Parts of this work were presented at the Annual Scientific Meeting of the Australian and New Zealand Bone and Mineral Society, Coolum, Australia, June 10–13, 2003, and the 30th European Symposium on Calcified Tissues, Rome, Italy, May 8-12, 2003.
Activation of Protease-Activated Receptor-2 Leads to Inhibition of Osteoclast Differentiation†
Article first published online: 22 DEC 2003
Copyright © 2004 ASBMR
Journal of Bone and Mineral Research
Volume 19, Issue 3, pages 507–516, March 2004
How to Cite
Smith, R., Ransjö, M., Tatarczuch, L., Song, S.-J., Pagel, C. N., Morrison, J. R., Pike, R. N. and Mackie, E. J. (2004), Activation of Protease-Activated Receptor-2 Leads to Inhibition of Osteoclast Differentiation. J Bone Miner Res, 19: 507–516. doi: 10.1359/JBMR.0301248
The authors have no conflict of interest.
- Issue published online: 2 DEC 2009
- Article first published online: 22 DEC 2003
- Manuscript Accepted: 8 OCT 2003
- Manuscript Revised: 12 SEP 2003
- Manuscript Received: 16 JUL 2003
- protease-activated receptor-2;
- osteoclast differentiation;
- prostaglandin G/H synthase;
PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption.
Introduction: Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the “tethered ligand” created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated.
Materials and Methods: Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR.
Results: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3-induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells.
Conclusion: These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation.
THE PROTEASE-ACTIVATED RECEPTORS (PARs) are a recently described family of receptors that couple to G-proteins to produce intracellular responses.(1) Proteolytic cleavage of the extracellular domains of the PARs reveals new amino-termini that act as “tethered ligands” that bind to and induce activation of the receptors. PAR-1, −3, and −4 are receptors for thrombin, whereas PAR-2 is activated by a number of proteases. Known mammalian activators of PAR-2 include trypsin, mast cell tryptase, membrane-type serine protease-1, factor Xa, and neutrophil proteinase 3.(2–6) Gingipain-R, a protease produced by Porphyromonas gingivalis, one of the major bacterial pathogens in periodontal disease, also activates PAR-2.(7) A synthetic receptor agonist peptide (RAP) corresponding to the sequence of the tethered ligand domain of PAR-2 (SLIGKV in human and SLIGRL in mouse) is able to activate the receptor and has been used experimentally to activate PAR-2 in the absence of proteolysis.(2, 8)
PAR-2 is expressed in a wide variety of tissues, including the gastrointestinal and respiratory tracts, pancreas, kidney, muscle, ovary, and skin.(2, 9–11) The functions of PAR-2 in different tissues include stimulation of cell proliferation and stimulation of smooth muscle contractility.(11-14) PAR-2 activation also leads to the release of prostanoids and cytokines including interleukin (IL)-6 and IL-8 in some tissues.(7, 15-17)
PAR-1 is expressed by osteoblasts in vitro and in vivo and mediates thrombin-induced osteoblast proliferation.(18–20) We have recently observed expression of PAR-4 by mouse primary osteoblasts, but have not yet identified a role for this receptor in bone.(21) We have also recently demonstrated that PAR-2 is expressed by osteoblasts in vivo and in vitro.(22) In an attempt to identify a role for PAR-2 in bone, we investigated the effect of RAP on osteoblast proliferation and on expression of alkaline phosphatase (ALP), a marker of osteoblast differentiation,(22) but found no effect on either of these processes. Another important role for cells of the osteoblast lineage is in regulation of osteoclast differentiation and activity.(23) An essential requirement for osteoclast differentiation is presentation (normally by bone marrow stromal cells or osteoblasts) of the cell surface protein known as RANKL, which binds to its receptor (RANK) on hemopoietic osteoclast progenitors.(24) Osteoblasts also express the secreted protein osteoprotegerin (OPG), which acts as a decoy receptor for RANKL and inhibits osteoclast differentiation. Osteotropic hormones, such as parathyroid hormone (PTH) and the active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and the inflammatory cytokine IL-11 can promote osteoclast differentiation by elevating RANKL production by osteoblasts.(24) An important intermediary step in the regulation of RANKL expression by PTH and 1,25(OH)2D3 is the induction of prostaglandin G/H synthase-2 (PGHS-2; also known as cyclo-oxygenase-2), resulting in synthesis of prostanoids.(25) Prostaglandin synthesis is also required for osteoclast differentiation in response to IL-11.(26) PTH and 1,25(OH)2D3 are known to stimulate expression of IL-6, which itself can stimulate osteoclast differentiation.(23) The mouse bone marrow culture system has been extremely valuable in the elucidation of pathways influencing osteoclast differentiation. In this system, bone marrow stromal cells provide the factors required for differentiation of osteoclasts from hematopoietic precursors.
