Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids (original) (raw)

Animals. C57Bl and Swiss Webster mice (Charles River Laboratories, Stone Ridge, New York, USA) were used. At 4 months of age, male Swiss Webster mice were electronically tagged (Biomedic Data System Inc., Maywood, New Jersey, USA) and kept in plastic cages (1 animal per cage) under standard laboratory conditions with a 12-hour dark/12-hour light cycle, a constant temperature of 20°C, and humidity of 48%. All mice were fed on a standard rodent diet (Agway RMH 3000; Arlington Heights, Illinois, USA) containing 22% protein, 5% fat, 5% fiber, 6% ash, 3.5 Kcal/g, 1.0 IU vitamin D3/g, 0.97% calcium, and 0.85% phosphorus with water ad libitum. The animals were weighed at the beginning and end of each experiment. The University of Arkansas for Medical Sciences (UAMS) Division of Laboratory and Animal Medicine approved the protocols.

Osteoclast survival assay. Bone marrow cells were harvested from femora of C57Bl mice (Charles River Laboratories) and cultured in αMEM media with 10% FBS (HyClone Laboratories, Logan, Utah, USA) for 2 days. Nonadherent cells were collected and aliquots of 0.2 × 106 cells were plated on 16-well chamber slides in αMEM media with 10% FBS containing 30 ng/ml human recombinant M-CSF (R & D Systems Inc., Minneapolis, Minnesota, USA) and 30 ng/ml receptor activator of NF-κB ligand (RANKL) (Amgen Inc., Thousand Oaks, California, USA) for 3–4 days. Media was then changed to fresh αMEM, M-CSF, and RANKL with 10% charcoal-stripped FBS, and the cells were incubated in triplicate for 1 hour with vehicle or 10–5 M alendronate before addition of 10–7 to 10–10 M dexamethasone for 24 hours. Floating cells were removed and the attached cells fixed with 2% paraformaldehyde for 15 minutes at room temperature. Floating cells average about 10 per well and attached cells 200 ± 40; thus, it is unlikely that exclusion of the floating cells interfered with the assessment. After washing with TBS, incubating with 3% hydrogen peroxide for 15 minutes, and blocking with 10% goat serum for 30–60 minutes, cells were incubated with active caspase-3 Ab (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) (1:50 in 2% goat serum in TBS) for 2 hours followed by biotinylated anti-rabbit Ab for 30 minutes and immunoperoxidase staining. Tartrate-resistant acid phosphatase (TRAPase) counterstaining was used to measure total osteoclast number. Apoptotic osteoclasts were identified as dark brown cells (active caspase positive) in each well and expressed as the percentage of total osteoclasts per well.

In addition, cells were incubated in triplicate for 1 hour with vehicle or 10–5 M alendronate before addition of 10–9 M dexamethasone, 10–8 M mifepristone (RU 486), or both for 24 hours, and aliquots of the supernatants from the wells were evaluated using fluorogenic substrates (Biomol Research Laboratories, Plymouth Meeting, Pennsylvania, USA) to measure the enzyme activity of caspase-3, caspase-8, and caspase-9.

Bone densitometry. Dual-energy x-ray absorptiometry (DEXA) was used to determine spinal bone mineral density (BMD) in live mice as previously described (21, 22). Over the past 3 years, the coefficient of variation of the measurement done on a plastic-embedded whole mouse skeleton was 1.8% (n = 202). BMD determinations were done at 2-week intervals to identify the peak adult bone mass of the mice, which was reached between 5 and 6 months of age (20–22). Before the experiment began, BMD measurements were repeated to allocate the animals into groups with equivalent spinal BMD values.

Glucocorticoid administration. Slow-release pellets (Innovative Research of America, Sarasota, Florida, USA) of placebo or 2.1 mg/kg/day of prednisolone were then implanted for 4, 10, or 27 days, as described previously (20). For dynamic histomorphometric measurements at the 10-day time point, tetracycline HCl (30 mg/kg body weight) was given intraperitoneally 6 and 2 days before sacrifice. At the time of sacrifice, bone marrow aspirates were obtained from the right femur for ex vivo marrow cell cultures, and the left distal femur and lumbar vertebrae (L1-L4) were prepared for histomorphometric analysis.

Detection and quantification of osteoblast and osteoclast progenitors. Femoral marrow cells were obtained as described previously (23). The number of osteoblast progenitors (CFU-OB) in the marrow isolate was determined by culturing cells at 2.0 × 106 cells per 10-cm2 well for 25–28 days with irradiated guinea pig feeder cells in phenol red–free αMEM containing 15% preselected FBS and 1 mM ascorbate-2-phosphate. One-half of the medium was replaced every 5 days. Colonies containing osteoblasts were visualized by Von Kossa staining.

