Analysis of human cellular retinol-binding protein II promoter during enterocyte differentiation

Liang Zhang1, Xueping E1, Kathryn E. Luker1, Jian-Su Shao1, Marc S. Levin1,3, Eunran Suh4, and Ellen Li1,2

Departments of 1 Medicine and 2 Biochemistry and Molecular Biophysics, Washington University- St. Louis School of Medicine and 3 Specialty Care, St. Louis Veterans Administration Medical Center, St. Louis, Missouri 63110; and 4 Department of Internal Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


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Cellular retinol binding protein II (CRBP II) is a vitamin A-binding protein that is expressed specifically in small intestinal villus absorptive cells. Previous studies have shown that retinoic acid upregulates endogenous human CRBP II gene expression in differentiated Caco-2 cells. To better characterize the regulation of human CRBP II expression, we analyzed the ability of receptor-selective agonists to enhance transcription from the 5'-upstream flanking region of the human CRBP II gene. Stable transfection experiments showed that the proximal 2.8-kb region of the human CRBP II gene is sufficient for retinoic acid inducibility in differentiated Caco-2 cells. However, direct sequence analysis and transient transfection experiments indicate that, unlike the rat CRBP II promoter, the human CRBP II promoter is not a direct retinoid X receptor target. The results indicate that the retinoic acid responsiveness of the human CRBP II promoter is mediated by an indirect mechanism and that this mechanism is associated with enterocyte differentiation.

gene regulation; retinoid X receptor; vitamin A; Caco-2 cells


    INTRODUCTION
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CELLULAR RETINOL BINDING PROTEIN II (CRBP II) is an abundant (0.4-1.0%) intestinal cytosolic protein that binds all-trans-retinol and all-trans-retinal (19, 20, 29). In adult animals, expression of CRBP II is restricted to villus-associated enterocytes in the proximal small intestine (5). The intestine-specific localization of CRBP II suggests that it is uniquely adapted for intestinal absorption and/or metabolism of retinol. The human colonic adenocarcinoma Caco-2 cell line develops into a polarized epithelium after reaching confluence. The cells express proteins characteristic of both enterocytes and colonocytes immediately after reaching confluence (9, 30), but with increasing time after confluence, the expression of enterocytic proteins become more prominent (9). Human CRBP II expression increases after human Caco-2 cell monolayers become confluent and undergo differentiation (17, 31). Because postconfluent differentiated Caco-2 cell monolayers express human CRBP II and lecithin/retinol acyl transferase, they have been used to study intestinal vitamin A uptake and metabolism (11, 17, 22, 31, 38). Stably transfected differentiated Caco-2 cells overexpressing CRBP II have increased uptake and esterification of all-trans-retinol (17, 22). Treatment of postconfluent, differentiated Caco-2 cells with all-trans-retinoic acid, 9-cis-retinoic acid, arachidonic acid, or its analog 5,8,11,14-eicosatetraynoic acid (ETYA) in serum-free medium further increases endogenous CRBP II mRNA levels (11, 38). Nuclear run-on experiments confirmed that the increase observed with ETYA and 9-cis-retinoic acid was due to increased transcription (11). These ligand studies suggest that retinoid X receptors (RXRs) are involved in the regulation of human CRBP II genes either as homodimers or partnered with other nuclear receptors such as the peroxisomal proliferator-activated receptors (PPARs).

The nuclear hormone receptors, including the RXRs, PPARs, and retinoic acid receptors (RARs) act via cis-acting DNA response elements (24). The response elements consist of direct repeats (DR) of two consensus sequences [5'-RG(G/T)TCA or a closely related sequence] separated by a spacer varying in length from one to five nucleotides. The response elements for RXR homodimers (RXRE) and RXR-PPAR heterodimers (PPRE) consist of direct repeats that are separated by a single base pair (i.e., DR1). RXR-RAR heterodimers bind to response elements (RAREs), which consist of direct repeats separated by two (DR2) or five (DR5) nucleotides.

The rat CRBP II promoter contains a RXRE consisting of five nearly perfect tandem repeats of the consensus sequence separated by a single nucleotide (Fig. 1) and is upregulated by retinoic acid only in the presence of RXR (25). This element also binds PPAR-RXR heterodimers and could potentially also serve as a PPRE (14, 38). The rat CRBP II promoter also contains two more proximal response elements, termed RE2 and RE3, which correspond to DR2 and DR1 elements, respectively. The rat CRBP II RXRE is not conserved in the mouse CRBP II promoter. Instead, the mouse CRBP II promoter contains a truncated cis-acting element consisting of only two direct repeats separated by a single nucleotide, which was termed RE1 (28). However, both RE2 and RE3 are conserved (28).


