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Pfizer Global Research and Development (H.Z.K., H.Q., D.T.C., W.A.G., V.M.P., M.L., L.P.A., C.A.G., D.D.T.), Groton Laboratories, Groton, Connecticut 06340; Canadian Institutes of Health Research Group in Skeletal Development and Remodeling (A.F.W., N.P., S.J.D., S.M.S.), and Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada N6A 5C1; and Radiobiology Division (Q.Z., W.S.S.J.), University of Utah School of Medicine, Salt Lake City, Utah 84108
Address all correspondence and requests for reprints to: Dr. H. Z. Ke, Osteoporosis Research, Mail Stop 8118W-216, Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut 06340. E-mail: huazhu_ke{at}groton.Pfizer.com.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Like other members of the P2X family, the P2X7 receptor is an ATP-gated ion channel. However, the P2X7 receptor has some distinguishing features, as reviewed by North (3). For example, it requires higher levels of ATP for activation compared with other P2X receptors. In addition, the P2X7 receptor has the ability to form large membrane pores when the concentration of divalent cations in the extracellular medium is lowered. P2X7 receptor expression and function have been studied in cells of hematopoietic origin, including monocytes, macrophages, and some lymphocyte populations (1, 3, 4). Osteoclasts, which are of hematopoietic origin, express multiple P2 receptors (2). Patch clamp electrophysiology, Ca2+ fluorescence, and dye-uptake studies have suggested the presence of P2X7 receptors in osteoclasts (2, 5, 6, 7). On the other hand, expression of the P2X7 receptor in cells of the osteoblast lineage is controversial. Two groups found no evidence for P2X7 receptor in osteoblasts (7, 8), whereas another group reported P2X7 receptor in a subpopulation of human osteoblasts (9).
In cells of hematopoietic origin, the P2X7 receptor has been reported to participate in diverse activities (3). These include lymphocyte proliferation, giant cell formation, cell death, killing of invading mycobacteria, and the posttranslational processing of IL-1. Further, ligation of the P2X7 receptor has been associated with activation of phospholipase D and activation of some forms of nuclear factor-
B (10, 11).
Although in vitro evidence indicates that the P2X7 receptor is expressed in osteoclasts and possibly osteoblasts, the role of the P2X7 receptor in bone development and remodeling in vivo is not known. The availability of genetically modified mice deficient in the P2X7 receptor [knockout (KO)] (12) provides a means for investigating this question. Peritoneal macrophages isolated from KO animals are defective in that they do not release IL-1 in response to ATP challenge (12). It has also been reported that KO mice exhibit altered leukocyte function and an attenuated inflammatory response (13). Here, we characterize a unique bone phenotype in KO mice using multiple approaches, including peripheral quantitative computerized tomography (pQCT), histomorphometry, bone marrow cell culture, patch clamp recording of ionic currents, and fluorescence methods to evaluate P2X7 pore formation. Two age groups, 2 and 9 months, were selected to determine how the absence of the P2X7 receptor affected bone mass, bone formation, and bone resorption in rapidly growing and adult mice. Our studies establish the presence of P2X7 receptors in osteoblasts and osteoclasts and reveal a critical role for this nucleotide receptor in bone formation and resorption.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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).
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). KO mice also had lower total bone content than their WT controls (Fig. 2C). The femoral shaft, a site containing only cortical bone, was also analyzed by pQCT. KO mice had smaller shaft diameters (Fig. 2B) and lower total bone content (Fig. 2D) than their WT controls, indicating reduced periosteal bone expansion. In general, male KO mice demonstrated a more obvious reduction in bone mass than female KO mice relative to their WT controls.
