Decreased Methylation and Transcription Repressor Sp3 Up-regulated Human Monoamine Oxidase (MAO) B Expression during Caco-2 Differentiation
2003; Elsevier BV; Volume: 278; Issue: 38 Linguagem: Inglês
10.1074/jbc.m305549200
ISSN1083-351X
AutoresWai Keong Wong, Kai Chen, Jean C. Shih,
Tópico(s)Pancreatic function and diabetes
ResumoMonoamine oxidase (MAO) A and B catalyze the oxidative deamination of neuroactive and dietary monoamines such as serotonin, tyramine, and phenylethylamine. Here we show that MAO B, but not MAO A, gene expression was induced during Caco-2 cell differentiation; thus this cell line was used as a model system to study the gene regulation unique for MAO B. Luciferase and gel shift assays showed that transcription factors Sp1 and Sp3 binding to -246 and -99 bp were responsible for the observed gene activation. Overexpression of Sp3 inhibited the induction of MAO B gene by Sp1, and the expression of Sp3 was decreased during Caco-2 cell differentiation. Computer analysis revealed a putative CpG island containing 22 potential CpG methylation sites between -261 and -58 bp. In vitro methylation of MAO B promoter with 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, up-regulated MAO B gene expression in both HeLa and Caco-2 cells. Sodium bisulfite sequencing showed a gradually reduced methylation of the CpG sites during Caco-2 cell differentiation. These results suggested that MAO B gene expression is selectively induced by a decreased Sp3/Sp1 ratio and reduced DNA methylation. This new information may provide insights on the tissue-specific expression of these two isoenzymes. Monoamine oxidase (MAO) A and B catalyze the oxidative deamination of neuroactive and dietary monoamines such as serotonin, tyramine, and phenylethylamine. Here we show that MAO B, but not MAO A, gene expression was induced during Caco-2 cell differentiation; thus this cell line was used as a model system to study the gene regulation unique for MAO B. Luciferase and gel shift assays showed that transcription factors Sp1 and Sp3 binding to -246 and -99 bp were responsible for the observed gene activation. Overexpression of Sp3 inhibited the induction of MAO B gene by Sp1, and the expression of Sp3 was decreased during Caco-2 cell differentiation. Computer analysis revealed a putative CpG island containing 22 potential CpG methylation sites between -261 and -58 bp. In vitro methylation of MAO B promoter with 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, up-regulated MAO B gene expression in both HeLa and Caco-2 cells. Sodium bisulfite sequencing showed a gradually reduced methylation of the CpG sites during Caco-2 cell differentiation. These results suggested that MAO B gene expression is selectively induced by a decreased Sp3/Sp1 ratio and reduced DNA methylation. This new information may provide insights on the tissue-specific expression of these two isoenzymes. Monoamine oxidase (MAO 1The abbreviations used are: MAO, monoamine oxidase; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; PC, pre-confluent.1The abbreviations used are: MAO, monoamine oxidase; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; PC, pre-confluent.; amine:oxygen oxidoreductase (deaminating, flavin-containing), EC 1.4.3.4) catalyzes the oxidative deamination of neuroactive, vasoactive, and dietary amines such as serotonin, norepinephrine, dopamine, tyramine, and phenylethylamine with the production of H2O2 (for review see Refs. 1Shih J.C. Chen K. Ridd M.J. Annu. Rev. Neurosci. 1999; 22: 197-217Crossref PubMed Scopus (996) Google Scholar and 2Shih J.C. Thompson R.F. Am. J. Hum. Genet. 1999; 65: 593-598Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). MAO is present in two isoforms, MAO A and MAO B, which are encoded by two closely linked genes on the X chromosome (3Lan N.C. Heinzmann C. Gal A. Klisak I. Orth U. Lai E. Grimsby J. Sparkes R.S. Mohandas T. Shih J.C. Genomics. 1989; 4: 552-559Crossref PubMed Scopus (175) Google Scholar, 4Grimsby J. Chen K. Wang L.J. Lan N. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3637-3641Crossref PubMed Scopus (231) Google Scholar) and share 70% amino acid sequence identity (5Bach A.W.J. Lan N.C. Johnson D.L. Abell C.W. Bemkenek M.E. Kwan S.-W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (690) Google Scholar). These two isoenzymes have distinct substrate specificity and inhibitor sensitivity. MAO A preferentially oxidizes serotonin and norepinephrine and is inhibited by low concentrations of clorgyline (6Johnston J.P. Biochem. Pharmacol. 1968; 17: 1285-1297Crossref PubMed Scopus (1429) Google Scholar). 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Karoum F. Gal J. Shih J.C. Nat. Genet. 1997; 17: 206-210Crossref PubMed Scopus (217) Google Scholar). The human brain MAOs have been implicated in a number of psychiatric and neurological disorders and targeted for drugs against depression and Parkinson's disease (19Saura J. Luque J.M. Cesura A.M. Da Prada M. Chan-Palay V. Huber G. Loffler J. Richards J.G. Neuroscience. 1994; 62: 15-30Crossref PubMed Scopus (307) Google Scholar, 20Oreland L. Monoamine Oxidase: Basic and Clinical Aspects. VSP Press, Utrecht1993: 219-247Google Scholar). MAO regulates the levels of serotonin in both the central and enteric nervous systems. It has been shown that serotonin plays important roles in the gut (21Gershon M.D. Aliment Pharmacol. Ther. 1999; 13: 15-30Crossref PubMed Google Scholar, 22Gershon M.D. Am. J. Physiol. 1998; 275: G869-G873PubMed Google Scholar) and is implicated for the management of irritable bowel syndrome (23Gershon M.D. Rev. Gastroenterol. Disord. 2003; 3: S25-S34PubMed Google Scholar). However, the mechanisms that govern the regulation of these two genes remain largely unknown. MAO A and B are co-expressed in most human tissues and are most abundant in the intestine (24Grimsby J. Lan N.C. Neve R. Chen K. Shih J.C. J. Neurochem. 1990; 55: 1166-1169Crossref PubMed Scopus (141) Google Scholar). However, fibroblasts and placenta express only MAO A (25Weyler W. Salach J.I. J. Biol. Chem. 1985; 260: 13199-13207Abstract Full Text PDF PubMed Google Scholar, 26Egashira T. Jpn. J. Pharmacol. 1976; 26: 493-500Crossref PubMed Scopus (46) Google Scholar). In contrast, platelets and lymphocytes express only MAO B (27Bond P.A. Cundall R.L. Clin. Chim. Acta. 1977; 80: 317-326Crossref PubMed Scopus (68) Google Scholar). In the human brain MAO A is found in catecholaminergic neurons; surprisingly MAO B, instead of MAO A, is in serotonergic neurons and astrocytes (28Westlund K.N. Denney R.M. Rose R.M. Abell C.W. Science. 1985; 230: 181-183Crossref PubMed Scopus (412) Google Scholar, 29Levitt P. Pintar J.E. Breakefield X.O. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6385-6389Crossref PubMed Scopus (459) Google Scholar). To understand the tissue-specific expression of MAO A and B, it is important to study the regulation of their gene expression. We have previously characterized the promoter regions of MAO A and MAO B genes and shown that they have a different promoter organization. The MAO A core promoter consists of three binding sites for the transcription factor Sp1 in reversed orientations and lacks a TATA box. In contrast, the MAO B core promoter contains a TATA box and two clusters of Sp1 binding sites separated by a CACCC box (30Zhu Q.S. Grimsby J. Chen K. Shih J.C. J. Neurosci. 1992; 12: 4437-4446Crossref PubMed Google Scholar, 31Zhu Q.S. Chen K. Shih J.C. J. Neurosci. 1994; 12: 7393-7403Crossref Google Scholar). Recently, we have shown that both transcription factors Sp1 and Sp3 interact with the two clusters of Sp1 binding sites within the MAO B core promoter. In addition, overexpression of Sp1 transactivates promoter activity through the proximal cluster of Sp1 binding sites (32Wong W.K. Chen K. Shih J.C. Mol. Pharmacol. 