Role of USF1 and USF2 as potential repressor proteins for human intestinal monocarboxylate transporter 1 promoter
Christos Hadjiagapiou,*
Alip Borthakur,*
Refka Y. Dahdal,
Ravinder K. Gill,
Jaleh Malakooti,
Krishnamurthy Ramaswamy, and
Pradeep K. Dudeja
Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois
Submitted 15 July 2004
; accepted in final form 26 January 2005
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ABSTRACT
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Butyrate, a short-chain fatty acid, is the major energy fuel for the colonocytes. We have previously reported that monocarboxylate transporter isoform 1 (MCT1) mediates uptake of butyrate by human colonic Caco-2 cells. To better understand the mechanisms of MCT1 expression and regulation in the human intestine, we examined the activity and regulation of MCT1 promoter in Caco-2 cells. The transcription initiation site in the MCT1 promoter was identified as a guanine nucleotide 281 bp upstream from the translation initiation site and is surrounded by a guanine-cytosine-rich area. The promoter was found to be highly active when transfected into Caco-2 cells, and its activity decreased with deletions at its 5'-end. Gel mobility shift experiments showed binding of the transcription factors upstream stimulatory factor (USF)1 and 2 to the site 114 to 119 of the MCT1 promoter. With the use of site-directed mutagenesis and promoter activity in Caco-2 cells, the USF proteins appeared to have a repressor role on the MCT1 promoter, which was further confirmed by cotransfecting expression vectors encoding USF1 and 2 in Caco-2 cells and determining endogenous MCT1 expression in USF2 overexpressed cells. The two potential SP1 binding sites found in the same region of the promoter were found not to be involved in its regulation.
short-chain fatty acids; transcriptional regulation; cis elements; mutagenesis; upstream stimulatory factors
THE MONOCARBOXYLATE TRANSPORTER ISOFORM 1 (MCT1) belongs to the family of monocarboxylate transporters comprising 14 MCT-related sequences that have been identified in mammals, each having a different tissue distribution and a broad spectrum of specificity (19). MCT activity has been found to be coupled with protons (19, 28, 30). MCTs are involved in the maintenance of internal pH in cells such as tumor cells, white skeletal muscle cells, and erythrocytes that derive ATP via glycolysis and need to remove lactate. In other mammalian tissues, the transport of monocarboxylic acids into cells is important for glycogenesis or respiratory metabolism (28). Recently, we reported that the MCT1 plays a key role in the uptake of butyrate by human colonic Caco-2 cells (18). Also, this transporter was shown to be responsible for the uptake of butyrate by Xenopus oocytes injected with the MCT1 cRNA as well as the transport of L-lactate in human and pig colonic luminal membrane vesicles (32).
The molecular identity of 14 monocarboxylate transporters (MCT114) has been reported (19). MCT1 was the first one cloned from Chinese hamster ovary cells during investigations of the mevalonate transporter (13). The MCT1 transporter differed from the mevalonate transporter by one amino acid and transported monocarboxylic acids such as, lactate and pyruvate, but not mevalonate. In addition, the MCT1 cDNA has been cloned from rat small intestine and skeletal muscle as well as from human heart (14, 23, 38). This integral membrane protein is predicted to have 494 amino acids and a molecular mass of 53 kDa. Hydropathy analysis of the predicted amino acid sequence from rat MCT1 cDNA suggests a structure of 12 transmembrane helixes with both the amino and carboxyl termini oriented intracellularly (29). The human MCT1 gene was localized to the chromosomal band 1p13.2-p12 (14). Although the regulation of the MCT1 has been studied in skeletal muscle, little is known about its regulation in the intestine. Recently, regulation of MCT1 by luminal leptin in Caco-2 bbe cells has been shown to involve increase in the intracellular pool of MCT1 protein (4). On the other hand, thyroid-stimulating hormone regulation of MCT1 in rat thyroid cell line FRTL-5 has been shown to be due to increased transcription, because both MCT1 mRNA and protein levels were elevated concordantly (12). Limited information on the transcriptional regulation of MCT1 in various tissues is due to the lack of information on its promoter. We recently cloned the human MCT1 promoter from Caco-2 cells (17) in parallel with another report of its cloning from AA/C2 cells (9), wherein the promoter has been shown to lack the TATA and CCAAT boxes and to have a long GC-rich area at its 3'-end.
In the current study, we report that the MCT1 promoter is very active when transfected into Caco-2 cells. Furthermore, we provide evidence that the transcription factors upstream stimulatory factors (USF)1 and 2 may act as negative regulatory factors when bound to a site located toward the 3'-end of the promoter.
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MATERIALS AND METHODS
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Cloning of MCT1 promoter sequence.