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Fig. 1.   Genomic organization of the 5'-end of the human cellular retinol binding protein (CRBP) II gene as determined by restriction mapping and sequencing. A: partial restriction enzyme analysis of the 5.5-kb HindIII genomic fragment containing exon 1 (shaded box). The restriction enzyme sites are indicated as follows: C, ClaI; E, EcoR1; H, HindIII; P, PstI; B, BglII; N, CoI. B: alignment of the 5'-flanking sequences of human, mouse, and rat CRBP II genes. Nucleotide is numbered so that +1 corresponds to the longest 5'-rapid amplification of cDNA ends as described in MATERIALS AND METHODS. The repeated sequence motifs 5'-RG(G/T)TCA or related motifs [5'-TGA(A/C)CY on the other strand] are in bold. Arrows mark cis-acting response elements labeled RE1, RE2, and RE3 in the mouse gene, and the repeated sequence motifs are numbered 1-6 (13). In the rat gene, there are 5 tandemly repeated motifs, which represent the rat CRBP II retinoid x receptor (RXR) response element (RXRE). No region of repeated sequence motifs homologous to the rat CRBP II RXRE and the mouse CRBP II RE1 are found within this 2.8-kb region of the human CRBP II promoter. The TATA box and the initiation codon (ATG) are also in bold.

When proliferating undifferentiated Caco-2 cells were transfected with a mouse CRBP II promoter [chloramphenicol acetyltransferase (CAT) reporter gene], there was no retinoic acid inducibility of CAT activity even after cotransfection with an RXR expression vector. The rat RXRE/thymidine kinase (tk)-CAT construct was also not responsive to retinoic acid when transiently transfected into preconfluent undifferentiated Caco-2 cells. However, cotransfection with an RXR expression vector restored retinoic acid inducibility. These results are consistent with observations that RXR-alpha levels increase with Caco-2 cell differentiation (39).

To further investigate transcriptional regulation of CRBP II, we cloned and sequenced 2.8 kb of the 5'-flanking region of the human CRBP II gene to determine whether it contains a cis-acting element similar to the rat CRBP II RXRE. We also studied retinoic acid inducibility of reporter constructs incorporating this segment of the human CRBP II promoter in transiently transfected undifferentiated Caco-2 cells and in stably transfected differentiated Caco-2 cells.


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Cell culture. COS-1 and Caco-2 cells from the American Type Culture Collection were maintained at 37°C, 5.5% CO2 in Dulbecco's modified Eagle's medium, 2 mM glutamine, 0.1 mM nonessential amino acids, 100 U/l penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal calf serum. Caco-2 cells were subcultured as described previously (17), and COS-1 cells were plated at 1 × 104 cells/cm2. The medium was changed every other day for preconfluent cells. Medium was changed daily to maintain postconfluent Caco-2 monolayers.

Effects of RAR and RXR agonist administration. Differentiated Caco-2 cell monolayers (14 days postconfluent) in T-25 flasks were washed with serum-free medium supplemented with 6 mM Na taurocholate, 10 mM HEPES, and ITS premix (final concentration: 5 µg/ml insulin, 5 µg/ml transferrin, and 38.8 nmol/l selenious acid) as described previously (11). The monolayers were then incubated for 24 h with ligand at 1 µmol/l or vehicle alone (0.022 M ethanol). The medium was removed, and the monolayers were washed with PBS and immediately harvested. The RXR-selective agonist 4-[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-napthyl)ethenyl]benzoic acid (LG-1069) (1) was a kind gift of Dr. Tim Willson (GlaxoSmithKline). The RAR-alpha agonist (E)-4-[2-(5,6, 7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)propen-1-yl] benzoic acid (TTNPB) (4) was a kind gift of Dr. Louise Foley (Hoffman-LaRoche). Although all-trans-retinoic acid is a RAR-specific agonist, when administered to cells it rapidly undergoes isomerization to 9-cis-retinoic acid, which is an agonist for both RXR and RAR.

Northern blot analysis and RT-PCR. Cells were scraped in TRIzol reagent (GIBCO-BRL, Grand Island, NY), and total RNA was isolated according to the manufacturer's recommendation based on improvements to the single-step RNA isolation method developed by Chomczynski and Sacchi (3). Northern blots were prepared and probed with radiolabeled cDNAs. The membranes were washed once with 1× SSC (0.15 M NaCl and 0.015 M sodium citrate)-0.1% SDS for 15 min at 25°C, once with 1× SSC-0.1% SDS for 15 min at 25°C, and twice with 0.1× SSC-0.1% SDS for 15 min at 65°C. The following cDNA probes were used: rat CRBP II cDNA (19), human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (17), and enhanced green fluorescent protein (EGFP) cDNA (11). The membranes were exposed to a PhosphorImager screen overnight. Signals were scanned and quantitated on a Storm PhosphorImager (Molecular Dynamics). The signal densities were normalized with respect to the GAPDH mRNA signal.

