The actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) are mediated through the nuclear vitamin D receptor (VDR). The regulation of VDR abundance plays an important role in determining the magnitude of the target cell response to 1,25(OH)2D3. The major physiological activity of 1,25(OH)2D3 is the regulation of calcium absorption in the small intestine, and the level of VDR is an important factor in this regulation. However, the characterization of VDR gene expression in the small intestine remains unknown. In the present study, we investigated the regulation of the human VDR (hVDR) gene expression in the small intestine. The 4.0 kb of the 5′-flanking region of the hVDR gene promoter was cloned and characterized by the measurement of luciferase activity and an electrophoretic mobility-shift assay (EMSA). With the EMSA, we found that Cdx-2 (a homeodomain protein-related caudal) binds to the sequence 5′-ATAAAAACTTAT-3′ at −3731 to −3720 bp (hVD-SIF1) relative to the transcription start site of the hVDR promoter. This sequence is very similar to the human sucrase-isomaltase footprint 1 (SIF1) element. With a competition analysis and specific antibodies for Cdx-2, we demonstrated that Cdx-2 is able to activate VDR gene transcription by binding to this element. The mutation of the hVD-SIF1 sequence in the hVDR gene promoter markedly suppressed the transactivation of the reporter gene in Caco-2 cells. In addition, the DNA fragment (−3996 to −3286) containing the hVD-SIF1 binding site increased transcription when placed upstream of the herpes simplex virus thymidine kinase promoter. These findings suggest that Cdx-2 plays an important role in the intestine-specific transcription of the hVDR gene.
1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the most active metabolite of vitamin D, functions to regulate mineral homeostasis.(1) Its effect is exhibited through vitamin D receptor (VDR), which is present in the vitamin D–responsive target organs such as intestine, kidney, and bone.(1-3) VDR is the most abundant in the small intestine and plays a key role in intestinal calcium absorption and cell differentiation.(4,5) In human and rodents after weaning, there is an onset of active calcium transport responsiveness to 1,25(OH)2 D3 as well as a marked increase in intestinal VDR mRNA and serum 1,25(OH)2D3.(6-10) In contrast, VDR levels and calcium transport activity in the intestine decrease with advancing age.(11,12) An interesting hypothesis involves the effect of aging or menopause on the intestinal VDR content as a contributing factor in the development of osteoporosis.(11) In addition, several hormones (retinoic acid, glucocorticoid, estrogen, and 1,25(OH)2D3, among others) and various physiological states have been shown to alter the tissue VDR level.(1) However, the factors controlling the VDR number in the small intestine are poorly understood.
The mechanisms underlying the regulation of VDR abundance include alterations in the rate of transcription of the VDR gene and/or the stability of VDR mRNA, which would be reflected as changes in VDR mRNA levels.(2) To gain a better understanding of the molecular mechanisms by which the VDR is regulated at the transcriptional level, we examined the structural characterization of the human VDR (hVDR) gene and preliminary functional activity of its promoter.(13) In the present study, we further analyzed the function of the hVDR promoter in the intestinal cells and identified the intestinal-specific cis-element of the hVDR gene promoter that interacts with a caudal-related homeodomain transcription factor, Cdx-2.
Several lines of evidence suggest that Cdx genes are important in intestinal gene transcription and epithelial cell differentiation.(14-18) Cdx-2 mediates the transcriptional activation of the intestine-specific gene, sucrase-isomaltase (SI), via an evolutionarily conserved DNA promoter element.(14) Other intestinal genes are also regulated by Cdx-2.(15,16) In addition, the forcedexpression of Cdx-2 in an undifferentiat intestinal epithelial cell line induced morphologic and molecular differentiation.(17) Thus, Cdx-2 is the first identified transcription factor, most likely acting in conjunction with a network of other factors, that is responsible for directing the development and differentiation of intestinal epithelial cells.(18) Based on the results of this study, we suggest that Cdx-2 interacts specifically with functional enhancer elements in the VDR gene in the small intestine and regulates its gene expression.
MATERIALS AND METHODS
Isolation and sequence of the hVDR gene promoter and reporter vector construction
The isolation of the hVDR gene was performed as described previously.(13) The mapping of λ-VDR1 was performed by restriction enzymes (EcoRI, Bam HI, SacI). The 5 ′-flanking region of the hVDR gene promoter was subcloned into pBluscript II SK and sequenced as described previously.(13) Each 5′ -deletion mutant was subcloned into the luciferase reporter vector pGL3 (Promega, Madison, WI, U.S.A.), and its sequences were determined as described previously.(13) Site-directed mutagenesis in the 5′-flanking region of the hVDR gene were performed as described previously.(19) The β-galactosidase expression vector pCMV-β (Clontech, Palo Alto, CA, U.S.A.) was used as an internal control. Each plasmid was purified with a Qiagen plasmid purification kit (Qiagen GmbH, Hilden, Germany).
