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. 2014 Apr 2;155(6):2064–2076. doi: 10.1210/en.2014-1107

Mouse and Human BAC Transgenes Recapitulate Tissue-Specific Expression of the Vitamin D Receptor in Mice and Rescue the VDR-Null Phenotype

Seong Min Lee 1, Kathleen A Bishop 1, Joseph J Goellner 1, Charles A O'Brien 1, J Wesley Pike 1,✉
PMCID: PMC4020932  PMID: 24693968

Abstract

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

The vitamin D receptor (VDR) is expressed in numerous tissues in higher vertebrates and mediates the diverse genomic actions of the vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a highly cell-specific manner (1–3). These actions involve binding of the VDR and its partner retinoid X receptor to the transcriptional control region(s) of linked genes, where the heterodimer functions to recruit coregulatory complexes that modulate changes in chromatin architecture that underlie altered gene expression (4, 5). The gene networks that are coordinately regulated by 1,25(OH)2D3 serve to control general functions of cellular growth, lineage progression and differentiation, and to direct specific mature cell functions inherent to cellular phenotype (6–12).

Despite the above insights, little is known of the components or of the mechanisms that control the expression of the Vdr gene in target tissues. Understanding these mechanisms is potentially important, because it is now well established that 1) VDR expression varies across various cell types, 2) the gene is strongly regulated, and 3) the presence of the VDR is an absolute determinant of cellular response to 1,25(OH)2D3. Down-regulation of VDR, for example, reduces the impact of 1,25(OH)2D3 on cellular growth and, in the extreme case of genetic deletion of the Vdr gene, can lead to increased tumor formation and exaggerated tumor cell growth (10, 11). VDR down-regulation also results in the loss of 1,25(OH)2D3 responsiveness in fully differentiated cell types as exemplified by mature osteoclasts (13), although this is now currently in dispute (14). VDR is also progressively down-regulated in the parathyroid gland as a result of chronic kidney disease, a process that removes the normal feedback mechanism whereby 1,25(OH)2D3 limits parathyroid hormone (PTH) secretion, thus contributing to secondary hyperparathyroidism characteristic of the renal osteodystrophy associated with chronic kidney disease (15, 16). Up-regulation of the VDR, on the other hand, sensitizes cells to the actions of 1,25(OH)2D3, as occurs during the maturation of the intestinal tract in rodents (17) and during human T-cell activation (18). VDR up-regulation also increases sensitivity to the antiproliferative effects of the hormone in tumor cells, supporting a potential therapeutic opportunity for the hormone and its analogs (19). Although certain pathways and transcription factors have been identified as playing a cell-specific role in Vdr expression, as exemplified by CAAT enhancer binding protein β (20, 21), cAMP response element binding protein (CREB) (21, 22), FBJ murine osteosarcoma viral oncogene homolog/Jun (23, 24), and Snail (25, 26), the roles and activities of these factors are yet to be fully understood. Finally, the tissue-specific determinants of Vdr gene expression are virtually unknown. Given the importance of the VDR in not only normal biology but also in a wide variety of disease states, a better understanding of the mechanisms associated with its expression is fully warranted.

The mouse and human VDR genes were cloned and structurally defined some years ago (24, 27–29). Recently, however, chromatin immunoprecipitation (ChIP)-tiled microarray and subsequently ChIP linked to deep sequencing analyses have revealed that the mouse Vdr gene is regulated through multiple enhancers located within 2 separate introns downstream of the gene's transcriptional start site and by sites −6 kb and perhaps −12 kb upstream (21, 30). These studies also demonstrated that an overlapping subset of these enhancers mediates not only the autoregulatory capabilities of the VDR to control its own expression in bone cells but the ability of the glucocorticoid/glucocorticoid receptor, retinoid/retinoic acid receptor, and protein kinase A/CREB pathways to induce Vdr expression as well (21, 30). Additional transcription factors have also been identified at the Vdr gene, including CAAT enhancer binding protein β and runt-related transcription factor 2; the precise roles of these factors in Vdr expression are currently unclear. Importantly, large mouse VDR bacterial artificial chromosome (BAC) clones, which contain the Vdr transcription unit, upstream and downstream intergenic segments and reporter, and whose boundaries were defined by the presence of active CTCF sites, fully recapitulate endogenous Vdr gene expression and regulation when introduced as stable constructs into host cells (21). These studies suggest the likelihood that an independent and fully functional Vdr gene locus is contained within this recombineered BAC clone.

In the studies reported here, we introduced 2 related mouse VDR BAC clones and a comparable human VDR BAC clone as transgenes into mice and evaluated their capacity to direct appropriate levels of expression of functional VDR protein and associated luciferase activity to tissue sites defined by endogenous Vdr gene expression. We also examined the ability of the transgenes to rescue both the gene expression responses to 1,25(OH)2D3 and dibutyryl-cAMP (db-cAMP) and the aberrant systemic, parathyroid gland, skeletal, alopecic, and skin phenotypes associated with loss of VDR expression in the VDR-null mouse. Validation of the behavior of these BAC clones in vivo suggests that tissue-specific determinants of Vdr gene expression and functions of both the mouse and human VDR proteins can now be dissected in vivo.