Endogenous human CDX2 gene expression in Caco-2 cells was assessed by semiquantitative RT-PCR using specific primers designed from different exons as described previously (8, 23). As an internal standard, RT-PCR was carried out with primers for actin. The primers for PCR amplification were as follows: human CDX2, forward primer 5'-CCCGGCGGCCAGCGGCGGAACCTGT-3', reverse primer 5'-GTCTAGCAGAGTCCACGCTCCTCAT-3'; actin, forward primer, 5'-GACTTCGAGCAGGAGATGGCCAC-3', reverse primer 5'-CTCCTGCTTGCTGATCCACATC-3'.

Single-stranded cDNA was synthesized for 1 h at 42°C in 25 µl containing 2 µg RNA, 0.5 µg oligo(dT)15, 100 units Maloney murine leukemia virus RT RNase H- point mutant (Promega, Madison, WI), 0.4 mM each dNTP, 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 7 mM MgCl2, 10 mM dithiothreitol, and 0.1 mg/ml bovine serum albumin. To amplify endogenous CDX2 cDNA, PCR reactions (50 µl) contained 5 µl of cDNA solution, 4 pmol primers, 10 mM dNTP, 1 units Taq polymerase (Promega), 10 mM Tris · HCl (pH 9), 50 mM KCl, 0.01% Triton X-100, 1.5 mM MgCl2, and 1 M betaine. To amplify actin, 0.1 µl of cDNA was used and betaine was omitted from the reaction. Conditions were: 94°C, 30 s; 55°C, 30 s; and 72°C, 45 s. For each primer, the reactions were performed for 28-45 cycles to overlap the range of cycles in which the amount of PCR product increased exponentially. The RT-PCR fragments were loaded on 2% agarose and analyzed by imaging densitometry (Eagle Eye II; Stratagene, LaJolla, CA). In addition, the fragments were cloned and sequenced to confirm their identity.

Cloning and analyzing the 5'-flanking genomic sequences of human CRBP II. A human P1 bacteriophage library was screened using PCR (Genome Systems, St. Louis, MO). Assuming that the intron/exon organization of human CRBP II gene would be similar to the mouse and rat Crbp II genes, the forward primer 5'-GCCACCCCCAAGATTGCA-3' and reverse primer 5'-CATGCCGGTTGTCCAGGC-3' were designed to generate a 163-bp product within exon 2 (corresponding to nucleotides 85-247 of the cDNA). Positive clones were then screened with the 32P-5'-end-labeled antisense oligonucleotide 5'-CCATTCTGGTCCCTCGTCAT-3' corresponding to nucleotides 1-20 of the cDNA. The positive P1 clone was partially mapped by restriction enzyme digestion. A 5.5-kb probe-positive HindIII fragment was cloned into the HindIII site of pBluescript II KS+ (Stratagene, La Jolla, CA) to generate plasmid pKSCPII. The 5'-flanking region in pKSCPII was sequenced on an ABI prism sequencer (Perkin- Elmer Biosystems) using the dye terminator method.

The transcriptional start site was mapped by 5'-rapid amplification of cDNA ends (RACE) (2) using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA) and following the manufacturer's recommendation. The first-strand cDNA synthesis was accomplished by reverse transcription of total RNA isolated from 14 day-postconfluent Caco-2 cells using a modified Moloney murine leukemia virus RT, SuperScript II RNase H- RT (Life Technologies, Rockville, MD), and the SMART II oligonucleotide and a modified oligo(dT) primer supplied by the kit. The 5'-cDNA fragment was amplified by PCR using the SMART II oligonucleotide as the 5'-primer and the antisense gene-specific primer 5'-TCACTTCTTTTTGAACACTTGAC-3' as the 3'-primer and was cloned. The transcriptional start site was determined by sequencing nine clones.