2001; 59: 852-859Crossref PubMed Scopus (43) Google Scholar). The promoter activation by Sp1 could be inhibited by overexpression of Sp3. Furthermore, we have demonstrated that MAO B, but not MAO A, gene expression is induced by the activation of protein kinase C and mitogenactivated protein kinase signaling pathway involving the transcription factors Sp1, Sp3, c-Jun, and Egr-1 (33Wong W.K. Ou X.M. Chen K. Shih J.C. J. Biol. Chem. 2002; 277: 22222-22230Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The mechanism underlying the gene regulation of MAO A and MAO B appears to be distinct and complex. This has led us to further investigate the transcriptional regulation of these genes. In the present study, we have found that the Caco-2 cell line (human colon adenocarcinoma) expressed both MAO A and MAO B. However, the expression of MAO B, but not MAO A, was progressively increased when the cells were undergoing differentiation. The Caco-2 cells undergo cell cycle arrest and differentiation after reaching confluence (12Geha R.M. Chen K. Shih J.C. J. Neurochem. 2000; 75: 1304-1309Crossref PubMed Scopus (38) Google Scholar, 13Zhou B.P. Wu B. Kwan S.W. Abell C.W. J. Biol. Chem. 1998; 273: 14862-14868Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Promoter deletion and reporter gene expression studies have identified the MAO B core promoter region (-246/-99 bp) as crucial for the induced gene expression during Caco-2 cell differentiation. In addition, the transcriptional repressor Sp3 for MAO B promoter was significantly down-regulated during the activation of MAO B gene expression. Within this promoter region, we have identified a 204-bp CpG island and 22 potential CpG methylation sites, suggesting that cytosine methylation plays a role in the transcriptional activation of MAO B gene. Indeed, sodium bisulfite sequencing showed a progressively decreased methylation status of the CpG sites in MAO B core promoter during Caco-2 cell differentiation. Northern blot analysis showed that treatment with 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor, up-regulated MAO B transcript levels in undifferentiated Caco-2 cells and HeLa cells. These results demonstrated that the up-regulation of MAO B gene expression is modulated by down-regulation of transcriptional repressor Sp3 and modifications of promoter methylation during Caco-2 cell differentiation. This is the first study demonstrating the regulation of MAO B gene expression by Sp3 level and DNA methylation downstream of signaling pathways. Cell lines and Reagents—The Caco-2 (human colonic adenocarcinoma) and HeLa (human cervical adenocarcinoma) cell lines were purchased from the American Type Culture Collection (ATCC) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 mm HEPES, 2 mml-glutamine, 100 units/ml penicillin, 10 μg/ml streptomycin, and 10% fetal bovine serum. Sodium bisulfite, hydroquinone, and 5-aza-2′-deoxycytidine were obtained from Sigma (St. Louis, MO). Polyclonal antisera against Sp1 and Sp3 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Human MAO B Promoter-luciferase Reporter Constructs—The BamHI/BamHI MAO B promoter fragment (-2099/-99 bp) was cloned into the polylinker site (BglII) upstream of the luciferase gene (LUC) in the pGL2-Basic vector (Promega, Madison, WI). Serial deletion constructs were generated by restriction enzyme digestion using the -2099/-99LUC as a template followed by Klenow fill-in and selfligation. The following restriction enzymes were used to generate the deletion constructs: XhoI/AspI (pGLB-1313); XhoI/BglII (pGLB-1180); XhoI/SpeI (pGLB-868); XhoI/ApaI (pGLB-425); XhoI/PstI (pGLB-246). MAO Catalytic Activity Assay—One hundred micrograms of total proteins were incubated with 100 μm14C-labeled serotonin (5-hy-droxytrytamine) (for MAO A) or 10 μm14C-labeled PEA (for MAO B) (Amersham Biosciences) in the assay buffer (50 mm sodium phosphate buffer, pH 7.4) at 37 °C for 20 min and terminated by addition of 100 μl of 6 n HCl. The reaction products were then extracted with benzene/ethyl acetate (1:1) (for MAO A) or water-saturated ethyl acetate/toluene (1:1) (for MAO B) and centrifuged at 4 °C for 10 min. The organic phase containing the reaction