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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Arthritis Rheum. 2010 Feb;62(2):534–541. doi: 10.1002/art.27219

Adenosine A1 Receptors (A1R) Regulate Bone Resorption II Adenosine A1R Blockade or Deletion Increases Bone Density and Prevents Ovariectomy-Induced Bone Loss

Firas M Kara 1, Stephen B Doty 2, Adele Boskey 2, Steven Goldring 2, Mone Zaidi 3, Bertil B Fredholm 4, Bruce N Cronstein 1
PMCID: PMC2853194  NIHMSID: NIHMS190575  PMID: 20112380

Abstract

Objective

Accelerated osteoclastic bone resorption plays a central role in the pathogenesis of osteoporosis and other bone diseases. Because identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and to the development of new treatments we studied the effect of adenosine A1 receptor blockade or deletion on bone density.

Methods

Bone mineral density (BMD) in adenosine A1 receptor knockout mice was analyzed by DEXA scan and the trabecular and cortical bone volume was determined by Micro CT. Mice were ovariectomized or sham-operated, and 5 weeks after surgery, when osteopenia had developed, several parameters were analysed by DEXA scan and MicroCT. Histological examination of bones from A1 knockout and wild type mice was carried out. Visualization of osteoblast function (bone formation) after Tetracycline double labeling was performed by fluorescence microscopy.

Results

MicroCT analysis of bones from A1KO mice showed significantly increased bone volume. Electron microscopy of bones from A1KO mice shows an absence of ruffled borders of osteoclasts and osteoclast bone resorption. Immunohistology demonstrates that although osteoclasts are present in the A1KO mice they are smaller and often not associated with bone. No morphologic changes in osteoblasts were observed and bone labeling studies reveal no change in bone formation rates in the A1KO mice.

Conclusion

These results suggest that the adenosine A1 receptor may be a useful target in treating diseases characterized by excessive bone turnover such as osteoporosis and prosthetic joint loosening.

INTRODUCTION

Homeostasis in bone relies on the balance between bone formation by osteoblasts and bone resorption by osteoclasts, processes which are tightly linked (1). When this balance is disturbed in favor of bone resorption it results in pathological bone destruction, as observed in osteoporosis (2) or inflammatory diseases like rheumatoid arthritis (3). Osteoblasts, the principal bone-forming cells are derived from mesenchymal stem cells (4). Osteoclasts are multinucleated giant cells (MNCs) responsible for resorbing bone, and form from the fusion of myeloid precursors of monocyte/macrophage lineage (5) under the control of systemic hormonal factors and local factors produced by supporting cells such as osteoblasts and bone marrow stromal cells. Among these factors, two critical extracellular regulators of osteoclast differentiation and activation recently have been recently identified: macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor K-B (NF-KB) ligand (RANKL) (6-10).

Adenosine is released from most cells and tissues as a result of ATP catabolism in response to stress such as hypoxia and inflammatory injury. Adenosine regulates numerous physiologic functions via interaction with one or more of at least four known receptors, designated as A1, A2A, A2B, and A3 (11). All these receptors are members of the family of G protein-coupled receptors. At least one adenosine receptor type, and generally more than one type, is expressed on nearly every cell type and tissue examined. The role of adenosine and adenosine receptors in the regulation of cells involved in bone metabolism and turnover has not been studied

In prior studies we found that adenosine A1 receptors (A1R) play an important role in promoting human monocyte fusion into giant cells in vitro (12). This prompted us to determine whether these receptors similarly regulate the formation of osteoclasts from myeloid precursors. In the associated manuscript we demonstrated that adenosine A1 receptor activation is required for appropriate formation and function of osteoclasts in vitro (Kara et al). Therefore, to determine how adenosine A1 receptors regulate bone homeostasis in vivo we examined the effect of adenosine A1 receptor deletion and blockade on bone density. Surprisingly, although osteoclast formation in vitro is defective in cells from adenosine A1 receptor knockout mice (A1R KO), in vivo these mice exhibit normal numbers of osteoclasts. However, A1R KO osteoclasts do not appear to be actively reabsorbing bone in vivo. No change in bone formation was observed in the A1R KO but, consistent with the diminished in vitro function of osteoclasts in the absence of A1R, there is a significant increase in bone density in the knockout mice. Moreover, administration of an adenosine A1R antagonist 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX) prevents ovariectomy-induced bone loss in a model of post-menopausal osteoporosis.

