Cloning of the Human Claudin-2 5′-Flanking Region Revealed a TATA-less Promoter with Conserved Binding Sites in Mouse and Human for Caudal-related Homeodomain Proteins and Hepatocyte Nuclear Factor-1α (2002)

Claudin-2 is a structural component of tight junctions in the kidneys, liver, and intestine, but the mechanisms regulating its expression have not been defined. The 5′-flanking region of the claudin-2 gene contains binding sites for intestine-specific Cdx homeodomain proteins and hepatocyte nuclear factor (HNF)-1, which are conserved in human and mouse. Both Cdx1 and Cdx2 activated the claudin-2 promoter in the human intestinal epithelial cell line Caco-2. HNF-1α augmented the Cdx2-induced but not Cdx1-induced transcriptional activation of the human claudin-2 promoter. In mice, HNF-1α was required for claudin-2 expression in the villus epithelium of the ileum and within the liver but not in the kidneys, indicating an organ-specific function of HNF-1α in the regulation of claudin-2 gene expression. Tight junction structural components, which determine epithelial polarization and intestinal barrier function, can be regulated by homeodomain proteins that control the differentiation of the intestinal epithelium. Claudin-2 is a structural component of tight junctions in the kidneys, liver, and intestine, but the mechanisms regulating its expression have not been defined. The 5′-flanking region of the claudin-2 gene contains binding sites for intestine-specific Cdx homeodomain proteins and hepatocyte nuclear factor (HNF)-1, which are conserved in human and mouse. Both Cdx1 and Cdx2 activated the claudin-2 promoter in the human intestinal epithelial cell line Caco-2. HNF-1α augmented the Cdx2-induced but not Cdx1-induced transcriptional activation of the human claudin-2 promoter. In mice, HNF-1α was required for claudin-2 expression in the villus epithelium of the ileum and within the liver but not in the kidneys, indicating an organ-specific function of HNF-1α in the regulation of claudin-2 gene expression. Tight junction structural components, which determine epithelial polarization and intestinal barrier function, can be regulated by homeodomain proteins that control the differentiation of the intestinal epithelium. sucrase isomaltase hepatocyte nuclear factor lactase-phlorizin hydrolase electrophoretic mobility shift assay phosphate-buffered saline glyceraldehyde-3-phosphate dehydrogenase Claudin-2 is a regulatory component of tight junctions in the liver, the kidneys, and the epithelium of the small and large intestines (1Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1695) Google Scholar, 2Rahner C. Mitic L.L. Anderson J.M. Gastroenterology. 2001; 120: 411-422Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). Claudin-2 expression has been demonstrated to be involved in the regulation of the intestinal barrier function by immune modulators (3Kinugasa T. Sakaguchi T., Gu, X. Reinecker H.C. Gastroenterology. 2000; 118: 1001-1011Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Claudins form a family of proteins composed of at least 24 members, which are expressed in an organ-specific manner and regulate the tissue-specific physiological properties of tight junctions (4Tsukita S. Furuse M. J. Cell Biol. 2000; 149: 13-16Crossref PubMed Scopus (403) Google Scholar, 5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell Biol. 2001; 2: 285-293Crossref PubMed Scopus (2008) Google Scholar). Tight junctions not only create a primary barrier to prevent paracellular passage of solutes and pathogens, but they also restrict the lateral diffusion of membrane lipids and proteins to maintain cellular polarity (5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell Biol. 2001; 2: 285-293Crossref PubMed Scopus (2008) Google Scholar, 6Schneeberger E.E. Lynch R.D. Am. J. Physiol. 1992; 262: L647-L661PubMed Google Scholar, 7Anderson J.M. Van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar, 8Madara J. Annu. Rev. Physiol. 1998; 60: 143-159Crossref PubMed Scopus (462) Google Scholar). Evidence is mounting that claudins are actively involved in the regulation of paracellular transport of ions through tight junctions (9Van Itallie C. Rahner C. Anderson J.M. J. Clin. Invest. 