Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein

Abstract

A line of mice deficient in vitamin D binding protein (DBP) was generated by targeted mutagenesis to establish a model for analysis of DBP's biological functions in vitamin D metabolism and action. On vitamin D-replete diets, DBP-/- mice had low levels of total serum vitamin D metabolites but were otherwise normal. When maintained on vitamin D-deficient diets for a brief period, the DBP-/-, but not DBP+/+, mice developed secondary hyperparathyroidism and the accompanying bone changes associated with vitamin D deficiency. DBP markedly prolonged the serum half-life of 25(OH)D and less dramatically prolonged the half-life of vitamin D by slowing its hepatic uptake and increasing the efficiency of its conversion to 25(OH)D in the liver. After an overload of vitamin D, DBP-/- mice were unexpectedly less susceptible to hypercalcemia and its toxic effects. Peak steady-state mRNA levels of the vitamin D-dependent calbindin-D9K gene were induced by 1,25(OH)2D more rapidly in the DBP-/- mice. Thus, the role of DBP is to maintain stable serum stores of vitamin D metabolites and modulate the rates of its bioavailability, activation, and end-organ responsiveness. These properties may have evolved to stabilize and maintain serum levels of vitamin D in environments with variable vitamin D availability.

Figures

Figure 1

Targeted disruption of the mouse DBP locus. (a) A fragment of mouse genomic DNA containing exons 4–8 of the DBP gene was used to design the targeting vector. A PGK-promoter/neomycin phosphotransferase cassette was inserted at the BamHI (M) site in exon 5 to disrupt the mDBP gene and provide for positive selection. A DTA cassette was ligated to the 5′ HindIII site for selection against random integration. (b) Restriction enzyme mapping distinguished the intact from the disrupted DBP allele. A mouse DBP exon 2 probe, located outside of the targeting vector itself, hybridized to an 8.8-kb EcoRI (R) fragment from the native DBP allele and a 5.7-kb EcoRI fragment from the disrupted mDBP allele. Restriction sites are: S, SalI; H, HindIII; B, BglII; R, EcoRI; C, ClaI; M, BamHI. (c) The targeting vector (a) was transfected into ES cells, G418 selection was applied, and surviving clones were analyzed by Southern blotting. Analysis of 8 representative ES cell lines among the 65 examined is shown. All eight clones contained the native 8.8-kb mouse DBP EcoRI fragment, and one clone (D1) also contained the 5.7-kb fragment, indicative of successful homologous recombination. DBP, vitamin D binding protein; DTA, diphtheria toxin A; ES, embryonic stem.

Figure 2

Functional inactivation of the mouse DBP locus by homologous recombination. (a) An autoradiogram of the Northern blot analysis of total RNA from livers of DBP+/+, DBP+/–, and DBP–/– mice hybridized with rat DBP cDNA is shown. The full-length 1.8-kb mDBP mRNA was detected in wild-type mice, present at diminished levels in heterozygous mice, and totally absent in mice homozygous for DBP gene activation. Balanced RNA loading was confirmed by the ethidium bromide staining of 18S rRNA (bottom). (b) Western analysis for serum DBP in 12 mice representing each of the three genotypes is shown. The antibody was a cross-reacting, polyclonal rabbit antiserum to rat DBP. The presence and relative levels of the 58-kDa DBP paralleled the mRNA levels in panel a. (c) Semiquantitative radial immunodiffusion analysis of sera from mice of all three genotypes using the polyclonal antiserum confirmed the absence of DBP in DBP–/– mice. (d) Serum saturation binding analysis using tracer 25(OH)[3H]D3 in the presence of increasing concentrations of cold 25(OH)D3is shown. The data were further analyzed by Scatchard plotting (inset). 25(OH)[3H]D3, 25(OH)[26,(27)-methyl-3H]vitamin D3.

Figure 3

Low serum 25(OH)D and 1,25(OH)2D levels and secondary hyperparathyroidism after mild dietary vitamin D deficiency in DBP–/– mice. Groups of DBP+/+, DBP+/–, and DBP–/– mice were fed either standard (vitamin D+) (a–c) or vitamin D–deficient (vitamin D–) (d–f) diets for 4 weeks. Serum 25(OH)D (a and d), 1,25(OH)2D (b and e), and PTH (c and f) levels were determined. Data displayed are the mean + SEM from 10 animals. The differences between DBP+/+ and DBP–/– groups were statistically significant in a, b (**P < 0.001), and f (*P < 0.01). PTH, parathyroid hormone.