Materials and Methods

Materials

Tetracycline, demeclocyline and DPCPX were purchased from Sigma Aldrich. DPCPX for in vivo studies was purchased from Toronto Research Chemicals, Ontario, Canada. Procollagen I from (Hybridoma Bank, U.Iowa).

Animals

Female 129/Sv Adenosine A1 knockout mice were originally developed as described elsewhere (13). Mice heterozygous for the null allele of the adenosine A1 receptor were bred in the NYU SoM animal facilities and homozygous null or homozygous wild type mice were isolated and bred continuously for these experiments. Genotyping was performed by polymerase chain reaction (PCR) on tail DNA. Using the primers 5-CCTGCTTCTGTTTCCCAAAG-3 (forward) and 5-CCACAAGGGAGAGAATCCAG-3 (reverse), which detect the wild-type A1 allele and 5-AGCACGTACTCGGATGGAAG-3 (forward) and 5-CCACAGTCGATGAATCCAGA-3 (reverse), which detect the mutated A1 allele. All protocols were approved by the NYU SoM Institutional Animal Care and Use Committee.

Ovariectomy

Two separate experiments were carried out in which either 4-6 week or 10-12 week old female mice were received a bilateral ovariectomy or a sham procedure at NYU SOM. C57Bl6 female mice, either 5 mice 4-6 week or 5 mice 10-12 week old, were subject to ovariectomy or a sham procedure in which the ovaries were exteriorized but not removed at Jackson Laboratories prior to their receipt at NYU School of medicine. Some animals of C57Bl6 group received either DPCPX, a selective adenosine A1 receptor antagonist (final dose 50mg/kg/day), or vehicle in their in their water. Mice were sacrificed by CO2 narcosis 5 weeks after the surgical procedure and Femurs were processed for MicroCT analysis

Bone Histology

Mouse femurs from 4 WT and 4 A1KO mice were excised, cleaned of soft tissue, placed into 10% formaldehyde for 24-48 hours and decalcified in EDTA. Paraffin-embedded histologic sections were stained with hematoxylin and eosin, by immunohistologic techniques for Procollagen I or for tartrate-resistant acid phosphatase (TRAP) activity. We measured the bone volume in a standard zone, situated at least 0.5 mm from the growth plate, excluding the primary spongiosa and trabeculae connected to the cortical bone, and enumerated the osteoclasts and trabecular area in the same zone as that used for assessing bone volume (original magnification, x10) using Bioquant software (14). For measuring the osteoid we use either the von Kossa stain (using calcified sections in methacrylate) or the Goldners stain (also on calcified sections).

In vivo Bone formation

In separate experiments, double labeling was carried out to examine the extent and rate of osteoblastic bone formation. Tetracycline (15 mg/kg of body weight) was administered in 4 WT and 4 A1KO mice i.p. at 8 days prior to sacrifice and demeclocyline (15 mg/kg of body weight) at 3 days prior to sacrifice. At sacrifice, the mouse long bones were fixed in 80% ethyl alcohol, followed by dehydration in graded alcohols and methacrylate embedding. The bone formation rate and the mineral apposition rate (MAR, in microns per day) were determined by dividing the mean of the width of the double labels by the interlabel time. Morphometry is performed by using a BioQuant imaging system (15).