2001; 107: 1319-1327Crossref PubMed Scopus (489) Google Scholar, 10Simon D.B., Lu, Y. Choate K.A. Velazquez H., Al- Sabban E. Praga M. Casari G. Bettinelli A. Colussi G. Rodriguez-Soriano J. McCredie D. Milford D. Sanjad S. Lifton R.P. Science. 1999; 285: 103-106Crossref PubMed Scopus (961) Google Scholar). The modulation of selective transport through tight junction may require the coordinated expression of distinct claudins in a particular cell type (1Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1695) Google Scholar, 11Morita K. Furuse M. Fujimoto K. Tsukita S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 511-516Crossref PubMed Scopus (978) Google Scholar). Therefore, the regulation of claudin expression may determine the fundamental ability of the intestinal epithelium to modulate water or ion transport and barrier function. However, the transcriptional events involved in the organ-specific expression of claudins have not been determined. Cdx1 and Cdx2 are members of the caudal-related homeobox gene family based on their sequence homology to the caudal gene of Drosophila melanogaster (12Charite J. de Graaff W. Consten D. Reijnen M.J. Korving J. Deschamps J. Development. 1998; 125: 4349-4358PubMed Google Scholar, 13Duprey P. Chowdhury K. Dressler G.R. Balling R. Simon D. Guenet J.L. Gruss P. Genes Dev. 1988; 2: 1647-1654Crossref PubMed Scopus (175) Google Scholar, 14James R. Erler T. Kazenwadel J. J. Biol. Chem. 1994; 269: 15229-15237Abstract Full Text PDF PubMed Google Scholar). In vitro and in vivostudies of Cdx1 and Cdx2 suggest that these transcription factors are important in the early differentiation and maintenance of intestinal epithelial cells. In vitro experiments show significant functional effects of Cdx genes on intestinal differentiation (15Suh E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Crossref PubMed Scopus (450) Google Scholar, 16Soubeyran P. Andre F. Lissitzky J.C. Mallo G.V. Moucadel V. Roccabianca M. Rechreche H. Marvaldi J. Dikic I. Dagorn J.C. Iovanna J.L. Gastroenterology. 1999; 117: 1326-1338Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), proliferation (15Suh E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Crossref PubMed Scopus (450) Google Scholar, 17Lynch J. Suh E.R. Silberg D.G. Rulyak S. Blanchard N. Traber P.G. J. Biol. Chem. 2000; 275: 4499-4506Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and intestine-specific gene transcription (18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (379) Google Scholar, 19Lambert M. Colnot S. Suh E. L'Horset F. Blin C. Calliot M.E. Raymondjean M. Thomasset M. Traber P.G. Perret C. Eur. J. Biochem. 1996; 236: 778-788Crossref PubMed Scopus (79) Google Scholar, 20Troelsen J.T. Mitchelmore C. Spodsberg N. Jensen A.M. Noren O. Sjostrom H. Biochem. J. 1997; 322: 833-838Crossref PubMed Scopus (144) Google Scholar, 21Mitchelmore C. Troelsen J.T. Spodsberg N. Sjostrom H. Noren O. Biochem. J. 2000; 346: 529-535Crossref PubMed Scopus (102) Google Scholar, 22Fang R. Santiago N.A. Olds L.C. Sibley E. Gastroenterology. 2000; 118: 115-127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Overexpression of Cdx2 in undifferentiated IEC-6 intestinal epithelial cells leads to the development of a differentiated phenotype (15Suh E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Crossref PubMed Scopus (450) Google Scholar). Cdx1 and Cdx2 have been shown to regulate intestine-specific gene transcription by binding to several intestine-specific promoters (18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (379) Google Scholar, 19Lambert M. Colnot S. Suh E. L'Horset F. Blin C. Calliot M.E. Raymondjean M. Thomasset M. Traber P.G. Perret C. Eur. J. Biochem. 1996; 236: 778-788Crossref PubMed Scopus (79) Google Scholar, 20Troelsen J.T. Mitchelmore C. Spodsberg N. Jensen A.M. Noren O. Sjostrom H. Biochem. J. 1997; 322: 833-838Crossref PubMed Scopus (144) Google Scholar, 23Drummond F. Sowden J. Morrison K. Edwards Y.H. Eur. J. Biochem. 