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Division of Endocrinology and Metabolism, Center for Osteoporosis and Metabolic Bone Diseases, Department of Internal Medicine, and the Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Address all correspondence and requests for reprints to: Robert S. Weinstein, M.D., University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 587, Little Rock, Arkansas 72205.
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Abstract |
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Introduction |
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We previously reported that glucocorticoid administration to mice dramatically reduces osteoblast number and bone formation due to a decrease in osteoblast precursors combined with stimulation of mature osteoblast apoptosis (4). Besides their effects on osteoblasts, glucocorticoid administration suppresses the number of osteoclast precursors. However, osteoclast number does not fall. This maintenance of osteoclast number, despite a reduction in osteoclast precursors, may be due to prolongation of mature osteoclast life span by glucocorticoid-mediated attenuation of osteoclast apoptosis (5). In vitro, glucocorticoids act directly on osteoclasts to attenuate the proapoptotic effects of bisphosphonates, and this effect is mediated by the glucocorticoid receptor because it could be reversed by the glucocorticoid receptor antagonist RU486 (5). Nevertheless, it is unknown whether glucocorticoids act directly on osteoclasts in vivo or whether glucocorticoid action on other cells or tissues may contribute to maintenance of osteoclast numbers via altered production of growth factors or cytokines.
In earlier work from our group, we found that osteoblast-specific expression of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), an enzyme that inactivates glucocorticoids, protected transgenic mice from glucocorticoid-induced apoptosis of osteoblasts and osteocytes and the resultant reduction in the number of cancellous osteoblasts, osteoid production, and bone formation (6). These findings demonstrate that excess glucocorticoids directly affect bone forming cells in vivo.
Using the same strategy to distinguish the direct from indirect effects of glucocorticoids on osteoclasts, in the studies described herein, we overexpressed 11ß-HSD2 in mice using the osteoclast-specific murine tartrate-resistant acid phosphatase (TRAP) promoter (7). We report that mice harboring the transgene exhibited decreased cancellous osteoclasts after glucocorticoid administration and were protected from the glucocorticoid-induced early, rapid loss of BMD.
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Materials and Methods |
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). The construct containing the murine TRAP promoter was kindly provided by Dr. G. D. Roodman (University of Pittsburgh, Pittsburgh, PA). Transgene DNA was excised from the plasmid vector, gel purified two times, and microinjected into fertilized FVB/N eggs at the University of Arkansas for Medical Sciences transgenic mouse facility. Transgenic founders were identified by Southern blotting using a probe consisting of the human 11ß-HSD2 cDNA. Transgenic offspring were identified by PCR using the following primer sequences: forward, 5'-CCTCTTGCAGCCTCTCTGAC-3' and reverse, 5'-CGGCCGTTACTAGTGGATCT-3'. Transgenic mice were maintained in the FVB/N genetic background by breeding male mice hemizygous for the transgene with wild-type females. All mice used in experiments were hemizygous for the transgene.
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Osteoclast culture
Bone marrow cells were harvested from the femora of untreated wild-type and transgenic mice at 3 or 7 months of age. Cells were suspended in 
MEM media with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and maintained in 75-cm2 flasks for 2 d. Nonadherent cells were collected and plated in 96-well plates (Corning Inc., Corning, NY) at density of 150,000 cells/well in MEM media supplemented with 10% FBS containing 30 ng/ml receptor activator of nuclear factor-
B ligand (RANKL) and 30 ng/ml macrophage-colony stimulating factor (M-CSF) (R & D Systems, Minneapolis, MN) for up to 6 d, at which point at least 80% of the dish surface was covered by multinucleated osteoclastic cells.
