 Sonifier (Branson Ultrasonics, Danbury, CT) on ice. The sonicator power was set to 3, and 30% duty cycle. Sonication was performed four times for 30 minutes with 1-minute breaks to prevent heat accumulation. This resulted in uniform chromatin shearing to approximately 200 to 500 bp. One million cells equivalent chromatin was used per immunoprecipitation, using 10 μg anti–HNF-4α, PU.1 (Cell Signaling, Boston, MA), or Oct2.1 (Santa Cruz Biotechnology) antibodies, or nonspecific anti-rabbit IgG. After reversing the cross-links and digestion with proteinase K, DNA was isolated using spin columns. qPCR primers were designed to amplify DNA sequences spanning the putative TFB sites and also sequences distant from binding sites as negative control. Transepithelial electrical resistance (TER) was measured across cells grown on 0.33 cm2 polycarbonate filters with 0.4-μm pores using an epithelial voltmeter (EVOM; Precision World Instruments, Sarasota, FL). TER values were normalized for the area of the filter and reduced by the contribution of the filter and bathing solution. Numeric values from three individual filters were pooled and expressed as means ± SEM. TER was in the 300 to 400 Ωcm2 range at the end of the experiment. Confluent monolayers of Caco-2 cells in 6-well cell culture plates were transfected overnight with 1 μg HNF-4α plasmid (#665; Emory DNA Custom Cloning Core Facility, Atlanta, GA) or empty vector (pCDNA3.1; Invitrogen, Life Technologies) using 8 μL Lipofectamine 2000 reagent in 2 mL Optimem I (both from Invitrogen, Life Technologies). Transfection media then was switched to culture media and cells were cultured as described. For statistical analysis, replicate data were expressed as averages ± SEM. Significance was determined using Student's t-test. Cell differentiation along the crypt–luminal axis (Figure 1A) is important for maintaining intestinal epithelial homeostasis and barrier function.19Lu Z. Ding L. Lu Q. Chen Y.H. Claudins in intestines: distribution and functional significance in health and diseases.Tissue Barriers. 2013; 1: e24978Crossref PubMed Google Scholar Immunofluorescence labeling and laser confocal microscopy was performed to evaluate claudin protein expression in the crypt–luminal axis of mouse colonic epithelial cells. Claudins showed differential expression in the crypt–luminal axis with claudin-2 and claudin-15 in crypt epithelial cells, whereas claudin-4 was expressed exclusively in the surface intestinal epithelium (Figure 1A). Of note, claudin-7 displayed a gradient with increased claudin-7 labeling in surface IECs compared with cells in the base of crypts. To establish an in vitro system that is suitable to study mechanisms of claudin protein change during intestinal epithelial differentiation, we used the Caco-2 model IEC line, which has been reported previously to differentiate in culture analogous to native IECs.20Saaf A.M. Halbleib J.M. Chen X. Yuen S.T. Leung S.Y. Nelson W.J. Brown P.O. Parallels between global transcriptional programs of polarizing Caco-2 intestinal epithelial cells in vitro and gene expression programs in normal colon and colon cancer.Mol Biol Cell. 2007; 18: 4245-4260Crossref PubMed Scopus (100) Google Scholar, 21Flandez M. Guilmeau S. Blache P. Augenlicht L.H. KLF4 regulation in intestinal epithelial cell maturation.Exp Cell Res. 2008; 314: 3712-3723Crossref PubMed Scopus (57) Google Scholar, 22Reisher S.R. Hughes T.E. Ordovas J.M. Schaefer E.J. Feinstein S.I. Increased expression of apolipoprotein genes accompanies differentiation in the intestinal cell line Caco-2.Proc Natl Acad Sci U S A. 1993; 90: 5757-5761Crossref PubMed Scopus (44) Google Scholar IECs were cultured at high cell density for 1 to 9 days and analyzed for claudin-7 expression and barrier function. By immunofluorescence staining, claudin-7 expression increased in culture as the cell height increased (Figure 1B). Moreover, claudin-7 was localized in the lateral plasma membrane and co-localized with the TJ protein zonula occludens protein 1 in the apical-most region of the lateral membrane. The development of barrier function was evaluated by measuring TER. After seeding at high density, epithelial monolayers of Caco-2 cells underwent differentiation and achieved a TER of 300 to 400 Ω*cm2 in 7 days. In addition, down-regulation of claudin-7 by siRNA (siCLDN7) in Caco-2 monolayers that already reached higher than 100 Ω*cm2 resulted in decreased TER compared with nonsilencing siRNA (Figure 1C). Analogous to the in vivo crypt–luminal claudin-7 gradient, immunoblots showed increasing claudin-7 protein as IECs differentiated in culture, with a peak after 5 days of differentiation (Figure 1D). Similarly, two markers of IEC differentiation, Cdx2 and the cyclin-dependent kinase inhibitor p21WAF1/Cip1, showed increased protein expression in differentiating IECs.23Guo R.-J. Suh E.R. Lynch J.P. The role of Cdx proteins in intestinal development and cancer.Cancer Biol Ther. 2004; 3: 593-601Crossref PubMed Scopus (203) Google Scholar, 24Quaroni A. Tian J.Q. Seth P. Ap Rhys C. p27Kip1 is an inducer of intestinal epithelial cell differentiation.Am J Physiol Cell Physiol. 2000; 279: C1045-C1057PubMed Google Scholar In addition, in intestinal crypt epithelial cells, claudin-2 protein was detected in cells after plating and was lost in confluent differentiated IECs. To test whether the observed claudin-7 increase is specific to Caco-2 cells, we cultured HT29/B6 cells and found increased claudin-7 protein levels after 5 days in culture compared with 2 days (Figure 1E). Because claudin-7 protein is increased during IEC differentiation, we next determined if increased protein was reflective of increased claudin-7 mRNA. Indeed, claudin-7 mRNA was increased 10-fold over the first 3 days and increased 20-fold by 6 days in culture (Figure 2A). Because increased mRNA levels during differentiation may reflect multiple processes, we next analyzed heteronuclear RNA (hnRNA) levels (Figure 2A). Analogous to mRNA, claudin-7 hnRNA was increased in differentiating IECs. The levels of unspliced hnRNA, also called "pre-mRNA," reflect the transcriptional activity of the gene. A comparison of mRNA and hnRNA structure of claudin-7 is shown in Figure 2B. These results suggest that transcriptional activation plays an important role in the increased expression of claudin-7 in differentiated IECs. Our results suggest that transcriptional control plays a role in the increased claudin-7 protein during colonic epithelial differentiation. To identify TFs that control gene expression during IEC differentiation and potential regulators of claudin-7 mRNA expression, we first compared the DNA binding activity of nuclear lysates from undifferentiated (2 days in culture) and differentiated (12 days in culture) Caco-2 cells (Figure 3A) using a TF/DNA array. The array consists of 345 unique consensus TFB sequences. Each sequence is recognized by a specific TF or by a family of closely related TFs. Our analysis found 93 sequences with ≥1.5-fold increas
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