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The Human Transient Receptor Potential Vanilloid Type 6 Distal Promoter Contains Multiple Vitamin D Receptor Binding Sites that Mediate Activation by 1,25-Dihydroxyvitamin D₃ in Intestinal Cells

Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706

Address all correspondence and requests for reprints to: J. Wesley Pike, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706. E-mail: pike{at}biochem.wisc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Transient receptor potential vanilloid type 6 (TRPV6) (ECAC2, CaT1) is the major ion channel in intestinal epithelial cell membranes responsible for calcium entry. Its expression is actively regulated at the transcriptional level by 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃]. In this report, we identify mechanisms integral to the regulation of TRPV6 by 1,25-(OH)₂D₃. Based upon the hormonal responsiveness of a 7-kb TRPV6 promoter fragment in intestinal cell lines, we used a chromatin immunoprecipitation (ChIP) scanning method to search for possible vitamin D receptor (VDR) and retinoid X receptor (RXR) regulatory regions within the TRPV6 locus. VDR/RXR binding was broad, ranging from –1.2 to –5.5 kb relative to the start site of TRPV6 transcription. These results were consistent with an in silico analysis that revealed putative regulatory elements (VDREs) located at –1.2, –2.1, –3.5, –4.3, and –5.5 kb. Despite the ChIP analyses, only regions of the TRPV6 gene that contained putative elements at –2.1 and –4.3 kb transferred 1,25-(OH)₂D₃ response to a heterologous promoter. Further study revealed that each of these two active regions contained composite VDREs comprised of two separate regulatory elements. Mutagenesis of the VDREs within the –2.1- and –4.3-kb region and the VDRE at –1.2 kb abrogated all response to 1,25-(OH)₂D₃ when examined within the natural TRPV6 promoter. A final ChIP assay revealed that VDR/RXR heterodimer binding to the TRPV6 gene was accompanied by both the recruitment of steroid receptor coactivator 1 as well as a broad change in histone 4 acetylation. These studies identify a mechanism by which 1,25-(OH)₂D₃ regulates the expression of TRPV6 in human intestinal cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
THE TRANSPORT OF calcium across epithelial cells such as those found lining the intestinal mucosa or in the distal renal nephron is a complex multistep process that involves the cellular uptake of calcium at the apical surface, the transfer of this cation through the cytoplasmic space, and its active extrusion across the basolateral cell membrane and into the blood (1). The components involved in the latter two phenomenon include calbindin D9K (D9K) and D28K (2) and the calcium-stimulated plasma membrane ATPase PMCA1b (3). Interestingly, those operable at the apical surface of the epithelial cell have only recently been identified, and include the two ion channels transient receptor potential vanilloid (TRPV) type 5 (TRPV5/CaT2/ECAC1) and type 6 (TRPV6/CaT1/ECAC2) (4, 5, 6). These cellular components are members of a large superfamily of non-voltage-gated cation channels that function as sensory receptors for temperature, touch, osmolarity, pheromones, and a wide range of additional external stimuli (7, 8). TRPV5 is expressed predominantly in the apical membranes of certain kidney cells whereas TRPV6 is expressed in a similar cellular location in the mucosa of the small intestine (8). Both genes are also expressed in additional tissues including bone cells and prostate tumor cells (9, 10). Because the entry of calcium into epithelial cells represents the rate-limiting step in transepithelial ion transport, the expression and regulation of the TRPV5 and TRPV6 genes in both the intestine and kidney, as well as in bone, are central to calcium homeostasis.