Materials and Methods

Construction of VDR BAC clones

WT-mVDR BAC and hemagglutinin (HA)-mVDR BAC clones, which were previously designated as mVDR BAC 2 and mVDR BAC 1, respectively, were constructed using BAC clone RP23–136G8 containing the mouse Vdr gene locus and upstream and downstream intergenic sequences, as previously described (21). To construct HA-hVDR BAC clone, BAC clone RP11–89H19 containing the entire human VDR gene, 82 kb of 5′-intergenic region and 48 kb of 3′-intergenic region, was obtained from the BACPAC Resource Center. The insertion of an HA-tag into the first translation start site and a reporter cassette into the 3′-untranslated region (UTR) was performed using the galactokinase system, as previous described (21). There are 2 internal NotI site in human VDR sequence in the BAC clone, one at 22 kb downstream of the VDR gene and the other within 3′-UTR. Because the latter is located upstream of the region in which the reporter cassette was inserted, the site (5′-GCGGCCGC-3′) was mutated to 5′-GCAGCCGC-3′ using the galactokinase system to avoid inappropriate digestion of the HA-hVDR BAC transgene during NotI-mediated linearization to generate the transgene. Accordingly, the 5′- and the 3′-flanking regions of human VDR gene in the transgene are approximately 82 and 22 kb, respectively (Figure 1A).

Figure 1.

Figure 1.

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Generation of mouse and human VDR transgenic mice. A, Schematic structures of mouse and human VDR BAC transgenes. The transgenes include the entire Vdr gene locus and the indicated sizes of its surrounding intergenic segments. Exons and introns are represented by black and gray boxes, respectively. Direction of transcription is indicated by an arrow at transcription start site. Insertion sites of a reporter cassette and HA-tag are shown in the 3′-UTR of each transgene and in the translation start site, the third exon of HA-mVDR BAC, and the fourth exon of HA-hVDR BAC, respectively. B and C, Luciferase activities in tissues of WT-mVDR Tg mice (B) and HA-hVDR Tg mice (C). Luciferase activity was normalized by protein amount. Neor, neomycin resistance gene; TK, human thymidine kinase promoter; LUC, luciferase; IRES, internal ribosome entry site.

Animal studies

All animal studies were reviewed and approved by Research Animal Care and Use Committee of University of Wisconsin-Madison. Mice were exposed to a 12-hour light, 12-hour dark cycle. C57BL/6 mice, obtained from Harlan, and transgenic mice were fed a standard rodent chow diet (number 5008). The VDR KO strain was produced by targeted ablation of the second zinc finger of VDR DNA-binding domain (31) and kindly provided by Dr H.F. DeLuca. This strain was fed a 20% lactose, high calcium, and high phosphate diet (TD.96348) to maintain the strain. The VDR-null phenotype was obtained in mice fed the normal rodent chow diet after weaning. Two- to 5-month-old mice of both genders were used equally for our studies and indicated separately for experiments assessing bone mineral density (BMD). Treatments with 1,25(OH)2D3 (10 ng/g body weight) or db-cAMP (100 μg/g body weight; Sigma) were performed by ip injection, and a mixture of ethanol and propylene glycol or PBS was used as vehicle controls, respectively. Tissues for RNA preparation were collected 6 or 1 hour after 1,25(OH)2D3 or db-cAMP injections, respectively.

Generation of BAC transgenic mice

To generate transgenic mice, 3 BAC clones, WT-mVDR BAC, HA-mVDR BAC, and HA-hVDR BAC, were independently linearized using NotI and used to prepare transgenic mice via standard pronuclear injection. The genotyping method is described in the Supplemental Materials and Methods and Supplemental Table 1. The transgenic strains were maintained as heterozygotes through outbreeding with C57BL/6 mice.

Generation of VDR-null mice containing BAC transgenes

As shown in Supplemental Figure 1A, VDR +/− mice expressing BAC-derived VDR and luciferase (VDR+/− Tg) were first obtained by pairing VDR +/− mice with transgenic mice (HA-mVDR Tg or HA-hVDR Tg) and the appropriate genotype mice were then bred with VDR +/− mice to generate VDR-null mice expressing BAC-derived VDR and luciferase (VDR−/− Tg). Genotyping methods using total RNAs or genomic DNAs obtained from tail clips are described in Supplemental Materials and Methods and Supplemental Figure 1.

Luciferase assay

Tissues were collected in Glo Lysis buffer (Promega) and homogenized using a PowerGen Model 125 Homogenizer (Fischer Scientific), and the lysates were cleared via centrifugation. Luciferase activities were measured using the Bright-Glo Luciferase Assay System (Promega) and normalized to protein amounts quantitated via Protein Assay (Bio-Rad).