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and Supplemental Table 2, which is published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). In addition, periosteal and endocortical circumferences were significantly decreased (by 14% and 15%, respectively) in male KO mice compared with WT controls at 2 months of age (Supplemental Table 2). At 9 months of age, KO male mice had significantly lower total bone content (-27%), total bone area (-28%), trabecular area (-27%), cortical content (-30%), cortical density (-8%), cortical area (-24%), cortical thickness (-11%), periosteal circumference (-15%), and endocortical circumference (-16%) compared with WT controls. The reduction in bone mass of the distal femoral metaphysis of KO female mice was less than that in KO male mice at 2 months of age relative to their gender- and age-matched controls. In contrast, the reduction in bone mass of female and male KO mice at 9 months of age was similar (Supplemental Table 2). At 9 months of age, KO female mice had significantly lower total bone content (-17%), total bone area (-16%), trabecular area (-18%), trabecular content (-22%), cortical content (-15%), cortical area (-12%), periosteal circumference (-8%), and endocortical circumference (-10%) compared with WT controls. These data indicate that P2X7 receptor deficiency leads to a significant reduction in both cortical and trabecular bone content and density stemming from inhibition of periosteal expansion in the distal femoral metaphysis.
The femoral shafts of KO male mice at 2 months of age exhibited a reduction of total bone content (-22%), total bone area (-25%), cortical content (-21%), cortical area (-21%), periosteal circumference (-13%), and endosteal circumference (-22%) compared with WT controls (Fig. 2D and Supplemental Table 3, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). These parameters were also significantly lower in adult KO male mice compared with WT controls. In KO female mice, total bone content, total bone area, cortical area, periosteal circumference, and endocortical circumference were decreased significantly compared with WT controls at 2 months of age (Fig. 2D and Supplemental Table 3). At 9 months of age, the KO female mice had significantly lower total bone content (-17%), total bone area (-18%), cortical content (-15%), cortical area (-12%), periosteal circumference (-9%), and endocortical circumference (-15%) compared with WT controls. These data show a very marked reduction in cortical bone area, mineral content, and density in P2X7 receptor-deficient male and female mice.
Histomorphometric Analysis of Tibias
To examine further the nature of these changes in bone structure, we carried out histomorphometric analyses. In trabecular bone of the proximal tibial metaphysis, both young and adult male and female KO mice exhibited increased osteoclast number per mm bone surface (OCN/BS) and percent osteoclast surface (OCS/BS) compared with their gender- and age-matched WT controls (Fig. 3 and Supplemental Table 4, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). These data indicate that P2X7 receptor deficiency leads to increased resorption of trabecular bone. In addition, there was a significant decrease in trabecular bone volume (BV/TV, -26%), mineral apposition rate (MAR, -15%), and bone formation rate/tissue volume referent (BFR/TV, -37%) in young male KO mice compared with WT controls (Supplemental Table 4). In female mice, BV/TV did not differ between KO and WT at 2 months of age but was significantly decreased in adult female KO mice compared with WT controls. Moreover, the BFR/BV, an index of bone turnover, was increased significantly in adult female KO mice (Supplemental Table 4).
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and Supplemental Table 5). The differences in cortical bone mass, structure, and periosteal bone formation parameters between KO and WT male mice at 9 months of age were similar to those observed at 2 months of age (Supplemental Table 5). In addition, endocortical MS/BS and BFR/BS were decreased significantly in 9-month-old KO male mice compared with WT controls. Endocortical eroded surface, an index of bone resorption, did not differ between KO and WT male mice at either age. In female mice, total tissue area, periosteal perimeter, cortical bone area, marrow cavity area, and endocortical perimeter were decreased significantly in KO mice compared with WT controls in adult but not in young mice (Supplemental Table 5). Periosteal MS/BS, MAR, and BFR/BS were decreased significantly in KO female mice compared with WT at 2 months (Fig. 4 and Supplemental Table 5). Adult female KO mice had significantly lower periosteal MS/BS than WT mice. Endocortical bone formation parameters and endocortical eroded surface did not differ between KO and WT female mice at either age. The histomorphometric analyses of the tibial shaft showed that reduction in cortical bone mass is due to inhibition of periosteal bone formation in KO mice of both sexes.