The ability of 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] to maintain calcium and phosphorus homeostasis via promotion of intestinal calcium absorption and renal calcium reabsorption was linked in early studies to its capacity to induce the expression of D9K and/or D28K as well as the calcium ATPase PMCA1b (2, 3). More recently, however, evidence has emerged to indicate that the regulation of TRPV5 and TRPV6 expression may be even more integral to the capacity of 1,25-(OH)₂D₃ to regulate the homeostasis of calcium. The most conclusive evidence derives from studies of 25-hydroxyvitamin D₃-1 hydroxylase-deficient (11) and vitamin D receptor (VDR)-deficient (12) mice, wherein these animals exhibit significant reductions in renal TRPV5 and intestinal TRPV5 and TRPV6 expression. Most importantly, these changes in mRNA expression correlate directly with a reduction in the absorption of calcium from the intestine and the reabsorption of the cation from the kidney. Of additional significance is the work of Song et al. (13), who demonstrated that 1,25-(OH)₂D₃ was able to induce TRPV6 and D9K gene expression in the intestine and TRPV5, D9K, and D28K expression in the kidney of wild-type mice in both a time- and dose-dependent manner. The ability of 1,25-(OH)₂D₃ to induce TRPV6 gene expression and to stimulate the transport of calcium has also been demonstrated in vitro in human colon-derived Caco2 cells (14). Collectively, these studies suggest that 1,25-(OH)₂D₃ regulates the expression of the TRPV genes and that these gene products are critical for intestinal absorption and renal reabsorption of calcium.

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] is a steroid hormone, the cellular actions of which are mediated through the VDR, a ligand-induced transcription factor that binds generally, but perhaps not exclusively, as a retinoid X receptor (RXR) heterodimer to vitamin D response elements (VDREs) located upstream of hormone-responsive gene promoters (15). This binding initiates a complex series of events within chromatin that culminates in altered gene expression. The molecular actions of 1,25-(OH)₂D₃ are typical of steroid hormones in general, including 17ß-estradiol, testosterone, the thyroid hormone, and the glucocorticoids (16, 17).

The molecular mechanism by which 1,25-(OH)₂D₃ induces up-regulation of the TRPV5 and TRPV6 genes is currently unknown. Putative vitamin D-responsive DNA sequences or VDREs reminiscent of those found in other vitamin D-regulated genes have been identified within the promoter region of the TRPV5 gene using computer-based strategies (18, 19). The functional relevance of these and other DNA sequence elements, however, has not been established, although a recent study described the activity of a proximal TRPV6 element (20). We report herein studies in two human colon carcinoma cell lines, which describe the mechanism whereby 1,25-(OH)₂D₃ regulates the expression of TRPV6. We show through the use of both VDR and RXR chromatin immunoprecipitation (ChIP) scanning methods as well as traditional promoter and mutational analyses that 1,25-(OH)₂D₃ is able to induce the expression of TRPV6 via at least four, and one minor, regulatory elements dispersed within the first 4.3 kb of the gene’s promoter. The functionality of the VDR/RXR binding sites in these regions was further established through additional ChIP analyses, which revealed that VDR/RXR binding correlated with the recruitment of steroid receptor coactivator 1 (SRC-1) and the enhancement of acetylation at histone 4. Our results point to at least five regions within the TRPV6 gene that collectively mediate the stimulatory actions of 1,25-(OH)₂D₃.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
1,25-(OH)₂D₃ Induces 25-Hydroxyvitamin D₃-24 Hydroxylase (CYP24A1) and TRPV6 Gene Expression in Intestinal Cells
Previous studies have indicated that 1,25-(OH)₂D₃ can induce the expression of CYP24A1 and TRPV6 in Caco2 cells (14). We therefore explored this induction process both in Caco2 as well as LS180 cells, the latter an equally well-studied colon cancer cell line. As seen in Fig. 1A, 1,25-(OH)₂D₃ induced the level of RNA transcripts, in a dose-dependent manner, for both TRPV6 and our control CYP24A1 gene in both Caco2 and LS180 cells. These results indicate that both cell lines are indeed useful models for exploring the regulation of TRPV6 by 1,25-(OH)₂D₃. To ensure that 1,25-(OH)₂D₃ induction was mediated via the VDR, we employed an interference assay using VDR short interfering RNA (siRNA) to knock down VDR expression (Fig. 1B). LS180 cells were first transfected with either nontargeted or cyclophilin B control siRNAs or an siRNA duplex that targeted the human VDR. A mock transfection control was also used. After 72 h, the cells were treated with either vehicle or 1,25-(OH)₂D₃ for an additional 6 h, and the isolated RNA was then analyzed for target gene expression. As can be seen in the figure, cyclophilin B and VDR siRNAs were effective in reducing the expression of cyclophilin B and VDR, respectively. Most importantly, whereas 1,25-(OH)₂D₃ was fully capable of inducing TRPV6 and CYP24A1 expression in mock-transfected cells as well as those transfected with nontargeted or cyclophilin B siRNAs, the reduction in VDR transcripts (and protein, data not shown) by VDR siRNA significantly reduced TRPV6 expression. This finding indicates that the VDR is indeed central to the ability of 1,25-(OH)₂D₃ to induce TRPV6 expression.

