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].

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.

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.

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.

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.

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.

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).

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.

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.