1996; 236: 670-681Crossref PubMed Scopus (68) Google Scholar, 24Drummond F.J. Sowden J. Morrison K. Edwards Y.H. FEBS Lett. 1998; 423: 218-222Crossref PubMed Scopus (42) Google Scholar). In intestinal epithelial Caco-2 cells, Cdx2 expression induces the expression of sucrase isomaltase (SI)1 and lactase-phlorizin hydrolase (LPH), two markers of intestinal differentiation (25Lorentz O. Duluc I. Arcangelis A.D. Simon-Assmann P. Kedinger M. Freund J.N. J. Cell Biol. 1997; 139: 1553-1565Crossref PubMed Scopus (256) Google Scholar). In the regulation of LPH expression, Cdx2 directly interacts with hepatocyte nuclear factor (HNF)-1α (21Mitchelmore C. Troelsen J.T. Spodsberg N. Sjostrom H. Noren O. Biochem. J. 2000; 346: 529-535Crossref PubMed Scopus (102) Google Scholar). HNF-1α and HNF-1β are related transcription factors that bind to DNA as homodimers or heterodimers (26Mendel D.B. Hansen L.P. Graves M.K. Conley P.B. Crabtree G.R. Genes Dev. 1991; 5: 1042-1056Crossref PubMed Scopus (249) Google Scholar). HNF-1α and HNF-1β are known to be important for liver-specific gene transcription but are also expressed in other organs, such as pancreas, kidney, stomach, and intestine (27Kuo C.J. Conley P.B. Hsieh C.L. Francke U. Crabtree G.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9838-9842Crossref PubMed Scopus (93) Google Scholar, 28Serfas M.S. Tyner A.L. Am. J. Physiol. 1993; 265: G506-G513PubMed Google Scholar, 29Boudreau F. Zhu Y. Traber P.G. J. Biol. Chem. 2001; 276: 32122-32128Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In this report, we examined the Cdx- and HNF-1α-mediated regulation of the claudin-2 promoter in the human intestinal epithelial cell line Caco-2 and determined the claudin-2 mRNA and protein expression in HNF-1α-deficient mice. These experiments identify claudin-2 as a target of Cdx homeoproteins and HNF-1α function in human intestinal epithelial cells. HNF-1α regulated the complex pattern of claudin-2 expression along the crypt-villus axis of the mouse ileum and was required for claudin-2 expression in the liver. Polyclonal antibodies recognizing claudin-1 and claudin-2 were obtained from Zymed Laboratories, Inc. (San Francisco, CA). Anti-Cdx1 and -Cdx2 polyclonal antibodies were previously described (30Silberg D.G. Furth E.E. Taylor J.K. Schuck T. Chiou T. Traber P.G. Gastroenterology. 1997; 113: 478-486Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 31Silberg D.G. Swain G.P. Suh E.R. Traber P.G. Gastroenterology. 2000; 119: 961-971Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). Antibodies for human HNF-1α and HNF-1β were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-rabbit and anti-goat antibodies were obtained from Amersham Biosciences and Santa Cruz, respectively. Mouse Cdx1 or Cdx2 expression vectors (pRc/CMV-Cdx1 or pRc/CMV-Cdx2) were previously described (15Suh E. Traber P.G. Mol. Cell. Biol. 1996; 16: 619-625Crossref PubMed Scopus (450) Google Scholar, 18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (379) Google Scholar). Human HNF-1α or HNF-1β expression vectors (32Bach I. Yaniv M. EMBO J. 1993; 12: 4229-4242Crossref PubMed Scopus (111) Google Scholar) and mouse HNF-1α expression vector (pBJ5mHNF-1α) (27Kuo C.J. Conley P.B. Hsieh C.L. Francke U. Crabtree G.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9838-9842Crossref PubMed Scopus (93) Google Scholar) were kindly provided by Dr. Marco Pontoglio (Institut Pasteur, Paris, France) and Dr. Gerald R. Crabtree (Stanford University School of Medicine, Stanford, CA), respectively. Cells from the human colon cancer-derived cell line Caco-2, the human hepatocellular carcinoma-derived cell line HepG2, and the mouse mesenchymal cell line NIH3T3 were obtained from the American Type Culture Collection (Manassas, VA). These cells were grown in Dulbecco's modified Eagle's medium (Cellgro; Mediatech Inc., Herndon, VA), supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10% (for HepG2 and NIH3T3) or 20% (for Caco-2) heat-inactivated fetal calf serum (Sigma) in a humidified 5% CO2 atmosphere at 37 °C. The human colon cancer-derived cell line T-84 cells were grown in Dulbecco's modified Eagle's medium with Ham's F-12 medium (1:1) with the antibiotics described above and 10% heat-inactivated fetal calf serum. A degenerate primer approach with primers 5′-TGG ATG GA(A/G) TGT GC(A/T/G/C) AC(A/T/G/C) CA(C/T)-3′ and 5′-GA GCA (G/A)GA (A/G)AA GCA (A/T/G/C)AG (A/G/T/C)AT(G/T/A)AT (A/G/T/C)CC-3′, corresponding to the mouse claudin-2 sequence, was used to amplify 407 bp of the open reading frame of human claudin-2. Data base searches with the putative human claudin-2 sequence identified several human claudin-2 expressed sequence tag clones, which were used to complement the 5′ and 3′ sequence. Additional PCR with the primers 5′-GCT TCT ACT GAG AGG TCT G-3′ and 5′-TTC TTC ACA CAT ACC CTG-3′, and DNA sequencing was utilized to confirm the expression of the full-length human claudin-2 sequence in T-84 cells. DNA and amino acid sequence of human claudin-2 has been submitted to GenBankTM and is available under the accession numberAF250558. The GenomeWalker kit (CLONTECH, Palo Alto, CA) was used to isolate the 5′-flanking region of the human claudin-2 gene. In brief, the first PCR was performed with gene-specific primer 1 (5′-CAA AAG CCC CAG AAG GCC TAG GAT GTA G-3′; +30 to +57 relative to the adenosine of the methionine start codon of the human claudin-2 cDNA; GenBankTM accession numbersAF250558 or AF177340) and adaptor primer 1. The second PCR was done with gene-specific primer 2 (5′-GGC AGA CCT CTC AGT AGA AGC GTC TTC-3′; −27 to −1; corresponding to the 493–519 sequence of AF177340) and adaptor primer 2. The longest PCR fragment was purified and subcloned into pCR2.1 vector (Invitrogen). The resulting plasmid was designated as pCR-hCL2p and sequenced. The KpnI/XhoI fragment of pCR-hCL2p was subcloned into the KpnI/XhoI site of the pGL3B vector (Promega, Madison, WI). Various length fragments of the 5′-flanking region of the human claudin-2 gene were amplified by PCR and subcloned into pGL3B. To obtain the −62 construct, theHindIII/XbaI fragment from the −84 construct was ligated to the EcoRI/HindIII-digested −84 construct with complementary 38-base oligonucleotides (designated as −62wt, from −62 to −31; sense, 5′-AAT TCA TAT TTA ATC TGG TTT ATG GAT TTT TTT TAG GT-3′; antisense, 5′-CTA GAC CTA AAA AAA ATC CAT AAA CCA GAT TAA ATA TG) with 5′-EcoRI and 3′-XbaI overhangs (underlined). To make mutant claudin-2 promoter constructs, mutated 38-base oligonucleotides were substituted for the wild type sequence. For Mut1, Mut2, and Mut1 + 2, 5′-AAT TCA TAT TTA ATC TGG TGG CTG GAT TTT TTT TAG GT-3′, 5′-AAT TCA TAT TTA ATC TGG TTT ATG GAT TTT TTG GCG GT-3′, and 5′-AAT TCA TAT TTA ATC TGG TGG CTG GAT TTT TTG GCG GT-3′ were used, respectively. (Only sense strands are shown; nucleotide substitutions are indicated with bold type.) For reporter assays, a DNA transfection mixture was prepared consisting of 1 μg of the reporter construct and 20 ng of pRL-CMV (Promega) as an internal control. The cells were split onto 6-well plates 18 h before transfection. The cell confluency at transfection was 40–60%. The individual DNA mixtures were transfected with LipofectAMINE Plus (Invitrogen) according to the manufacturer's protocol. For cotransfection experiments, 0.5 μg of the expression vector was transfected along with reporter vectors. pcDNA3.1 vector (Invitrogen) was used to equalize the amount of transfected DNA. The cells were harvested 48 h after transfection, and the luciferase activity was measured using the dual-luciferase reporter assay system (Promega) and a luminometer. Transfection efficiencies were normalized to Renilla luciferase activity of the pRL-CMV vector, and the results are expressed as the mean relative luciferase activity ± S.D. of at least three independent experiments. Nuclear proteins were prepared as previously described (33Awane M. Andres P.G., Li, D.J. Reinecker H.C. J. Immunol. 