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) and could be isolated from KO mice (Fig. 5A). This establishes that multinucleated osteoclasts can form in the absence of P2X7 receptors, but does not rule out differences in the rate of formation. Accordingly, osteoclastogenesis was assessed in bone marrow cultures from WT and KO mice (Fig. 5B). The number of tartrate-resistant acid phosphatase (TRAP)-positive, osteoclast-like, multinucleated cells did not differ significantly between WT and KO mice, although there was an age-related increase in osteoclast formation in both WT and KO mice (Fig. 5C). These results indicate that the P2X7 receptor had a minimal effect on osteoclastogenesis and that the P2X7 receptor was not essential for the fusion of osteoclast precursors.
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). The current-voltage relationship of the BzATP- and ATP-activated current showed slight inward rectification and reversal close to 10 mV (Fig. 6B), consistent with the properties reported for P2X7 current in rabbit osteoclasts (6). Thus, WT but not KO murine osteoclasts express functional P2X7 receptors in their plasma membrane.
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). To determine whether functional P2X7 receptors are expressed by osteoblasts, pore formation was assessed. In buffer nominally free of divalent cations, BzATP induced pore formation in approximately 30% of calvarial osteoblasts from WT animals. In contrast, BzATP had no significant effect on ethidium bromide uptake by osteoblasts from KO animals (Fig. 7B). The finding that a population of murine osteoblastic cells express functional P2X7 receptors is in keeping with a previous report indicating that a subpopulation of human osteoblasts express this receptor (9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In vitro studies on bone cells isolated from WT mice established the presence of functional P2X7 receptors on both osteoclasts and a subpopulation of osteoblastic cells. Previous studies have provided functional evidence for P2X7 receptor in osteoclasts based on pore formation and pharmacological characterization (5, 6, 7). However, it is now realized that P2X2 and P2X4, as well as P2X7 receptors, can give rise to membrane pores and that widely used agonists and antagonists are not selective for the P2X7 receptor (3), raising doubt as to the precise identity of the receptor subtype in osteoclasts. Comparison of cells from WT and KO mice enabled us to unequivocally verify the presence of functional P2X7 receptors in osteoclasts. This approach also allowed us to establish the presence of P2X7 receptors in cells of the osteoblast lineage. This finding is consistent with that of Gartland and co-workers (9).
P2X7 receptor activation leads to a number of signaling events, including depolarization of the membrane potential, influx of Ca2+ and Na+, as well as stimulation of several kinase cascades, including c-Jun N-terminal kinase and p38 (3, 15, 16). In skeletal tissues, the P2X7 receptor is suggested to play a role in intercellular communication between osteoblasts and osteoclasts (7). Deletion of the P2X7 receptor could uncouple an important regulatory mechanism by which osteoclasts and osteoblasts normally regulate their activities within the bone environment.
A key finding in this study was that deletion of the P2X7 receptor markedly reduces periosteal bone formation. Periosteal mineralizing surface, mineral apposition rate, and bone formation rate in the tibial shafts were significantly decreased in male KO mice, consistent with reduced periosteal osteoblast number and activity. These findings were confirmed by pQCT analysis of the femoral shaft, which revealed significantly lower total bone content, total tissue area, and periosteal circumference in KO mice. The more obvious reduction in bone mass in male vs. female mice may indicate an interaction between sex steroids and P2X7 signaling. In this regard, 17ß-estradiol has been reported to inhibit P2X7 receptor activation when assessed by electrophysiological methods (17).
The mechanism by which the P2X7 receptor regulates periosteal bone formation requires further investigation. The presence of P2X7 receptors in cells of the osteoblast lineage might indicate a direct regulatory role of this receptor in bone formation. At the tissue level, as first postulated by Frost (18) and confirmed by other investigators (19), mechanical loading controls bone formation and periosteal bone modeling. During growth, increased body weight results in additional mechanical loading on the periosteal surface, leading to periosteal bone formation. In WT mice, total tissue area and periosteal circumference in the long-bone shaft increased during the growth period, a process that was reduced in KO mice. This suggests that mice lacking the P2X7 receptor have decreased sensitivity to mechanical loading. Nucleotides are released from many cell types in response to mechanical stimulation and have been suggested to mediate mechanotransduction in bone (2), perhaps accounting for the decreased sensitivity of KO mice to loading. A recent report has demonstrated the occurrence of a natural mutation in the cytoplasmic domain of the P2X7 receptor in several strains of mice including C57BL/6. The P451L mutation reduces the sensitivity of the P2X7 receptor to nucleotides, which may result in underestimation of the severity of the KO phenotype (20).
