The impact of VDR expression and regulation in vivo

Abstract

The vitamin D receptor (VDR) mediates the pleiotropic biological actions of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). These actions include orchestration of mineral homeostasis which is coordinated by the kidney, intestine, bone and parathyroid gland wherein the VDR transcriptionally regulates expression of the genes involved in this complex process. Mutations in human VDR (hVDR) cause hereditary vitamin D resistant rickets, a genetic syndrome characterized by hypocalcemia, hyperparathyroidism and rickets resulting from dysregulation of mineral homeostasis. Expression of the VDR is regulated by external stimuli in a tissue-specific manner. However, the mechanisms of this tissue-specificity remain unclear. Studies also suggest that phosphorylation of hVDR at serine 208 impacts the receptor's transcriptional activity. These experiments were conducted in vitro, however, and therefore limited in their conclusions. In this report, we summarize (1) our most recently updated ChIP-seq data from mouse tissues to identify regulatory regions responsible for the tissues-specific regulation of the VDR and (2) our studies to understand the mechanism of hormonal regulation of Vdr expression in bone and kidney in vivo using transgenic mouse strains generated by mouse mini-genes that contain comprehensive genetic information capable of recapitulating endogenous Vdr gene regulation and expression. We also defined the functional human VDR gene locus in vivo by using a human mini-gene comparable to that in the mouse to generate a humanized VDR mouse strain in which the receptor is expressed at normal levels (normal expressor). The present report also shows that a humanized mouse model in which the VDR is expressed at levels about 10-fold lower than the normal expressor mouse rescued the VDR-null phenotype despite its reduced transcriptional activity relative to wildtype expression. We also generated an additional humanized mouse model expressing hVDR bearing a mutation converting serine 208 to alanine (hVDR-S208A). In spite of the mutation, target gene expression induced by the ligand was unchanged relative to a mouse strain expressing comparable levels of wildtype hVDR. Further characterization also showed that serum calcium and parathyroid hormone levels were normal and alopecia was not observed in this hVDR-S208A mouse strain as well. Taken together, our in vivo studies using ChIP-seq analyses and the mini-gene transgenic mice improve our understanding of the tissue-specific regulatory mechanisms of controlling VDR expression and the mechanisms of action of the VDR.

Keywords: 1,25-dihydroxyvitamin D(3); Bacterial artificial chromosome; ChIP-seq; Hereditary vitamin D resistant rickets; Humanized VDR mouse model; Vitamin D receptor.

Fig. 1

Schematic structures of the targeting vector and Cyp27b1 gene locus for Cyp27b-null mice and the mini-genes for transgenic mouse models. (A) Schematic structures of the targeting vector and the Cyp27b1 gene locus in the mice produced in the process to generate Cyp27b1-null mice in which exon 7 through 9 of the gene are deleted are presented (see Materials and methods). Numbered hatched boxes represent each exon. FRT sites and loxP sites are shown double gray triangles and a black triangle, respectively. Selectable markers, diphtheria toxin A (DTA) and neomycin (neo) resistance genes, are indicated. (B, C) Schematic structures of mouse (B) and human (C) VDR mini-genes are presented. Each mini-gene includes the entire mouse or human VDR gene locus and its surrounding intergenic segments. Location of each enhancer (S1, S3 and U1) of mouse VDR mini-gene is indicated by an arrow (B). The S208A mutation was introduced through nucleotide mutagenesis (AGT to GCT) as indicated (C). Exons and introns are represented by black and gray boxes, respectively. Direction of transcription is indicated by an arrow at the TSS. Insertion sites of HA-tag (HA) and a cassette containing an IRES-driven luciferase (LUC) reporter and a TK promoter (TK)-driven neomycin resistance gene (Neo^r) are shown in the translation start site and in the 3′-UTR, respectively. The sizes of upstream and downstream intergenic sequence included in the mini-genes are indicated.

Fig. 2

Tissue specific regulation of endogenous Vdr expression. (A) Wildtype mice were intraperitoneally injected with vehicle (Veh), 1,25(OH)₂D₃ (1,25), FGF23 or PTH and expression of endogenous Vdr gene in the indicated tissues was measured by qPCR. (B) Expression of endogenous Vdr gene was measured in the indicated tissues obtained from Cyp27b1-null mice (C27KO) and wildtype littermates (WT). The expression level represents relative quantitation (RQ) normalized to Gapdh (ΔΔCT) and data are displayed as the means for each group ± SEM (5–7 mice per group). *p < 0.05 compared with vehicle-treated samples (A) or wildtype littermates (B).