Electron microscopy of osteoclasts

Femurs of five animals were fixed in 2.5% paraformaldehyde plus 0.5% glutaraldehyde in 0.05M sodium cacodylate buffer (pH 7.4) for 12 hours at ambient temperature. After rinsing three times for 20 min in the same buffer, the material was post fixed for 1 hour in 1% osmium tetroxide (in 0.1 M sodium cacodylate buffer), dehydrated in graded ethyl alcohol series and embedded In epon (Embed-812, EM Sciences). Thin sections (80-90 nm) of calcified bone were collected in distilled water containing one drop of brom-thymol blue, with pH at 8.0 or greater, to prevent mineral dissolution from the thin sections. Sections were stained with lead citrate and alcoholic uranyl acetate, and examined in a Philips CM-12 electron microscope.

Measurement of Bone Mineral Density

We assessed the bone mineral density (BMD; g/cm2) of the whole skeleton of 6 month-old mice with a LUNAR PIXIMUS bone densitometer (Lunar Corp., Madison, WI). The instrument was calibrated before each scanning session using a Phantom with known BMD according to the manufacturer's guidelines. 12 WT and 7 A1KO mice were anesthetized by an intraperitoneal injection of ketamine (90 μg/g body weight) and xylazine (10 μg/g body weight) and then were placed in prone position on the specimen tray to scan the entire skeleton.

Micro-CT

Femurs of 4 WT and 4 A1KO were measured in Micro CT as described (16) by using a MS-8 (MS-8, GE Healthcare, London, Ontario, Canada) at 18 μm isotropic resolution-scans calibrated by air, water and a mineral standard material phantom and the Parker algorithm for digital reconstruction (17). Parameters were calculated using instrument-supplied software.

Luminex multiplex assays

Multi-analyte profiling was performed on the Luminex-200 system and the XMap Platform (Luminex Corporation). Calibration microspheres for classification and reporter readings as well as sheath fluid were also purchased from Luminex Corporation. Acquired fluorescence data were analyzed by the Beadview software. Plasma concentrations of OPG, RANKL and Osteocalcin were determined by the plex panel (Millipore). All analyses were performed according to the manufacturers’ protocols.

Statistical Analysis

Statistical significance for differences between groups was determined by use of Student's T test or ANOVA followed by Tukey's post hoc testing, as appropriate. All statistics were calculated using GraphPadâ Software running on a PC.

RESULTS

Skeletal phenotype of A1 knockout mice

To determine whether the adenosine A1 receptor-mediated defect in osteoclast formation and function observed in vitro is reflected in vivo we studied the skeleton of A1KO mice compared to WT mice. The A1KO mice and their normal littermates did not differ in external appearance, bodyweight or organ weight (data not shown), as previously described. There were no abnormalities in tooth eruption and the relative size and shape of the bones in the A1KO mice did not differ from those of control mice. Using high-resolution MicroCT studies we observed that 6-month old A1KO mice had significantly greater trabecular and cortical bone density than wild type mice (Figure 1A). There was greater trabecular bone volume (TV/BV), trabecular number and trabecular thickness with a concomitant reduction in trabecular separation (Figure 1B) in the femurs of A1KO than WT mice. Cortical bone, total area, BMC and outer perimeter were also increased in A1KO mice (data not shown). Dual X-ray absorptiometry (DEXA scan) of whole mice confirmed the increased bone mineral density in the A1KO mice (Figure 1C). Alizarin blue staining in A1KO mice demonstrated increased cartilage remnants (blue staining) in bone, a characteristic of osteopetrosis resulting from defective resorption (Figure 1D).

FIGURE 1. Adenosine A1 receptor knockout mice have increased bone mass.

FIGURE 1

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(A) 5 Femurs from 6-month old control (wild type) and A1KO mice were examined by MicroCT. Two-dimensional (top) and three-dimensional (bottom) reconstruction of femurs revealed increased bone mass in A1KO mice compared with wild type littermates. (B) Histograms of three-dimensional trabecular structural parameters in the secondary spongiosa of the proximal femur: bone volume fraction (BV/TV), trabecular number (Tb.N), Trabecular thickness 3D (TB.TH. 3D), trabecular separation (Tb.Sp), cortical bone area and outer perimeter. (C) Whole Bone mineral density and mineral content assessed by DEXA scan in 6-month A1KO mice and wild type littermates, as described. Bone mineral density (BMD) and bone mineral content (BMC) from 12 WT and 7 A1KO mice is plotted. There was no difference in the body weight of WT and A1KO mice (25.4 ± 0.6 grams N=12 vs 27.2 ± 1.3 N=7). (D) A1KO mice had increased cartilage remnants (blue staining) in bone, a characteristic of osteopetrosis resulting from defective resorption.