The effects of P2X7 receptor deletion on cancellous bone formation were less clear than its effects on periosteal bone formation. In young male KO mice, there was a significant decrease in mineral apposition rate and bone formation rate/tissue volume referent in the proximal tibial cancellous bone. However, there was no significant difference in formation parameters of cancellous bone in adult male or young female KO mice. Taken together, these data indicate that the P2X7 receptor plays an important role in regulating periosteal and possibly trabecular bone formation.
Mice lacking the P2X7 receptor exhibit increased osteoclast surface and osteoclast number on the trabecular bone of the proximal tibial metaphysis. Because WT mouse osteoclasts express the P2X7 receptor, the phenotype observed in KO mice could reflect a primary defect in osteoclasts, although the nature of this defect remains to be clarified. In vitro studies indicated no difference in osteoclastogenesis in bone marrow from WT and KO mice, suggesting that increased osteoclast surface and number in vivo were not due to an increased rate of osteoclast formation. Although the P2X7 receptor has been suggested to play a role in the fusion of macrophages (14), the presence of increased numbers of osteoclasts in KO animals indicated that the receptor was not required for the fusion of osteoclast precursors. Deletion of the P2X7 receptor may lead to less apoptosis in response to ATP or other as yet unknown ligands, resulting in prolonged osteoclast survival and increased osteoclast number. This is in keeping with the known role of the P2X7 receptor in mediating cell death in other systems (3).
In summary, a unique skeletal phenotype has been described, revealing critical roles for P2X7 receptors in regulating bone formation and resorption. The KO model has permitted us to demonstrate unambiguously the expression of functional P2X7 receptors in bone cells. Whereas longitudinal bone growth was not altered in KO mice, there were unique site-specific alterations in bone formation and resorption, consistent with a role for nucleotides in transducing mechanical stimuli. Loss of P2X7 receptors increased osteoclastic bone resorption, while decreasing osteoblastic bone formation. Thus, P2X7 receptor agonists may be useful as a combined antiresorptive and anabolic therapy in skeletal disorders such as osteoporosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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For pQCT and histomorphometry, male and female wild-type (WT) and KO littermate mice were weighed and necropsied at 2 or 9 months of age. There were 12–17 mice per sex per genotype in each group. All mice were given an sc injection of calcein (0.5 mg) for fluorescent bone labeling at 12 and 2 d before necropsy. The right femoral length was measured for each mouse after necropsy using an electronic digital caliper. Serum calcium and phosphate were determined for adult (9-month-old) male and female mice using VetACE Biochemical Analyzer (Alfawassermann, Inc., West Caldwell, NJ).
pQCT
The right femur from each mouse was analyzed at two sites: the distal femoral metaphysis, a trabecular and cortical bone site, and the femoral shaft, a cortical bone site, to obtain volumetric mineral area and content and density of trabecular, cortical, and total bone (21). Further, periosteal circumference (closely related to periosteal bone formation) and endocortical circumference (closely related to endocortical bone formation and resorption) were determined by pQCT. The methods for pQCT analysis have been reported previously (21). Briefly, excised femurs were scanned with a pQCT system (Stratec XCT Research M, Norland Medical Systems, Inc., Fort Atkinson, WI) with software version 5.40. A 1-mm thick cross-section of the distal femoral metaphysis was imaged 2.5 mm proximal from the distal end, and a 1-mm thick cross-section of the midfemoral shaft was imaged 7 mm proximal from the distal end, each with a voxel size of 0.10 mm.