Transient transfection assays. A 2.8-kb human CRBP II promoter fragment (bp -2,742-+58) was generated by PCR amplification of pKSCPII using the forward primer 5'-ATCCTCGAGTCAGCATAATGTCTGTGAGA-3' and the reverse primer 5'-ATCAAGCTTTGGCCACTGGTTCGGTGA-3'. The amplified fragments were digested with XhoI and HindIII and subcloned into the XhoI and HindIII sites of the promotorless pGL2 luciferase reporter vector (Promega) to form the phCRBP II-2,742-+58-luc reporter construct for transient transfections. The RXR expression vector and the parental vector pEF-BOS (27, 32) were obtained from Dr. Daniel Kelly (Washington University, St. Louis, MO). The pCRBPII×2RXRE tk-luciferase plasmid was a generous gift from Dr. Ozato (National Institute of Child Health and Human Development, Bethesda, MD) and was used as a control reporter construct for the RXR cotransfection experiments. It was constructed by placing two copies of the rat pCRBP II-RXRE DNA element upstream of the tk sequence (26), which in turn was inserted in the pGL2-Basic vector (Promega). The expression vectors for mouse Cdx2 (pRc-Cdx-2), mouse Cdx1 (pRc-Cdx-1), the control vector pRcCMV, and the -183HSILuc luciferase reporter construct have been previously described (40, 41). -183HSILuc contains the evolutionarily conserved sucrase isomaltase gene promoter (-183-+54 bp). The beta -galactosidase expression vector pEF1/V5-His/LacZ (Invitrogen, Carlsbad, CA) was used as an internal control for transfection efficiency.

COS-1 cells (1 × 105) or Caco-2 cells (4 × 105) were plated in six-well tissue culture plates and maintained for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum. The cells (at 40-60% confluence) were transfected using Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer's instructions, with 0.5 µg of reporter plasmid, 1 µg of expression vector (expression vector and control vector total), and 1 µg of the beta -galactosidase expression vector pEF1/V5-His/LacZ (Invitrogen).

Twenty hours after transfection, the medium was changed to a serum-free medium containing either 1 µM all-trans-retinoic acid or vehicle (ethanol) alone. After an additional 24 h, the cells were washed twice with PBS and harvested with lysis buffer supplied with the luciferase assay kit (Promega). Luciferase activity was assayed on a Monolight luminometer (Pharmingen) according to the manufacturer's instructions. beta -Galactosidase activity was assayed as described by Rosenthal (33). The luciferase activity was normalized against the beta -galactosidase activity. Transfections were performed in triplicate for each construct, and the results were expressed as the average fold induction over the pGL2-Basic vector.

Stably-transfected Caco-2 cells. The 2.8-kb human CRBP II (bp -2,742-+58) promoter fragment was generated by PCR amplification as described in Transient transfection assays. The 5'-serial deletions of this promoter, human CRBP II (bp -745-+58), human CRBP II (bp -150-+58), and human CRBP II (bp -103-+58), were generated by PCR amplification using the forward primers 5'-CCGCTCGAGCTCCCTCCAGGCTCAGCTTGC-3', 5'-CCGCTCGAGTACTGGATGAAATGTTCTGTG-3', and 5'-CCGCTCGAGTTGTGCTTCTGCCCTTTG-3', respectively. The PCR fragments were subcloned into the XhoI and HindIII sites of pEGFP-1 (Clontech) to generate phCRBP II-2,742-+58-EGFP-1, phCRBP II-745-+58-EGFP-1, and phCRBP II-150-+58-EGFP-1 reporter constructs.

Caco-2 cells (4 × 105) were plated in six-well plates and were transfected 24 h after plating using Fugene 6 with 2 µg of the reporter constructs. Following removal of the DNA, fresh medium was added and the cells were incubated for 24 h at 37°C. Twenty-four hours after transfection, the cells were subjected to selection in G-418-supplemented media (0.8 mg/ml active drug). The medium was replaced with fresh G-418-supplemented medium every 3 days. Frozen stocks of pooled G-418-selected cells were prepared after 4 wk of selection.

Fluorescence microscopy. Stably transfected Caco-2 cells were plated on chamber slides and grown until 7 days after confluence in G-418-supplemented medium (0.8 mg/ml). The slides were washed with PBS and fixed in 2% formaldehyde-0.2% glutaraldehyde in PBS for 10 min at room temperature and then washed twice with PBS. The cells were mounted in VectorMount permanent mounting media (Vector Laboratories, Burlingame, CA) and observed by fluorescence microscopy using a Zeiss Axiostop 2 microscope.