Figure 4

Bone mineralization defect after mild dietary vitamin D deficiency in DBP–/– mice. Sections of femurs from age- and sex-matched groups of vitamin D–deficient (vitamin D−) DBP+/+ and DBP–/– mice were stained with Masson's trichrome. Representative photomicrographs from a DBP+/+ (a) and a DBP–/– (b) mouse are shown. Osteoid seams were characteristically thicker in the DBP–/– group (arrowhead). This difference was not observed in mice fed vitamin D–sufficient chow. (c–f) Quantitative histomorphometric analyses of mice on vitamin D–deficient diets demonstrated significant abnormalities in OS/BS (male animals, n = 5; trend similar among females, but not significant; c) and osteoid thickness (both sexes compared, n = 10; d) in DBP–/– mice. The bones of mice from both groups were labeled by two injections of the fluorochrome (calcein) at a 7-day interval, and bone sections were subjected to quantitative histomorphometric analyses to determine the amount of mineralization during this period. The MAR (both sexes compared, n = 7, 8; e) and the MS/BS (both sexes compared, n = 6; f) were indicative of a quantitative mineralization defect in DBP–/– mice. DBP+/+ and DBP–/– mice maintained on normal diets showed no significant differences in any of these parameters (not shown). MAR, mineral apposition rate; MS/BS, mineralizing surface/bone surface; OS/BS, osteoid surface/bone surface.

Figure 5

Accelerated clearance of 25(OH)[3H]D3 from the plasma of DBP–/– mice. (a) 25(OH)[3H]D3 was preincubated with aliquots of serum from either DBP+/+ or DBP–/– mice, and these were injected intravenously into mice of homologous genotype. Plasma was sampled at the indicated times after injection, and tritium counts were obtained. Data were normalized to the calculated total plasma volume and expressed as a percentage of total cpm injected. Data for the time interval from 0 to 24 h represent the mean ± SEM of five replicate experiments, and data in the inset depict the mean ± SEM of four experiments examining the 0–40-min time interval. (b) 25(OH)[3H]D3 was preincubated with aliquots of DBP+/+ or DBP–/– serum and injected intravenously into mice of the same respective genotype. Urine was collected for 24 h using metabolic cages and was counted. Data are the mean ± SEM of six determinations (P < 0.01). (c) Aliquots of plasma from one of the 0–40-min studies in a were extracted in organic solvent and analyzed by TLC. Data were expressed as a percentage of total cpm chromatographed. The percentage of cpm migrating in the 25(OH)D region (left) and the polar region (right) of the chromatograph for each time point were plotted.

Figure 6

Accelerated entry of serum [3H]vitamin D into the liver and its conversion to polar metabolites. [3H]vitamin D3 was preincubated with aliquots of DBP+/+ or DBP–/– serum and injected intravenously into mice in the respective groups. Plasma samples (a) and livers (b) were harvested at the indicated times after injection, and tritium counts were obtained. Data were expressed as a percentage of total cpm injected normalized to total plasma volume (P < 0.05 at 20 and 40 min; a) or per gram of liver (b), and represent the mean ± SEM from three independent experiments. (c) Aliquots of plasma from the 1-min time point in a were extracted and subjected to TLC. The position of a 25(OH)D standard was localized by ultraviolet visualization, and the percentage of total chromatographed cpm migrating in the 25(OH)D region was plotted. (d) The percentage of total chromatographed cpm migrating in the polar region was plotted. The data are the mean ± SEM of two to three independent experiments.

Figure 7

Relative resistance to vitamin D3 toxicity demonstrated by DBP–/– mice. DBP+/+ and DBP–/– mice were injected with toxic doses of vitamin D or with vehicle alone. (a) Serum calcium levels were determined 7 days after injection, and results are expressed as a percentage of the serum calcium in the vehicle-injected control groups. The differences were significant in both comparisons: DBP+/+ group (P < 0.001) and DBP–/– group (P < 0.01) compared with vehicle (not shown), and the calcium increase in the DBP+/+ group compared with the DBP–/– group (P < 0.05, shown). Seven days after injection, kidney sections were fixed and stained with von Kossa to detect calcium deposits. Representative kidney sections from DBP+/+ mice injected with vehicle (b) or vitamin D (c), and DBP–/– mice injected with vehicle (d) or vitamin D (e) are shown (×10). The arrows (c and e) point to calcium deposits in the renal cortex, and these regions are shown in the insets (×20).

Figure 8

Accelerated activation of calbindin-D9K gene expression by 1,25(OH)2D in DBP–/– mice. (a) Vitamin D–deficient DBP+/+ and DBP–/– mice were injected intravenously with 50 ng 1,25(OH)2D3. Animals were sacrificed at the indicated times after injection, and RNA was isolated from the most proximal centimeter of small intestine. The RNA was analyzed by Northern blots hybridized with 32P-labeled calbindin-D9K and [32P]rpL32 (loading control) probes. A representative autoradiogram is shown. (b) Relative band intensities were quantitated by PhosphorImager and were normalized for RNA loading. The data presented are expressed as a percentage of the maximal calbindin-D9K mRNA levels in response to 1,25(OH)2D3. The mean ± SEM from four independent experiments is shown.