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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ABSTRACT |
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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
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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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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- 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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METHODS |
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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- 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 HCloning 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 HTransient 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
-galactosidase expression vector pEF1/V5-His/LacZ (Invitrogen,
Carlsbad, CA) was used as an internal control for transfection efficiency.
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.
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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RESULTS |
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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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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.
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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
II2,742-+58-luc was cotransfected in
undifferentiated Caco-2 cells or COS-1 cells, with either the parental
expression vector pEF-BOS or the RXR-
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-
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-
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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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 II2,742-+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).
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DISCUSSION |
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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- 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-
mRNA levels in
postconfluent differentiated Caco-2 cells (11, 28). Since overexpression of RXR-
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-
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Boehm, MF,
Zhang L,
Badea BA,
White SK,
Mais DE,
Berger E,
Suto CM,
Goldman ME,
and
Heyman RA.
Synthesis and structure-activity relationships of novel retinoid X receptor-selective retinoids.
J Med Chem
37:
2930-2941,
1994[ISI][Medline].
2.
Chenchik, A,
Diachenko L,
Moqadam F,
Tarabykin V,
Lukyanov S,
and
Siebert PD.
Full-length cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-ligated cDNA.
Biotechniques
21:
526-534,
1996[ISI][Medline].
3.
Chomczynski, P,
and
Sacchi N.
Single step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extractions.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
4.
Crettaz, M,
Baron A,
Siegenthaler G,
and
Hunziker W.
Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.
Biochem J
272:
391-397,
1990[ISI][Medline].
5.
Crow, A,
and
Ong DE.
Cell-specific immunohistochemical localization of a cellular retinol-binding protein (type two) in the small intestine of rat.
Proc Natl Acad Sci USA
82:
4707-4511,
1985[Abstract].
6.
Drummond, F,
Sowden J,
Morrison K,
and
Edwards YH.
The caudal-type homeobox protein Cdx-2 binds to the colon promoter of the carbonic anhydrase 1 gene.
Eur J Biochem
236:
670-681,
1996[Abstract].
7.
Duprey, P,
Chowdhury K,
Dressler GR,
Balling R,
Simon D,
Guenet JL,
and
Gruss P.
A mouse gene homologous to the Drosophila gene caudal is expressed in epithelial cells from the embryonic intestine.
Genes Dev
2:
1647-1654,
1988[Abstract].
8.
Ee, HC,
Erler T,
Bhathal PS,
Young GP,
and
James RJ.
Cdx-2 homeodomain protein expression in human and rat colorectal adenoma and carcinoma.
Am J Pathol
147:
586-592,
1995[Abstract].
9.
Engle, MJ,
Goetz GS,
and
Alpers DH.
Caco-2 cells express a combination of colonocytes and enterocyte phenotypes.
J Cell Physiol
174:
362-369,
1998[ISI][Medline].
10.
Fang, R,
Santiago NA,
Olds LO,
and
Sibley E.
The homeodomain protein Cdx-2 regulates lactase gene promoter activity during enterocyte differentiation.
Gastroenterology
118:
115-127,
2000[ISI][Medline].
11.
Haas, J,
Park EC,
and
Seed B.
Codon usage limitation in the expression of HIV-1 envelope glycoprotein.
Curr Biol
6:
315-324,
1996[ISI][Medline].
12.
Houde, M,
Laprise P,
Jean D,
Blais M,
Asselin C,
and
Rivard N.
Intestinal epithelial differentiation involves activation of p38 mitogen-activated protein kinase that regulates the homeobox transcription factor cdx2.
J Biol Chem
276:
21885-21894,
2001
13.
Houle, M,
Prinos P,
Iuliamnella A,
Bouchard N,
and
Lohnes D.
Retinoic acid regulation of Cdx1: an indirect mechanism for retinoids and vertebral specification.
Mol Cell Biol
20:
6579-6586,
2000
14.
James, R,
Erler T,
and
Kazenwadel J.
Structure of the murine homeobox gene cdx-2. Expression in embryonic and adult intestinal epithelium.
J Biol Chem
269:
15229-15237,
1994
15.
Kliewer, SA,
Umesono K,
Noonan DJ,
Heyman RA,
and
Evans RE.
Convergence of 9-cis retinoic acid and peroxisome proliferator signaling pathways through heterodimer formation of their receptors.
Nature
358:
771-774,
1992[ISI][Medline].
16.
Lambert, M,
Colnot S,
Suh E,
L'Horset FL,
Blin C,
Calliot ME,
Raymondjean M,
Thomasset M,
Traber PG,
and
Perret C.
Cis-acting elements and transcription factors involved in the intestinal specific expression of the rat calbindin-D9k gene: binding of the intestine-specific transcription factor Cdx-2 to the TATA box.
Eur J Biochem
236:
778-788,
1996[Abstract].
17.
Levin, MS.
Cellular retinol-binding proteins are determinants of retinol uptake and metabolism in stably transfected Caco-2 cells.
J Biol Chem
268:
8267-8276,
1993
18.
Levin, MS,
and
Davis AE.
Retinoic acid increases cellular retinol binding protein II mRNA and retinol uptake in the human intestinal Caco-2 cell line.
J Nutr
127:
13-17,
1997
19.
Levin, MS,
Li E,
Ong DE,
and
Gordon JI.
Comparison of the tissue-specific expression and developmental regulation of two closely linked rodent genes encoding cytosolic retinol-binding proteins.
J Biol Chem
262:
7118-7124,
1987
20.
Li, E,
Demmer LA,
Sweetser DA,
Ong DE,
and
Gordon JI.
Rat cellular retinol-binding protein II: use of a cloned cDNA to define its primary structure, tissue-specific expression, and developmental regulation.