Assessment of gene expression by reverse transcription and real-time PCR
Total RNA from murine tibia, lumbar vertebra (L6), liver, and spleen was extracted using Ultraspec reagent (Biotecx Laboratories, Inc., Houston, TX) following instructions of the manufacturer. RNA was also extracted from osteoclasts formed under culture conditions described above at various times. Equal amounts of RNA (2 µg) from each sample were reverse transcribed using a high-capacity cDNA archive kit (Applied Biosystems, Forster City, CA). The first-strand cDNA was subsequently amplified by real-time PCR using TaqMan universal PCR master mix (Applied Biosystems). The primer and probe sets used were: human 11ß-HSD2 forward, 5'-GCTTCAAGACAGAGTCAGTGAGAAA-3', reverse 5'-AGGTTGGCCAGCAGCAATT-3', probe, 5'-FAM-CGTGGGTCAGTGGGA-NFQ-3'; the mouse housekeeping gene, ribosomal protein S2 (ChoB), forward, 5'-CCCAGGATGGCGACGAT-3', reverse, 5'-CCGAATGCTGTAATGGCGTAT-3', probe, 5'-FAM-TCCAGAGCAGGATCC-NFQ-3'; mouse osteocalcin forward, 5'-GCTGCGCTCTGTCTCTCTGA-3', reverse, 5'-TGCTTGGACATGAAGGCTTTG-3', probe, 5'-FAM-AAGCCCAGCGGCC-NFQ-3'; predesigned primer/probe sets for murine calcitonin receptor, TRAP, RANKL, and osteoprotegerin (OPG) were commercially available (Applied Biosystems), where all primers and probes were synthesized. PCR amplification and detection were carried out on an ABI Prism 7300 real-time PCR system (Applied Biosystems) as follows: denaturation at 95 C for 10 min and 40 cycles of amplification including denaturation at 95 C for 15 sec and annealing/extension at 60 C for 1 min. Gene expression was quantified using the comparative threshold cycle (Ct) method by subtracting the ChoB Ct value from the Ct values of the gene of interest because amplification efficiencies of these genes were equal.
Assessment of gene expression by Western blotting
Protein was extracted from lumbar vertebra L6 of untreated female wild-type and transgenic mice of 6 months of age by homogenizing the bone in protein lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and various protease inhibitors]. Osteoclast progenitors in the nonadherent bone marrow cells from the femora of the same mice were cultured for 6 d in the presence of RANKL and M-CSF, and cell layer protein extracted at d 6 of culture, when at least 80% of the dish surface was covered by multinucleated osteoclastic cells. Protein extracts were quantified using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). Protein samples were boiled for 5 min and 50 µg protein from each extract was fractionated by SDS-PAGE on a 10% gel under reducing conditions. The fractionated proteins were transferred to HyBond polyvinyl difluoride membranes (Amersham Biosciences Corp., Piscataway, NJ). The blots were blocked in TBS-Tween 20 (TBS-T) buffer [20 mM Tris base, 0.1375 M NaCl, 0.1% Tween 20, 0.01% Thiomerosal (pH 8.0)] containing 5% skim milk for 3 h at room temperature, incubated with rabbit antihuman 11ß-HSD2 polyclonal antibody (Alpha Diagnostic International, San Antonio, TX) at 1:1000 dilution in TBS-T overnight at 4 C and then incubated for 3 h at room temperature with bovine antirabbit IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:2000 dilution in TBS-T. Signals on the blot were visualized using ECL Advance Western blotting detection kit (Amersham) and scanned in a VersaDoc imaging system (model 3000; Bio-Rad).
Immunohistochemistry
L1–4 from 3-month-old wild-type and transgenic mice were fixed overnight in modified phosphate-buffered formalin (SurgiPath, Richmond IL) and decalcified in 5% EDTA for 1 wk. The vertebrae were then embedded in paraffin and 5-µm longitudinal sections were taken. After paraffin removal and rehydration, antigens were recovered by microwaving the sections in 0.1 M citrate buffer (pH 6.0), for 4 min at 48 C in a H2800 microwave processor (Energy Beam Sciences, Inc., East Granby, CT). The sections were then treated with 0.05% pepsin in 0.1 N HCl, at 37 C for 30 min followed by quenching in 3% H2O2 in methanol for 5 min. Sections were washed twice in PBS and blocked for 20 min in 2.5% normal horse serum. Antihuman 11ß-HSD2 antibody (Alpha Diagnostic International) was diluted to 7.5 µg/ml in PBS and incubated with the sections for 1 h at 37 C followed by rinsing and additional incubation for 20 min with biotinylated antirabbit IgG. RTU Elite ABC reagent (VectaStain; Vector Laboratories, Burlingame, CA) was applied for 20 min, washed thoroughly, and developed with diaminobenzidine (Lab Vision, Fremont, CA). Diaminobenzidine staining was enhanced by a 2-min incubation in 0.15% copper sulfate. Sections were counterstained with 2% methyl green for 2 min, dehydrated, and mounted. Sections of human kidney were used as positive controls. The specificity of the anti-11ß-HSD2 antibody has been confirmed previously by demonstrating that it stained human kidney tubules and collecting ducts, which express high levels of this protein, but not glomeruli, which do not (6).
