Impaired Vitamin D Signaling in T Cells From a Family With Hereditary Vitamin D Resistant Rickets

Abstract

The active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), mediates its immunomodulatory effects by binding to the vitamin D receptor (VDR). Here, we describe a new point mutation in the DNA-binding domain of the VDR and its consequences for 1,25(OH)₂D₃ signaling in T cells from heterozygous and homozygous carriers of the mutation. The mutation did not affect the overall structure or the ability of the VDR to bind 1,25(OH)₂D₃ and the retinoid X receptor. However, the subcellular localization of the VDR was strongly affected and the transcriptional activity was abolished by the mutation. In heterozygous carriers of the mutation, 1,25(OH)₂D₃-induced gene regulation was reduced by ~ 50% indicating that the expression level of wild-type VDR determines 1,25(OH)₂D₃ responsiveness in T cells. We show that vitamin D-mediated suppression of vitamin A-induced gene regulation depends on an intact ability of the VDR to bind DNA. Furthermore, we demonstrate that vitamin A inhibits 1,25(OH)₂D₃-induced translocation of the VDR to the nucleus and 1,25(OH)₂D₃-induced up-regulation of CYP24A1. Taken together, this study unravels novel aspects of vitamin D signaling and function of the VDR in human T cells.

Keywords: HVDRR; T cells; vitamin A; vitamin D; vitamin D receptor.

Figure 1

The R80W mutation abolishes the transcriptional activity without affecting the structure or expression of VDR^R80W (A) Localization of the C238T mutation to exon 3 in the VDR gene resulting in the R80W in the C-terminal part of the second zinc finger in the DBD of the VDR. The amino acids surrounding the R80 in the VDR from different species with the R80W at the bottom. (B) Family tree where circles represent females, squares represent males, homozygous VDR^R80W marked as filled black, heterozygous VDR^WT/R80W marked as filled black and white, and homozygous VDR^WT marked as filled white. (C, D) In silico structure of the zinc fingers of the VDR interacting with DNA. (C) VDR^WT with the R80 in red and with the hydrogen bonds between R80 and the DNA backbone indicated as blue lines. (D) The same region of VDR^R80W with the W80 in blue. (E) VDR mRNA, (F) VDR protein and (G) CYP24A1 mRNA in VDR^WT (black circles) and VDR^R80W (white squares) CD4⁺ T cells activated in the presence of the indicated concentration of 25(OH)D₃. (E–G) The expression levels are given as fold change normalized to the average expression level of VDR^WT in control cells activated in the absence of 25(OH)D₃. (E) Mean ± SD (VDR^WT n = 6; VDR^R80W n = 1). (F) Relative VDR protein expression as determined by the density of the VDR bands from Western blotting analysis of VDR^WT and VDR^R80W. The upper panel gives the density of VDR^R80W (white squares) normalized to the average density of VDR^WT (black circles) from control cells. The lower panel shows one representative Western blotting analysis out of five independent experiments of VDR and GAPDH (loading control) from three controls and the patient. Mean + SD (VDR^WT n = 13; VDR^R80W n = 1 repeated 5 times). (G) Mean ± SD (VDR^WT n = 3; VDR^R80W n = 1). n.s. means not significant.

Figure 2

The R80W mutation abolishes normal 1,25(OH)₂D₃-mediated gene up- and down-regulation Regularized log2 normalized gene expression in (A) VDR^WT and (B) VDR^R80W CD4⁺ T cells activated in the presence or absence of 25(OH)D₃. The red and blue lines indicate the 1.5-fold threshold for up- and down-regulated genes, respectively. Genes that are at least 1.5-fold up- or down-regulated by 25(OH)D₃ are colored red or blue, respectively, and the number of genes up- or down-regulated by 25(OH)D₃ is indicated in the respective corners. Heatmap of genes up- (C) and down-regulated (D) by 25(OH)D₃ in VDR^WT CD4⁺ T cells. Selected genes are indicated to the right of the heatmaps. (E) CYB27B1 mRNA and (F) 1,25(OH)₂D₃ production in VDR^WT (black circles) and VDR^R80W (white squares) CD4⁺ T cells activated in the presence of the indicated concentration of 25(OH)D₃. (E) The expression levels are given as fold change normalized to the average expression level of VDR^WT in control cells activated in the absence of 25(OH)D₃. Mean ± SD (VDR^WT n = 6; VDR^R80W n = 1). (F) Mean ± SD (VDR^WT n = 6; VDR^R80W n = 1).