Caco-2 cells were obtained from the American Type Culture Collection (HTB-37; Rockville, MD, U.S.A.), and HepG2 cells and COS-7 cells were obtained from Riken Cell Bank (Tokyo, Japan). The Caco-2, HepG2, and COS-7 cells were cultured at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (GIBCO BRL, Gaithersburg, MD, U.S.A.) containing 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml of streptomycin. The Caco-2 medium also contained 1% nonessential amino acids (GIBCO BRL).
Transfection and luciferase assays
Cells were transfected using TransIT-LT1 lipofection reagent (Pan Vera Corp., Madison, WI) with 0.3 μg of the luciferase reporter plasmids and 0.2 μg of pCMV-β per 5 × 105 cells.(20) In the Cdx-2 studies, 0.25 μgof the mouse Cdx-2 expression vector pRc/CMV-Cdx-2 (kindly provided by P.G. Traber)(14) was used for cotransfection as described above with 0.25 μg of the luciferase reporter plasmids and 0.1 μg of pCMV- β. In mock cotransfections, pRc/CMV-Cdx-2 was replaced by pRc/CMV. Cells were harvested 24–36 h later in cell lysis buffer, and the lysate was assayed for luciferase activity, β-galactosidase activity, and protein concentrations.(21) Luciferase activity was normalized to the activity of β -galactosidase.
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays (EMSAs) were done using standard methods.(22) A double-stranded nucleotide DNA fragment was synthesized with the annealing of two oligonucleotides. This fragment was labeled with [γ-33P] ATP (110 TBq/mmol; ICN Biomedicals, Costa Mesa, CA, U.S.A.) using T4 polynucleotide kinase (Takara, Shiga, Japan). Extracts were incubated with the radiolabeled probe in binding buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 0.7 mM phenylmethylsulfonyl fluoride, 2.0 mg/ml aprochinin, 2.0 mg/ml pepstatin, 2.0 mg/ml leupeptin) in a final volume of 20 ml for 30 minutes at room temperature. The reaction mixture was then subjected to electrophoresis on a 4% polyacrylamide gel with 1× TAE (40 mM Tris-HCl, 40 mM acetic acid, 1 mM EDTA) as electrode buffer at a constant current of 30 mA for 1.5 h. The gel was dried and analyzed with a Fujix Bio-imaging analyzer BAS-1500 (Fuji-film, Tokyo, Japan).
Northern blot analysis
Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction.(23) Total RNA was denatured by heating for 5 minutes at 70°C in a solution containing 10 mM MOPS (pH 7.0), 5 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, and 50% (v/v) formamide, and subjected to electrophoresis in a 1.0% agarose gel containing 2.2 M formaldehyde. Resolved RNA was transferred to a Hybond-N+ membrane (Amersham, Buckinghamshire, U.K.) and covalently cross-linked to the membrane by exposure to ultraviolet light. Northern analysis was also performed with membrane blots containing size-fractionated polyadenylated RNA from several human tissues (Multiblots I and II; Clontech). Hybridization with32P-labeled cDNA probes was performed in a solution containing 50% formamide, 5× SSPE (saline-sodium phosphate-EDTA), 2× Denhardt's solution, and 1% SDS, after which the membranes were analyzed with the Fuji BAS-1500 system. Probes were labeled with [α-32P]dCTP (110 TBq/mmol; ICN) with the use of a Megaprime labeling system (Amersham).
Analysis of the hVDR gene promoter in various cell lines
To determine the intestinal specific cis-element of the hVDR promoter, 5′ deletions of the hVDR promoters were transfected into Caco-2, which was derived from a colonic adenocarcinoma and, in some respects, models a differentiated enterocyte in cell culture. The transfection of pVDR-4000 markedly stimulated (by 60-fold) the expression of luciferase activity in the Caco-2 cells (Fig. 1). In contrast, other constructed vectors (pVDR-3290, pVDR-1500, and pVDR-100) caused the elevation (by 10- to 20-fold) of the luciferase activity in Caco-2 cells. In the non-intestinal cell line COS-7 and HepG2 cells, the transfection of pVDR-4000 did not show such a marked increase in the luciferase activity. These data suggest that the element from −3996 to −3286 is important for the intestinal-specific transcription of the hVDR gene in the intestine.