The MCT1 promoter region was cloned using the GenomeWalker DNA system (Clontech, CA) and the PCR method. Primers were designed from the Human genome sequence upstream from the 5'-untranslated region of MCT1 cDNA. The promoter region was obtained as two PCR fragments that were joined together by overlapping PCR. The amplification conditions for fragment 1 were heating at 94°C for 2 min, followed by 7 cycles at 94°C for 30 s and at 72°C for 4 min, 28 cycles at 94°C for 30 s, and at 67°C for 4 min. Primers used for this amplification were CH100 and AP1 for first-round PCR and CH101 and AP2 for nested PCR. Fragment 2 was obtained by denaturation at 96°C for one cycle of 3 min and 28 cycles of amplification at 95°C for 30 s, 67°C for 30 s, and 72°C for 1 min. Primers used to isolate this fragment were GI519 and GI520. The resulted fragments were cloned into pGEM-T vector and sequenced. The primers used for the overlap PCR were CH519 and CH101, and the conditions were the same as those used for amplification of fragment 2.
The oligonucleotides used were as follows: GI519, 5'-TACCCACGCAGCTAGCCAG TCACGTCGC -3'(119*, R); GI520, 5'-TGGTAGCTCCGCGCCGCTCAGCAG-3' (177*, F); CH100, 5'-TGTTCAGGGCAGTGCCCACTTCACAGGGCTGA-3' (F); CH101, 5'-CCCACTTCACAGGGCTGAGGATTAGAGAGGATAA -3' (871*, F); AP1, 5'-GTAATACGACTCACTATAGGGC -3' (R); and AP2, 5'-ACTATAGGGCACGCGTGGT -3' (R), where * is from the transcription initiation site, F is the forward primer, and R is the reverse primer.
Transcription initiation site.
The transcription initiation site was determined by primer extension. An antisense oligonucleotide, CH519, 5'-TACCCACGCAGCTAGCCAGTCACGTCGC-3' (10 pmol) was end labeled with [
-32P]ATP and T4 polynucleotide kinase for 10 min. The labeled probe was purified with QIAquick nucleotide removal kit (Qiagen). Fifteen micrograms of total Caco-2 RNA and 1.5 x 105 counts/min (cpm) of 32P end-labeled oligonucleotide were heated at 75°C for 10 min and placed in a 42°C water bath for 5 min. Primer extension was carried out in a buffer containing (in mM) 100 Tris·HCl (pH 8.3), 10 DTT, 3 MgCl2, 50 KCl, and 0.4 dNTP using 200 U SuperScript II RT (GIBCO-BRL) at 42°C for 60 min. The reaction mixture was extracted with phenol/chloroform, ethanol precipitated, and resuspended in 7 µl of sequencing stop solution. The products were analyzed on a 6% polyacrylamide, 7 M urea denaturing gel and visualized by autoradiography. The size of the extended primer was determined by a parallel sequencing run using the same oligonucleotide as the one used for primer extension.
Construction of reporter plasmids.
Construction of reporter plasmids for functional analysis of the MCT1 promoter was carried out using the pGL2-Basic vector (Promega, Madison, WI) that consists of a promoterless luciferase reporter gene. The MCT1 promoter was removed from pGEM-T vector using Sal1 and NcoI restriction endonucleases and was blunt ended with Klenow. The blunt-ended fragment was inserted into SmaI site of pGL2 and both its forward and reverse directions were obtained. Deletions of the 5'-end of the promoter sequence were done using the PCR method. The PCR conditions were heating at 95°C for one cycle of 3 min, 20 cycles heating at 95°C for 30 s, annealing at 67°C for 30 s, and extension at 72°C for 1 min. Final extension was done at 72°C for 5 min. Primers used to make deletion pGL2/625 were CH528 and CH519, for pGL2/378 were CH529 and CH519, for pGL2/229 were CH530 and CH519, and for pGL2/5 were CH531 and CH519. The resulting fragments were cloned into pGEM-T vector, sequenced, and subcloned at the SmaI site of pGL2 vector.
The oligonucleotides were as follows: CH528, 5'-GACTCGAGTGGCTCTATGGTGGCAAGTTGCAT-3'; CH529, 5'-ATCTCGAGATGCTGCCCATTCCCCGCTCCTCAGT-3'; CH530, 5'-GACTCGAGAGATTGCCTAGAGCTCGTCAGACA-3'; CH531, 5'-ATCTCGAGCTAGAGGGCGCGCGCGGCTGAAAGCGTGTGG-3'; and CH519, 5'-TACCCACGCAGCTAGCCAGTCACGTCGC-3'.
Site directed mutagenesis.
