Vitamin D-binding protein influences total circulating levels of 1,25-dihydroxyvitamin D3 but does not directly modulate the bioactive levels of the hormone in vivo

Abstract

Mice deficient in the expression of vitamin D-binding protein (DBP) are normocalcemic despite undetectable levels of circulating 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. We used this in vivo mouse model together with cells in culture to explore the impact of DBP on the biological activity of 1,25(OH)(2)D(3). Modest changes in the basal expression of genes involved in 1,25(OH)(2)D(3) metabolism and calcium homeostasis were observed in vivo; however, these changes seemed unlikely to explain the normal calcium balance seen in DBP-null mice. Further investigation revealed that despite the reduced blood levels of 1,25(OH)(2)D(3) in these mice, tissue concentrations were equivalent to those measured in wild-type counterparts. Thus, the presence of DBP has limited impact on the extracellular pool of 1,25(OH)(2)D(3) that is biologically active and that accumulates within target tissues. In cell culture, in contrast, the biological activity of 1,25(OH)(2)D(3) is significantly impacted by DBP. Here, although DBP deficiency had no effect on the activation profile itself, the absence of DBP strongly reduced the concentration of exogenous 1,25(OH)(2)D(3) necessary for transactivation. Surprisingly, analogous studies in wild-type and DBP-null mice, wherein we explored the activity of exogenous 1,25(OH)(2)D(3), produced strikingly different results as compared with those in vitro. Here, the carrier protein had virtually no impact on the distribution, uptake, activation profile, or biological potency of the hormone. Collectively, these experiments suggest that whereas DBP is important to total circulating 1,25(OH)(2)D(3) and sequesters extracellular levels of this hormone both in vivo and in vitro, the binding protein does not influence the hormone's biologically active pool.

Figures

Figure 1

Molecular structures of 1α,25 (OH)2D3, MC1288 [20-epi-1α,25(OH)2D3] and KH1060 [20-epi-22-oxa, 24a, 26a, 27a-tri-homo-1α,25(OH)2D3].

Figure 2

Serum concentrations of calcium and 1,25(OH)2D3 in wild-type and DBP-null mice. A, Serum calcium content. Serum was collected from 8-wk-old female DBP-null mice and their comparable controls maintained on a standard synthetic diet and analyzed for calcium content by atomic absorption spectrophotometry. The histograms reflect the means ± sem (n = 5). B, Serum 1,25(OH)2D3 content. Serum was collected from eight week old female DBP-null mice and their comparable controls as above and evaluated for 1,25(OH)2D3 content by RIA as described in the Materials and Methods. The histograms reflect the means ± sem (n = 5). *, P < 0.05 vs. wild-type control levels of 1,25(OH)D3.

Figure 3

Target gene expression levels in the intestine and kidney of wild-type and DBP-null mice. A, Kidney content of mRNA transcripts for Cyp24a1 and Cyp27b1. Kidneys were isolated from both wild-type and DBP-null mice (age and sex as in Fig. 2) and the isolated RNA subjected to qRT-PCR to quantitate levels of Cyp24a1 and Cyp27b1 as described in Materials and Methods. Specific mRNA content was normalized to mβ-actin. The histograms reflect the means ± sem (n = 5). *, P < 0.05 vs. wild-type levels of Cyp24a1 or Cyp27b1 transcripts. B, Intestinal and kidney content of mRNA transcripts for calcium-regulating target genes. The intestines and kidneys were isolated from both wild-type and DBP-null mice (age and sex as above) and the isolated RNA subjected to qRT-PCR as above to quantitate levels of intestinal TRPV6, CaD9K, and PMCA1b and renal TRPV5 and CaD28K mRNA. Content was normalized as in A. The histograms reflect the means ± sem (n = 5). *, P < 0.05 vs. wild-type levels of Cyp24a1, Cyp27b1 or CaD28K transcripts.