Western blot analysis

Tissues were collected in Tissue Lysis buffer containing 10mM Tris-HCl (pH 8), 300mM KCl, 1mM EDTA, 2mM DTT, and protease inhibitor cocktail and homogenized using a PowerGen Model 125 Homogenizer (Fischer Scientific), and the protein content was determined in the supernatant after centrifugation. Lysates were denatured and subjected to 12% SDS-PAGE. Proteins were transferred to Immobilo-PSQ polyvinylidene difluoride membranes (Millipore) and subjected to Western blot analysis using anti-VDR (1:2000 dilution; 9A7), anti-VDR (1:1000 dilution; C-20, SC-1008, Santa Cruz Biotechnology, Inc), anti-HA (1:1000 dilution; HA.11, Covance), anti-β-actin (1:5000 dilution; A5441, Sigma), or anti-β-tubulin (1:5000 dilution; sc-9104, Santa Cruz Biotechnology, Inc) antibodies. Appropriate horseradish peroxidase-conjugated antibodies to the primary antibodies were used as secondary antibodies. Images were developed using ECL Plus Western Blotting Detection System (GE Healthcare).

Immunohistology

Mice were perfused with PBS and then with 4% paraformaldehyde and the collected tissues were then immersed overnight in 4% paraformaldehyde and then overnight in 30% sucrose. The tissues were processed to prepare frozen tissue blocks using Neg-50 (Richard-Allan Scientific) and cut into 10-μm sections with HM5050E microtome (Microm). Sections were immunostained using primary antibodies, anti-VDR (C-20, SC-1008; Santa Cruz Biotechnology, Inc) or antiluciferase (Promega) (2 μg/mL) and then developed with secondary antibodies, donkey antirabbit IgG labeled with Alexa Fluor 555 or donkey antigoat IgG labeled with Alexa Fluor 488 (1:200 dilution; Invitrogen), respectively. The stained tissue sections were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing VECTASHIELD (Vector Laboratories). The tissue sections were stained with Hematoxylin Stain Solution (3530–32; Ricca Chemical Co) and Eosin-Y (Fisher Scientific). The stained tissue sections were mounted with Permount (Fisher Scientific). Images for histological assay were taken using ECLIPSE Ti-S microscope (Nikon) and QICAM 12-bit Mono Fast 1394 Cooled camera (QImaging).

Reverse transcription-polymerase chain reaction

Tissues were collected in TRI reagent (Molecular Research Center) and homogenized using a PowerGen Model 125 Homogenizer (Fischer Scientific) to prepare total RNAs following the manufacturer's protocol. Total RNAs were subjected to reverse transcription using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Expression levels of genes were measured by quantitative real-time PCR using target gene-specific primers and FastStart SYBR Green Master (Roche). Primer sequences for quantitative real-time PCR are available upon request.

Serum analysis

Blood was collected from euthanized mice via cardiac puncture. Calcium and phosphate concentrations were determined in isolated serum using QuantiChrom Calcium and QuantiChrom Phosphate Assay kits (BioAssay Systems) as indicated by the manufacturer. Serum PTH, and fibroblast growth factor (FGF)23 concentrations were measured in EDTA-plasma using Mouse Intact PTH ELISA kits (Immutopics) and FGF-23 ELISA kits (Kainos Laboratories) according to the manufacturer.

Bone mineral density

Twelve-week-old mice were anesthetized with a mixture of isoflurane and oxygen and scanned using a Lunar PIXImus instrument. BMDs of total body and femur were analyzed using Lunar PIXImus 2.10 software.

Statistical analysis

All data are presented as the mean ± SEM. Comparisons between the samples from VDR-null mice expressing the BAC transgenes and their control mice were made through a one-way ANOVA with Turkey post hoc test to define significance (P < .05). A two-way ANOVA was used to identify significant differences (P < .05) between samples from 1,25(OH)2D3, or db-cAMP-injected wild-type and transgenic mice.

Results

Generation of VDR transgenic mice

To explore the ability of mouse and human VDR BAC clones to mimic the properties of the endogenous Vdr gene in vivo, we introduced 2 similar mouse VDR BAC clone constructs (WT-mVDR BAC and HA-mVDR BAC) and 1 human VDR BAC clone construct (HA-hVDR BAC) as transgenes into mice and generated 3 independent strains for each construct. The human construct and 1 mouse construct contain an HA-tag inserted into the amino-terminal end of the VDR for unequivocal detection. The features of these constructs are depicted in Figure 1A. Direct luciferase analysis of selected tissues from each of the 3 strains derived from the WT-mVDR BAC transgene, as documented for 1 strain in Figure 1B, revealed measureable levels of luciferase activity in the intestinal tract, kidney, and bone and lower levels of luciferase activity in pancreas and spleen; reporter activities in muscle, liver, and brain were more than 1000-fold lower than kidney. All strains, including those generated using the HA-hVDR BAC (see Figure 1C), expressed luciferase activity reflecting a similar pattern.