To determine the functionality of the two SP1 sites in the MCT1 promoter, mutations were performed in each site. The site closer to the transcription initiation site is referred to as the proximal SP1 site (M1), and the other site is referred to as the distal SP1 site (M2). The mutations were carried out using the GeneEditor in vitro site-directed mutagenesis system (Promega). The MCT1 promoter fragment was removed from the pGL2/229 construct and inserted into blunt ended XhoI site of the pGEM11Zf vector. The new construct was denatured, and the generated single-strand DNA templates were used for annealing of the phosphorylated mutant and selection of oligonucleotides in 20 mM Tris·HCl (pH 7.5), 10 mM MgCl2, and 50 mM NaCl buffer. The reaction was heated at 75°C for 5 min and allowed to slowly cool to 37°C. The mutant strand was synthesized in buffer containing (in mM) 10 Tris·HCl (pH 7.5), 0.5 dNTP, 1 ATP, and 2 DTT. The synthesis was carried out at 37°C for 60 min in the presence of T4 DNA polymerase and T4 DNA ligase in a 30-µl volume. Competent BMH 7118 mutS cells were transformed with 1.5 µl of second strand reaction and allowed to grow overnight in 5 ml LB media supplemented with 150 µl of antibiotic selection mix. Plasmids were extracted from the overnight grown cultures, and 5 ng were used to transform JM109 competent cells, which were plated on LB plates supplemented with 100 µg/ml ampicillin. Overnight grown colonies were used to extract plasmids that were sequenced for the presence of mutations. The mutated promoter fragment was removed from pGEM 11Zf and inserted into SacI/HindIII sites of pGL2.
The mutant oligonucleotides were as follows: M1, 5'-CGGATGTCTGTGAAAAGGGGAGGGGGCG-3' and M2, 5'- GACGCCGGTCACAAAACGGGGAGGGGGCG-3'.
Cell culture and transfections.
Caco-2 cells were grown routinely in 100-mm dishes. They were maintained in a high-glucose DMEM medium (GIBCO-BRL) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The cells were kept at 37°C in a 5% CO2-95% air environment. Cells were seeded at 2.5 x 105 per well in 12-well plates the day before transfection to achieve 9095% confluency by the day of transfection. Cells were cotransfected with 1.6 µg DNA construct and 0.4 µg pRSV-
-gal, the latter was used as internal control for transfection efficiency. Transfections were done using Lipofectamine 2000 (GIBCO-BRL) in an OptiMEM medium for 4 h. Media were replaced with normal Caco-2 media 4 and 24 h after transfection. The cells were washed 48 h after transfection, once with 2 ml of PBS followed by the addition of 400 µl lysis buffer, incubated for 10 min at room temperature, scraped, and stored at 80°C. Luciferase activity was measured according to Luciferase reporter kit (Promega). In other experiments, pSG5 expression vectors harboring USF1 and USF2 (generous gifts from Dr. Michelle Sawadago, Houston, TX) were cotransfected with the promoter construct in pGL2, and luciferase activity was measured after 48 h as described above.
Nuclear extracts.
Nuclear extracts were obtained from Caco-2 cells after washing the cells twice with 10 ml of PBS at room temperature. One milliliter of lysis buffer [in mM: 10 HEPES (pH 7.9), 10 KCl, 2 MgCl2, 0.1 EDTA, 1 DTT, and 1 PMSF] was added to the T75 flask, and cells were scraped off, transferred into an Eppendorf tube, and incubated on ice for 10 min. Fifty microliters of 10% Nonidet P-40 were added, vortexed for 10 s, and centrifuged at 1,000 g for 5 min at 4°C. The pellet was washed with 100 µl lysis buffer, resuspended in 200 µl of nuclear extraction buffer [in mM: 50 HEPES (pH 7.9), 50 KCl, 300 NaCl, 1 EDTA, 1 DTT, and 1 PMSF, with 10% glycerol] and incubated on ice for 3 h. It was centrifuged at 1,000 g for 5 min at 4°C and aliquoted in 20 µl and stored at 80°C.
Gel mobility shift assay.
Gel mobility shift assay was carried out using the Promega kit. Polyclonal antibodies against SP1, USF1, USF2, and MyoD proteins as well as USF (5'-CACCCGGTCACGTGGCCTACACC-3') consensus oligonucleotide were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). SP1 (5'-ATTCGATCGG GGCGGGGCGAGC-3') and AP2 (5'-GATCGAACTGACCGCCCGCGGCCCGT-3') consensus oligos were purchased from Promega, whereas MZF1 (5'-GATCTAAAAGTGGGGAGAAAA-3'), MyoD (5'-CCCAACAGCTGCTGCCTGA-3'), and CH552 (5'-CAAGCCGGTCACGTGGCGGGGAGGGGGCG-3', spans distal SP1 site) consensus oligonucleotides were synthesized by GIBCO-BRL. After being annealed, the double-stranded DNA was end labeled with [
-32P]ATP and T4 polynucleotide kinase (Promega) and purified with QIAquick nucleotide removal kit (Qiagen). The DNA protein-binding reactions were carried out in a binding buffer consisting of 10 mM Tris·HCl (pH 7.5), 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 4% glycerol, and 0.05 mg/ml poly(dI-dC). Nuclear proteins (45 µg) were added to the binding buffer and incubated for 10 min at room temperature. Then, radiolabeled oligonucleotide (50,000 cpm) was added, and the incubation was further extended for 20 min at room temperature. In competition assays, 100-fold molar excess of unlabeled oligonucleotide was added at the start of the reaction. Supershift assays were done by the addition of 2 µg of antibody at the end of the binding reaction, and the incubation was further extended for 30 min at room temperature.