Figure 4

Concentrations of 1,25(OH)2D3 in intestinal tissue of wild-type and DBP-null mice. Serum was collected from DBP-null mice and their wild-type littermates (age and sex as in Fig. 2) and subjected to solid-phase extraction chromatography and RIA as described in Materials and Methods. The histograms reflect the means ± sem (n = 5). *, P < 0.05 vs. wild-type levels of 1,25(OH)2D3.

Figure 5

The effects of DBP on the biological activity of 1,25(OH)2D3 and the 1,25(OH)2D3 analog KH1060 in osteoblastic MC3T3-E1 cells. A, Serum increases the concentration of 1,25(OH)2D3 necessary for activation. MC3T3-E1 cells were treated in the presence or absence of serum with increasing concentrations of either 1,25(OH)2D3 or KH1060. RNA isolated at 6 h was subjected to RT-PCR analysis to detect Cyp24a1 and mβ-actin mRNA transcript levels. Analysis of cells induced in the absence of serum included a single group that was incubated in the presence of serum and 1,25(OH)2D3 to control for overall efficacy. This experiment is representative of six to eight similar experiments. B, The effect of serum on the biological potency of 1,25(OH)2D3 is due to DBP. MC3T3-E1 cells were treated in the presence [FBS (+)] or absence [FBS (−)] of serum or in the presence of serum obtained from either wild-type (S-DBP+/+) or DBP-null (S-DBP−/−) mice. RNA was isolated and evaluated as in A. Cyp24a1 transcripts were normalized to those of mβ-actin. EC50 and r2 values for each condition are as follows: FBS (+), 2.0 × 10−8 m (0.99); FBS (−), 3.0 × 10−11 m (0.95); S-DBP+/+, 2.6 × 10−9 m (0.99); S-DBP−/−, 6.0 × 10−11 m (0.97). The data are representative of several similar experiments.

Figure 6

The effect of DBP on vitamin D ligand binding to VDR on MC3T3-E1 cells in culture. A, The influence of serum on 1,25(OH)2D3 binding to the VDR in intact cells in culture. MC3T3-E1 cells were incubated in triplicate with tritiated 1,25(OH)2D3 (1 nm, 166 Ci/mmol) or a 250-fold molar excess of radioinert 1,25(OH)2D3 as outlined in Materials and Methods. At the times indicated, cells were harvested, and the level of total and nonspecific 1,25(OH)2D3 binding activity was determined by hydroxyapatite assay. Specific VDR binding is depicted. Each point represents the mean + sem for a triplicate determination. These data are representative of at least three similar independent evaluations. B, The influence of serum on the interaction between selected vitamin D ligands and the VDR. MC3T3-E1 cells were treated in the presence (left panel) or absence (right panel) of serum with increasing concentrations of 1,25(OH)2D3, MC1288, or KH1060. Cells were harvested at 6 h and subjected to Western blot analysis using the anti-VDR monoclonal antibody 9A7. EC50 and r2 values for each condition are as follows: 1,25(OH)2D3 [FBS (+)], 2.8 × 10−9 m (0.99) and [FBS (−)], 1.1 × 10−11 m (0.99); KH1060 [FBS (+)], 3.2 × 10−11 m (0.94) and [FBS (−)], 1.6 × 10−11 m (0.99); MC1288 [FBS (+)], 6.4 × 10−11 m (1.0) and[FBS (−)], 4.5 × 10−11 m (0.99). Data from at least three different experiments were quantitated using densitometry and presented as fold induction over vehicle treatment. C, The influence of serum on 1,25(OH)2D3-induced VDR localization to the Cyp24a1 and OPN gene promoters. MC3T3-E1 cells were treated in the absence or presence of serum with increasing concentrations of 1,25(OH)2D3. Cells were harvested after 6 h and subjected to ChIP analysis using antibodies to either VDR or nonspecific IgG. Immunoprecipitated DNA was evaluated for the presence of vitamin D response element-containing Cyp24a1 (left panel) or OPN (right panel) promoter DNA using oligonucleotide primers indicated in Materials and Methods. PCR products were quantitated using densitometry and the values plotted as fold induction over vehicle controls. These data are representative of at least three similar comparable evaluations by ChIP.