Histological and histomorphometric analysis of femurs in A1KO mice

To better characterize the bone phenotype in the A1KO and WT mice we examined the long bones of the mice histomorphometrically. Surprisingly, as shown in figure 2A, there were numerous TRAP-positive osteoclasts in the femoral metaphyses of both the A1KO and WT mice. The increased BV/TV observed in A1KO mice by MicroCT and increased bone density observed by DEXA scan was confirmed by histomorphometric quantitation of BV/TV. As with the MicroCT, there was an increase in the trabecular number and decreased trabecular separation in the femurs of the A1KO compared to the WT femurs.

FIGURE 2. Osteoclast numbers are comparable in wt and A1 knockout mice.

FIGURE 2

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(A) Representative histological sections of a femur stained for TRAP activity, as a marker of osteoclasts, counterstained with hematoxylin. (B) Trap+ cells were counted in four 10x fields per slide and normalized to the bone volume, N=4. Bone Volume / Tissue volume (BV/TV) and Trabecular number (Tb N) are increased in the A1KO mice, while the Trabecular separation (Tb Sp) is decreased in the A1KO. The Osteoclast number/bone perimeter (OC/B.Pm) was not different from the WT.

Surprisingly, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells per square millimeter of tissue area was similar in the wild type and knockout mice (Figure 2B). Moreover, unlike results obtained in vitro, osteoclasts in A1KO mice were multinucleated, stained strongly with TRAP and morphologically resembled WT osteoclasts (Kara et al.).

A1KO osteoblast number and function are normal

As described above, histological analysis of femoral bone sections confirmed the increased bone density observed by MicroCT and DEXA scan and the A1KO mice had increased cartilage remnants, a characteristic of osteopetrosis due to defective resorption. We next determined whether there was an effect of adenosine A1 receptor deletion on osteoblast number, function or bone formation. Osteoid width was similar in A1KO and wt mice (Figure 3A) and there was no obvious difference in the morphology of osteoblastic bone formation by electron microscopy (data not shown). Bone formation rate and mineral apposition rates, determined following tetracycline labeling, we measured the rate of bone deposition in A1KO and control littermates in vivo. We twice injected six to eight-week-old mice with Tetracycline and demeclocyline, with an interval of two days between injections. We then killed the mice after two days, sectioned the bone and examined it by fluorescence microscopy. A1KO and wild-type mice showed no different in double labeling with no difference in mean mineral apposition rate (Figure 3B), indicating a normal rate of osteoblastic bone formation in the A1KO mice. Histomorphometric analysis of procollagen 1-positive osteoblasts did not reveal any difference in the number or density of these cells between wild type and knockout mice (Figure 3C), Further evidence to support this hypothesis is provided by the finding that RANKL and OPG concentrations, and the RANKL: OPG ratio in the serum of 12 WT and 7 A1KO mice also did not differ (Figure 3D). These findings confirm the hypothesis that the increase in bone density in the A1KO mice results from osteoclastic bone resorption rather than from an increase in bone formation.

FIGURE 3. Normal bone formation in A1KO mice.

FIGURE 3

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(A) Femurs were fixed and processed for von Kossa staining. Osteoid widths were not significantly different in the femurs of A1KO as compared to the WT mice. (B) Mice were fed tetracycline and demeclocycline, as described before isolation and processing of femurs for examination under fluorescent light. Mineral apposition rate (MAR) and bone formation rate (BFR) in A1KO mice were calculated as described in Materials and Methods. Results are presented as mean ± SD, n = 4 in each group of mice. (C) The Osteoblast number/ trabecular area (Ob/Tr.a) and the Osteoblast number/bone perimeter (Ob/B.Pm) were the same in WT compare to A1KO mice. (D) RANKL and OPG concentrations, and the RANKL: OPG ratio in serum of 12 WT and 7 A1KO mice, as determined by Luminex assay, were not different.