RNA extraction and real-time RT-PCR analysis.
Caco-2 cells were transfected with pSG5-USF2 or the empty vector using Lipofectamine 2000 (Invitrogen) and were used 48 h posttransfection for RNA extraction. Total RNA from the cells was prepared using Aurum total RNA mini kit (Bio-Rad, Hercules, CA) according to manufacturer's instructions. An equal amount of RNA for each sample was reverse transcribed and amplified in one-step reaction using Brilliant SYBR Green QRT-PCR master mix kit (Stratagene, La Jolla, CA) and using Mx 3000 (Stratagene). Human MCT1 was amplified with gene-specific primers (sense primer: 5'-TCTGTGTCTATGCGGGATTCTT-3'; antisense primer: TTGAGCCGACCTAAAAGTGGT-3').
-actin was amplified as an internal control using gene-specific primers (5) (sense primer: 5'-CATGTTTGAGACCTTCAACAC-3'; antisense primer: 5'-CCAGGAAGGAAGGCTGGAA-3'). The quantitation of the amplification was expressed as a ratio of 2
Ct-MCT1/2
Ct-
-actin, where
Ct-MCT1 and
Ct-
-actin represent the difference between the threshold cycle of amplification of treated (USF2 transfected) and control (empty vector transfected) RNA for both MCT1 and
-actin, respectively.
RESULTS
Cloning of MCT1 promoter and identification of transcription initiation site. The primers used to clone the human MCT1 promoter region were designed based on the initially cloned sequence of the MCT1 5'-untranslated region (Accession no. AL158844). Using human genomic DNA as a template and applying the PCR method, we isolated a 962-bp genomic region (Fig. 1). To determine the transcription initiation site of MCT1 gene in Caco-2 cells, the antisense radiolabeled oligonucleotide CH519 (+92 to +119) was extended employing the primer extension method and using total RNA and RT enzyme. The transcription initiation site was identified by running a sequencing ladder, generated from a corresponding genomic clone and the antisense oligo CH 519, on the same gel. Results are shown in Fig. 2. An extension product was observed when the RNA from Caco-2 cells was used, whereas the lane with yeast tRNA did not show any extension product. The single-primer extension product compared with the sequencing ladder indicated that the transcription start was at a guanine (G) nucleotide, 281 bases from the translation initiation codon, and was surrounded by cytosine- and guanine-rich areas.

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Fig. 1. 5'-Upstream sequence of human monocarboxylate transporter isoform 1 (MCT1) gene. The 5'-untranslated region of the MCT1 initially cloned (Accession no. AF239919) is shown in lowercase letters. The transcription start site is marked as +1. Possible transcription regulation sites on the (+) or () strands are shown single or double underlined, respectively. PE primer used for primer extension is the CH519 oligonucleotide.
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Fig. 2. Identification of the transcription start site. Total RNA from Caco-2 cells (20 µg) or the yeast (20 µg) was annealed to 32P-labeled antisense primer CH519 and reverse transcribed. The extension products were electrophoresed along with a sequencing ladder generated from the corresponding region of the gene and the CH519 primer. Lane 1 shows the results from yeast tRNA and lane 2 from Caco-2 RNA. The arrow shows the primer extension product, and * is the transcription start nucleotide. The sequence ladder shown is of the complimentary strand.
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The sequence analysis of the region upstream from the transcription initiation site revealed the presence of multiple consensus sequences that may be potential binding sites for nuclear factors and sites for its transcriptional regulation. These binding sites include SP1 (at 14 and 107), AP1 (287 and 491), AP2 (84 and 507), CdxA (653 and 794), MZF-1 (360 and 691), and USF (117 and 731). Also, there are two motifs for GATA-1 at 572 and 818 and Nkx2 at 536 and 863, one motif for NF-
B at 597 and one for GATA-2 at 836.
Activity of MCT1 promoter in Caco-2 cells.
Functional analysis of MCT1 promoter was carried out by transient transfection in Caco-2 cells, a cell line shown by us recently to express the MCT1 gene (18). The 962-bp promoter fragment (871/+91) was cloned in both the forward and reverse orientation at the SmaI site of pGL2 basic, upstream from the luciferase reporter gene. The activity of the reporter gene was measured 48 h after transfection. Results are shown in Fig. 3. It can be clearly seen that the forward orientation of the promoter had a much higher luciferase gene expression compared with the promoterless vector and positive control pGL2-promoter. The MCT1 promoter activity was >250-fold higher than the pGL2-basic activity and fivefold higher than the pGL2-promoter activity that is driven by the SV40 promoter. The promoter fragment in the reverse orientation showed similar activity as the pGL2-basic.