product was extracted, and its radioactivity was obtained by liquid scintillation spectroscopy. Northern Blot Analysis—Total RNA were purified using TRIzol reagents (Invitrogen). Thirty micrograms of total RNA was loaded onto each gel lane. Electrophoresis, transfer onto BrightStar nylon membrane, and hybridization were carried out using NorthernMax according to the manufacturer's protocol (Ambion). The human MAO A and MAO B cDNA probes and an internal control probe encoding human β-actin were labeled by random-priming technique using the Multiprime kit (Amersham Biosciences) following the manufacturer's instructions. Membrane hybridized with the MAO A or MAO B probe was autoradiographed for 72 h. When the β-actin control probe was used, membrane was autoradiographed for 1 h. Western Blot Analysis—Cells were harvested and washed with phosphate-buffered saline. The protein concentration was determined by the Bradford protein assay (Bio-Rad). For MAO A and B detection, 100 μg of total proteins was separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After the transfer, membranes were blocked at room temperature for 2 h with 5% bovine serum albumin in TTBS (10 mm Tris/HCl, pH 7.5, 150 mm NaCl, and 0.05% Tween 20). The membranes were then incubated with anti-MAO A or anti-MAO B antibodies (1:1000) in 0.5% bovine serum albumin in TTBS overnight at room temperature. After incubation with the secondary antibody at room temperature for 2 h, the bands were visualized by horseradish peroxidase reaction DAB (Sigma). Nuclear Extraction and Electrophoretic Mobility Gel Shift Assay— Cells were washed with cold phosphate-buffered saline, harvested by scraping, and pelleted. Cells were resuspended in a 5-pellet volume of buffer A (10 mm KCl, 20 mm HEPES, 1 mm MgCl2, 0.5 mm DTT, and 0.5 mm phenylmethylsulfonyl), incubated on ice for 10 min, and centrifuged for 10 min. Pellets were then resuspended in a 3-pellet volume of buffer A plus 0.1% Nonidet P-40, incubated on ice for 10 min, and centrifuged for 10 min. The pellets were resuspended in buffer B (10 mm HEPES, 400 mm NaCl, 0.1 mm EDTA, 1 mm MgCl2, 1 mm DTT, 0.5 mm phenylmethylsulfonyl fluoride, and 15% glycerol) and incubated on ice for 30 min with gentle shaking. Nuclear proteins were then centrifuged for 30 min and dialyzed for 4 h at 4 °C against 1 liter of buffer D (20 mm HEPES, 200 mm KCl, 1 mm MgCl2, 0.1 mm EDTA, 1 mm DTT, 0.5 mm phenylmethylsulfonyl fluoride, and 15% glycerol). Protein extracts were cleared by centrifugation at 4 °C for 15 min. Protein concentrations were determined by using a Bio-Rad protein assay. MAO B promoter DNA fragment (-246/-99 bp) was radiolabeled by Klenow fill-in and purified by gel electrophoresis (5% polyacrylamide) and eluted in TE. For DNA-protein binding, 5 μg of nuclear extracts were diluted in binding buffer (40 mm Hepes (pH 8.0), 50 mm KCl, 1 mm dithiothreitol, 0.1 mm EDTA, 10% glycerol, 10 μg/ml of poly(dI-dC) (Sigma)) with a total volume of 20 μl. Antibodies against Sp1 or Sp3 were added (when required), and the mixture was incubated for 20 min at room temperature. Labeled probes (0.2 ng) were added to the mixture and incubated for additional 20 min at room temperature. The samples were then run on a 5% nondenaturing polyacrylamide gel in 1× Tris borate/EDTA at 150 V for 3 h. Gels were dried and visualized by autoradiography. Sodium Bisulfite Genomic Sequencing—10-μg aliquots of EcoRI-digested genomic DNA from cells were denatured in 0.3 m NaOH at 37 °C for 15 min in a total volume of 50 μl. Sulfonation and hydrolytic deamination reactions were then carried out by adding 450 μl of 2.5 m sodium bisulfite (Na2S2O5)/10 mm hydroquinone solution (pH 5.0) to the digested genomic DNA and incubating at 50 °C for 4 h in the dark. Bisulfite-converted DNA was purified using a QIAquick spin purification kit (Qiagen) and eluted with TE according to the manufacturer's protocol. Desulfonation reactions were then performed in 0.3 m NaOH at 37 °C for 15 min, and DNA was precipitated