Detection of VDR expression in VDR transgenic mice

To further explore the basic properties of these 3 independent transgenes and their VDR products, we chose a single representative mouse strain derived from each construct and assessed the overall presence of the VDR in selected tissues by Western blot analysis and both VDR and luciferase coexpression by immunohistology. As can be seen in Figure 2, the VDR protein is detected using an anti-VDR antibody in several regions of the intestinal tract (duodenum, jejunum, ileum, and colon) and in kidney in each of the 3 transgenic strains and wild-type littermates; total VDR protein level in each of the individual mouse strains was generally increased several fold over wild-type VDR levels. VDR protein expression from the strains containing the HA-mVDR BAC and HA-hVDR BAC transgenes was also detected in these tissues by an antibody to the HA-tag. Finally, VDR was not detected by Western blot analysis in either the liver or the quadriceps muscle (quads). These results are supported further by the immunohistology documented in Figure 3, which also reveals a several-fold elevation in the expression of the VDR in duodenum and kidney tubules derived from each of 3 transgenic strains as compared with the nontransgenic wild-type strain. The VDR was also expressed in the parathyroid gland as well (Figure 3). Importantly, expression levels of the VDR in these tissues were correlated directly with the relative levels of luciferase protein detected by immunohistology in the 3 transgenic strains but not in the wild-type strain. As in Figure 2, neither VDR nor luciferase protein was detected in the liver or muscle in any of the strains of mice examined. To confirm the coexpression of both proteins in the tissues, isotype controls for anti-VDR and antiluciferase antibodies were applied to the immunostaining assay for WT-mVDR Tg mice, as described in Supplemental Materials and Methods. The results of these experiments revealed coexpression of VDR and luciferase proteins in duodenum, jejunum, ileum, and colon, kidney, and parathyroid gland as well as keratinocytes, hair follicles, and β-cells of the pancreas (data not shown). These data suggest that BAC-derived VDR and luciferase expression recapitulate the tissue- and cell type-specific expression of endogenous VDR protein in the tissues examined.

Figure 2.

Figure 2.

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Tissue-specific expressions of BAC-derived VDR in transgenic mice. VDR and HA-VDR expressions in tissues obtained from the transgenic mice were detected by Western blot analysis. Protein amounts analyzed are 20 μg for intestine and 100 μg for kidney, liver, and quad. β-Tubulin was used as a loading control. Duo, duodenum; Jej, jejunum; Ile, ileum; WT, wild-type littermate; Tg, transgenic mouse.

Figure 3.

Figure 3.

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Cell type-specific VDR and luciferase expressions in transgenic mice. VDR and luciferase expression in duodenum, kidney, parathyroid gland, liver, and muscle (quadriceps) in each transgenic mouse and VDR-null (VDR KO) and wild-type (C57BL/6) mice were detected by immunohistology. Fluorescent signals of DAPI-stained nuclei, VDR, and luciferase are shown as blue, red, and green colors, respectively. Original magnifications are ×100 for duodenum and kidney and ×200 for parathyroid gland, liver, and muscle. Arrows indicated microvilli in duodenum, renal tubules in kidney, and parathyroid glands. Arrowheads indicated intestinal smooth muscle in duodenum, glomeruli in kidney, follicles in thyroid glands, and blood vessels in liver.

Regulation of VDR gene expression by 1,25(OH)2D3 and db-cAMP in VDR transgenic mice

Previous studies have suggested that mouse Vdr mRNA production is up-regulated by both 1,25(OH)2D3 and PTH in normal mouse calvaria but not intestine or kidney in vivo (32). The ability of these 2 hormones to regulate luciferase expression from the WT-mVDR BAC clone stably integrated into the osteoblast genome has been confirmed (21). We therefore explored whether the transgenic mouse strain expressing VDR from this construct was equally sensitive to both 1,25(OH)2D3 and a PTH surrogate (db-cAMP). Accordingly, wild-type and transgenic WT-mVDR BAC mouse strains were injected with a single ip dose of either 1,25(OH)2D3 (10 mg/kg body weight) or db-cAMP (100 mg/kg body weight) (33), and the expression of Vdr mRNA was examined after 6 or 1 hour, respectively. As can be seen in Figure 4, basal levels of Vdr mRNA were increased several fold in the duodenum, kidney, and calvarial tissue of the transgenic strain as compared with the wild-type strain and induced further in calvaria but not in kidney or duodenal extracts by both 1,25(OH)2D3 and db-cAMP. Cyp24a1 (kidney) and Tnfsf11 (calvaria) mRNA levels representing positive response controls were also raised in response to 1,25(OH)2D3 and db-cAMP, respectively. Surprisingly, despite an increase in VDR expression in the kidney, this increase relative to wild-type littermate kidney did not serve to potentiate the induction of Cyp24a1 by 1,25(OH)2D3 (Figure 4). Increases in basal VDR expression were also ineffectual in raising serum calcium levels in each of the 3 transgenic mouse strains as well (Supplemental Figure 2). These data indicate that Vdr mRNA expression from the WT-mVDR BAC transgene was similarly inducible by both 1,25(OH)2D3 and db-cAMP relative to that of the endogenous Vdr gene, but this increase in VDR expression did not enhance Cyp24a1 induction by 1,25(OH)2D3.