Functional assessment of the transgene by osteoclast survival
Osteoclast progenitors in nonadherent bone marrow cell populations were maintained for 3 d under conditions described above. Media were then replaced with fresh MEM, RANKL, and M-CSF supplemented with dexamethasone or vehicle. In some experiments, 2 x 10–5 M alendronate was also added to the medium, alone or 30 min after dexamethasone. Osteoclast apoptosis was evaluated 18 h after the addition of dexamethasone and/or alendronate by measuring cell layer caspase-3 activity, resulting from cleavage of 7-amino-4-trifluoromethylcoumarin, a fluorogenic substrate (Biomol Research Labs, Plymouth Meeting, PA), as previously described (5). We have shown previously that bisphosphonates increase active caspase-3 immunostaining, caspase-3 activity, and caspase-9 activity in our osteoclast culture system (5). Under these culture conditions, caspase-3 activity in conditioned medium was negligible. The fluorescent signal generated was detected on a Fluorocount plate reader (Packard Bioscience, Co., Meriden, CT).
BMD Determinations
Starting at 3 months of age, sequential measurements of spinal BMD in live mice were done by dual-energy x-ray absorptiometry on a QDR-2000 Plus densitometer (Hologic, Waltham, MA) using customized software as previously described (8). The measurement was performed at 2- and 4-wk intervals over a 10-wk period.
Evaluation of the basal skeletal phenotype
At 6.5 months of age, the basal skeletal phenotype of the wild-type and transgenic mice was assessed by measuring body weight, bone geometry (femoral length; femoral distal growth plate width; and L5 height, depth and width) and BMD. L1-L4 from wild-type and transgenic mice were fixed and embedded undecalcified in methyl methacrylate and stained for quantitative histological analysis as previously described (4, 5, 9). The histomorphometric examination was done with a computer and digitizer tablet (version 3.00; OsteoMetrics, Decatur, GA) interfaced to a Zeiss Axioscope (Carl Zeiss, Thornwood, NY) with a drawing tube attachment. All measurements were two dimensional, confined to the secondary spongiosa, and made at x400 magnification (plan-neofluar objective, numerical aperture 0.75). The terminology used is that recommended by the Histomorphometry Nomenclature Committee of the American Society for Bone and Mineral Research (10).
For static measurements, cortical width was measured as previously described (5). The cancellous bone area was expressed as the percentage of tissue area, including bone and marrow as described previously (9). The trabecular width and spacing (the distance between the midpoints of adjacent trabeculae) were measured directly. The osteoblast perimeter was expressed as the number of osteoblasts lining osteoid per millimeter cancellous bone perimeter. The osteoclast perimeter was expressed as the number of osteoclasts per millimeter cancellous bone perimeter.
For dynamic measurements, the rate of mineral apposition was calculated as the mean distance between the midpoints of the two tetracycline labels divided by the interdose duration (5 d). The rates of bone formation was calculated as previously described (9).
Prednisolone administration
At 6.5 months of age, male wild-type and transgenic mice were allocated into four groups with equivalent basal spinal BMD values (13 mice per group) and given sc, slow-release pellets (Innovative Research of America, Sarasota, FL) of prednisolone (1.4 mg/kg·d) as dispensed from a 90-d pellet used to ensure constant prednisolone delivery (4) or placebo for 7 d. The prednisolone dose was based on previous dose-response studies (4). Tetracycline HCl (30 mg/kg body weight) was given ip 6 and 2 d before the animals were killed. Spinal BMD of these mice was determined by dual-energy x-ray absorptiometry at d 0 and 7 of the experiment, L1-L4 were processed for histomorphometry, and L6 was used for analysis of total RNA by RT-PCR as described above.
Statistics
To evaluate treatment effects for the various measurements, ANOVA methods were used with interaction tests included where appropriate. Levene’s test was used to test for homogeneity of variance of a given measurement. The Shapiro-Wilk test was performed to investigate the assumption of normality. When either the homogeneity of variance or normality assumptions were not satisfied, transformations were done or the Kruskal-Wallis nonparametric test was used. Bonferroni methods were used to control the experiment-wise error rate when there were multiple comparisons of interest. All comparisons were defined a priori. P < 0.05 were considered significant.
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Results |
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), which is expressed in mono- and multinucleated osteoclasts (7). The transgenic mice were generated and maintained in the FVB/N genetic background. Offspring hemizygous for the TRAP-11ß-HSD2 transgene were obtained at the expected Mendalian frequency. Real-time PCR analysis of mRNA from tibia, L6, liver, and spleen of 4-month-old male wild-type and transgenic mice indicated that expression of the transgene was detected only in transgenic mice and in bone but not soft tissues (Fig. 1B). The specificity of the transgene was confirmed by the differentiation-dependent increase in the mRNA levels of the transgene in osteoclasts in vitro (Fig. 1C), the presence of the expected 41-kDa human 11ß-HSD2 protein in mature osteoclasts derived from transgenic mice (Fig. 1D), and immunostaining of vertebral bone demonstrating the human 11ß-HSD2 protein in osteoclasts of the transgenic mice but not in other cells or the wild-type animals (Fig. 1, E–G). Because osteoclasts constitute only a small fraction of total bone cells, we were unable to detect the 41-kDa human 11ß-HSD2 protein band in bone extracts from transgenic mice (data not shown).