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Fig. 3. Expression of the MCT1 promoter-luciferase construct in Caco-2 cells. The MCT1 promoter (871/+91), in the reverse or forward orientation, was inserted upstream from the luciferase gene in pGL2 vector, and its activity was determined by transient transfection in Caco-2 cells. Transfection efficiency corrections were made by cotransfection with pRSV- -gal vector. Promoter activity was measured by fold increase over the promoterless control pGL2-basic. Positive control is the pGL2-promoter construct driven by the SV40 promoter. Results are the means of 3 different experiments ± SE.
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Deletion analysis of MCT1 promoter.
In an effort to determine regions that play an important role in the promoter activity, a number of consecutive deletions was generated at the 5'-end of the MCT1 promoter. The newly generated fragments were placed upstream from the luciferase reporter gene in pGL2-basic and transiently transfected into Caco-2 cells. The results are shown in Fig. 4. The longest fragment, pGL2/871, showed the highest luciferase activity, and the activity decreased as the length of the promoter fragment was reduced. The promoter activity was reduced by 25% with the expression of pGL2/625 construct, by 65% with the pGL2/378, and by 83% with the pGL2/229 construct. The shortest deletion construct, pGL2/5, showed the lowest luciferase activity similar to the control promoterless pGL2-basic. These results demonstrated that the basal regulatory elements to sustain the minimal promoter activity are present within the deletion construct pGL2/229.

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Fig. 4. Deletion analysis of MCT1 promoter. Deletions of the 5'-end of MCT1 promoter were inserted upstream from the luciferase gene in pGL2 vector and transient transfection of Caco-2 cells was performed. Transfection efficiency correction was made by cotransfection with pRSV- -gal vector. Promoter activity was measured by fold increase over the control pGL2-Basic. Results are the means of 3 different experiments ± SE.
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Functional analysis of putative SP1 transcription factor binding sites.
The two SP1 sites present in the MCT1 promoter region were examined for their probable involvement in the regulation of the promoter activity. The two sites present in pGL2/229 construct were mutated as described in the section "site-directed mutagenesis" in methodology, and the mutants were transiently transfected into Caco-2 cells. The expression of the pGL2/229 construct in which the SP1 site proximal to transcription initiation site was mutated (M1) exhibited promoter activity similar to the control, pGL2/229, whereas the expression of distal SP1 site mutant (M2) showed more than twofold increase in luciferase activity (Fig. 5). To further characterize the involvement of distal SP1 site in transcriptional regulation of the MCT1 promoter activity, gel mobility shift assay was employed using the oligonucleotide CH552 that encompasses the distal SP1 site sequence. As shown in Fig. 6, a protein from Caco-2 nuclear extracts did bind to the probe (lane 2) and was competed out with 100-fold molar excess of unlabeled CH552 (lane 3). This oligonucleotide also encompasses an AP2 binding site, but the unlabeled AP2 consensus sequence at 100-fold molar excess failed to compete out CH552/protein complex formation (lane 3). When mutated CH552 (M2) was used as an end-labeled probe, no DNA/protein interaction was detected with Caco-2 nuclear extract (Fig. 6, bottom lane 6), and when used as a competitor at 100-fold molar excess, unlabeled M2 did not compete out protein binding to CH552 (lane 7). Similarly, M1 as labeled probe virtually did not show DNA/protein interaction (lane 5). To determine whether the SP1 protein was involved in the DNA/protein complex formation, a consensus SP1 oligonucleotide was used as a competitor to the binding of labeled CH552 to the nuclear protein. As shown in Fig. 7A (lane 5), the SP1 oligo failed to compete out the DNA-protein interactions, indicating that the binding factor to this site was not the SP1 protein. Further computer database search of the area spanning the distal SP1 site revealed the presence of other potential DNA binding core sequences for MZF1, USF, and MyoD transcription factors. Oligonucleotides containing the consensus binding sites for these proteins were used as competitors to determine the identity of the protein(s) binding to labeled CH552. The results in Fig. 7A showed that the oligonucleotides for MZF1 and MyoD did not compete out protein binding to CH552 (lanes 6 and 7, respectively), whereas, interestingly, the USF consensus oligo competed out this interaction (lane 4). Furthermore, antibodies against SP1, USF1, USF2, and MyoD were tested for protein complex, and the results are shown in Fig. 7B. As can be seen, SP1 and MyoD antibodies had no effect on complex formation or its mobility did not supershift the interaction (lanes 3 and 4, respectively) in agreement with the results obtained in competition studies shown in Fig. 7A. On the other hand, both USF1 and USF2 antibodies supershifted the DNA-protein complex (lanes 1 and 2, respectively). Supershifting ability of USF-2 was found to be more as evidenced from higher intensity of the band in lane 2 compared with lane 1. These results suggest that USF proteins are in fact the nuclear factors interacting with CH552, which contains a consensus USF binding site (CACGTG). As evident from mutational studies (Fig. 5), USFs appeared to act as a repressor of the MCT1 promoter, because mutational disruption of its binding motif showed increased luciferase activity.