Histomorphometry of Tibias
An image analysis system (Osteomeasure, Inc., Atlanta, GA) was used for histomorphometric analysis. For studies of trabecular bone, undecalcified, methyl methacrylate-embedded longitudinal sections of the proximal tibial metaphysis at thicknesses of 4 and 10 µm were prepared for trabecular bone histomorphometry as described previously (22). Trabecular bone volume (BV/TV), bone resorption parameters [osteoclast number per mm bone surface (OCN/BS) and percent osteoclast surface (OCS/BS)], and bone formation and turnover parameters [mineralizing surface (MS/BS), mineral apposition rate (MAR), bone formation rate/tissue volume referent (BFR/TV), bone formation rate/bone volume referent (BFR/BV), bone formation rate/bone surface referent (BFR/BS)] were determined. These parameters have been described previously (22, 23). For studies of cortical bone, cross-sections of the tibial shaft at a thickness of 20 µm were prepared. Total tissue area, periosteal perimeter, cortical bone area, marrow cavity area, endocortical perimeter, and dynamic histomorphometric parameters including MS/BS, MAR, and BFR/BS were determined on periosteal and endocortical surfaces (21, 22, 23). Additionally, endocortical eroded surface (E-ES/BS) was determined as an index for endocortical bone resorption.
Mouse Bone Marrow Culture
To determine the effects of deleting the P2X7 receptor on osteoclastogenesis, a bone marrow culture system was used (24). Tibias of WT and KO male mice at 2 and 9 months of age were removed and cut using a diamond wafer blade (no. 11-4244, Buehler, Lake Bluff, IL) at points just below the epiphyseal growth plate of the proximal tibia and above the tibio-fibular junction. Marrow from the tibias of 10 mice from the WT and KO groups was flushed out of the bone into complete tissue culture media (phenol red-free 
-MEM) with 15% fetal bovine serum and antibiotics (Invitrogen, Carlsbad, CA). Extruded marrow pieces were triturated approximately 20 times and then filtered through a 100-µm mesh cell strainer (Falcon no. 2360, Becton Dickinson and Co., Franklin Lakes, NJ) to remove bone fragments and vascular remnants. The cell suspension was diluted to 10 ml with complete media, counted using a hemocytometer, and plated at 1 x 105 cells per cm2 in 24-well plates with 25 ng/ml each of RANKL and M-CSF (R \|[amp ]\| D Systems, Minneapolis, MN) and 10-8 M 1,25-dihydroxyvitamin D3 (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) and cultured in 5% CO2 at 37 C. Marrow cultures were fed by replacing one-half the media with fresh complete media on d 3 and 5 and were stained for tartrate-resistant acid phosphatase (TRAP) on d 6 using the leukocyte acid phosphatase kit (Sigma, St. Louis, MO). TRAP-positive cells with three or more nuclei were counted as osteoclasts.
Osteoclast Isolation and Electrophysiology
Osteoclasts were isolated using methods described previously (6), and currents were recorded in whole-cell configuration of patch clamp. Electrode solution contained (in mM): CsCl, 136; HEPES, 20; MgCl2, 1; TEACl, 10; CaCl2, 0.4; EGTA, 1; adjusted with CsOH to pH 7.2; 280–290 mOsm/liter. Cells were superfused continuously (1–2 ml/min) in Na+ solution containing (in mM): NaCl, 135; KCl, 5; glucose, 10; CaCl2, 1; NaHEPES, 20; pH 7.4, 280–290 mOsm/liter. Currents were recorded using an Axopatch-1D amplifier (Axon Instruments, Foster City, CA), filtered (-3 dB at 1 kHz) and digitized at 2–5 kHz using pClamp 6.0 (Axon Instruments). Experiments were performed at room temperature (22–25 C). Nucleotides were dissolved in Na+ solution and applied to individual osteoclasts using pressure ejection from micropipettes (5–10 µm diameter) positioned 30–50 µm from the cell (Picospritzer II, General Valve Corp., Fairfield, NJ).