1999; 162: 5337-5344PubMed Google Scholar). Cytosolic fractions obtained during this procedure were separated for Western blot analysis. The double-stranded oligonucleotides, −62wt, Mut1, Mut2, and Mut1 + 2, were used as probes or cold competitors to analyze the interaction between Cdx protein and DNA. The HNF-1 wild type probe from −67 to −51 of the human claudin-2 gene sequence consisted of complementary 29-nucleotide oligonucleotides (sense, 5′-AAT TCC TGG TCA ATA TTT AAT CTG T-3′, and antisense, 5′-CTA GAC AGA TTA AAT ATT GAC CAG G-3′) with 5′-EcoRI and 3′-XbaI overhangs (underlined). Mutant HNF complementary oligonucleotides were as follows: sense, 5′-AAT TCC TAA TTC AGG TTT AAT CTG T-3′, and antisense, 5′-CTA GAC AGA TTA AAC CTG AAT TAG G-3′ (nucleotide substitutions are indicated with bold type). The probes were labeled with Klenow enzyme by fill-in incorporation with nucleotide triphosphates, including [α-32P]dATP. The binding reaction was performed as previously described (34Traber P.G., Wu, G.D. Wang W. Mol. Cell. Biol. 1992; 12: 3614-3627Crossref PubMed Scopus (114) Google Scholar). For a competition assay, a 100-fold excess of unlabeled oligonucleotide was added to the reaction. To perform supershift assay, the binding mixtures were incubated for 10 min at room temperature in the presence of 1 μl of antibodies. The samples were fractionated on 4% nondenaturing polyacrylamide gel in 0.5× TBE buffer. The resultant DNA-protein complexes were detected by autoradiography. The protein concentration of each sample was quantified by the Bradford method. The samples were electrophoresed through a 4–20% gradient SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The blots were blocked overnight at 4 °C with 10% dry milk in PBS containing 0.1% Tween 20 (PBS-T), followed by the incubation for 3 h at room temperature with primary antibodies diluted in blocking buffer at 1:1000. After washing in PBS-T for 30 min, the blots were incubated with secondary antibodies diluted in blocking buffer for 45 min at room temperature. The hybridized bands were detected by an ECL kit (Amersham Biosciences) according to the manufacturer's instructions. Total RNA was isolated from tissues using Trizol reagent (Invitrogen). Total RNA (30 μg) was electrophoresed in a 1% agarose formaldehyde gel and transferred to a nylon membrane (Magna NT, MicroSeparations Inc., Westbrough, MA) by capillary blotting. The probes were labeled with [α-32P]dCTP using a Rediprime random primer labeling kit (Amersham Biosciences). The membranes were hybridized with radiolabeled probes in Quickhyb solution (Stratagene, La Jolla, CA) at 65 °C for 1 h. The membranes were washed with 0.1% SDS, 2× sodium chloride sodium citrate buffer at room temperature for 15 min and at 65 °C for 10 min. The blots were analyzed by autoradiography. The probes used to detect claudin-1, claudin-2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were described previously (3Kinugasa T. Sakaguchi T., Gu, X. Reinecker H.C. Gastroenterology. 2000; 118: 1001-1011Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Other probes were: Cdx1, a 0.9-kbHindIII/XbaI fragment of pRc/CMV-Cdx1; Cdx2, a 0.9-kb HindIII fragment of pRc/CMV-Cdx2; and HNF-1α, a 0.4-kb SmaI fragment of pBJ5mHNF1α. The HNF-1α probe derives from a unique sequence in HNF-1α cDNA and does not cross-hybridize with HNF-1β. Northern blots were and gene-specific mRNA expression were normalized to mRNA expression in the samples and expressed as the mean from three independent experiments. the HNF-1α were obtained from Dr. J. of Y.H. F.J. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar). HNF-1α and wild type were obtained by of the experiments were performed in with of and by the on at The liver and were and washed with of 2 from the and the ileum were For small were in and in dry For RNA small of tissues were at °C. of tissues were The were and in at °C for 10 min followed by in PBS at 4 °C for 30 min as previously described C. Mitic L.L. Anderson J.M. Gastroenterology. 2001; 120: 411-422Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar). The were blocked with 5% serum in PBS for 1 h at 20 °C and incubated with primary antibodies or serum diluted at with blocking solution for 3 h at room temperature. After three with the were incubated at room temperature with anti-rabbit Laboratories, CA) diluted at with blocking solution for 1 h in the and analyzed with an The 5′-flanking region of the claudin-2 gene isolated from the human and T-84 claudin-2 cDNA sequence were by sequence with the human A that the gene human claudin-2 is on to The claudin-2 mRNA expressed in T-84 cells contains an open reading frame of Human and mouse claudin-2 have a sequence of on the mRNA and on the amino acid with the mouse claudin-2 