Osteoclast-specific expression of 11ß-HSD2 transgene did not affect skeletal growth
At 6.5 months of age, body weight, geometry of the femur and L6, BMD, and vertebral cancellous histomorphometry of the wild-type and transgenic mice were indistinguishable (Table 1). Furthermore, serial BMD determinations performed every 2–4 wk during this period showed that both genotypes reached peak BMD at 5.5 months of age (data not shown). There were no gender differences. These findings established that expression of 11ß-HSD2 in osteoclasts did not alter skeletal development, peak adult bone mass, or the number of osteoblasts and osteoclasts.
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, in the osteoclasts derived from wild-type mice, treatment with dexamethasone reduced osteoclast apoptosis as indicated by the 22–26% decrease in caspase-3 activity at the lowest concentrations tested (10–10 and 10–9 M) (P < 0.01). In contrast, in the osteoclasts derived from the transgenic mice, the effects of dexamethasone at 10–10 and 10–9 M were attenuated. With higher doses of dexamethasone, the difference between wild-type and transgenic mice was lost. The lack of effect at the higher doses may be due to overwhelming the capacity of the transgenic 11ß-HSD2 enzyme, similar to what occurs in patients receiving high doses of glucocorticoids or with the increased cortisol production that accompanies ectopic production of adrenocorticotropic hormone by tumors. In both cases, the human renal 11ß-HSD2 may be quenched, resulting in hypertension and hypokalemia (11).
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). In contrast, at the lower concentrations of dexamethasone (10–10 and 10–9 M), the antiapoptotic effects of the hormone on osteoclasts obtained from the TRAP-11ß-HSD2 mice was abrogated. Consistent with the results shown in Fig. 2A, the lowest dose of dexamethasone required to antagonize alendronate-induced caspase-3 activity in TRAP-11ß-HSD2 mice was 10–8 M, a dose 100-fold greater than that required in wild-type controls.
Expression of 11ß-HSD2 in osteoclasts prevented prednisolone-induced maintenance of vertebral cancellous osteoclasts and loss of spinal BMD
Having established the absence of basal skeletal phenotype, we went on to challenge 6.5-month-old wild-type and transgenic mice with prednisolone administration for 7 d. In wild-type mice, prednisolone administration resulted in a 102% increase in the mRNA level of the calcitonin receptor, an osteoclast-specific gene (12) previously shown to be stimulated by glucocorticoids (13), in vertebrae (P < 0.001, Fig. 3). This effect, however, was prevented in transgenic mice. In contrast, prednisolone caused a practically identical decrease (94 and 96%) in the mRNA levels of osteocalcin, an osteoblast marker (14, 15), in both wild-type and transgenic mice (P < 0.001). These results demonstrated that the blockade of glucocorticoid action by the TRAP-11ß-HSD2 transgene in vivo was effective and cell type specific. Prednisolone administration did not alter mRNA levels of RANKL and OPG in either genotype (Fig. 3), suggesting that changes in the expression of these mediators of bone resorption (16, 17) were not involved in bone loss during this experiment.
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) and a decrease in the osteoid perimeter, osteoblast number, and bone formation rate (Fig. 4, B, C, and E) in mice of both genotypes.
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). In striking contrast, in the transgenic mice in which glucocorticoid action on mature osteoclasts was blocked by expression of 11ß-HSD2, prednisolone reduced osteoclast number by 50%. This latter result is most likely due to a loss of the antiapoptotic effect of glucocorticoids on mature osteoclasts such that the prednisolone-mediated reduction in osteoclast precursors now results in decreased osteoclast number. Furthermore and in agreement with the changes in osteoclast number, wild-type mice receiving prednisolone demonstrated a 9.4% decrease in spinal BMD (P < 0.001; Fig. 4F), whereas the loss of BMD was prevented in the transgenic mice. |
Discussion |
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Glucocorticoid administration causes a rapid decline in bone mass due to a striking remodeling imbalance in the number of osteoblasts and osteoclasts (4). Glucocorticoids inhibit both osteoblastogenesis and osteoclastogenesis, yet cancellous osteoclast numbers are maintained or increased, whereas osteoblasts decrease precipitously (5). We previously obtained evidence that the dichotomous effects on osteoblasts and osteoclasts number may be explained by prolongation of osteoclast survival and curtailment of osteoblast survival. In the results presented in the present report, we obtained additional evidence that this prolongation is due to direct effects of glucocorticoids on osteoclasts. Indeed, consistent with the contention that glucocorticoids prolong osteoclast life span despite a reduction in osteoclast precursors, glucocorticoid administration to TRAP-11ß-HSD2 transgenic mice resulted in a reduction in osteoclast number and preservation of bone density. These results provide the first in vivo evidence that glucocorticoids act directly on osteoclasts, and this action is the major cause of glucocorticoid-induced early bone loss. In addition, our findings that basal bone geometric, densitometric, and histomorphometric measurements were similar in both young and adult wild-type and TRAP-11ß-HSD2 transgenic mice suggests that endogenous glucocorticoid action in TRAP-expressing osteoclasts is not required for normal skeletal development.