View larger version (71K): [in this window] [in a new window]   Fig. 1. 1,25-(OH)2D3 Induces Expression of CYP24A1 and TRPV6 in Human Caco2 and LS180 Cells A, Cells were seeded into six-well plates and treated for 12 h with either vehicle (NT) or increasing concentrations of 1,25-(OH)2D3 (10–10 to 10–7 M). Cells were harvested and used to prepare total RNA. RNA was reverse transcribed and subjected to PCR with primers (see Table 2) designed to amplify the appropriate DNA fragments of CYP24A1 (27 cycles), TRPV6 (27 cycles), or ß-actin (25 cycles). B, VDR siRNA causes a decrease in CYP24A1 and TRPV6 target gene expression. LS180 cells were seeded into six-well plates, transfected, and allowed to grow for 72 h. Cells were then treated for an additional 6 h with ethanol vehicle (–) or 10–7M 1,25-(OH)2D3 (+). RNA was isolated, reverse transcribed, and analyzed via PCR using the number of cycles indicated: CYP24A1 (27 cycles), TRPV6 (27 cycles), Cyclophilin B (18 cycles), VDR and ß-actin (25 cycles). CycloB, Cyclophilin B.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Transcriptional Activation of the Native TRPV6 Promoter by 1,25-(OH)2D3 A, Schematic diagram of the hTRPV6 (–1.3 kb/+165) luc and the hTRPV6 (–7 kb/+160) luc promoter constructs. B, 1,25-(OH)2D3 induces hTRPV6 (–7 kb/+160) luc but not hTRPV6 (–1.3 kb/+165) luc. LS180 cells were cotransfected with pCH110-ßgal and either pGL3-basic, hTRPV6 (–1.3 kb/+165), or hTRPV6 (–7 kb/+160). Cells were treated with either vehicle (NT) or increasing concentrations of 1,25-(OH)2D3 (10–9 M to 10–7 M) and evaluated for both luciferase and ß-gal activity as described in Materials and Methods. Each point represents the normalized relative light unit average ± SEM of a quadruplicate set of transfections and is representative of at least two independent experiments. Statistical analysis: one-way ANOVA with Dunnett’s posttest (**, P < 0.001; *, P < 0.05) C, LS180 cells were cotransfected with 50 nM siRNA duplexes composed of either nontargeting scrambled sequence, cyclophilin B (positive control), or human VDR and either pCH110-ßgal and (C) rCyp24a1-LUC or (D) hTRPV6 (–7 kb/+160). Cells were treated and processed as described in panel B. CycloB, Cyclophilin B; si, short interfering.

View larger version (71K): [in this window] [in a new window]   Fig. 3. ChIP Analysis Reveals 1,25-(OH)2D3-Induced Localization of VDR and RXR to a Broad Upstream Region of the TRPV6 Gene A, Schematic diagram of the upstream region of the TRPV6 gene with the gene positions and sequences of putative VDREs as determined by the CONSITE program. B, Specific locations of the amplification primers used to detect upstream fragments of the TRPV6 promoter are indicated by arrows. +1 (TSS) represents the most 5'-nucleotide found in the mRNA transcript (216 bp from the start site of translation). C, ChIP analysis of 1,25-(OH)2D3-induced VDR and RXR binding to regions of the TRPV6 and Cyp24 promoters. Caco2 and LS180 cells were seeded into 100-mm plates and allowed to become confluent. Cells were then treated for 3 h with either vehicle or 1,25-(OH)2D3 (10–7 M). Cross-linked cell lysates were subjected to immunoprecipitation with antibodies to VDR, RXR, or IgG as indicated in Materials and Methods. DNA precipitates were isolated and then subjected to PCR (28 cycles) using the primers indicated in panel B, the sequences of which are indicated in Table 3. Detection of input DNA (0.2%) was performed before precipitation using the appropriate primers. D, VDR/RXR occupancy at the CYP24A1 and TRPV6 promoters in response to 1,25-(OH)2D3 is reduced in the presence of VDR siRNA. LS180 cells were transfected with the indicated RNA duplexes for 72 h, treated for an additional 3 h with ethanol vehicle or 10–7M 1,25-(OH)2D3, and then subjected to ChIP analysis using antibodies to VDR, RXR, or IgG. The DNA precipitates were isolated and then treated as described in panel C. cycloB, Cyclophilin B; ORF, open reading frame.