Sequence in the hVDR gene promoter
We found several potential binding site transcription factors at − 3996 to −3286 bp in the VDR promoter (Fig. 2A). At −3731 to −3720 bp relative to the transcription start site, the human SI footprint 1 (SIF1)-like sequence, which is known to be as the caudal-related homeodomain protein Cdx-2 binding site, was identified.(14) In addition, the nuclear extract of Caco-2 cells also protected this region by DNAse I foot printing assay (data not shown). However, no homologous sequence for the other known intestinal-specific transcription factor binding elements (e.g, GAGA and HNF-1) were identified.
Binding of Caco-2 nuclear extract to the SIF1 of the hVDR promoter
The SIF1 of the hVDR gene (hVD-SIF1) was investigated by an EMSA with various oligonucleotides as probes and competitors (Fig. 2B). The EMSA demonstrated the formation of a complex between an oligonucleotide containing the hVD-SIF1 and Caco-2 cells nuclear protein (Fig. 3A, lane 2). The formation of this complex was inhibited in the presence of the human sucrase-isomaltase SIF1 oligonucleotide (hSI-SIF1) (Fig. 3A, lanes 5 and 6). Similar observations were obtained in the hVD-SIF1 (Fig. 3A, lanes 3 and 4). In addition, the oligonucleotide containing pig lactase-phlorizin hydrolase intestinal-specific motif (CE-LPH1), which is another Cdx-2 binding sequence, also completely inhibited the DNA binding (Fig. 3A, lanes 7 and 8). To confirm that the hVD-SIF1 sequence 5′-ATAAAAACTTAT-3′ is the target sequence of Cdx-2, we performed EMSAs with oligonucleotides containing specific mutations of this sequence (hVD-SIFM1 and hVD-SIFM2) as competitors of hVD-SIF1. The hVD-SIFM1 oligonucleotide, in which TA in the 5 ′ flanking region of the candidate SIF1 was changed to GC, inhibited the DNA binding. The mutation of the first and second nucleotides (TA→ GC) in 5′ and 3′ of the hVD-SIF1 sequence, hVD-SIFM2, which is the core sequence of the binding of Cdx-2 in the human SI complex gene, did not affect the binding activity (Fig. 3A, lanes 9–12).
We next analyzed whether the DNA–protein complex observed in EMSA using the hVD-SIF1 oligonucleotide is due to Cdx-2 protein binding. When the hVD-SIF1 oligonucleotide was incubated with the nuclear extract of COS-7 cells transfected into the mouse Cdx-2 expression vector, we observed the same protein–DNA complex in EMSA (Fig. 3B, lane 6), but not in the COS-7 cells expressing the control vector (Fig. 3B, lane 5). The formation of this complex was markedly inhibited in the presence of the Cdx-2 antibody (cdx2) (Fig. 3B, lanes 3, 7, and 10), but not the c-Fos antibody (c-fos) (Fig. 3B, lane 4).
Effect of the SIF1 element in the hVDR gene promoter
To elucidate the functional role of the SIF1 sequence in the hVDR promoter, we constructed the vector containing the mutation (M1 and M2) on the core sequence of this element (Fig. 2B). As shown in Fig. 4, the basic promoter activity was decreased to 40% and 70%, respectively, relative to that in the wild-type promoter. The results of the functional analysis were consistent with those of the EMSA. These observations clearly indicated that the core sequence for Cdx-2 binding is important in the transactivation of the hVDR gene promoter.
Effects of the hVD-SIF1 element in transcriptional activation
To examine the function of the hVD-SIF1 element in transcriptional activation, we performed transient-transfection experiments with COS-7 cells which neither express SIF1 binding protein nor support SI gene transcription. A minimum intestine-specific promoter containing the hVD-SIF1 element and the GC box (base −103 to +77) was linked to the luciferase gene and used as a reporter construct in cotransfection experiments with an expression vector for Cdx-2. The coexpression of the Cdx-2 with the chimeric promoter-reporter gene construct in COS-7 cells markedly stimulated the transcription of the hVDR promoter. However, we did not observe the transcriptional activation in Caco-2 cells transfected into the Cdx-2 expression vector. The result may be due to the presence of the large amounts of endogenous Cdx-2 protein in Caco-2 cells.
Experiments were conducted to determine whether the hVD-SIF1 element was capable of activating a heterologous promoter. The herpes simplex virus thymidine kinase promoter was chosen because it has a transcriptional activity in multiple cell types. The DNA fragment containing the hVD-SIF1 binding site increased transcription when placed upstream of the thymidine kinase promoter. In addition, the vectors containing reversed sequence (3 ′-5′ direction) stimulated the luciferase activity (Fig. 5).