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Human CRBP II levels in postconfluent differentiated Caco-2 cells are upregulated by a RXR-specific agonist but not by a RAR-specific agonist. Endogenous human CRBP II expression increases dramatically after Caco-2 cells reach confluence. RXR binds only 9-cis-retinoic acid, and RAR binds both 9-cis-retinoic acid and all-trans-retinoic acid (24). It has previously been shown that retinoic acid upregulates human CRBP II expression and retinol uptake by postconfluent differentiated Caco-2 cells (11). Since both 9-cis-retinoic acid and all-trans-retinoic acid rapidly undergo isomerization within the cell, these compounds cannot be used to distinguish between RXR- and RAR-mediated upregulation. Synthetic receptor-selective agonists, such as the RXR-specific agonist LG-1069 (1) and the RAR-specific agonist TTNPB (4), were used for determining which receptors mediate retinoic acid inducibility of the human CRBP II promoter. We found that there was a two- to threefold upregulation of human CRBP II mRNA levels (Fig. 2) when Caco-2 cell monolayers were incubated with 1 µM LG-1069. However, no significant increase was observed when the monolayers were incubated with 1 µM TTNPB. These results suggest that the upregulation observed with all-trans-retinoic acid and 9-cis-retinoic acid is mediated by RXRs and not by RARs.


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Fig. 2.   Northern blot analysis of the effect of specific receptor agonists on endogenous human CRBP II mRNA levels in postconfluent differentiated Caco-2 cells. LG-1069 is a RXR-selective agonist, and (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)propen-1-yl] benzoic acid (TTNPB) is a retinoic acid receptor (RAR)-selective agonist. The CRBP II mRNA signal is normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal. Values are means ± SD of at least 3 separate experiments performed in triplicate, with the value for vehicle alone (ethanol) normalized to 1 (* P < 0.01)

Comparison of the human CRBP II 5'-flanking sequence with that of the mouse and rat CRBP II genes. A P1 bacteriophage clone containing both exon 1 and exon 2 sequences of the human CRBP II gene was isolated. A 5.5-kb HindIII fragment containing exon 1 was subjected to partial restriction enzyme analysis (Fig. 1A). Sequence analysis revealed that this fragment contained 2,857 nucleotides upstream of exon 1. To map the human CRBP II transcription start site, 5'-RACE experiments were carried out as described in METHODS. A cDNA fragment that was ~450 bp in size was amplified and cloned. The 5'-nucleotide in seven of the nine clones was a cytidine, and the 5'-nucleotide in two of the nine clones was the subsequent nucleotide, which was a thymidine (Fig. 1B). On the basis of this analysis, we have designated the cytidine at position +1 as the transcriptional start site. Comparisons of the proximal human CRBP II promoter sequences with the mouse and rat CRBP II promoter sequences reveal a high degree of sequence conservation in the 5'-flanking region between nucleotide -352 and the transcriptional start site at position +1 (Fig. 1B). The TATA box and the transcriptional start site are conserved across all three species.

Motifs 1-4 corresponding to the 5'-RG(G/T)TCA or related motifs that form the response elements RE2 and RE3 in the rat and mouse promoters are contained within the proximal region of the promoter that is highly conserved across species (Fig. 1B). However, neither RE2 nor RE3, which are conserved in the mouse and rat CRBP II promoters, are conserved in the human CRBP II promoter. The human sequence corresponding to that of the mouse and rat RE3 differs by a single A right-arrow C base change in motif 3, which has previously been shown to impair retinoic acid inducibility of promoter activity and to abolish both RXR-RXR homodimer and RXR-RAR heterodimer binding (25). There are also changes in motif 4, but the changes result in a sequence that corresponds to the 5'-TGA(C/A)CR-3' motif. The sequences corresponding to the mouse and rat CRBP II RE1 lie more distal to the region of high sequence homology. Alignment of the sequences could not detect an RE1 element in human CRBP II promoter (Fig. 3B). Although Suruga et al. (38) reported that a RXRE could be amplified by PCR from the human CRBP II gene, an equivalent sequence could not be found within 2.8 kb upstream of exon 1 by direct sequence analysis.


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Fig. 3.   Expression of enhanced green fluorescent protein (EGFP)-1 in 7-day-postconfluent, stably transfected Caco-2 cells. The cells were grown and fixed as described in MATERIALS AND METHODS and observed by fluorescence microscopy. A: undifferentiated proliferating Caco-2 cells stably transfected with promoterless pEGFP-1. B: differentiated Caco-2 cells stably transfected with the promoterless pEGFP-1. C: undifferentiated Caco-2 cells stably transfected with phCRBP II-2,742-+58-EGFP-1. D: differentiated Caco-2 cells stably transfected with phCRBP II-2,742-+58-EGFP-1. E: undifferentiated Caco-2 cells stably transfected with phCRBP II-745-+58-EGFP-1. F: differentiated Caco-2 cells stably transfected with phCRBP II-745-+58-EGFP-1. G: undifferentiated Caco-2 cells stably transfected with phCRBP II-150-+58-EGFP-1. H: differentiated Caco-2 cells stably transfected with phCRBP II-150-+58-EGFP-1. I: undifferentiated Caco-2 cells stably transfected with phCRBP II-103-+58-EGFP-1. J: differentiated Caco-2 cells stably transfected with phCRBP II-103-+58-EGFP-1. Magnification, ×50.