Proc Natl Acad Sci USA
83:
5779-5783,
1986[Abstract].
21.
Li, E,
and
Norris AW.
Structure/function of cytoplasmic vitamin A binding proteins.
Annu Rev Nutr
16:
205-234,
1996[ISI][Medline].
22.
Lissoos, TW,
Davis AE,
and
Levin MS.
Vitamin A trafficking in Caco-2 cells stably transfected with cellular retinol binding proteins.
Am J Physiol Gastrointest Liver Physiol
268:
G224-G231,
1995
23.
Lorentz, O,
Duluc I,
Arcangelis AD,
Simon-Assmann P,
Kedinger M,
and
Freund JN.
Key role of the Cdx2 homeobox in extracellular matrix-mediated intestinal cell differentiation.
J Cell Biol
139:
1553-1556,
1997
24.
Mangelsdorf, DJ,
and
Evans RM.
The RXR heterodimers and orphan receptors.
Cell
83:
841-850,
1995[ISI][Medline].
25.
Mangelsdorf, DJ,
Umesono K,
Kliewer SA,
Borgmeyer U,
Ong ES,
and
Evans RM.
A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR.
Cell
66:
555-561,
1991[ISI][Medline].
26.
Medin, JA,
Minucci S,
Driggers PH,
Lee IJ,
and
Ozato K.
Quantitative increases in DNA binding affinity and positional effects determine 9-cis retinoic acid induced activation of the retinoid X receptor beta homodimer.
Mol Cell Endocrinol
105:
27-35,
1994[ISI][Medline].
27.
Mizushima, S,
and
Nagata S.
pEF-BOS, a powerful mammalian expression vector.
Nucleic Acids Res
18:
5322,
1990[ISI][Medline].
28.
Nakshatri, H,
and
Chambon P.
The directly repeated RG(G/T)TCA motifs of the rat and mouse cellular retinol-binding protein II genes are promiscuous binding sites for RAR, RXR, HNF-4, and ARP-1 homo- and heterodimers.
J Biol Chem
269:
890-902,
1994
29.
Ong, DE.
A novel retinol-binding protein from rat. Purification and partial characterization.
J Biol Chem
259:
1476-1482,
1984
30.
Pinto, MS,
Robine-Leon S,
Appay MD,
Kedinger M,
Triadou E,
Dussaulx E,
Lacroix B,
Simon-Assman P,
Haffen K,
Fogh J,
and
Zweibaum A.
Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture.
Biol Cell
47:
323-330,
1983[ISI].
31.
Quick, TC,
and
Ong DE.
Vitamin A metabolism in the human intestinal Caco-2 cell line.
Biochemistry
29:
11116-11123,
1990[ISI][Medline].
32.
Robinson, CE,
Wu X,
Morris DC,
and
Gimble JM.
DNA bending is induced by binding of the peroxisome proliferator-activated receptor gamma 2 heterodimer to its response element in the murine lipoprotein lipase promoter.
Biochem Biophys Res Commun
244:
671-672,
1998[ISI][Medline].
33.
Rosenthal, N.
Identification of regulatory elements of cloned genes with functional assays.
Methods Enzymol
152:
704-720,
1987[ISI][Medline].
34.
Sladek, FM,
Zhong WM,
La E,
and
Darnell JEJ
Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.
Genes Dev
4:
2353-2365,
1990[Abstract].
35.
Suh, ER,
Chen L,
Taylor J,
and
Traber PG.
An intestine-specific homeobox gene regulates proliferation and differentiation.
Mol Cell Biol
16:
619-625,
1996[Abstract].
36.
Suh, ER,
and
Traber PG.
A homeodomain protein related to caudal regulates intestine-specific gene transcription.
Mol Cell Biol
14:
7340-7351,
1994[Abstract].
37.
Suh, E,
Wang Z,
Swain GP,
Tenniswood M,
and
Traber PG.
Clusterin gene transcription is activated by caudal-related homeobox genes in intestinal epithelium.
Am J Physiol Gastrointest Liver Physiol
280:
G149-G156,
2001
38.
Suruga, K,
Mochizuki K,
Suzuki R,
Goda T,
and
Takase S.
Regulation of cellular retinol-binding protein type II gene expression by arachidonic acid analogue and 9-cis retinoic acid in Caco-2 cells.
Eur J Biochem
262:
70-78,
1999
39.
Suzuki, R,
Suruga K,
Goda T,
and
Takase S.
Peroxisome proliferator enhances gene expression of cellular retinol-binding protein, type II in Caco-2 cells.
Life Sci
62:
861-871,
1998[ISI][Medline].
40.
Taylor, JK,
Levy T,
Suh ER,
and
Traber PG.
Activation of enhancer elements by the homeobox gene Cdx2 is cell line specific.
Nucleic Acids Res
25:
2293-2300,
1997
41.
Traber, PG,
Wu GD,
and
Wang W.
Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.
Mol Cell Biol
12:
3614-3627,
1992[Abstract].
42.
Troelson, JT,
Mitchelmore C,
Spodsberg N,
Jensen AM,
Noren O,
and
Sjostrom H.
Regulation of lactase-phlorizin hydrolase gene expression by the caudal-related homeodomain protein Cdx-2.
Biochem J
322:
833-838,
1997[ISI][Medline].
43.
Ye, H,
Kelly TF,
Samadani U,
Lim L,
Rubio S,
Overdier DG,
Roebuck KA,
and
Costa RH.
Hepatocyte nuclear factor 3/fork head homolog 11 is expressed in proliferating epithelial and mesenchymal cells of embryonic and adult tissues.
Mol Cell Biol
17:
1626-1641,
1997[Abstract].
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