View larger version (37K): [in this window] [in a new window]   Fig. 4. DNA Fragments Containing the Putative –2.1- and –4.3-kb VDREs Are Active in 1,25-(OH)2D3-Induced Transcription A, LS180 cells were transfected with pCH110-ßgal and either tkluc, –5.5, –4.3, –3.5, –2.1, or –1.2tkluc (as illustrated to the left) and their activities evaluated in the absence (NT, vehicle) or presence of increasing concentrations 1,25-(OH)2D3 (10–9 M to 10–7M) as described in Materials and Methods. Each point represents the normalized relative light unit average ± SEM of a quadruplicate set of transfections and is representative of at least three independent experiments. Fold induction is indicated above each group as compared with the control groups. Statistical analysis: one-way ANOVA with Dunnett’s posttest (**, P < 0.001; *, P < 0.05). B, LS180 cells were transfected with pCH110-ßgal and either tkluc, –4.3tkluc, –4.3/–2.1tkluc, –3.5/–2.1tkluc, or –2.1tkluc (as illustrated to the left) and their activities evaluated in the absence or presence of 1,25-(OH)2D3 (10–7 M) as described in panel A. Fold induction is indicated above each group as compared with the control groups. Each point represents the normalized relative light unit average ± SEM of a quadruplicate set of transfections and is representative of at least two independent experiments. C, LS180 cells were transfected with pCH110-ßgal and either tkluc, –1.3TRPV6luc, or –1.3TRPV6luc to which was fused the –5.5-, –4.3-, –4.3-, and –2.1-kb; the –3.5-, –3.5-, and –2.1-kb; or the –2.1-kb regions as illustrated to the left, and their activities were evaluated in the absence or presence of 1,25-(OH)2D3 (10–7 M) as described in panel A. Fold induction is indicated above each group as compared with the control groups. Each point represents the normalized relative light unit average ± SEM of a quadruplicate set of transfections and is representative of at least two independent experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Point Mutations Reveal Duplex VDREs in the –4.3- (A) and –2.1-kb (B) Regions of the TRPV6 Gene Wild-type –4.3tk-LUC and –2.1tk-LUC reporters were subjected to site-directed mutagenesis to alter half-sites in the sequence of the two putative VDREs identified using in silico analysis. The organization and sequence of these constructs are illustrated in the left panels of both (A) and (B). The transcriptional activity of each construct after transfection into LS180 cells is documented in the right panels in both (A) and (B). Each point represents the normalized relative light unit average ± SEM of a quadruplicate set of transfections and is displayed as fold induction representative of three independent experiments. Statistical analysis was performed using a one-way ANOVA with Tukey posttest and divided/displayed into statistical groups (P < 0.05); a, b, or c. mut, Mutant.

View larger version (79K): [in this window] [in a new window]   Fig. 6. The VDR/RXR Heterodimer Interacts with All Wild-Type hTRPV6 VDREs A, Purified VDR and RXR bind to TRPV6-derived VDREs. Labeled duplex DNA probes derived from the TRPV6 DNA sequences corresponding to –5.916/–5.486 (–5.5), –4.347/–4.312 (–4.3A), –4.303/–4.265 (–4.3B), –3.532/–3.495 (–3.5), –2.183/–2.146 (–2.1A), –2.130/–2.100 (–2.1B), or –1.292/–1.254 kb (–1.2), or mutated half-site sequences (AGG to TCC or TGG to CCC) were incubated in the presence of 1,25-(OH)2D3 with purified VDR (10 ng) and RXR (5 ng) as described in Materials and Methods. B, TRPV6 VDREs all compete for VDR/RXR binding in the presence of an authentic VDRE derived from OPN. hVDR (10 ng) and hRXR (5 ng) were incubated with labeled mOPN VDRE and increasing concentrations of excess unlabeled hTRPV6 VDREs (1-, 10-, 100-fold excess, indicated by triangles) as described in Materials and Methods. Complexes were resolved on 6% nondenaturing polyacrylamide gels, dried, and visualized using autoradiography. The positions of the VDR/RXR complex and free probe are indicated in the figure. mut, Mutant; wt, wild type.