Fig. 3

Genetic and epigenetic features of mouse Vdr gene locus. (A) Representative ChIP-seq tracks of triplicate samples for the VDR at mouse Vdr gene locus in MC3T3-E1 cells (MC3T3), kidney and intestine obtained from Cyp27b1-null mice (C27KO) and wildtype littermates (WT) are presented. Binding of the VDR in vehicle- and 1,25(OH)₂D₃-treated samples is shown in yellow and blue, respectively. Overlaps in binding are shown in green. (B, C) Representative ChIP-seq tracks of triplicate samples for Lys 4-monomethylated histone H3 (B, H3K4me1) and Lys 27-acetylated histone H3 (C, H3K27ac) at mouse Vdr gene locus in MC3T3-E1 cells (MC3T3) and kidney and intestine from Cyp27b1-null mice (C27KO) and wildtype littermates (WT) treated with vehicle are presented in yellow. ChIP-seq data of the same histone marks in kidney and intestine obtained from C57BL/6 mice (ENCODE Consortium) are also displayed in gray (BL6). Exons and introns of Vdr and adjacent Tmem106c genes are shown in boxes and lines, respectively. Transcriptional direction of a gene is indicated by an arrow at TSS and genomic location and scale are provided. Maximum height of tag density for the data track is indicated on each track. CCCTC-binding factor binding sites (ENCODE Consortium) commonly found in kidney and intestine are indicated below the tracks with arrows (C, CTCF). Potential enhancer regions in each sample are highlighted in red boxes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Regulation of Vdr mini-gene expression by hormones in the transgenic mice. (A) Binding of the VDR, phosphorylated CREB (pCREB) or RAR was determined by ChIP-chip and ChIP-seq analyses using MC3T3-E1 cells (Bone) or kidney from wildtype mice (Kidney). Major and minor binding of the indicated transcription factors at S1, S3 and U1 in the samples is summarized as large and small circles, respectively. Exons and introns of the Vdr gene are shown in boxes and lines, respectively. Transcriptional direction of a gene is indicated by an arrow at TSS and the scale is provided at the bottom of the gene. (B, C) The transgenic mice generated by wildtype (WT) and U1- (U1KO), U1 and S1- (U1/S1 KO) and S1 and S3-deleted (S1/S3 KO) mouse VDR mini-genes were intraperitoneally injected with vehicle (Veh), 1,25(OH)₂D₃ (1,25), db-cAMP (cAMP) or atRA and the expression of the Vdr gene from the mini-genes (HA-Vdr) in calvaria (B) and kidney (C) was measured by qPCR. The expression level was calculated as a relative quantitation (RQ) normalized to Gapdh (ΔΔCT). Data are displayed as the means of fold induction relative to vehicle-treated samples for each group±SEM (5 mice per group). *p < 0.05 compared with vehicle-treated sample.

Fig. 5

Characterization of hVDR-low expressor mice. (A) Levels of hVDR transcripts and proteins were measured in the indicated tissues obtained from hVDR-normal and –low expressor mice by qPCR and western blot analysis, respectively. Level of β-tubulin was used as a loading control. *p < 0.05 compared with hVDR-normal expressors. (B) The hVDR-normal and –low expressor were intraperitoneally injected with vehicle (Veh) or 1,25(OH)₂D₃ (1,25) and the expression of Cyp24a1 gene in the indicated tissues was measured by qPCR. *p < 0.05 compared with vehicle-treated samples, ^#p < 0.05 compared with hVDR-normal expressors received the same treatment. The expression level (A, B) is relative quantitation (RQ) normalized to Gapdh (ΔΔCT) and data are displayed as the means for each group±SEM (5–7 mice per group). (C, D) Serum calcium and PTH levels (C) and total body BMDs (D, males) in hVDR-low expressor mice were measured and compared with those in VDR-null mice (VDR KO) and wildtype littermates (WT). Each value is the average of 5–7 mice per strain±SEM. *p < 0.05 compared with wildtype littermates, ^#p < 0.05 compared with VDR-null mice. (E) The images show gross appearance of a representative mouse of wildtype (WT), VDR-null mice (VDR KO) or hVDR-low expressor mice at 6 month of age.

Fig. 6

Characterization of hVDR-S208A mini-gene mice. (A, B) Levels of hVDR transcripts (A) and proteins (B) were measured in the indicated tissues obtained from hVDR-low expressor and hVDR-S208A mice by qPCR (A) and western blot analysis (B), respectively. Level of β-tubulin was used as a loading control. No difference in the expression level was found in all tissues examined. (C) The hVDR-low expressor and hVDR-S208A mice were intraperitoneally injected with vehicle (Veh) or 1,25(OH)₂D₃ (1,25) and the expression of Cyp24a1 gene in the indicated tissues was measured by qPCR. *p < 0.05 compared with vehicle-treated samples. No difference was found between two mouse strains. The level of transcript (A, C) represents relative quantitation (RQ) normalized to Gapdh (ΔΔCt) and data are displayed as the means for each group ± SEM (5–7 mice per group). (D) Serum calcium and PTH levels in hVDR-low expressor and hVDRS208A mice were measured. Each value is the average of 5 mice per strain ± SEM. No difference was found in the levels of the serum factors. (E) The images show gross appearance of a representative mouse of hVDR-low expressor and hVDR-S208A mice at 6 month of age.