The PAR-2 activators neutrophil proteinase 3, factor Xa, and mast cell tryptase are likely to be present in bone under inflammatory conditions, which lead to increased resorption. This fact, together with the observation that prostanoids and IL-6 are produced in response to PAR-2 activation in other cell types, led us to hypothesize that PAR-2 activation plays a role in osteoblast-mediated osteoclast differentiation. This study was undertaken with the aim of investigating this hypothesis. Contrary to expectations, PAR-2 activation was found to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Further studies were undertaken to elucidate some aspects of the mechanism of this effect.
MATERIALS AND METHODS
Animals and materials
Tissue culture media, media supplements, and reagents were purchased from GIBCO-BRL/Life Technologies (Paisley, UK). Collagenase was purchased from Roche (Mannheim, Germany). Thermanox coverslips, multi-well plates, and culture flasks were purchased from Nunc (Roskilde, Denmark). PCR primers were synthesized by Geneworks (Adelaide, Australia). Deoxynucleoside triphosphates (dNTPs), 1-kb DNA ladder, PCR buffer, 25 mM MgCl2, oligo(dT) primer, and TaqDNA polymerase were purchased from Promega (Madison, WI, USA). Mouse PAR-2-activating peptide (RAP: SLIGRL) was synthesized as a carboxy amide and purified by HPLC by AusPep (Melbourne, Australia). Human PTH(1–34), IL-11, ALP staining kit, and other biochemical reagents were purchased from Sigma (Poole, UK), and 1,25(OH)2D3 was purchased from ICN Biomedicals (Aurora, OH, USA). RANKL was kindly provided by Dr Julian Quinn (St. Vincent's Institute of Medical Research, Melbourne, Australia). PAR-2 null mice were generated by Drs John Morrison and Mary Stevens.(27) Experiments making use of animal tissues were approved either by the Animal Care and Use Committee of the University of Umeå or the Animal Welfare Committee of the Department of Biochemistry and Molecular Biology, Monash University.
Mouse bone marrow cultures
Bone marrow cells were isolated and cultured essentially as previously described.(28, 29) Briefly, 6- to 9-week-old C57BL6/J mice and wildtype and PAR-2-null 129/Sv-+P+Tyr-c+Mgf-S1J/J mice (designated wildtype and PAR-2-null) were killed by cervical dislocation. In all experiments, except those shown in Fig. 3, C57BL6/J mice were used. For experiments shown in Fig. 3, wildtype and PAR-2-null mice were used. The tibias and femurs were removed and dissected free from adhering soft tissues. The bone extremities were removed, and the marrow cavity was flushed with α-MEM supplemented with 10% heat-inactivated FCS and antibiotics. The marrow cells were collected into tubes, washed, and cultured in α-MEM (supplemented with 10% FCS and antibiotics) at 2 × 106 cells/ml in 24-well plates for osteoclast differentiation assays (0.5 ml/well) or in 6-well plates for RNA extraction. For enzyme histochemical experiments, each well contained a 13-mm-round Thermanox coverslip. Cultures were fed every 3 days by replacing 0.4 ml medium with fresh medium. Test substances [PTH (10 nM); 1,25(OH)2D3 (10 nM); IL-11 (10 nM); RAP (100 μM); PTH (10 nM) and RAP (100 μM); 1,25(OH)2D3 (10 nM) and RAP (100 μM); and IL-11 (10 nM) and RAP (100 μM)] were added after a 24-h attachment period and at each time of medium change. For the experiment shown in Fig. 4, cultures were washed with prewarmed PBS (37°C) for 3–5 minutes between changes of medium. Cultures were maintained for various times up to 7 or 8 days at 37°C in a humidified atmosphere of 5% CO2 in air.