Figure 4.

Figure 4.

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Recapitulation of hormone-regulated expression of BAC transgene. Expressions of Cyp24a1 (A), Vdr (B and D), and Tnfsf11 (C) in the indicated tissues obtained from 1,25(OH)2D3-injected (A and B) or db-cAMP-injected (C and D) WT-mVDR Tg mice were measured by RT-PCR. The expression levels were normalized to β-actin (Actb) (A and B) or glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (C and D). Each value is the average of 3–4 mice per strain, and error bar represents SEM. *, P < .05 compared with vehicle-treated control; #, P < .05 compared with wild-type littermate. WT, wild-type littermate; Tg, transgenic mouse; Veh, vehicle control; 1,25, 1,25(OH)2D3; cAMP, db-cAMP.

Mouse and human VDR BAC transgenes restore VDR expression in VDR-null mice

The above studies suggest that both the mouse and human VDR BAC transgenes are capable of mimicking the expression behavior of the endogenous mouse Vdr gene. To confirm whether the VDR produced from these transgenes was independently expressed at sufficient levels to be biologically active, we crossed the HA-mVDR BAC and HA-hVDR BAC transgenic mice with VDR-null mice to generate a VDR-null cross that contained either the HA-mVDR BAC or the HA-hVDR BAC transgene (designated VDR−/− mTg or VDR−/− hTg, respectively) and examined whether presence of the transgenes in these crosses could restore VDR expression and transcriptional response to 1,25(OH)2D3 and in so doing rescue the phenotype of VDR deficiency. The genotyping scheme used to obtain the appropriate genetic crosses is documented in Supplemental Figure 1; both males and females from the VDR−/− mTg or VDR−/− hTg crosses were fertile. As observed in Figure 5A, Western blot analysis using both anti-VDR and anti-HA antibodies revealed that although the VDR was undetectable in the tissues of VDR-null mice, expression of the VDR from both the mouse and human VDR transgenes restored VDR expression levels in the tissues examined. Human VDR expression appears to be substantially elevated when evaluated by the anti-VDR antibody 9A7, likely due to this antibody's elevated affinity for the human protein. Western blot analysis of the mouse and human proteins using an alternative anti-VDR antibody (C-20) (data not shown) and an anti-HA-tag antibody (Figure 5A) suggests that human VDR expression was only slightly elevated relative to the mouse construct and that both were similar to that of the endogenous gene. Neither the mouse nor the human VDR was expressed in the liver. As seen in Figure 5B, VDR and luciferase protein expression from both the mouse and human crosses were also detected by immunohistology in the duodenum, kidney, and parathyroid glands but not in liver or muscle. Human VDR expression appeared to be slightly elevated. These results indicate that the VDR BAC transgenes retain their ability to express recombinant VDR and the reporter in a tissue-appropriate fashion in the VDR-null mouse background.

Figure 5.

Figure 5.

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Recovery of VDR expression in VDR-null mice by VDR BAC transgene. Tissue- and cell type-specific expression of BAC-derived VDR in VDR-null mice was examined by Western blot analysis (A) and immunohistology (B). A, Protein amounts analyzed are 20 μg for jejunum and ileum and 100 μg for kidney and liver. β-Actin was used as a loading control. B, Fluorescent signals of DAPI-stained nuclei, VDR, and luciferase are shown as blue, red, and green colors, respectively. Original magnifications are ×100 for duodenum, kidney, and liver and ×200 for parathyroid gland and muscle. Genotypes of endogenous VDR allele are indicated by +/+ or −/−, representing that the indicated mouse contains wild-type or mutated VDR alleles, respectively, and those of BAC transgene by (−), mVDR, or hVDR, representing that the indicated mouse contains no BAC clone, HA-mVDR BAC clone, or HA-hVDR BAC clone as a transgene, respectively. Arrows indicated microvilli in duodenum, renal tubules in kidney, and parathyroid glands. Arrowheads indicated intestinal smooth muscle in duodenum, glomeruli in kidney, follicles in thyroid glands, and blood vessels in liver.