CFU-OB replication in vitro was determined as described previously (23). One aliquot of cells was used to determine the number of CFU-OB per 106 marrow cells in the initial isolate, as described above. A second aliquot was used to establish replicate cultures of cells in type I collagen gels at 5 × 106 cells in 1 ml of gel, which were then maintained in the absence or presence of 10 nM prednisolone for 7 days. The cells were then dispersed using bacterial collagenase, and the number of CFU-OB within each gel was determined. To calculate the fold increase in CFU-OB during culture in the collagen gels, the number of CFU-OB obtained per gel (after 7 days of culture) was divided by the number of CFU-OB initially incorporated into the collagen gel.

The number of osteoclast progenitors within the marrow isolate was determined by coculturing 75,000 marrow cells with 8,000 UAMS-32 stromal/osteoblastic cells for 8 days in a 2-cm2 well in the presence of 10% FBS in phenol red–free αMEM supplemented with 10 nM 1,25(OH)2D3 to stimulate osteoclast formation, as described previously (24). Replicate cultures (n = 4–6) were established from each animal. Osteoclastic cells were enumerated after staining for TRAPase; both mononucleated and multinucleated cells were counted.

Bisphosphonate administration. To examine the impact of an antiresorptive agent on the loss of bone density that accompanies glucocorticoid excess, we pretreated the mice with subcutaneous injections of 0.75 mg/kg/day of alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate; obtained from C.W.G.M. Löwik, University Hospital, Leiden, The Netherlands) dissolved in saline or saline alone beginning 3 days before prednisolone or placebo administration and then continued as daily injections during glucocorticoid administration. That this dose of alendronate is adequate has been shown by our previous findings that administration of one-third of this amount of alendronate prevented the increase in urinary free deoxypyridinoline excretion and serum osteocalcin that occurs in mice after ovariectomy (25).

Histomorphometric analysis. The lumbar vertebrae were fixed, embedded undecalcified in methyl methacrylate, and stained as described previously (20–22). The histomorphometric examination was done with a computer and digitizer tablet (OsteoMetrics Inc., Atlanta, Georgia, USA) interfaced to a Zeiss Axioscope (Carl Zeiss Inc., Thornwood, New York, USA) with a drawing tube attachment. The identity of each specimen was concealed from the histomorphometry reader. All measurements were two-dimensional, confined to the secondary spongiosa, and made at ×400 magnification (numerical aperture 0.75). The terminology used was that recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (26).

Static measurements of cancellous bone. Cancellous bone area, trabecular width, and osteoid area, perimeter, and width were measured as described previously (20). The osteoblast perimeter was expressed as a percentage of the total cancellous perimeter and also as the number of osteoblasts palisading osteoid per millimeter of cancellous perimeter. Likewise, the osteoclast perimeter was expressed as a percentage of the total cancellous perimeter covered by TRAPase-positive osteoclasts and as the number of osteoclasts per millimeter of cancellous perimeter. The ratio of osteoclasts to osteoblasts was also expressed as both the percentage and number of cells.

Dynamic measurements of cancellous bone. The rate of mineral apposition was calculated as the mean distance between the midpoints of the two tetracycline labels divided by the interdose duration (4 days). The mineralizing perimeter and rate of bone formation per cancellous perimeter (square micrometer per micrometer per day) were calculated as described previously (20, 21).

Measurement of apoptosis in undecalcified bone sections. Apoptosis was detected by in situ nick-end labeling (ISEL) using Klenow terminal deoxynucleotidyl transferase (Oncogene Research Products, Cambridge, Massachusetts, USA) as described previously (27). Sections were counterstained with 0.5–3% methyl green. Plastic-embedded sections of vertebrae taken from orchidectomized adult mice were used as a positive control. Omitting the transferase made negative controls. Apoptotic osteoblasts were identified as ISEL-positive cells lining the osteoid-covered cancellous perimeter.

Statistics. In the osteoclast apoptosis assay, drug effects were examined using one-way ANOVA. In addition, a dose-response test for a linear trend between vehicle and dexamethasone was done using linear contrast coefficients. To evaluate the changes in ex vivo marrow cell cultures and 4- and 10-day BMD measurements, we used two-way ANOVA. If variances were unequal, a Satterthwaite approximation was used to determine the degrees of freedom. Differences between group means with the CFU-OB replication assay were evaluated with Student t tests. Serial changes in BMD were analyzed using a mixed effects model of repeated measures (28). Histomorphometric data were examined by one-way ANOVA. Comparisons of interest were specified a priori and their P values adjusted with Bonferroni’s correction (29). Pearson correlation coefficients were calculated to test for an association between two independently measured variables. P values less than 0.05 were considered significant.