Figure 7

Uptake and kidney tissue content of tritiated 1,25(OH)2D3 after injection into wild-type and DBP-null mice. A, The kinetics of 1,25(OH)2D3 uptake into the blood. A single dose of 1,25(OH)2D3 (10 ng/g bw) containing 1 μCi tritiated 1,25(OH)2D3 was injected ip into 8-wk-old female DBP-null mice and their comparable controls. Blood was drawn at the times indicated and the presence of tritiated ligand assessed as described in Materials and Methods. The histogram values represent the mean ± sem (n = 3). These results are representative of two similar experiments. B, The kinetics of 1,25(OH)2D3 uptake into kidneys. A single dose of 1,25(OH)2D3 (10 ng/g bw) containing 1 μCi tritiated 1,25(OH)2D3 was injected ip into 8-wk-old female DBP-null mice and their comparable controls. Individual kidneys were harvested at the time points indicated and processed as described in Materials and Methods, and the presence of tritiated ligand was assessed as in A. The histogram values represent the mean ± sem for each group of mice at each time point (n = 3). *, P < 0.01 relative to wild-type control.

Figure 8

Dose-dependent induction of intestinal and kidney target genes by 1,25(OH)2D3 in wild-type and DBP-null mice. A single dose of 1,25(OH)2D3 (0–100 ng/g bw as indicated) was injected ip into 8-wk-old female DBP-null mice and their comparable controls. The intestine and kidneys were harvested at 6 h and processed to obtain total RNA as described in Materials and Methods. A, Analysis of Cyp24a1 (left panel) and TRPV5 (right panel) transcripts induced in kidney. Total RNA was subjected to qRT-PCR analysis using the primers documented in Fig. 2 and Materials and Methods, normalized to mβ-actin levels and plotted as a function of injected 1,25(OH)2D3 concentration. Each point represents the mean ± sem (n = 5). EC50 and r2 values for each condition are as follows: kidney Cyp24a1 (DBP+/+), 0.33 ng/g bw (0.95); (DBP−/−), 0.23 ng/g bw (0.96); kidney TRPV5 (DBP+/+), 0.09 ng/g bw (0.91); (DBP−/−), 0.02 ng/g bw (0.82). B, Analysis of Cyp24a1 (left panel) and TRPV6 (right panel) transcripts induced in intestine. Measurements were assessed as in A. Each point represents the mean ± sem (n = 5). EC50 and r2 values for each condition are as follows: intestinal Cyp24a1 (DBP+/+), 0.30 ng/g bw (0.77); (DBP−/−), 0.44 ng/g bw (0.88); intestinal TRPV6 (DBP+/+), 1.15 ng/g bw (0.55); (DBP−/−), 0.05 ng/g bw (0.67).

Figure 9

Induction of calcemic responses by 1,25(OH)2D3 in wild-type and DBP-null mice. Eight-week-old DBP-null mice and controls maintained on a standard diet were treated with single doses of 1,25(OH)2D3 at the concentrations indicated. Blood was drawn at 24 and 48 h and serum calcium content determined by atomic absorption spectrophotometry as described in Materials and Methods. A, Experiment 1 examines 1,25(OH)2D3 at 0.01 ng/g bw. *, Different from DBP+/+ controls at the equivalent time points, P < 0.05. B, Experiment 2 examines 1,25(OH)2D3 at 1 ng/g bw. The histograms represent the mean ± sem (n = 5) for each treatment group as indicated. *, Different from DBP+/+ controls at the equivalent time points, P < 0.05; **, different from DBP−/− controls at the equivalent time points, P < 0.05.