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Fig. 5. Expression of the MCT1 promoter mutants for SP1 consensus sites in Caco-2 cells. The two SP1 sites of the MCT1 promoter present in deletion pGL2/229 were mutated and expressed in Caco-2 cells. Transfection efficiency correction was performed by cotransfection with RSV- -gal vector. Promoter activity was assessed by fold increase over the control pGL2-Basic. pGL2/M1 indicates the pGL2/229 construct mutated at the proximal SP1 site, pGL2/M2 indicates the pGL2/229 construct mutated at the distal SP1 site. Results are the means of 3 different experiments ± SE.
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Fig. 6. Binding of nuclear proteins to the region spanning the distal SP1 site. 32P-labeled double-stranded oligonucleotide encompassing the distal SP1 site was incubated with or without nuclear extracts (NEs) from Caco-2 cells or Hela cells. NEs were incubated with the binding buffer alone or in the presence of competitor for 10 min at room temperature. Labeled probe [50,000 counts/min (cpm)] was added, and the incubation was further extended for 20 min at room temperature. In competition experiment, the unlabeled oligo was added at 100-fold molar excess compared with the radioactive probe. The reaction was stopped with the addition of 1 µl of stop dye and assayed on a 4% nondenatured polyacrylamide:bisacrylamide gel. The top picture shows binding of Hela (left lanes) or Caco-2 (right lanes) nuclear protein(s) to labeled CH552, which was competed out by cold CH552 but not by consensus AP2 oligonucleotide. The bottom picture shows the binding of Caco-2 nuclear extracts to labeled CH552 (lanes 14 and 7, lane 1 without nuclear extract) and labeled mutant M1 (lane 5) and M2 (lane 6) and competition by unlabeled CH552 (lane 3), and no competition by AP2 (lane 4) and M2 (lane 7).
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Fig. 7. Gel mobility shift assay and supershift assay. A: NEs were incubated with binding buffer alone or in the presence of the indicated competitor oligonucleotide for 10 min at room temperature. Labeled CH552 probe (50,000 cpm) was added to the reaction mixture, and the incubation was extended by 20 min at room temperature. The reaction was stopped with the addition of 1 µl of stop dye and assayed on a 4% nondenatured polyacrylamide/bisacrylamide gel. Competitor oligonucleotides were added to the reaction at 100-fold molar excess than the labeled probe. Lane 1 was without NE. B: NEs were incubated with binding buffer alone or in presence of the indicated antibody (2 µg) for 10 min at room temperature. Labeled CH552 probe (50,000 cpm) was added to the reaction mixture, and the incubation was extended by 20 min at room temperature. The reaction was stopped with the addition of 1 µl of stop dye and assayed on a 4% nondenatured polyacrylamide/bisacrylamide gel.
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Promoter activity in Caco-2 cells overexpressing USF-1 and USF-2.
Expression vector (pSG5) containing the coding sequence for human USF1 or murine USF2 was used in cotransfection experiments with the MCT1 promoter construct pGL2/871 in Caco-2 cells, and the luciferase activity was determined. As shown in Fig. 8A, cotransfection of USF1 caused a 36% decrease in reporter gene activity compared with the promoter alone or with the empty vector, whereas that of USF2 resulted in an 84% decrease in promoter activity. Repressor activity of both USF1 and USF2 on the promoter was found to be dose dependent (Fig. 8B). Cotransfection of USF2 with the M2 promoter construct, wherein the USF binding site was mutated, however, had no effect on promoter activity (results not shown). These results are also consistent with the gel mobility supershift assay (Fig. 7B), in which it is shown that supershift was much more pronounced with USF2 antibody than with USF1 antibody. Cotransfection experiments further confirmed the role of USFs as repressor proteins for the MCT1 promoter.

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Fig. 8. Repression of MCT1 promoter activity in Caco-2 cells by upstream stimulatory factors (USF)1 and 2. A: Caco-2 cells were cotransfected with the MCT1 promoter region in the pGL2 vector and either the pSG5 expression vector alone or those containing the coding region for human USF1 or murine USF2. Luciferase activity was measured 48 h after transfection. Normalized luciferase activity was calculated as described in Fig. 4 and expressed as per mg protein. Results are the means of 3 different experiments. B: the same experiment was done using increasing amounts of USF1 and USF2 plasmid DNA (0.4, 0.8, and 1.6 µg/well, respectively, in 24-well plate) cotransfected with pGL2/871 promoter construct.
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Endogenous MCT1 expression in Caco-2 cells overexpressing USF2.