We did not detect significant decreases in the amount of cortical or cancellous bone, by histomorphometry, in the wild-type mice that received prednisolone and lost spinal BMD. However, histomorphometry and BMD measure quite different aspects of bone. The histomorphometric measurements were obtained from one to three longitudinal sections of L1–4, whereas the murine spinal BMD analysis is an integral measurement of all the vertebrae from below the skull to the base of the tail, a much larger skeletal sample. In addition, the coefficient of variation for repeated histomorphometric measurements of microarchitecture is more than 7-fold greater than that for murine BMD (18). The main advantages of histomorphometric measurements lie in quantification of osteoid, bone cells, and the rate of bone formation as demonstrated by the glucocorticoid-induced decrease in osteoid, osteoblasts, and bone formation rate in the wild-type and transgenic mice and the decrease in osteoclasts found only in the TRAP-11ß-HSD2 transgenic animals.
We used the 11ß-HSD enzyme system as a unique tool to distinguish the direct effects of excess glucocorticoids on bone cells from the indirect effects that occur in almost every other tissue. This system is also a natural prereceptor controller of corticosteroid action (19). Variations in the role of this enzyme system in the metabolism of synthetic glucocorticoids and species-specific differences in substrate specificity do, however, exist as evidenced by observations that dexamethasone is readily metabolized by 11ß-HSD2 in rat liver, human kidney, and human osteoblasts but is less effectively inactivated in human lung epithelial cells (20, 21, 22). We previously reported that conditional expression of 11ß-HSD2 in murine osteocytic cells effectively blocks dexamethasone-induced apoptosis (6). However, it is possible that the 11ß-HSD2 system responds to synthetic glucocorticoids in some tissues differently from others and that the results of the in vitro experiments shown here may have been more dramatic if performed with prednisolone rather than dexamethasone (21). Nonetheless, the key observation remains that expressing 11ß-HSD2 with the TRAP promoter prevented the action of glucocorticoids on osteoclasts in vivo.
Glucocorticoid administration enhances expression of RANKL and inhibits production of the decoy receptor for RANKL (osteoprotegerin) by stromal cells and osteoblasts in vitro (16). Therefore, changes in these cytokines have been proposed as candidates mediating the proresorptive effects of glucocorticoids on osteoclasts (17). However, we did not detect changes in RANKL and OPG expression in bone obtained from wild-type and transgenic mice after 7 d of prednisolone administration. Our results demonstrating the blockade of glucocorticoid-induced promotion of osteoclast survival and prevention of bone loss in TRAP-11ß-HSD2 transgenic mice suggest that direct effects of glucocorticoids on osteoclasts are more important than these mediators in the early, rapid loss of bone mass that follows glucocorticoid administration.
In conclusion, the evidence presented herein demonstrates that osteoclasts are direct targets of glucocorticoid action and that this direct action prolongs the life span of these cells. Moreover, our results indicate that direct action(s) of glucocorticoids on osteoclasts is the major cause of the early, rapid loss of bone in states of glucocorticoid excess.
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Acknowledgments |
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Disclosure Statement: R.S.W. is a lecturer for Merck and a consultant for Radius. Other authors have nothing to disclose.
First Published Online August 24, 2006
1 D.J. and C.A.O. have contributed equally to this work. 
Abbreviations: BMD, Bone mineral density; ChoB, ribosomal protein S2; Ct, threshold cycle; FBS, fetal bovine serum; 11ß-HSD2, 11ß-hydroxysteroid dehydrogenase type 2; L, lumbar vertebrae; M-CSF, macrophage-colony stimulating factor; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-B ligand; TBS-T, TBS-Tween 20; TRAP, tartrate-resistant acid phosphatase.
Received April 11, 2006.
Accepted for publication August 15, 2006.
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