View larger version (37K): [in this window] [in a new window]   Fig. 7. 1,25-(OH)2D3-Inducible Transcriptional Activity of hTPRV6 (–7 kb/+160) Containing VDRE Mutations Point mutations (AGG to TCC or TGG to CCC) were created in the VDREs within hTRPV6 (–7 kb/+160) at –5.5, –4.3A, –4.3B, –3.5, –2.1A, –2.1B, –1.2, and various double, triple, and quadruple mutations as seen in the schematic depiction on the left. LS180 cells were cotransfected with pCH110-ßgal and either pGL3, hTRPV6 (–1.3/+165), hTRPV6 (–7 kb/+160), or hTRPV6 (–7 kb/+160) containing the mutations described above. Cells were treated with either vehicle or 10–7M 1,25-(OH)2D3 and then processed as described in Materials and Methods. Each point represents the normalized relative light unit average ± SEM of a quadruplicate set of transfections and is displayed as fold induction from three independent experiments. Statistical analysis was performed using a one-way ANOVA with Tukey posttest and divided/displayed into statistical groups (P < 0.05) a, b, or c. mut, Mutant.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Sequence Conservation within the TRPV6 Promoter Region A 10-way vertebrate alignment of the TRPV6 promoter region (chromosome 7 (q34)) is illustrated (May 2004 assembly; UCSC Genome Browser, http://genome.ucsc.edu/). The continuous line below depicts the corresponding regions wherein the locations of the multiple VDREs are indicated by numbered (1–5) boxes (the TSS is indicated). Small boxes are indicative of the presence of a single VDRE whereas the large boxes are indicative of duplex VDREs. The specific DNA sequences seen in human, mouse, and rat TRPV6 genes are indicated across the –2.1- and –4.3-kb regions, with the sequence of the two VDREs indicated in bold type.

View larger version (90K): [in this window] [in a new window]   Fig. 9. SRC-1 and Histone Acetylation Accompany VDR/RXR Binding to the hTRPV6 Promoter LS180 cells were seeded into 100-mm plates and allowed to grow to 90% confluency. They were then treated for up to 2 h with ethanol vehicle or 10–7 M 1,25-(OH)2D3 and subjected to ChIP analysis using antibodies to VDR, SRC-1, GRIP-1, p/CIP, AcH4, or IgG. Input DNA was 0.2%. Immunoprecipitated DNA was subjected to PCR amplification (30 cycles) using the primer pair indicated at the top of each set of ChIP analyses as described in Fig. 3. An amplification of the DNA using primers to CYP24A1 was also examined as a control. ORF, Open reading frame.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

We used a combinatorial approach to define the components and the mechanism integral to the ability of 1,25-(OH)₂D₃ to stimulate TRPV6 gene expression. The finding that an extended version of the TRPV6 promoter was responsive to 1,25-(OH)₂D₃ in transfection studies, together with the observation that this induction was VDR dependent, enabled us to search for VDR/RXR binding sites using a ChIP scanning approach. The DNA binding of the receptor to a rather broad region of the TRPV6 promoter from –1.2 to –5.5 kb in two different intestinal cell lines was consistent with the in silico analysis that we carried out, which revealed the presence of four putative VDREs located at –1.2, –2.1, –3.5, –4.3, and –5.5 kb relative to the TSS. Despite the VDR/RXR binding studies, however, only the DNA fragments that contained putative elements located at –1.2, –2.1, and –4.3 kb were capable of mediating a significant transcriptional response to 1,25-(OH)₂D₃ when cloned upstream of either the TK promoter or a TRPV6 promoter containing –1.3 kb of the most proximal sequence. These activities within the –2.1- and –4.3-kb fragments, however, enabled us to precisely identify the VDREs located therein. Interestingly, two functionally independent VDREs were identified within each of these regions of the TRPV6 gene, such that they contributed almost 85% of the overall activity observed within the native TRPV6 promoter. The final 15% was contributed via the activity found in the VDRE at –1.2 kb. Thus, although the VDR and its partner were capable of interacting with all of the putative VDREs identified in the TRPV6 gene, only five appear to be functional in this combination of assays. These conclusions are supported by the results we obtained in a final ChIP study, in which we show that localization of the VDR/RXR heterodimer to each of these four regions within the TRPV6 gene promoter leads to both the broad recruitment of SRC-1 across this region and to extensive acetylation on histone 4. Our studies therefore point to a definitive biological role for each of these regions within the TRPV6 gene in modulating TRPV6 mRNA output by 1,25-(OH)₂D₃.