Because there was a significant decrease in the MCT1 promoter activity in Caco-2 cells overexpressing USF2, we investigated the relative abundance of MCT1 mRNA in Caco-2 cells 48 h posttransfection with USF2, compared with empty vector-transfected cells as control. MCT1 mRNA level was quantified by real-time PCR relative to the level of
-actin mRNA as an internal control. As shown in Table 1, relative abundance of MCT1 in USF2-transfected cells decreased by 40% compared with control cells, indicating a significant downregulation of MCT1 expression by USF2.
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DISCUSSION
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The regulation of human MCT1 promoter was investigated in an effort to better understand the mechanisms and regulation of short-chain fatty acid (SCFA) absorption in the human intestinal epithelial cells. The limited availability of the luminal SCFA has been implicated in inflammation of the intestine, which has been shown to be alleviated by the instillation of SCFA at the site of inflammation (2, 20, 34). Because MCT1 plays a key role in the uptake of SCFA in the intestine (18, 32), identifying the regulatory factors of MCT1 promoter will help us better understand the mechanism(s) of its regulation and its potential involvement in inflammatory conditions of the bowel. In an effort to identify the transcription factors and cis elements involved in the regulation of MCT1 expression, Caco-2 cell line was used as an experimental model. Human MCT1 promoter region was cloned using PCR method, its transcription initiation site identified, and the promoter activity was studied. The transcription initiation site was found to be a guanine nucleotide, 281 bp upstream from the translation start codon, and was surrounded by a highly GC-rich area. Two SP1 consensus sites and potential binding sites for AP1, AP2, and MZF1 were found in this region. Other transcription factor binding sites detected were for Nkx2, GATA1, GATA2, USF, CdxA, and NF-
B.
The MCT1 promoter sequence was also recently described in another report (9). In this report, the transcription initiation site was determined using total RNA from human colonic AA/C1 cells which was 81 nucleotides upstream from the transcription start site reported in this study. This difference is attributed to the TATA-less promoter and to the presence of a long GC-rich area at its 3'-end. A significant difference between the two reports relates to the MCT1 promoter activity, which seems to be significantly higher in Caco-2 cells compared with AA/C1 cells. In MCT1 promoter-transfected Caco-2 cells, a >250-fold increase in luciferase activity was observed by us compared with the promoterless pGL2-Basic, whereas in AA/C1 cells the reporter gene activity was shown to be increased only by
25-fold compared with the empty vector (pGL3) (9). Also, we showed that deletions at the 5'-end of the promoter-reduced promoter activity, when its length was sequentially reduced from 871/+91 to 5/+91. Furthermore, the minimal promoter region (229/+91) in our study showed similar activity in Caco-2 to that of the full-length promoter transfected in AA/C1 cells. These data suggest that the MCT1 promoter shows cell type-specific characteristics of regulation that may also be related to the state of cellular differentiation.
In an effort to understand the transcriptional regulation of MCT1 promoter, we decided to examine whether the two potential SP1 binding sites detected immediately upstream to the transcription initiation site were involved in promoter regulation. These sites are found to be present in the highly GC-rich region of the promoter, and in general, they act as stimulators of promoter activity. We mutated the two sites in the 229/+91 construct. The expression of the construct mutated in the distal SP1 site (M2), in Caco-2 cells, combined with the results from gel mobility shift assay showed that a nuclear factor from Caco-2 cells, binding to an oligonucleotide spanning the distal SP1 site, was acting as a repressor of promoter activity, whereas the proximal SP1 site was not involved in promoter regulation. With additional studies using mobility shift assays and supershift analysis, we were able to show conclusively that USF1 and USF2 were the transcription factors binding to this cis element. USF1 and USF2 are ubiquitously expressed basic helix-loop-helix-leucine zipper (bHLHzip) transcription factors that share nearly identical COOH-terminal bHLHzip motifs and bind as both homodimers and heterodimers to DNA sequences centered on CAC(G/A)TG, known as E-box (37). This CACGTG motif is present in the MCT1 promoter overlapping with the putative distal SP1 site, and the mutations in this motif in the M2 construct disrupt the binding of USF proteins to this site. This is in agreement with our results showing that the mutant M2 failed to bind to Caco-2 nuclear extracts and at 100-fold molar excess failed to compete out the labeled DNA-protein binding. A repressor role of USF, more particularly of USF2, on the MCT1 promoter activity was further confirmed by cotransfection experiments in Caco-2 cells where we showed that USF2 cotransfection resulted in a drop of promoter activity by 84%. The effect was less pronounced with USF1, causing promoter activity to decrease by 36%. The repressor role of USF is also conclusively evident in our studies using real-time PCR to show that, in USF2 overexpressed Caco-2 cells, there was 40% decrease in endogenous MCT1 mRNA expression. Although USF1 and USF2 genes are ubiquitously expressed in human, the relative abundance of USF1 and USF2 transcripts and protein levels vary among different cell types (36). In current studies, we did not quantitate the relative expression of USF1 and USF2 in the Caco-2 nuclear extract; however, the antibody supershift analysis and cotransfection studies suggested that USF2 was the major transcription factor regulating MCT1 expression. Although in most instances, USFs have been shown to act as activators of transcription (3, 11, 22, 24, 39); however, the role of USFs as a repressor is not unique to MCT1 promoter only, because there are reports that these proteins can also repress transcription of target genes independently or as components of a repressor complex (15, 16, 27, 35).
