A potent analog of 1alpha,25-dihydroxyvitamin D3 selectively induces bone formation

Abstract

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is a principal regulator of calcium and phosphorus homeostasis through actions on intestine, kidney, and bone. 1,25(OH)(2)D(3) is not considered to play a significant role in bone formation, except for its role in supporting mineralization. We report here on the properties of 2-methylene-19-nor-(20S)-1alpha,25(OH)(2)D(3) (2MD), a highly potent analog of 1,25(OH)(2)D(3) that induces bone formation both in vitro and in vivo. Selectivity for bone was first demonstrated through the observation that 2MD is at least 30-fold more effective than 1,25(OH)(2)D(3) in stimulating osteoblast-mediated bone calcium mobilization while being only slightly more potent in supporting intestinal calcium transport. 2MD is also highly potent in promoting osteoblast-mediated osteoclast formation in vitro, a process essential to both bone resorption and formation. Most significantly, 2MD at concentrations as low as 10(-12) M causes primary cultures of osteoblasts to produce bone in vitro. This effect is not found with 1,25(OH)(2)D(3) even at 10(-8) M, suggesting that 2MD might be osteogenic in vivo. Indeed, 2MD (7 pmol/day) causes a substantial increase (9%) in total body bone mass in ovariectomized rats over a 23-week period. 1,25(OH)(2)D(3) (500 pmol three times a week) only prevented the bone loss associated with ovariectomy and did not increase bone mass. These results indicate that 2MD is a potent bone-selective analog of 1,25(OH)(2)D(3) potentially effective in treating bone loss diseases.

Figures

Figure 1

Weanling male rats were obtained from the low-vitamin D colony of Harlan–Sprague–Dawley and housed in overhanging wire cages in a vivarium provided with a 12-h light/12-h dark cycle. The fluorescent lighting was shielded to block UV light. Animals were fed a purified 0.47% calcium, 0.3% phosphorus vitamin D-deficient diet as described by Suda et al. (8) ad libitum for 1 week and then the same diet containing 0.02% calcium and 0.3% phosphorus diet for 3 weeks. During the final week, the indicated doses of either 1,25(OH)2D3 or 2MD (6) were delivered in 0.1 ml of propylene glycol/ethanol (95:5) i.p. each day for 7 days. Control animals received vehicle. (a) Twenty-four hours after the final dose, animals were killed by decapitation and blood was taken to determine serum calcium levels (mg/dl). (b) The first 10 cm of duodenum was used to determine intestinal calcium transport by the everted sac technique as described (8). Each group contained at least five animals, and the values represent the mean ± SEM.

Figure 2

Regulation of RANKL and osteoclast formation by 1,25(OH)2D3 and 2MD. (a) RNA analysis. Murine osteoblastic ST2 cells were cultured in MEMα supplemented with 10% FBS. Cells were plated at densities of 5 × 105/ml in 100-mm dishes and treated for 24 h with the indicated concentrations of vehicle, dexamethasone (10−7 M), dexamethasone (10−7 M) plus 1,25(OH)2D3, or dexamethasone (10−7 M) plus 2MD at the indicated concentrations. Total RNA was isolated by using the Trizol reagent, and 20 μg was used for Northern blot analysis, using end-labeled probes for RANKL, osteoprotegerin, 24OHase, or β-actin. Densitometric measurements were plotted as a function of ligand concentration. (Left) Autoradiograms. (Right) Fold quantitation of densitometric analyses. (b) Induction of osteoclast formation in whole bone marrow cultures treated with vehicle, 1,25(OH)2D3, or 2MD at the indicated concentrations. Bone marrow cells were isolated under sterile conditions from the tibiae and femurs of 6-week-old C57B6 mice (Harlan–Sprague–Dawley) and plated at 1 × 106 cells/well in 48-well plates as described (10). Cells were cultured in phenol red-free αMEM with 10% charcoal–dextran-treated FBS in the presence or absence of compounds. Fresh medium containing the individual compounds was supplemented three times during the 8- to 10-day culture period. Cells were then fixed and stained for TRAP activity as described (10). (c) Quantitation of osteoclast number/well induced by 1,25(OH)2D3 or 2MD at the indicated concentrations in either whole bone marrow or cocultures of ST2 and spleen cells. Spleen cells were also isolated under sterile conditions, and 1 × 106 cells were plated together with 5 × 104 ST2 cells/well in 48-well plates as described. Values represent the number of TRAP-positive, multinucleated (>3 nuclei) osteoclasts/well ± SE for triplicate determinations. (d) Quantitation of total resorption capacity of osteoclasts. The ability of osteoclasts generated from murine bone marrow cells to resorb bone was assessed by first stimulating osteoclast formation with either 1,25(OH)2D3 or 2MD for 14 days on synthetic bone disks (BD Biosciences). Adherent cells were removed by using 5% sodium hypochlorite, and the disks were examined for the presence of resorption lacunace or pits by using dark-field microscopy. Quantitation of the total resorbed surface area was carried out by using Scion image software and was indicated as total surface area resorbed (mm2/disk). Seven different fields on each disk were analyzed to determine total resorption area. The two experimental conditions were carried out in triplicate. Values represent mean ± SE.

Figure 3

Effects of 1,25(OH)2D3 and 2MD on bone formation in osteoblast cultures in vitro. (a) Primary human osteoblasts were isolated as described (9) and cultured in 6-well plates at a density of 3 × 105 cells/ml in DMEM/F-12 medium with 10% FBS. Confluent cultures were treated with 1,25(OH)2D3 or 2MD at the concentrations indicated on days 0, 3, and 6 followed by treatment with ascorbic acid (50 μg/ml) and β−glycerophosphate (10 mM) on days 9 and 12. Cells were stained on day 14 by using the Von Kossa staining technique to detect the presence of calcified matrix/bone (11). The dark brown to black stain is indicative of calcified bone nodules. (Upper) Von Kossa-stained cultures are indicated. (Lower) Microscopic images (×10) are shown. (b) Primary fetal mouse calvarial cells were isolated from wild-type and VDR null mice as described (25). Confluent cultures were treated with vehicle, 1,25(OH)2D3 (10−8 M), or 2MD (10−10 M) and evaluated after 14 days as in a. (c) Bone nodule area (mm2) was quantitated in each culture as described in Materials and Methods. Numbers indicate mean ± SE.

Figure 4

Induction of bone formation in ovariectomized rats by 2MD in vivo. Retired breeders received from Harlan–Sprague–Dawley were sham-operated or ovariectomized. The rats were fed a 0.47% calcium, 0.3% phosphorus diet supplemented with vitamins A, D, E, and K as described by Suda et al. (8), which provides 75 units (3 μg) of vitamin D2/week per animal. Before ovariectomy, the individual BMD were assessed by using dual photon absorptiometry (DPXα General Electric, Lunar). Five weeks after the initial BMD readings were made, oral administration of either 2MD or 1,25(OH)2D3 (6) in 0.1 ml of Wesson oil was initiated at the doses and frequencies indicated. Ovariectomized and sham-operated control animals received 0.1 ml of Wesson oil. (a) Serum calcium levels were determined by atomic absorption spectrometry at the indicated times for vehicle-treated sham-operated rats and ovariectomized rats treated with vehicle, 2MD, or 1,25(OH)2D3 at the indicated dose. (b) Total body bone mass was assessed in each group at 13 weeks and again at 23 weeks as above. *, Significance at P < 0.001.