Tissue distributions and developmental changes in VDR and Cdx-2
The distributions of VDR and Cdx-2 transcripts were determined with the use of a human multiple tissue RNA filter (in Materials and Methods). As shown in Fig. 6A, VDR mRNA was expressed in various tissues. The relative intensity of the VDR transcript was strongest in the small intestine and colon. The VDR transcript was abundant in kidney, lung, placenta, and pancreas. In addition, VDR mRNA was detected in the human intestine-like Caco-2 cells (Fig. 6B). In contrast, Cdx-2 mRNA was detected only in the small intestine, colon, and pancreas (Fig. 6A).
In addition, The VDR mRNA levels were detectable during neonatal development in the small intestine and gradually increased from low levels at 7–14 days postpartum to approximately adult levels at 21–28 days of age in the suckling rats (data not shown). In contrast, the intestinal Cdx-2 mRNA levels were not changed (data not shown).
The regulation of VDR abundance plays an important role in determining the magnitude of the target cell response to 1,25(OH)2D3.(1) The mechanisms controlling tissue VDR levels are not entirely clear and could be dependent on several factors.(24) Using an intestinal cell line that expresses Cdx-2 as a model system, we demonstrated that the Cdx-2 binding sites (hVD-SIF1) in the upstream region of the hVDR gene promoter regulate the expression of the VDR gene in the small intestine.
A previous study showed that the hVDR promoter lacks consensus TATA or CAAT boxes, and that this region is GC-rich with five binding motifs for the transcription factor SP1 lying between nucleotide −72 and −34 relative to the transcription start site.(13) Potential binding sites for other transcription factors are also evident. To evaluate the transcriptional capacity of the hVDR promoter, we cloned a series of 5′-deletion fragments of the gene into a luciferase reporter gene and transfected them individually into three mammalian cell lines, Caco-2, HeLa, and ROS 17/2.8 cells. The 5′ deletion of the hVDR promoter from −103 bp to −34 bp that removed four of the five GC boxes near the initiator resulted in a 10-fold drop in activity. These elements play an important role in the activity of the hVDR promoter. However, the VDR gene is predominantly expressed in the intestine and colon. To examine further the intestinal-specific cis-element in the VDR gene, we cloned a larger fragment of the hVDR gene promoter and determined its activity. The analysis of the promoter of the hVDR gene by an in vitro foot printing assay identified several transcriptional factors (data not shown). These factors were found mainly in the intestine, liver, and kidney. We showed here that the hVDR promoter contains AT-rich subdomains that bind proteins present in the intestine and colon. The EMSA experiments using specific antisera identified the homeobox protein Cdx-2 as a major component of the DNA–protein complex in Caco-2 cells. Mutations of the SIF1 element that decreased Cdx-2 binding also resulted in decreased hVDR promoter activity in Caco-2 cells. These findings suggest that Cdx-2 mediates the activation of the hVDR promoter in the intestine and colon.
Cdx-2 has been implicated in the control of cell proliferation and differentiation in the intestine as well as in the regulation of intestinal-specific gene transcription, at least for the SI, calbinbin-D9k, and lactase genes.(14-17) Cdx-2 is also able to bind and activate the rat insulin I gene promoter.(25) In addition, recent reports indicate that Cdx-3 is involved in glucagon gene expression in both the endocrine pancreas and in enteroendocrine cell lines expressing glucagon. In both cell types, Cdx-2/3 was shown to transactivate the glucagon gene promoter.(26,27)
The sequence of the hVD-SIF1 element is quite similar to that of the hSI-SIF1. The core region of the Cdx-2 binding site in the SI promoter is an inverted repeat ATAAA, separated by two nucleotides. Each half-site is capable of interacting with a single molecule of Cdx-2, forming a monomer. The Cdx-2 protein also binds as a dimer to the whole element, as influenced by the redox state. Recent studies have demonstrated that the interaction of Cdx-2 with the SIF1 element may occur through either a monomer or homodimer conformation, with the extent of homodimer formation dependent on the redox state of the binding reaction.(28) The redox regulation of homeodomain binding appears to be an increasingly common mode of regulating cooperative DNA binding and appears to be dependent on the presence of a specific cysteine residue in homeodomain proteins.