Mouse and human VDR BAC transgenes normalize the expression of 1,25(OH)2D3-sensitive genes

Given the ability of the VDR BAC transgenes to direct expression of the VDR in a tissue-specific manner in receptor-deficient mice (in the tissues examined), we next assessed whether BAC clone transgenic expression was capable of restoring the appropriate level of expression of key genes in VDR-null mice. As can be seen in Figure 6A, although Cyp27b1 mRNA expression was highly elevated in the VDR-null mouse, due to the inability of 1,25(OH)2D3 to suppress Cyp27b1 expression in the absence of the VDR, the level of this gene was decreased to normal wild-type levels after VDR expression from each of the mouse and human VDR transgenes. Likewise, although basal renal Cyp24a1 expression was strongly reduced in VDR-null mice, highlighting the dependency of this gene on both VDR and endogenous 1,25(OH)2D3 for its basal expression in this tissue, its levels were significantly increased after VDR expression from both the mouse and human VDR transgenes. Interestingly, the human VDR transgene appeared less capable of increasing the basal level of Cyp24a1 mRNA than the mouse gene despite slightly elevated levels of human VDR. A similar sensitivity to the transgenes was observed for the 1,25(OH)2D3 target gene S100g, which encodes the calcium binding protein calbindin D9k. These results suggest that restoration of either mouse or human VDR expression in tissues such as the kidney and in other tissues as well (data not shown) are capable of reestablishing genetic response to 1,25(OH)2D3.

Figure 6.

Figure 6.

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Recovery of serum calcium and phosphate homeostasis in VDR-null mice by VDR BAC transgene-mediated restoration of transcriptional response to 1,25(OH)2D3. A, Expressions of Cyp27b1, Cyp24a1, and S100g (calbindin D9k) in the kidney were measured by RT-PCR. The expression levels were normalized to Gapdh. Each value is the average of 3–5 mice per strain, and error bar represents SEM. *, P < .05 compared with wild-type control (+/+); #, P < .05 compared with VDR-null control (−/−). B, Recovery of defects in serum was examined by measuring the levels of serum calcium, phosphate, PTH, and FGF23. Each value is the average of 3–9 mice per strain, and error bar represents SEM. *, P < .05 compared with wild-type control (+/+); #, P < .05 compared with VDR-null control (−/−). ND, not determined. C, Thyro-parathyroid glands obtained from 2-month-old mice were stained with hematoxylin and eosin (H&E). Representative images of each strain are shown. Original magnification is ×40. Each genotype is described in Figure 5.

Mouse and human VDR BAC transgenes rescue the biological phenotype of VDR-null mice

VDR-null mice are both hypocalcemic and hypophosphatemic and display consequential phenotypic aberrations that include systemic FGF23 deficiency (34), hyperparathyroidism, and parathyroid gland hyperplasia (31). They also display specific bone growth defects, reduced BMD, and hypertrophic chondrocyte disorganization at the growth plate as well (31). The absence of the VDR also results in a disrupted hair cycle, alopecia, and the presence of dermal cysts (31); additional more subtle physiologic defects as well as aberrant pathological responses have been noted as well (35–39). Much work has gone into exploring individual aspects of these phenotypic abnormalities. We therefore examined whether expression of the VDR from the VDR transgenes could rescue specific aspects of this complex biological phenotype. The results depicted in Figure 6B clearly show that although VDR-null mice were both hypocalcemic and hypophosphatemic relative to wild-type mice, expression of the VDR from either the mouse or the human transgene normalized blood calcium and phosphate levels. Likewise, the enormously elevated PTH levels and dramatically suppressed FGF23 levels apparent in the VDR-null mice were both restored to the blood levels observed in wild-type mice. As expected, although VDR-null mice displayed hyperplastic parathyroid glands, as documented in Figure 6C, this defect was also normalized upon transgenic expression of either the mouse or the human VDR transgene. These results suggest that the appropriate expression of transgenic mouse or human VDR in intestine, kidney, bone, and parathyroid gland restores the ability of 1,25(OH)2D3 to direct transcriptional responses that integrate the actions of these tissues to maintain calcium and phosphate homeostasis. As previously described (31, 40) and documented in Figure 7, disrupted calcium and phosphorus homeostasis in the VDR-null mouse also leads to the development of an aberrant skeletal phenotype, as characterized by reduced skeletal BMD, a developmental growth defect in the tibia, and in the development of a disorganized and disaggregated growth plate. Each of these defects was fully rescued, however, after expression of the VDR from either the mouse or human VDR transgene, as documented in Figure 7. Finally, loss of the VDR also leads to defects in the skin, which results in an arrest of the hair cycle, alopecia, and the appearance of dermal cysts (7, 31, 41). Although each of these abnormalities in the skin is present in the VDR-null mice, as documented in Figure 7, hair cycling is restored when the VDR is reexpressed as a result of the presence of either the mouse or the human transgene, and the dermal cysts are eliminated as well. Taken together, these results suggest that appropriate expression of either the mouse or human VDR transgenes across multiple tissues is capable of rescuing virtually all of the physiological abnormalities examined here that arise as a result of the loss of the VDR.