ChIP scanning techniques, as used here, facilitate the identification of transcription factor-chromatin DNA interactions specifically in the context of intact cells. An important feature of this approach is that there is no limit to where within a gene locus one is able to search. We coupled ChIP scanning to a standard transcriptional analysis wherein small DNA fragments containing the binding sites of interest were first cloned upstream of a minimal promoter and their activities then examined in response to 1,25-(OH)₂D₃. We completed our evaluation by identifying the specific sites of transcription factor interaction using site-directed mutagenesis and DNA bandshift analysis and by examining the activity of the individual VDREs in the context of the wild-type TRPV6 promoter. Interestingly, ChIP scanning analyses are rapidly being replaced by ChIP-DNA microarray (ChIP-on-chip) techniques wherein immunoprecipitated DNA from an initial ChIP analysis is amplified, labeled, and used to screen a high-density DNA microarray containing oligonucleotides that tile a specific region of interest at very high resolution. The regions so identified can then be cloned and evaluated as described above. ChIP-on-chip techniques have been applied not only to the analysis of specific gene loci, but to chromosome-wide and genome-wide scans for individual as well as transcription factor groups (28, 29, 30, 31, 32). Indeed, we have recently employed such an approach to identify and characterize the vitamin D-regulatory elements located in the genes for the VDR (33) and receptor activator of nuclear factor (NF)-B ligand (RANKL) (Kim, S., and J. W. Pike, unpublished).

Our results raise an interesting question as to why the VDREs located at –3.5 and –5.5 kb do not appear to contribute to the ability of 1,25-(OH)₂D₃ to activate the TRPV6 promoter, yet are capable of interacting with the VDR and its heterodimer partner as assessed by both ChIP analysis and bandshift assays. One answer may be that chromatin structure limits the accessibility of the VDR heterodimer to these elements in living cells. Our ChIP studies modestly support that possibility in that an abbreviated interaction between VDR was noted at the –5.5- and –3.5-kb regions of the TRPV6 gene. This result, however, was limited to the studies carried out in Caco2 cells and was not seen in total using LS180 cells. Further studies of nucleosome position and chromatin structure upstream of the TRPV6 gene will be required to resolve this issue.

Localization of VDR and RXR to binding sites on the TRPV6 gene in response to 1,25-(OH)₂D₃ correlates with the recruitment of the chromatin-remodeling coactivator SRC-1 and the modification of histone 4. The coappearance of RXR indicates that this gene is regulated in a traditional sense by the VDR/RXR heterodimer (34). The temporal relationship between the appearance of SRC-1 and VDR/RXR heterodimer binding suggests that the latter plays a pivotal role in this recruitment, although we did not conduct sequential ChIP studies to support this speculation. Whether coactivation of TRPV6 is limited to the recruitment of SRC-1 is unknown, although it seems clear that neither GRIP-1 nor p/CIP participates in a significant way. Finally, the TRPV6 gene is broadly acetylated across a wide expanse of the TRPV6 gene, extending from the TSS to the –5.5-kb region. It seems likely that acetylation is precipitated in response to 1,25-(OH)₂D₃ specifically at the VDR/RXR binding sites and that this change in chromatin structure may spread, in some way, across the entire region upstream of –4.3 kb. Whether this speculation can be demonstrated directly will require additional high-resolution studies. Future experiments are now aimed at examining the temporal patterns of both coactivator recruitment and histone modification using ChIP-on-chip analyses.