Figure 3

The R80W mutation does not affect binding to 1,25(OH)₂D₃ and RXR but significantly affects the subcellular localization of VDR^R80W (A) Relative VDR protein expression as determined by the density of the VDR bands from Western blotting analysis of VDR^WT and VDR^R80W CD4⁺ T cells activated in the absence (-) or presence (+) of 1,25(OH)₂D₃ (10 nM). The upper panel gives the density of VDR^WT (black circles) and VDR^R80W (white squares) normalized to the average density of the VDR^WT bands from control cells activated in the absence of 1,25(OH)₂D₃. The lower panel shows one representative Western blotting analysis out of five independent experiments of VDR and GAPDH (loading control) from control and patient cells. Mean ± SD (VDR^WT n = 13; VDR^R80W n = 1 repeated 5 times; *p < 0.05). (B–D) Subcellular localization of VDR^WT and VDR^R80W in CD4⁺ T cells activated in (B) the absence (-) or presence (+) of 1,25(OH)₂D₃ (10 nM) and in (C, D) the absence (-) or presence (+) of RA (1 µM) and 1,25(OH)₂D₃ (10 nM). The upper panels show the fraction of VDR located to the nucleus and the cytosol in grey and black columns, respectively, as determined by the density of the VDR bands from the Western blots. The lower panels show one representative Western blotting analysis of VDR and GAPDH (loading control) from the cytosolic (C) and nuclear (N) fraction of CD4⁺ T cells activated in the absence (-) or presence (+) of 1,25(OH)₂D₃ and RA as indicated. (B) Mean ± SD (VDR^WT n = 14; VDR^R80W n = 1 repeated 5 times; *p < 0.05). (C, D) Mean ± SD (VDR^WT n = 8; VDR^R80W n = 1 repeated 3 times; *p < 0.05). (E) Relative mRNA expression of CYP24A1 in VDR^WT (black circles) and VDR^R80W (white squares) CD4⁺ T cells activated in the absence (-) or presence (+) of RA (1 µM) and 1,25(OH)₂D₃ (10 nM). The expression levels are given as fold change normalized to the average expression level of CYP24A1 in control cells activated in the absence of RA and 1,25(OH)₂D₃. Mean ± SD (VDR^WT n = 6; VDR^R80W n = 1 repeated twice; *p < 0.05).

Figure 4

Reduced responsiveness to 1,25(OH)₂D₃ in T cells from heterozygous family members (A) Subcellular localization of the VDR in VDR^WT/R80W heterozygous CD4⁺ T cells activated in the absence (-) or presence (+) of 1,25(OH)₂D₃ (10 nM). The upper panel shows the fraction of VDR located to the nucleus and the cytosol in grey and black columns, respectively, as determined by the density of the VDR bands from the Western blotting analysis. The lower panels show one representative Western blotting analysis out of four independent experiments of VDR and GAPDH (loading control) from the cytosolic (C) and nuclear (N) fraction. Mean ± SD (VDR^WT/R80W n = 4; ^#p < 0.05). (B) CYP24A1 and (C) CD38 mRNA expression, (D) CD38 cell surface expression, (E) IFN-γ and (F) IL-13 production in VDR^WT (black circles), VDR^WT/R80W (gray triangles) and VDR^R80W (white squares) CD4⁺ T cells activated in the presence of the indicated concentration of 1,25(OH)₂D₃. The expression levels are given as fold change normalized to the average expression level of the given molecule in cells activated in the absence of 1,25(OH)₂D₃ in each group. Mean ± SD (VDR^WT n = 13; VDR^WT/R80W n = 4; VDR^R80W n = 1; *VDR^WT p < 0.05 compared to VDR^WT in the absence of 1,25(OH)₂D₃, ^#VDR^WT/R80W p < 0.05 compared to VDR^WT/R80W in the absence of 1,25(OH)₂D₃).

Figure 5

The R80W mutation abolishes 1,25(OH)₂D₃-mediated suppression of RA-induced gene up-regulation Relative mRNA expression of (A) α4, (B) β7, (C) CCR9, (D) VDR and (E) LZTFL1 in VDR^WT (black circles) and VDR^R80W (white squares) CD4⁺ T cells activated in the absence (-) or presence (+) of RA (1 µM) and 1,25(OH)₂D₃ (10 nM). The expression levels are given as fold change normalized to the average expression level of the target gene in each group activated in the absence of RA and 1,25(OH)₂D₃. Mean ± SD (VDR^WT n = 6; VDR^R80W n = 1 repeated twice; *p < 0.05).