Figure 7.

Figure 7.

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Recovery of defects in bone and skin of VDR-null mice by VDR BAC transgene. A, BMDs of total body and femur of 12-week-old male and female mice were measured using PIXImus densitometer. Each value is the average of 6–10 mice per strain, and error bar represents SEM. *, P < .05 compared with wild-type control (+/+); #, P < .05 compared with VDR-null control (−/−). B and C, Tibia was obtained from 2-month-old mouse. Images of tissue sections stained with H&E (B) and tibia appearance (C) were taken. Original magnification is ×40 for H&E stained-samples. D and E, Images of appearance of 5-month-old mice (D) and H&E-stained skin sections obtained from 2-month-old mice (E) were taken. Original magnification is ×200 for H&E-stained samples. Representative images of each strain are shown. Each genotype is described in Figure 5.

Discussion

We began a series of experiments several years ago using a functional genomics approach involving both ChIP-tiled microarray and ChIP linked to deep sequencing analysis coupled with the use of stably integrated BAC clones in host cells to identify the boundaries of the mouse Vdr gene locus and to define its internal regulatory features (21, 30). These studies resulted in the discovery of CTCF sites that appeared to serve as boundary elements limiting activation induced changes in histone H4 modification to the Vdr gene locus and the identification of several enhancers located both within introns as well as upstream of the Vdr transcriptional start site that selectively mediated response to 1,25(OH)2D3, retinoic acid, glucocorticoids, and PTH through binding of the VDR, retinoic acid receptor, glucocorticoid receptor, and CREB. All of these functional features of the endogenous Vdr gene locus were recapitulated after stable transfection of a recombineered BAC clone that contains not only the gene and its regulatory components but a detectable reporter function as well (21). Although these advances facilitated our understanding of Vdr gene regulation in bone cells, it left open the question of whether the BAC clone was capable of expressing appropriate endogenous-like activity in vivo or whether it contained sufficient genetic information to direct appropriate tissue-specific expression of the VDR to vitamin D target tissues. In addition, although the human gene is conserved relative to the mouse, unique features of this gene are also apparent (27, 42, 43), prompting the question of whether a comparable human transgene was capable of appropriate expression and activity in the mouse as well.

In this report, we show that when introduced into mice, both mouse and human VDR BAC transgenes are capable of mediating the expression of endogenous levels of the VDR in major target tissues known previously to contain the VDR. These tissues include the intestine, kidney, and bone as well as pancreatic β-cells and keratinocytes but not liver or muscle, tissues for which the presence of receptor remains highly controversial. Importantly, this tissue distribution of VDR expression was also supported by the simultaneous coexpression of luciferase, an additional marker that was inserted into the 3′-UTR of the Vdr gene and linked directly via an internal ribosome entry site to Vdr transcription. These transgenes also restored tissue-specific expression of the VDR in the VDR-null mouse, prompting the recovery of VDR-mediated gene expression, rescue of altered systemic mineral homeostasis, and resulting skeletal abnormalities, normalization of the aberrant endocrine production of PTH and FGF23, prevention of parathyroid gland hyperplasia, rescue of the block in hair cycling, and prevention of the development of dermal cysts. The fact that the systemic calcium imbalance and the skeletal abnormalities in the receptor-deficient mice were restored after transgene expression suggests a normal action of the VDR in the intestine, kidney, and bone. Restoration of VDR-dependent negative feedback mechanisms that are inherent to PTH expression in the parathyroid gland (44) and Cyp27b1 expression in the proximal tubules of the kidney (45) and the parathyroid gland hyperplasia that occurred as well provides evidence for additional direct actions of the VDR in the parathyroid gland and the kidney. Finally, the fact that the alopecia was also normalized suggests that the unique actions of the VDR in skin were similarly restored. This feature of VDR action is particularly interesting, because it is distinct from other VDR activities in that it appears to be a ligand-independent function of the VDR, although activation via an unknown ligand has not been ruled out (46). Importantly, orchestration of many of these biological events requires the simultaneous up- and down-regulation of numerous genes by 1,25(OH)2D3 that likely involve multiple functional activities of the VDR. Although not every aspect of the VDR-null mouse phenotype was explored in these transgenic rescue mice, our results suggest the likelihood that we have defined segments of DNA from both the mouse and human genome that contain not only the VDR transcription units but also the regulatory genetic information necessary to direct appropriate and regulated VDR gene expression to all vitamin D target tissues in vivo. The data also suggest that the human gene carries similar information such that its regulatory features are manifested in the mouse as well. These findings strongly support a functional genomics approach to defining appropriate transgenic segments for mouse transgenesis.