In conclusion, we have identified five significant VDREs located in an upstream region of the TRPV6 gene that we believe mediate the cis actions of 1,25-(OH)₂D₃. Our studies point to a mechanism whereby 1,25-(OH)₂D₃ is able to modulate intestinal expression of TRPV6 through a number of elements. Why this gene requires the actions of so many regulatory elements will require further study. The regulation of this and other genes in both intestinal as well as in kidney epithelium is likely to come under further scrutiny in attempts to understand the apparent tissue- and possibly gene-selective properties of vitamin D analogs that exhibit reduced calcemic potential.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Reagents
1,25-(OH)₂D₃ was obtained from Solvay (da Weesp, The Netherlands). DMEM was purchased from Cellgro (Herndon, VA). Oligonucleotide primers were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Anti-VDR, RXR, SRC-1, GRIP-1, and p/CIP antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiacetyl lysine H4 antibodies were obtained from Upstate Biotechnology, Inc. (Charlottesville, VA). Lipofectamine Plus was obtained from Invitrogen (Carlsbad, CA).

Plasmids
Full-length hVDR and hRXR were cloned into the pET-29b vector obtained from Novagen (Darmstadt, Germany) and expressed with C-terminal 6xHis tags. The rCyp24-luc reporter plasmid and the pCH110-ßgal ß-galactosidase reporter plasmid were previously described (35). All reporter plasmids were prepared by cloning the appropriate TRPV6 DNA fragments obtained through amplification of LS180 cell DNA or BAC clone DNA into either the pGL3-basic or ptkluc vectors using primers as indicated in Table 1. Because the start site of the TRPV6 gene has not been confirmed, the numbering system we have employed here designates the most 5'-nucleotide found in the mRNA transcript (216 bp from the start site of translation) as +1. Mutagenesis of the VDREs was performed using the QuikChange Site-Directed Mutagenesis kit from Stratagene (San Diego, CA). All inserts were sequenced for verification.

Cell Culture
Human Caco2 cells were cultured in DMEM supplemented with 1% pen/strep and 20% fetal bovine serum (FBS) obtained from Life Technologies, Inc., Rockville, MD). LS180 cells were cultured in MEM supplemented with 10% FBS, 1% nonessential amino acids, 1% sodium pyruvate, and 1% pen/strep from Life Technologies, Inc. LS180 cells were maintained using non-heat-inactivated FBS and not exposed to trypsin during passage. 1,25-(OH)₂D₃ was added to the culture medium in ethanol (0.1% maximum final concentration).

Transfection Analysis
Caco2 cells were seeded into 24-well plates at a concentration of 3 x 10⁴ cells per well and transfected 2 d later (at 80% confluency). LS180 cells were seeded into 24-well plates by dilution (100-mm plate transferred into four 24-well plates) because single cell suspensions were not possible. Both cell types were transfected using Lipofectamine Plus as indicated by the manufacturer. Individual wells were transfected with 300 ng of DNA made up of 250 ng of either prCyp24a1-luc, modified ptk-luc, or specific human TRVP6 promoter reporter plasmids and 50 ng pCH110-ßgal. After transfection, the cells were cultured in medium supplemented with 20% FBS with or without 1,25-(OH)₂D₃. Cells were harvested 24 h after stimulation, and the lysates were assayed for luciferase and ß-galactosidase (ß-gal) activities as described (35). Luciferase activity was normalized in all cases using ß-gal activity.

RNA Isolation and Analysis
Caco2 or LS180 cells were seeded into six-well plates at subconfluent densities (80%) and treated for up to 24 h without or with the indicated concentration of 1,25-(OH)₂D₃. Total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH). Total RNA was reverse transcribed using a SuperScript III RNase H Reverse transcriptase kit obtained from Invitrogen (Carlsbad, CA) and then subjected to amplification using standard methods (see also ChIP Assays). The sequences of the RT-PCR primers are documented in Table 2.

DNA Bandshift Analysis
Duplex oligonucleotide probes made up of the mouse osteopontin VDRE and the seven putative human TRPV6 VDREs (wild type and mutant) were end labeled using [³²P]-deoxy-ATP (New England Nuclear, Boston, MA). Probes were incubated in 10 mM HEPES (pH 7.4), 1 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.5 mM dithiothreitol, 0.7 mM phenylmethylsulfonylfluoride, and 150 mM KCl at room temperature for 30 min with 10 ng of purified hVDR or 10 ng hVDR and 5 ng of hRXR in the presence of 1,25-(OH)₂D₃ (10^–6 M). Complexes were resolved on 6% nondenaturing polyacrylamide gels, dried, and then visualized using autoradiography.