The 2.8-kb human CRBP II promoter does not contain a functional RARE. To investigate whether functional RAREs are present in the human CRBP II 5'-flanking region, the reporter plasmid phCRBP II-2,742-+58-luc was cotransfected in undifferentiated Caco-2 cells or COS-1 cells, with either the parental expression vector pEF-BOS or the RXR-alpha expression vector. There was no significant retinoic acid induction of luciferase expression in either Caco-2 cells or COS-1 cells cotransfected with the phCRBP II-2,742-+58-luc reporter construct and the parental expression vector pEF-Bos. Cotransfection of the RXR-alpha expression vector did not confer retinoic acid inducibility to the phCRBP II-2,742-+58-luc reporter construct (Table 1). The pCRBPII×2RXRE tk-luciferase construct, which contains two copies of the RXRE from the rat CRBP II promoter, was used as a positive control. Similar to the results reported by Nakshatri and Chambon (28) for their rat RXRE/tk-CAT reporter construct, no retinoic acid induction of luciferase activity was observed when this vector was cotransfected with the parental expression vector pEF-BOS in either Caco-2 cells or COS-1 cells. However, when cotransfected with the RXR-alpha expression vector, retinoic acid induction of luciferase activity was observed in both cells. These results are consistent with the observation that the RXRE in the rat CRBP II promoter is not conserved in the human CRBP II promoter.

                              
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Table 1.   Overexpression of RXR does not upregulate the human CRBP II promoter in transiently transfected Caco-2 and COS-1 cells

There are two potential explanations for why retinoic acid induction of the human CRBP II luciferase construct was not observed in transiently transfected undifferentiated proliferating Caco-2 cells. Since human CRBP II mRNA levels are barely detectable in proliferating undifferentiated Caco-2 cells (11), it is possible that differentiation of the Caco-2 cells is associated with alterations in the levels of other transactivating nuclear factors that regulate the human CRBP II promoter activity. Alternatively, an RXRE similar to the rat CRBP II RXRE may be located beyond 2.8 kb of exon 1 in the human CRBP II promoter.

The 2.8 kb human CRBP II promoter is retinoic acid responsive in postconfluent differentiated stably transfected Caco-2 cells. Transient transfection of Caco-2 cells can only be reliably achieved in proliferating undifferentiated cells. Consequently, these contransfection experiments could not be performed in postconfluent differentiated Caco-2 monolayers. To investigate whether the 2.8-kb fragment of the human CRBP II 5'-flanking region can recapitulate the retinoic acid upregulation of the endogenous human CRBP II gene in differentiated Caco-2 cells, the reporter plasmid phCRBP II-2,742-+58-EGFP-1 was stably transfected into Caco-2 cells and maintained under G-418 selection. The cells were grown to confluence and maintained for 7 days postconfluence. Strong green fluorescence was observed in 7-day-postconfluent, differentiated Caco-2 cells stably transfected with phCRBP II-2,742-+58-EGFP-1 but not in 7-day-postconfluent, differentiated Caco-2 cells stably transfected with the promotorless vector pEGFP-1 (Fig. 3). In preconfluent, differentiated Caco-2 cells stably transfected with phCRBP II-EGFP-1 as well as the promoterless vector pEGFP-1, the fluorescence was barely detectable (Fig. 3). Fluorescence was observed, although at a lower intensity, in postconfluent Caco-2 cells, which were transfected with the phCRBP II-103-+58-EGFP-1 construct (Fig. 3).

To compare the retinoic acid inducibility of the 2.8-kb fragment of the proximal human CRBP II promoter with the endogenous human CRBP II gene, human CRBP II, EGFP-1, and human GAPDH mRNA levels were measured in differentiated 14-day-postconfluent, differentiated Caco-2 cells stably transfected with the phCRBP II-2,742-+58-EGFP-1 construct. Northern blot analyses demonstrated that retinoic acid treatment induced a fourfold increase in EGFP-1 mRNA levels and a threefold increase in endogenous human CRBP II mRNA levels (P < 0.02; n = 3; Fig. 4). Thus this 2.8-kb segment of the human CRBP II promoter recapitulates the retinoic acid response of the endogenous human CRBP II gene in differentiated Caco-2 cells.