with 3 m sodium acetate/ethanol and resuspended in 100 μl of water. PCR amplification was performed in 50 μl of reaction mixture containing 5 μl of bisulfite-treated genomic DNA, 0.5 μm primers, 100 μm dNTPs, 2 mm MgCl2, 50 mm KCl, 10 mm Tris-HCl (pH 8.3), and 2.5 units of Taq polymerase (Invitrogen). The PCR conditions were: 97 °C for 4 min (1 cycle) and 95 °C for 1 min, 56 °C for 1 min, 72 °C for 2 min (30 cycles). The following primers were used: 5′-GCCTTCCTGACTTAATCAC-3′ (MAO B, forward -752) and 5′-CCTCGATCCCAGTCCTGCC-3′ (MAO B, reverse -55). Amplified DNA was subcloned into vector pCR2.1 using a TOPO-TA cloning kit (Invitrogen) and sequenced. Transient Transfection and Luciferase Assay—Transfections were performed using SuperFect transfection reagent (Qiagen) following the manufacturer's instructions. Exponentially growing cells were plated at a density of 4 × 105 cells/well in 6-well plates (Costar, Cambridge, MA) in 2 ml of DMEM, 10% fetal bovine serum and grown until 60% confluent (24–36 h). Plasmids were mixed with 100 μl of serum-free and antibiotic-free medium, and 10 μl of SuperFect reagent. Following a 15-min incubation at room temperature, 600 μl of DMEM (10% fetal bovine serum and antibiotics) were added to the DNA-SuperFect complexes. The cells were washed once with phosphate-buffered saline and incubated with DNA-SuperFect complexes. After 2-h incubation, the cells were washed with phosphate-buffered saline and incubated with DMEM (10% fetal bovine serum and antibiotics). Cells were harvested 48 h later with Luciferase Assay lysis buffer (Promega). The cell lysates were then assayed for luciferase using the Promega dual luciferase assay system following the manufacturer's instructions. For DNA methylation studies, promoter fragments for MAO B(-246/-99 bp) were digested with PstI and HindIII, gel-isolated, and then in vitro methylated with SssI methylase (New England Biolabs) in the presence of 160 μm S-adenosylmethionine according to manufacturer's protocol. The procedure for mock methylation is identical to the one for DNA methylation except no methylase was included. Complete methylation of the fragments was confirmed by the HpaII and HhaI methylation-sensitive restriction enzyme digestion. The luciferase vector was digested with PstI and HindIII and ligated with an equimolar concentration of methylated or unmethylated promoter fragments. One microgram of methylated or unmethylated MAO B promoter-reporter constructs was co-transfected into the cells with 20 ng of pRL-TK (internal control, Promega). For the co-transfection experiments, the Sp1 expression plasmid (pCMV-Sp1) was kindly provided by Dr. Robert Tjian, and the Sp3 expression plasmid (pCMV-Sp3) was a generous gift from Dr. Guntram Suske. The total amount of DNA for each transfection was kept constant by the addition of the empty expression vector pCMV. CpG Analysis—The presence of a CpG island was analyzed with the EMBOSS program CpGplot (available at www.hgmp.mrc.ac.uk/Software/EMBOSS/Apps/cpgplot.html). This program detects regions of genomic sequences that are rich in the CpG pattern known as CpG islands. The program defines a CpG island as a region where the moving average of percentage of G + C nucleotides is greater than 50 and the moving average of observed/expected CpG is greater than 0.6 within a minimum of 200 bases. Statistical Analysis—All values were presented as means ± S.E. Student t test was used for statistical analysis, and differences were considered significant when p < 0.05. Increased MAO B but Not MAO A Gene Expression during Caco-2 Cell Differentiation—To determine the expression of MAO A and MAO B during Caco-2 cell differentiation, we cultured the Caco-2 cells at various growth stages (pre-confluence; confluence, day 0; post-confluence, days 2, 7, and 14). Radiochemical and enzymatic assays using enzyme-specific 14C-labeled substrates serotonin (5-hydroxytrytamine) and phenylethylamine for MAO A and MAO B, respectively, indicated that both MAO A and