To better understand how morphometric studies could demonstrate increased bone density despite an absence of any change in either osteoblast or osteoclast number or morphology we examined WT and A1KO bone by transmission electron microscopy. We found, as we had found in vitro, that there appeared to be a defect in osteoclast bone resorption. In the osteoclasts of the A1KO mice there was a marked loss of ruffled borders and bone resorption as compared to the WT mice (Figure 4).

FIGURE 4. Absence of ruffled borders in A1KO osteoclasts.

FIGURE 4

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Bone from wild type and A1 knockout mice was fixed and prepared for electron microscopy by standard methods. There is a normal ruffled border with resorption of bone in the WT osteoclasts (arrowhead in upper and lower panels) with features of a normal activated, mineral-resorbing osteoclast. There is no ruffled border in the A1KO osteoclasts and a clear zone (CZ) is present at sites of osteoclast adhesion to bone.

Adenosine A1 receptor knockout and blockade prevents ovariectomy-induced bone loss

Because blockade or deletion of adenosine A1 receptors diminishes osteoclast function in vitro and A1KO mice have increased bone density we next determined whether pharmacological blockade of adenosine A1 receptors prevents pathologic bone loss in a murine model of post-menopausal bone loss following ovariectomy. We therefore determined the effect on ovariectomy-induced bone loss of the systemic administration of a selective adenosine A1 receptor (DPCPX, 50mg/kg/d in the drinking water (18), over 1000 references to date,(http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=DetailsSearch&term=dpcpx+A1+receptor&log$=activity), antagonist administered at a dose previously demonstrated to give receptor selective effects (18, 19). Compared with the sham-operated animals the ovariectomized mice suffered marked trabecular bone loss after as little as four weeks following the procedure, as determined by MicroCT and DEXA scan scan (Figure 5A). Treatment with DPCPX completely prevented ovariectomy-induced bone loss and modestly increased bone density in the sham-ovariectomized mice (Figure. 5B). Results of DEXA scan scan of these mice further confirmed that administration of DPCPX prevented OVX-induced bone loss (Figure 5C), as compared to the vehicle-treated ovariectomized mice. Similarly, the adenosine A1 knockout mice did not suffer bone loss following ovariectomy (Figure 5C). There was no significant change in serum RANKL or osteoprotegerin level or their ratio in treated mice (Figure 6) consistent with the hypothesis that the major effect of DPCPX treatment in these mice is on osteoclasts and their function.

FIGURE 5. DPCPX prevents bone loss in ovariectomized mice.

FIGURE 5

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Mice were ovariectomized or sham ovariectomized, as described and treated with vehicle or DPCPX (50mg/kg/d) for 5 weeks, as described, before sacrifice. (A) Representative 2-dimensional (top panels) and 3-dimensional MicroCT reconstructions of femurs from sham ovariectomized or ovariectomized (OVX) mice treated with vehicle or DPCPX are shown. (B) MicroCT-derived quantification of the bone volume as a percentage of total volume (BV/TV), trabecular number (Tb N), and trabecular space (Tb Sp) is plotted. (C) BMD of the whole mice was measured by using a small animal dual-energy x-ray absorptiometry apparatus. Shown are the means ± SEM of 5 mice for each group.

FIGURE 6. Normal Levels of RANKL, Osteoprotegerin (OPG) and osteocalcin in ovariectomized (OVX) or sham ovariectomized mice treated with DPCPX.

FIGURE 6

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Mice were ovariectomized or sham ovariectomized, as described and treated with vehicle or DPCPX (50mg/kg/d) for 5 weeks, before sacrifice and collection of serum. (A) RANKL and OPG concentrations and the RANKL: OPG ratio in the serum of animals was determined by luminex (B) osteocalcin concentration determined by commercial ELISA. Each bar represents results from 5 mice for each measurement.