Osteoblast Culture and RT-PCR
Osteoblasts were isolated from calvaria by sequential enzymatic digestion (25) and populations II to V cultured in -MEM with 10% fetal bovine serum and antibiotics (Life Technologies, Inc., Gaithersburg, MD). For RT-PCR, total RNA was extracted using RNeasy Minikit (QIAGEN, Chatsworth, CA) and treated with DNase I (Amersham Pharmacia Biotech, Arlington Heights, IL) for 10 min at 37 C. First-strand cDNA was synthesized from 10 µg of total RNA using oligo(dT)12-18 primer and Superscript II RNase H- Reverse Transcriptase (Life Technologies, Inc.) for 50 min at 42 C and 15 min at 70 C. The control reaction was processed in the absence of reverse transcriptase. Forward primer 5'-CGAGTTGGTGCCAGTGTGGA and reverse primer 5'-CCTGCTGTTGGTGGCCTCTT were designed using the mouse P2X7 sequence AJ009823. PCR was performed in a final volume of 25 µl containing the template cDNA, 1 U Taq DNA polymerase, 5 µM of each primer, and 1.25 mM of each deoxynucleoside triphosphate under the following conditions: 36 cycles at 95 C for 30 sec, 64 C for 30 sec, and 68 C for 1 min. The products were then kept at 68 C for 10 min and stored at 4 C. Amplification products were electrophoresed on 1% agarose gel containing 5 µg/ml ethidium bromide and visualized using ImageMaster VDS (Pharmacia Biotech, Piscataway, NJ).
Pore Formation Assay
To investigate pore formation in calvarial osteoblasts, we used divalent cation-free solution consisting of (in mM): NaCl, 140; KCl, 5; NaHEPES, 20; glucose, 10; pH 7.4, 290 mOsm/liter. Cells were incubated for 12 min at 37 C with 20 µg/ml ethidium bromide in the absence or presence of 300 µM BzATP. Cells were then washed and incubated with 5 µg/ml Hoechst 33342 for 5 min at 37 C to reveal nuclei. Fluorescence was visualized using an Axiovert S100 microscope (Carl Zeiss, Thornwood, NY).
Statistics
Data are expressed as means ± SEM with sample sizes (n) indicating the number of animals, cell preparations, or individual cells. ANOVA was followed by the Tukey or Fisher’s protected least significant difference (PLSD) test to compare differences among groups. P < 0.05 was considered a significant difference.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address for A.F.W.: Department of Cell Physiology, University of Nijmegen, Nijmegen, The Netherlands.
Abbreviations: BFR/BS, Bone formation rate/bone surface referent (µm2/µm/d x 100); BFR/BV, bone formation rate/bone volume referent (%/yr); BFR/TV, bone formation rate/tissue volume referent (%/yr); BV/TV, trabecular bone volume/total tissue volume (%); BzATP, 2'- and 3'-O-(4-benzoylbenzoyl)-ATP; E-ES/BS, endocortical eroded surface/ endocortical bone surface (%); KO, knockout; MAR, mineral apposition rate (µm/d); MS/BS, mineralizing surface/bone surface (%); OCN/BS, osteoclast number/mm bone surface (no./mm); OCS/BS, osteoclast surface/total bone surface (%); pQCT, peripheral quantitative computerized tomography; TRAP, tartrate-resistant acid phosphatase; WT, wild-type.
Received for publication January 21, 2003. Accepted for publication March 28, 2003.
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J. Korcok, L. N. Raimundo, X. Du, S. M. Sims, and S. J. Dixon P2Y6 Nucleotide Receptors Activate NF-{kappa}B and Increase Survival of Osteoclasts J. Biol. Chem., April 29, 2005; 280(17): 16909 - 16915. [Abstract] [Full Text] [PDF]
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 C. H. Turner and A. G. Robling Mechanical Loading and Bone Formation IBMS BoneKEy, September 1, 2004; 1(9): 15 - 23. [Abstract] [Full Text] [PDF]
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 J. F. Hiken and T. H. Steinberg ATP downregulates P2X7 and inhibits osteoclast formation in RAW cells Am J Physiol Cell Physiol, August 1, 2004; 287(2): C403 - C412. [Abstract] [Full Text] [PDF]
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