MAO B were expressed as early as 2 days before cells reached confluence (Fig. 1A). Interestingly, MAO B enzymatic activity increased when the cells reached confluence (day 0) and continued to increase progressively after confluence during cellular differentiation (days 7 and 14). In contrast, the enzymatic activity of MAO A remained relatively unchanged during the course of differentiation. These enzymatic activities were specific to MAO A and MAO B as indicated by the selective inhibition of these enzymes by clorgyline and deprenyl, respectively (data not shown). Northern and Western blot analyses revealed that the specific increase of MAO B enzymatic activity correlated with the increased level of MAO B mRNA transcript and protein (Fig. 1, B and C) at confluent and post-confluent states. In contrast, the levels of MAO A transcript and protein remained relatively unchanged. The major MAO A transcripts of 5 and 2 kb, and MAO B transcript of 3 kb were detected with their specific probes as early as 2 days before confluence, indicating that both MAO genes were constitutively expressed in the proliferating Caco-2 cells. The induced level of MAO B mRNA transcript was followed by the increased level of MAO B protein. These data suggested that MAO B, but not MAO A, gene expression was selectively induced during Caco-2 cell differentiation. Identification of Essential Regulatory Region for MAO B Gene Activation—Promoter deletion and reporter gene assay were performed to map the regulatory region responsible for the increased MAO B gene expression during Caco-2 cell differentiation. The 5′-flanking regulatory sequence of MAO B gene was gradually truncated to generate a series of promoter fragments. The deleted promoter fragments were then ligated to the upstream sequence of firefly luciferase reporter gene (pGL2-Basic) and transiently transfected into pre-confluent (2 days before confluence) or differentiating (days 7 and 14) Caco-2 cells. The MAO B promoter activity was determined by the relative expression level of firefly luciferase reporter gene using the expression level of co-transfected Renilla firefly luciferase reporter gene as an internal control to normalize the transfection efficiency. As shown in Fig. 2, increased MAO B promoter activity was found to correlated with the states of differentiation except the promoter construct pB-2099/-246 that lacked the core promoter. This promoter construct had similar activity to that of the control promoter-less vector (pGL2-Basic), indicating that the promoter region between -246 and -99 was essential for the induced MAO B promoter activity during Caco-2 cell differentiation. (A similar correlation between MAO B promoter activity and level of gene expression was also found in HeLa (human cervical adenocarcinoma), 1242-MG (human glioma), and HepG2 (human hepatocarcinoma) cell lines (data not shown). These data indicated that Caco-2 cell is a suitable model system to study the MAO B gene regulation.) Interestingly, the pattern of progressively increasing promoter activity was increased progressively from pre-confluence to days 7 and 14, similar to increasing levels of MAO B catalytic activity in RNA and protein levels (Fig. 1). This result indicates that MAO B promoter activity was correlated with the differentiation states of Caco-2 cells, and the regulatory region between -246 and -99 was essential for the MAO B gene up-regulation. Characterization of Nuclear Factors Binding to MAO B Core Promoter during Differentiation—EMSA, coupled with supershift assays, was performed to characterize the transcription factors that interact with the MAO B core promoter region. The nuclear proteins from pre-confluent (PC), confluent (day 0), and post-confluent (days 7 and 14) were extracted and incubated with 32P-labeled MAO B promoter fragment between -246 and -99 bp. Five shifted bands (I, II, III, IV, and V) were observed. Interestingly, the intensity of complexes I and III gradually decreased (lanes 1–4) and disappeared in