The results of these studies also facilitated an examination of tissues, in which the presence of VDR has been questioned. Neither VDR expression nor luciferase expression and/or activity was detected in liver, quadriceps muscle, or brain, in any substantial way, supporting the conclusion that under normal physiological conditions, these tissues do not express detectable levels of VDR protein and are therefore not direct targets of vitamin D hormone action. This issue has been questioned recently (38, 47–50), despite evidence accumulated over several decades through binding studies, scintillation autoradiography, Western blot analysis, and mRNA analysis. The potential presence of the VDR is particularly relevant in muscle, because vitamin D deficiency is known to lead to muscle weakness, which increases the tendency for falls in older humans. Indeed, vitamin D treatment has been shown to restore muscle integrity (50). Actions in liver and brain have also been described (38, 51). With regard to the former, the vitamin D hormone has been shown recently to oppose the profibrotic program directed by TGFβ-activated stellate cells found in this tissue. Interestingly, although these cells express the VDR, there is no evidence here that their quiescent, nonactivated counterparts are VDR-positive. We therefore hypothesize that the disease-inducing activation of stellate cells towards a myofibroblastic phenotype may prompt an up-regulation of the VDR gene. Indirect actions of 1,25(OH)2D3 in the liver have also been noted, however. For example, the vitamin D hormone has been shown to induce intestinal expression of FGF19, which acts, after systemic delivery, directly on the liver (52). Importantly, although we cannot rule out low expression of the VDR from unique subsets of additional highly dispersed, low abundance cell types such as neurons, the early use of tissue scintillation autoradiography (using tritiated 1,25(OH)2D3) did not identify subsets of specialized cells in either the liver or muscle (53, 54). This is consistent with the application of more recent techniques such as immunohistology analyses, which similarly do not confirm the existence of the VDR in muscle (55). Resolving these issues continue to be of considerable importance.

Although not as extensively characterized as in the mouse, the human VDR gene locus differs from the mouse in several ways. These differences include the presence of additional exons at the 5′ end of the gene (24), which have been suggested to encode an N-terminally extended form of the VDR protein in some cell types (42) and the presence of a second, alternative upstream promoter (42), a possibility supported by detection of functional genomic activity within this region (21). Additional differences include the observation that the regulatory regions in the human are not fully overlapping with those of the mouse gene (21). In the studies described here, the human gene appears to retain the capacity to be expressed in all the appropriate tissues in the mouse and to rescue the VDR-null phenotype. Only 1 difference has been noted, that of its inability to normalize fully basal level of expression of Cyp24a1 in the kidney. Regardless of this issue, normalization of the VDR-null mouse phenotype by the human gene suggests the creation of a “humanized” mouse model. The benefits of such a model include the ability to explore human VDR gene regulation and to examine the properties of the human protein in vivo. With regard to the latter possibility, properties of the human VDR have been the subject of extensive in vitro mutagenesis over the past several decades aimed at studying DNA binding, activation, ligand binding, and phosphorylation sites (56–59). These properties also included the in vitro evaluation of natural mutations that occur in the syndrome of hereditary 1,25(OH)2D3-resistant rickets (60–63). The biological effects of these mutations as well as those derived from hereditary 1,25(OH)2D3-resistant rickets can now be examined in the human VDR in vivo by establishing appropriate transgenic mouse models. In addition, there are numerous examples where mouse and human transcription factors differentially bind certain synthetic ligands, which alter in turn the protein's function. In the case of the VDR, for example, it has been shown that a 26,23-lactone derivative of 1α,25-dihydroxyvitamin D3, TEI-9647, functions as an agonist on the rodent VDR and as an antagonist on the human VDR (64, 65). These differences could have significant therapeutic relevance during drug screening and could be appropriately defined in the humanized VDR mouse.

In summary, the current studies advance our understanding of the expression and regulation of the mouse and human VDR genes in mice in vivo. The studies provide a clear definition of the span of DNA surrounding the VDR gene that is required for endogenous-like expression in vivo and provide a basis for additional studies of VDR gene expression and of the VDR protein itself in vivo.

Acknowledgments

We thank members of the Pike laboratory for their contributions to this work, David Nehls and Regina Berget for animal husbandry, and Dr Ricki Colman for acquiring and analyzing scanned images for BMD.

Present address for K.A.B.: Maine Medical Center Research Institute, Scarborough, ME 04074.

This work was supported by the National Institute of Arthritis, Musculoskeletal, and Skin Diseases Grant AR-045173 and the National Institute of Diabetes, Digestive, and Kidney Diseases Grant DK-072281 (to J.W.P.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
BAC
bacterial artificial chromosome
BMD
bone mineral density
ChIP
chromatin immunoprecipitation
CREB
cAMP response element binding protein
DAPI
4′6-diamidino-2-phenylindole
db-cAMP
dibutyryl-cAMP
FGF
fibroblast growth factor
HA
hemagglutinin
1,25(OH)2D3
1,25-dihydroxyvitamin D3
PTH
parathyroid hormone
UTR
untranslated region
VDR
vitamin D receptor.

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