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Fig. 4.   Northern blot analysis of the effect of 9-cis-retinoic acid on CRBP II and EGFP-1 mRNA levels in postconfluent, differentiated Caco-2 cells stably transfected with phCRBP II-2,742-+58-EGFP-1. EGFP mRNA was measured to measure the human CRBP II promoter activity of the EGFP-1 reporter construct. Endogenous human CRBP II mRNA levels were measured using a rat CRBP II cDNA probe to measure endogenous human CRBP II promoter activity. The CRBP II and EGFP data were normalized to the corresponding GAPDH signal, with the units for vehicle alone set to 1 (* P < 0.02).

Retinoic acid upregulation of the CRBP II promoter could potentially occur via an indirect mechanism. The caudal-related homeodomain transcription factor Cdx1 has been implicated in transducing the retinoic acid effect on Hox gene transcription (13). Both the caudal-related homeodomain transcription factors Cdx1 and Cdx2 play important roles in intestinal gene expression and intestinal epithelial cell differentiation (7, 14, 35). In Caco-2 cells, human CDX1 expression is barely detectable, and human CDX2 is the predominant caudal-related homeodomain transcription factor (23, 41). CDX2 levels increase shortly after Caco-2 cells reach confluence (10, 43). Cdx2 binding elements have been identified in the gene promoters of enterocytic genes, including sucrase isomaltase, lactase-phlorizin hydrolase, carbonic anhydrase 1, calbindin-D9k, and clusterin (6, 10, 16, 37, 42). In the calbindin-D9k gene (16) and the clusterin gene (37), Cdx2 binding sites have been localized within the TATA box. A similar ATAAA consensus sequence was identified within the TATA box of the human CRBP II promoter. This sequence is evolutionarily conserved in the rat and mouse CRBP II promoter (Table 2).

                              
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Table 2.   Comparison of the caudal-related element sequences

These observations suggest the hypothesis that CDX2 directly induces CRBP II expression and that retinoic acid could indirectly induce CRBP II expression in differentiated Caco-2 cells by upregulating CDX2 expression. As shown in Fig. 5, CDX2 expression is indeed upregulated by retinoic acid treatment of 14- day-postconfluent, Caco-2 cells. This increase was detected by RT-PCR after 35 and 45 cycles (Fig. 5). By densitometry, the increase was estimated to be approximately fourfold. Using previously described human CDX1-specific primers (23), we were unable to detect an amplified fragment when RT-PCR was performed on oligo(dT)-primed cDNA, although a weak band was observed when RT-PCR was performed on cDNA primed with a gene-specific primer (data not shown). However, we were unable to detect any upregulation of the human CRBP II promoter under conditions in which we observed upregulation of the sucrase-isomaltase promoter (-183HSILuc luciferase reporter plasmid) in preconfluent Caco-2 cells transiently transfected with either a mouse Cdx1 or mouse Cdx2 expression vector (Table 3). We were only able to carry out the transfection assays using low amounts of the mouse Cdx1 and mouse Cdx2 expression vectors, because we observed significant cell death when we increased the concentration of the expression vectors further (0.5-1.0 µg).


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Fig. 5.   The effect of retinoic acid on human CDX2 expression in 14- day-postconfluent, differentiated Caco-2 cells. Top: RT-PCR with primers designed to detect human CDX2 were performed on oligo(dT)-primed cDNA prepared from untreated (lane 1) and retinoic acid-treated (lane 2) Caco-2 cells for 45 cycles. Bottom: RT-PCR with primers designed to detect actin were performed on oligo(dT)-primed cDNA prepared from untreated (lane 1) and retinoic acid-treated (lane 2) Caco-2 cells for 28 cycles.


                              
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Table 3.   Overexpression of the Cdx2 and Cdx1 expression vectors does not upregulate the hCRBP II promoter in transiently transfectedCaco-2 cells


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CRBP II expression is limited to the villus absorptive cells of the proximal intestine in the adult animal (5). Its expression in Caco-2 cells is associated with differentiation of the cells after they reach confluence (17, 31). Differentiated Caco-2 cell monolayers incubated in the presence of retinoic acid have increased human CRBP II expression and increased retinol uptake compared with monolayers incubated in the absence of retinoids (11). Since a RXR-specific agonist but not a RAR-specific agonist increased human CRBP II expression, activation of RXR but not RAR is involved in the upregulation. However, sequence analysis and cotransfection experiments with RXR expression vectors indicate that the retinoic acid inducibility occurs independent of the direct effect of RXR on the human CRBP II promoter, in contrast to the rat CRBP II promoter. Furthermore, retinoic acid inducibility of the human CRBP II promoter is observed only in postconfluent differentiated Caco-2 cells and not in proliferating undifferentiated Caco-2 cells.