day 14 (lane 4, Fig. 3A). To further characterize the complexes, we performed a supershift assay by using antibodies raised against transcription factors Sp1 and Sp3 proteins, and nuclear proteins from pre-confluent cells. The complex II was supershifted by the addition of Sp1 antibody (lane 2), whereas the complexes I and III were supershifted by the addition of Sp3 antibody (lane 3) (Fig. 3B). Addition of both Sp1 and Sp3 antibodies supershifted complexes I–III. The gradually decreased intensity of complexes I and III suggested that the expression of Sp3 was reduced during Caco-2 cell differentiation. Western blot analysis was performed to determine the expression levels of Sp1 and Sp3 in Caco-2 cells. The nuclear proteins from pre-confluent (PC), confluent (day 0), and post-confluent (days 7 and 14) Caco-2 cells were extracted and analyzed. As shown in Fig. 3C, although the expression level of Sp1 decreased when cells reach confluence, its expression level appeared relatively the same during differentiation from day 0 to day 14. In contrast, the expression level of Sp3 gradually decreased throughout the course of differentiation. This decreased expression level of Sp3 protein may explain the reduced intensity of complexes I and III in EMSA. Functional Analysis of Sp1 and Sp3 on MAO B Promoter Activity in Caco-2 Cells—The decreased expression of transcription factor Sp3 correlated inversely with the increased MAO B gene expression during Caco-2 cell differentiation. The MAO B core promoter consisted of two clusters of overlapping binding sites for transcription factors Sp1 or Sp3. To determine the functional roles of Sp1 and Sp3 in the up-regulation of MAO B gene expression in Caco-2 cells, the core promoter-luciferase reporter gene construct (pB-246/-99) was transiently cotransfected with increasing amounts of cDNA plasmids expressing the human Sp1 or Sp3 in pre-confluent Caco-2 cells. Overexpression of Sp1 enhanced MAO B promoter activity in a dose-dependent manner (Fig. 4). In contrast, overexpression of Sp3 resulted in a slight decrease of promoter activity. Because the binding sites were overlapped, competition between Sp1 and Sp3 for binding to these sites may influence the overall MAO B promoter activity. To determine whether an interplay exists between Sp1 and Sp3 on promoter activity, transient co-transfection experiments were performed using a constant amount of Sp1 expression plasmid along with gradually increasing amounts of Sp3 expression plasmids. As expected, expression of Sp1 enhanced MAO B promoter activity. However, the Sp1-mediated promoter activation was gradually reduced by increasing amounts of Sp3 expression (Fig. 4). The result of Sp3 suppression of MAO B promoter was found in other cell lines such as 1243-MG (human glioma), HepG2 (human hepatocytoma), and HeLa (human cervical adenocarcinoma) (32Wong W.K. Chen K. Shih J.C. Mol. Pharmacol. 2001; 59: 852-859Crossref PubMed Scopus (43) Google Scholar). These findings suggested that variation in the ratio of Sp1 and Sp3 expression might influence the regulation of MAO B gene expression. Reduced Promoter Methylation during Caco-2 Cell Differentiation—MAO B promoter region between -246 and -99 bp was GC-rich. We analyzed the CpG dinucleotide distribution and found a potential CpG island within the middle of this region using the program CpGPlot (Fig. 5A). Twenty-two CpG sites were located in this promoter region. The effect of in vitro methylation on the promoter activity of MAO B gene was tested in a transient expression assay using the pB-246/-99 promoter-reporter gene construct. After methylation with SssI methylase, which methylated cytosine residues of CpG dinucleotides, the methylated and unmethylated reporter constructs were transiently transfected into pre- and post-confluent Caco-2 cells and assayed for promoter activity. As shown in Fig. 5B, in vitro methylation almost completely suppressed MAO B promoter activity compared
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