Discussion

Our results clearly demonstrate that adenosine A1 receptors play a role in regulating bone turnover and this effect is primarily mediated by regulation of osteoclast differentiation and function. When studied in vitro adenosine A1 receptor blockade or deletion diminishes osteoclast differentiation and function (Kara et al). Mice lacking adenosine A1 receptors have increased bone density and blockade of adenosine A1 receptors prevents ovariectomy-induced bone loss. Moreover, our results indicate that adenosine A1 receptor blockade diminishes osteoclast function by increasing degradation of TRAF6, a downstream signaling protein for RANK that is critical for osteoclast differentiation and function (Kara et al).

When studied in vivo we found no evidence for a change in osteoblast number, morphology or function in adenosine A1KO mice or following ingestion of a selective adenosine A1 receptor antagonist. The osteoprotegerin/RANKL ratio does not differ between wild type and A1KO or DPCPX-treated and untreated mice. Moreover, tetracycline labeled bone formation rates and width of osteoid (measured histomorphometrically with either von Kossa or Goldner's stain) were similar in wild type and A1KO mice. These observations provide further support for the hypothesis that the effect of adenosine A1 receptor deletion or blockade on bone metabolism is not mediated primarily through effects on osteoblast function. As osteoclast and osteoblast function are tightly linked more subtle effects on their interactions resulting from A1 receptor occupancy cannot be ruled out.

In contrast to our findings with a selective adenosine A1 receptor antagonist (Kara et al), ingestion of caffeine, a non-selective and fairly weak adenosine receptor antagonist, has been reported to be an important risk factor for osteoporotic fractures (20). A Swedish study in middle-aged and elderly women has shown that consuming caffeine in amounts equivalent to approximately 4 cups (600 ml) of coffee or more per day leads to an increased risk of osteoporotic fractures when calcium intake is low (21). In contrast to the selective adenosine A1 receptor antagonist, DPCPX, caffeine blocks A2A and A2B receptors and thus its effect on bone mineral density is likely to arise from blockade of other adenosine receptors. In accord with this hypothesis we have observed that adenosine A2A and A2B knockout mice have diminished bone density (F. Kara and B.N. Cronstein, Unpublished).

The observations reported here indicate that adenosine A1 receptors represent a novel target for the treatment of pathologic bone loss, such as post-menopausal osteoporosis, prosthetic joint loosening, Paget's disease and some forms of tumor-associated bone loss. Although adenosine A1 receptors are ubiquitous in their expression it is possible to block these receptors without overwhelming toxicity, as demonstrated by the development of adenosine A1 receptor antagonists for the treatment of congestive heart failure which are currently in the late stages of clinical development. Thus, A1 receptor antagonists may represent a promising new class of antiresorptive drugs for the treatment of osteoporosis and other bone diseases associated with increased osteoclast activity.

Disclosure

Intellectual Property

Dr. Cronstein holds or has filed applications for patents on the use of adenosine A2A receptor agonists to promote wound healing and use of A2A receptor antagonists to inhibit fibrosis; use of adenosine A1 receptor antagonists to treat osteoporosis and other diseases of bone; the use of adenosine A1 and A2B Receptor antagonists to treat fatty liver, and; the use of adenosine A2A receptor agonists to prevent prosthesis loosening.

Consultant (within the past two years)

King Pharmaceutical (licensee of patents on wound healing and fibrosis above). CanFite Biopharmaceuticals, Savient Pharmaceuticals, Bristol-Myers Squibb, Roche Pharmaceuticals, Cellzome, Tap (Takeda) Pharmaceuticals, Prometheus Laboratories, Regeneron (Westat, DSMB), Sepracor, Amgen, Endocyte, Protalex, Allos, Inc., Combinatorx, Kyowa Hakka. Honoraria/Speakers’ Bureaus: Tap (Takeda) Pharmaceuticals. Stock: CanFite Biopharmaceuticals received for membership in Scientific Advisory Board. The NIH Core Center grant AR046121

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