Upregulation of the CRBP II promoter associated with Caco-2 cell monolayers is observed even when the promoter was truncated to within 103 bp upstream of the transcriptional start site. The results suggest that retinoic acid upregulation of human CRBP II expression in postconfluent differentiated Caco-2 cells occurs via an indirect mechanism, and the mechanism is linked with enterocyte differentiation. RXR-alpha mRNA levels are detectable in Caco-2 cells at 70% confluence but are 1.6-fold greater by 14 days postconfluence (28). Retinoic acid treatment does not alter RXR-alpha mRNA levels in postconfluent differentiated Caco-2 cells (11, 28). Since overexpression of RXR-alpha did not restore retinoic acid inducibility of the human CRBP II promoter in undifferentiated proliferating Caco-2 cells, it is unlikely that the increased RXR-alpha levels in postconfluent Caco-2 cells are sufficient to account for the retinoic acid responsiveness observed with differentiation. Other nuclear receptors, such as hepatocyte nuclear factor 4 (HNF-4) and apolipoprotein AI regulatory protein (ARP-1), have been shown to compete with RAR and RXR for RE3 in the mouse and rat CRBP II promoters (28). HNF-4 is expressed in the small intestine as well as the liver, kidney, and pancreas (34). HNF-4 mRNA levels are increased in differentiated Caco-2 cells but are not modulated by retinoic acid treatment (11). Furthermore, overexpression of HNF-4 has been shown to diminish retinoic acid responsiveness of the mouse and rat CRBP II promoters. Thus it is unlikely that HNF-4 serves as an intermediary for conferring retinoic acid responsiveness to the human CRBP II gene.

Retinoic acid treatment of differentiated Caco-2 cells resulted in increased human CDX2 expression. The evolutionarily conserved region of the human CRBP II promoter contains a potential cdx2 binding site ATAAA within the TATA box. Of note, this consensus sequence is also present in the TATA box of the promoters of two homologous proteins that are expressed in the small intestinal villus absorptive cells: intestinal fatty acid binding protein and liver fatty acid binding protein. This consensus sequence is not present in the promoter of a highly homologous CRBP, which is not expressed in intestinal epithelial cells (21). However, despite increased expression of a human sucrase-isomaltase reporter, expression of the human CRBP II luciferase reporter construct was not increased when mouse cdx1 or cdx2 was overexpressed in undifferentiated Caco-2 cells. The achievable levels of mouse cdx1 or cdx2 expression in undifferentiated Caco-2 cells were limited by reduced cell viability when cells were transfected with greater quantities of the expression vectors. Since differentiation of intestinal cells affects cdx2 signaling pathways by altering cdx2 phosphorylation (12), it is also possible that hypophosphorylation of overexpressed mouse cdx2 in undifferentiated preconfluent Caco-2 cells limited its transcriptional activity.

In summary, we have cloned and sequenced 2.8 kb of the human CRBP II promoter and have found that the rat CRBP II RXRE is not conserved in this segment of human CRBP II promoter. Retinoic acid upregulation of human CRBP II promoter activity is not induced by retinoic acid in transiently transfected undifferentiated Caco-2 cells but is observed in stably transfected differentiated Caco-2 cells. These results suggest that retinoic acid upregulation of human CRBP II promoter activity is mediated indirectly by an intermediary, which is also dependent on enterocyte differentiation.


    ACKNOWLEDGEMENTS

We thank Dr. Daniel Kelly for a critical reading of this manuscript and Aditya Pandey for assistance with Northern blot analysis. Fluorescence microscopy was performed in the Morphology Core of the Washington University Digestive Diseases Research Core Center.


    FOOTNOTES

This work was supported by the Washington University Digestive Diseases Research Core Center (DK-52574) and National Institutes of Health Grants DK-40172 (E. Li), DK-50446 (M. Levin), and CA-81342 (E. Suh).

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number AF338345.

Present address for K. E. Luker: Laboratory of Molecular Radiopharmacology, Mallinkrodt Institute of Radiology, Campus Box 8225, Washington University School of Medicine, St. Louis MO 63110.

Address for reprint requests and other correspondence: E. Li, Division of Gastroenterology, Washington Univ. School of Medicine, Campus Box 8124, 660 South Euclid Ave., St. Louis, MO 63110 (E-mail: eli{at}im.wustl.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.

First published January 16, 2002;10.1152/ajpgi.00041.2001

Received 30 January 2001; accepted in final form 14 January 2002.


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