Studies have also reported antiproliferative roles of USF1 and USF2, which were shown to be cell-type dependent (21, 31, 37). In the intestinal epithelial cells, USFs have been reported to be involved in the regulation of various genes. For example, human dipeptidyl peptidase IV gene was shown to be activated by USF1 (11). Glut 5, the fructose transporter in mature villi of mouse was found to be activated by USF in response to dietary fructose (7). A recent analysis revealed that USF binding motifs (E-boxes) are commonly found in genes highly expressed in colon and that USF and c-Myc interplay to bind to genes that are upregulated or downregulated in colorectal cancer (3). However, there are very few studies showing involvement of USFs in the regulation of intestinal epithelial transporters. Studies on the regulation of expression of nutrient transporters are of particular importance for those nutrients whose functions are beyond their recognized role in providing energy and in metabolism. In this regard, butyrate, a key SCFA in the colonocytes, is such a nutrient with diversified roles in maintaining colonocyte health, proliferation, and epithelial integrity (6, 10). Known cellular effects of butyrate in the colonocytes depend on its intracellular availability, which determines its ability to maintain tissue homeostasis in the colonic epithelium. However, the results of our present studies showing the regulation of MCT1 expression by USF are insufficient to draw any inference that USF might have a direct role in the differentiation dependent expression of MCT1 in Caco-2 cells.
It has also been shown that the glucose-response elements identified in the promoters of several glycolytic and lipogenic enzymes including L-pyruvate kinase (24), fatty acid synthase (39), and glucokinase (22) are also modulated by USF in various cell types. Increased liver levels of USF1 associated with fasting and refeeding of rats have been reported to be important for the regulation of fatty acid synthase gene (39). In another report (6), the fructose transporter Glut 5 expression pattern in the mature villus of mouse small intestine and sensitivity to dietary fructose have been suggested to be controlled by USF. Therefore, there is a precedent for USF playing an important role in the response of cellular genes to various nutrients. Adaptation to nutritional environment by the absorptive cells of the intestinal epithelium is known to be achieved through the modulation of expression of specialized nutrient transporters (10). Although such reports are not available with regard to regulation of MCT1 expression in any tissues, it is not unlikely that USF might play a similar role in the regulation of MCT1, at least in the colonic epithelium, in response to the availability of SCFAs. In this regard, a recent study (8) has shown induction of MCT1 expression in AA/C1 cells by its substrate butyrate. Our preliminary studies (1) also suggested stimulation of the MCT1 promoter activity by butyrate in Caco-2 cells. It is possible that butyrate-induced stimulation of MCT1 promoter may be secondary to downregulation of USF expression in colonocytes. Additional detailed studies are required to investigate the potential involvement of the role of USF, if any, in the activation of the MCT1 promoter by butyrate.
Hormonal regulation of MCT1 has also been shown in two recent reports (4, 12). Thyroid-stimulating hormone stimulation of lactate uptake in rat thyroid cells was due to upregulation of MCT1 transcription (12). On the other hand, luminal leptin stimulation of butyrate uptake in Caco-2.bbe cells has been shown to be due to increased intracellular pool of MCT1 protein (4). It will be of interest to extend these studies in context to the MCT1 promoter to reveal the involvement of USF, or any other transcription factor, in mediating these hormonal effects. There has also been accumulating evidence to support the role of SP1 and USF in mediating insulin response in various systems (25, 33).
In summary, we have shown the high activity of MCT1 promoter in Caco-2 cells and have provided evidence for the transcriptional regulation of this gene by USF1 and USF2. We speculate that USF regulation of this gene might be a response to nutritional environment or related to differentiation of intestinal epithelial cells. Our future studies will focus on further elucidating the in vivo roles of USF in the regulation of intestinal epithelial MCT1 expression and determining the roles of other cis elements of the MCT1 promoter in the transcriptional and hormonal regulation of the MCT1 gene and SCFA absorption.
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GRANTS
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These studies were supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54016 (to P. K. Dudeja), DK-68324 (to P. K. Dudeja), DK-33349 (to K. Ramaswamy), and DK-67990 (to K. Ramaswamy).
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FOOTNOTES
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Address for reprint requests and other correspondence: P. K. Dudeja, Professor of Physiology in Medicine, Univ. of Illinois at Chicago, Medical Research Service (600/151), Jesse Brown VA Medical Center, 820 South Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* C. Hadjiagapiou and A. Borthakur contributed equally to this work. 
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