promoter in that the promoters of the human and mouse claudin-2 genes a homology of for the region of to The mouse claudin-2 cDNA accession by the putative transcriptional start site at bp of the start The transcriptional site is within a Y. T. J. H. J. H. T. T. T. Y. A. Y. S. K. S. 2001; PubMed Scopus Google Scholar, 1997; PubMed Scopus Google Scholar). The promoters of the human and mouse claudin-2 genes have the putative transcriptional site However, a is at to and two are at to bp and −67 to −62 bp that regulatory to gene transcription are The human claudin-2 promoter contains two sites for the intestine-specific homeodomain protein family Cdx (18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (379) Google Scholar), designated and The promoter has also binding sites for HNF-1 and S. J. 1996; PubMed Scopus Google Scholar), as as putative C. G. M. Mol. Cell. Biol. PubMed Scopus Google Scholar), Cell. 1990; Full Text PDF PubMed Scopus Google Scholar), and 1992; PubMed Google Scholar) the first site and the and sites are conserved in human and mouse claudin-2 promoters To identify the involved in regulating claudin-2 gene 5′-flanking and to were into the reporter plasmid pGL3B. were transfected into intestinal epithelial cell line Caco-2, cell line HepG2, or cell line In Caco-2 cells the claudin-2 promoter fragments containing to −62 bp of the 5′-flanking region a in relative luciferase activity above that after transfection with the control reporter construct In the promoter only a transfected into HepG2 and NIH3T3 cells of the putative site and the site the promoter activity of the site in the construct not the promoter activity in Caco-2 cells. However, of the sites in a of promoter activity in Caco-2, HepG2, and NIH3T3 cells To the function of the two Cdx were into or + shown in in the or site the promoter activity to 30 and of that with −62 wild type construct, respectively. sites were mutated + promoter activity was to of the wild type construct, with the construct Cdx we determined the ability of Cdx1 and Cdx2 to the claudin-2 promoter. shown in Cdx2 but not Cdx1 protein was in nuclear proteins from Caco-2 cells. expression with Cdx1 or Cdx2 or in in the expression of these proteins in the of Caco-2 cells 48 h after transfection expression of Cdx1 not the expression of Cdx2, Cdx2 Cdx1 protein expression in the shown in Cdx1 in a of the promoter activity by the −62 construct containing Cdx sites in Caco-2 cells relative to the activity of pGL3B In Cdx2 the activity of the construct to of pGL3B vector Overexpression of Cdx1 and Cdx2 not the activity above the by Cdx2 of the or site the ability to to Cdx2 promoter by Cdx2 were and of that in −62 construct, respectively. Mut1 and were to Cdx1 In the of Cdx sites Cdx1 Cdx2 a significant of reporter gene transcription in Caco-2 cells The ability of Cdx2 to a of claudin-2 promoter activity in with Cdx1 was for Caco-2 cells. demonstrated in Cdx1 and Cdx2 claudin-2 promoter activity in and Cdx1 a and Cdx2 a promoter activity in Caco-2 cells. To the interaction between Cdx2 and the two Cdx and were performed with nuclear proteins from Caco-2 cells. These experiments were in Caco-2 cells was shown that complexes can be by binding of in nuclear proteins from Caco-2 cells R. Santiago N.A. Olds L.C. Sibley E. Gastroenterology. 2000; 118: 115-127Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). shown in three DNA-protein complexes and were binding were with radiolabeled wild type oligonucleotide containing Cdx sites oligonucleotide with an but a mutated with the of three unlabeled Mut1 oligonucleotide with a mutated but an only the of complex 3 and In supershift assays, only complex A to two distinct complexes In not the mobility of the complexes The complex A was also with radiolabeled oligonucleotide used as a probe that this complex is with not form on the Cdx sites this complex was detected and by three mutated oligonucleotides and in the site the of complex the that of complex is on this site and the gene promoter two Cdx sites may be to the of Cdx2 homodimers (18Suh E. Chen L. Taylor J. Traber P.G. Mol. Cell. Biol. 1994; 14: 7340-7351Crossref PubMed Scopus (379) Google Scholar).