Isolation of Proteins
Human VDR and RXR proteins were produced in BL21(DE3) codon Plus RIL cells (Stratagene, La Jolla, CA) using the bacterial expression vectors pET-hVDR and pET-hRXR. Soluble full-length hVDR and hRXR protein was purified to homogeneity using sequential Ni-NTA and SP-Sepharose column chromatography (35).

ChIP Assays
ChIP was performed as described previously (35, 38, 39). Briefly, Caco2 cells or LS180 cells were plated at 80% confluency and the next day subjected to a medium change and treated with or without 1,25-(OH)₂D₃ for the times indicated. Treated cells were then washed and exposed to a cross-linking reaction with 1% formaldehyde for 10 min. After the fixative was neutralized with glycine, cells were extracted sequentially in 1) 5 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 8.0), 85 mM KCl, and 0.5% Nonidet P-40 and 2) 50 mM Tris-HCl (pH 8.1), 10 mM EDTA, and 1% sodium dodecyl sulfate (SDS), and the insoluble pellet was then subjected to sonication to produce chromatin fragments averaging 500 bp of DNA in size (as assessed by agarose gel electrophoresis) using a Fisher model 100 Sonic Dismembranator (Fisher Scientific, Pittsburgh, PA). The sonicated extract was collected, diluted into ChIP buffer (16.7 mM Tris-HCl, pH 8.1; 150 mM NaCl; 0.01% SDS; 1.1% Triton X-100; 1.2 mM EDTA) and then precleared for 1 h with salmon sperm DNA- and BSA-pretreated Zysorbin (Zymed Laboratories, Inc., South San Francisco, CA). Immunoprecipitations were performed overnight at 4 C with the indicated antibodies. Precipitates were then washed and the cross-links were reversed during a 6-h incubation in 1% SDS, 0.1 M NaHCO₃, and 0.2 M NaCl at 65 C. DNA fragments were purified from chromatin using QIAGEN QIAquick Spin Kits (Valencia, CA) and then subjected to PCR using primers designed to amplify appropriate fragments of either the proximal human CYP24A1 promoter region (–252 to –51) or various regions of the human TRPV6 promoter (see Table 3). Analyses for each primer set were carried out in a predetermined linear range of DNA amplification and the PCR products resolved on 2% agarose gels and visualized using ethidium bromide. DNA acquired before precipitation was used as input to assess the initial presence of the gene fragments.

	ACKNOWLEDGMENTS

 
We thank members of the Pike laboratory for their helpful contributions to this work. We also thank Dr. Kenneth E. Thummel for the kind gift of LS180 cells. We especially acknowledge Miwa Yamazaki for her technical assistance.

	FOOTNOTES

 
This work was supported by National Institutes of Health Grant DK-52453 (to J.W.P.).

Disclosure: The authors state that they have nothing to declare.

First Published Online March 30, 2006

¹ M.B.M and M.W. contributed equally to this work.

Abbreviations: ACH4, Tetra acetylated histone 4; ChIP, chromatin immunoprecipitation; CYP24A1, 25 hydroxyvitamin D₃-24 hydroxylase; D9K, calbindin D9K; D28K, calbindin D28K; FBS, fetal bovine serum; GRIP-1, glucocorticoid receptor-interacting protein 1; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; OPN, osteopontin; p/CIP, p300/cAMP response element binding protein-binding protein interacting protein; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; siRNA, short interfering RNA; SRC-1, steroid receptor coactivator-1; TK, thymidine kinase; TRPV, transient receptor potential vanilloid; TSS, start site of transcription; VDR, vitamin D receptor; VDRE, vitamin D response element.

Received for publication January 18, 2006. Accepted for publication March 20, 2006.

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NURSA Molecule Pages Link:

Nuclear Receptors:   VDR  |  RXRα

Coregulators:   SRC-1  |  GRIP1  |  AIB1

Ligands:   Calcitriol

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