Replacement of the AT-rich sequences in the 3′ half-site clearly disrupts Cdx-2 binding to the hVD-SIF1 element and suppresses the activation of the VDR promoter, suggesting that the interaction of Cdx-2 with its target sequences is specific. Although we detected only a single Cdx-2 complex with the hVD-SIF1 probe and failed to observe evidence of multiple Cdx-2 complexes when using intestinal extracts, this may potentially be attributable to the redox state of the binding reactions or to the structure of the VDR gene Cdx-2 binding site. The multiple Cdx-2 complexes were obtained in the proglucagon gene Cdx-2 binding site when islet extract was used.(26)
VDR regulation shows pronounced tissue- and cell-specific variations.(24) Tissue-specific developmental patterns of VDR mRNA expression and tissue- andspecies-specific patterns of glucocorticoid and 1,25(OH)2D3 responsiveness have been reported.(4,29,30) There are many examples of tissue-specific regulation in the vitamin D–endocrine system in addition to VDR.(24) The mechanisms of the tissue-specific VDR regulation are not fully understood. The mechanisms contributing to these variations could be dependent on several factors, including the state of differentiation of target cells, differences in the intracellular signaling pathways activated in these cells by the regulator, and differences in the interaction with the downstream nuclear proteins involved in the regulation of gene transcription in various target cells.(24) Intestinal-specific transcription factor Cdx-2 may be involved in tissue-specific regulation of the VDR.
In the small intestine, the regulation of VDR gene expression has been well studied regarding the calcium absorption in the neonatal development.(12) VDR controls the expression of calbindin-D9k, which is important to intestinal calcium transport, because changes in the amount of VDR mRNA are known to be associated with intestinal calcium transport.(11)
A recent study suggested that Cdx-2 regulates the expression of the calbindin-D9k gene.(15) The alignment of the sequences of rat calbindin-D9k element A, the SI gene SIF1 element, and the lactase gene CE-LPH1 with this consensus sequence reveals two binding sites in a direct repeat in the calbindin-D9k gene, two binding sites in an inverted repeat in the SI gene, and one binding site in the lactase gene.(15) Cdx-2 binds to the 3′ half-site of the calbindin-D9k element A, and another protein, probably belonging to the same homeodomain family, binds to the 5′ half-site.(15) The author concluded that VDR and Cdx-2 may be factors for intestine-specific calbindin-D9k gene expression.(15)
Recent studies suggest that the coactivator protein that interacts with the Cdx-2 activation domain might provide a second level of regulatory control over Cdx-2–responsive genes.(31) Indeed, developmental changes in VDR and calbindin-D9k occurred after weaning in rats, while the intestinal Cdx-2 mRNA levels were not changed (data not shown). This may involve coactivation proteins which are able to regulate Cdx-2 function on the VDR gene promoter. In addition, the establishment of stable intestinal epithelial IEC-6 cell lines expressing high levels of mouse Cdx-2 was followed by proliferation arrest and differentiation in association with the expression of Cdx-2.(17) The proliferation arrest was terminated after several days, despite the continuing expression of Cdx-2. Although no induction of the Cdx-2–responsive SI gene was observed after 40 days of culture, IEC-6 cells expressing Cdx-2 for more than 50 days did express SI gene transcripts, suggesting an indirect link between the prolonged expression of Cdx-2 and the activation of endogenous target gene expression.(17) We suspected that a coactivator for Cdx-2 may allow cell-specific and spatial regulation of Cdx-2–dependent genes in a manner independent of Cdx-2 expression. Indeed, when the Cdx-2 activation domain was linked to the Gal4 DNA binding domain, the chimeric protein was able to activate Gal4 enhancer constructs in an intestinal cell line but was unable to activate transcription in NIH-3T3 cells.(31) These data suggest that there are cell-specific factors that allow the Cdx-2 activation domain to function in the activation of enhancer elements. Thus, identification of cell-specific factors may be important to clarify the regulation of the VDR gene expression in the small intestine.
Finally, the identification of Cdx-2 as a major component of the hVDR gene expression provides an important first step toward the identification of the specific binding proteins that regulate VDR promoter specificity in intestinal cells. It is possible that 1,25(OH)2D3 and glucocorticoid may regulate intestinal-specific transcription factors such as Cdx-2. If this is true, we can explain why VDR regulation shows pronounced tissue- and cell-specific variations. Further study is needed to clarify the regulation of intestinal VDR gene expression.
We thank Dr. P.G. Traber for kindly providing the mouse Cdx-2 expression vector pRc/CMV-Cdx-2 and the mouse Cdx-2 antiserum. We also thank Dr. Hisato Kondo at Osaka University for kindly providing the minimum promoter of herpes simplex virus thymidine kinase gene. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, the Setsuro Fujii Memorial Foundation, the Uehara Memorial Foundation, and the Salt Science Research Foundation.