Characterization and quantitation of osteoclasts
Osteoclasts were identified by TRACP staining. At the end of the culture period (7 or 8 days), coverslips were washed with PBS, fixed with 4% paraformaldehyde in PBS, and dried. Fixed cells were incubated for 15 minutes at 37°C in freshly prepared and filtered substrate solution (0.1 M sodium acetate pH 5.2, 0.9 mM N-N-dimethylformamide, 1.4 M naphthol AS-TR phosphate, 0.8 M sodium tartrate, and 0.9 M fast red TR salt). Cells were washed with PBS. TRACP+ multinucleated cells containing three or more nuclei were counted as osteoclasts.
Osteoclast populations were also investigated by their ability to resorb bone. Bone resorption pit assays were performed by culturing mouse bone marrow cells on horse cortical bone slices (200 μm thick) placed in 24-well plates. After being cultured for 8 days, the slices were trypsinized and fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde in PBS (pH 7.4). After fixation, the bone slices were washed in distilled water, dehydrated in ethanol, air-dried, and mounted on scanning electron microscopy (SEM) stubs (ProSciTech). The bone slices were sputter-coated in gold and examined in a Phillips SEM 505 scanning electron microscope. Images (three fields/bone slice) were recorded and analyzed for resorption pit area using Imagepro Plus image analysis software.
Mouse calvarial osteoblast cultures
Calvarial osteoblasts were derived by sequential collagenase digestion of calvariae removed from neonatal C57BL6/J mice as previously described.(22) Calvariae were removed from neonates within 12 h of birth and stripped of periosteum before incubation for 20 minutes at 27°C in 3 ml of enzyme solution containing 0.1% collagenase, 0.05% trypsin, and 4 mM EDTA in calcium- and magnesium-free PBS. The digestion was repeated to obtain six cell populations. The cells released during each digest were collected by centrifugation in 3 ml of FCS and resuspended in DMEM (without phenol red) supplemented with 10% FCS, L-glutamine, and antibiotics for plating into tissue culture flasks. Medium was changed every second day, and cultures were maintained in a humidified atmosphere at 37°C under 5% CO2 in air. Cells were cultured to confluence, trypsinized, and pooled before plating in 6-well plates for RNA extraction. Subconfluent pooled cells were deprived of serum overnight. Cells were treated with PTH (10 nM); 1,25(OH)2D3 (10 nM); IL-11 (10 nM); RAP (100 μM); PTH (10 nM) and RAP (100 μM); 1,25(OH)2D3 (10 nM) and RAP (100 μM); or IL-11 (10 nM) and RAP (100 μM) for 24 h before RNA extraction.
Mouse bone marrow stromal cell cultures
Bone marrow stromal cells were isolated as described.(30) Briefly, bone marrow was isolated from mouse femurs as described above for osteoclast differentiation assays. Single-cell suspensions were prepared by aspiration of the marrow preparation through a 21-gauge needle for 3 minutes. Cells were cultured in DMEM (containing 10% FCS and antibiotics) in 25-cm2 flasks and grown until confluent. Adherent cells were passaged and grown to confluence for RNA isolation.
Mouse macrophage RAW 264.7 cell cultures
The mouse macrophage RAW 264.7 osteoclastogenic cell line was cultivated in α-MEM (supplemented with 10% FCS and antibiotics), and the cultures were maintained in a humidified atmosphere at 37°C under 5% CO2 in air. The cells were cultured at 2 × 104 cells/ml in 24-well plates for osteoclast differentiation assays (0.5 ml/well) or in 6-well plates for RNA extraction. The cells were cultured continuously for 7 days in the absence or presence of RANKL (2, 10, or 50 ng/ml) for osteoclast differentiation assays. RAP (100 μM), where included, was present simultaneously for the entire culture period. Osteoclasts were assessed as TRACP+ cells with three or more nuclei.
RNA extraction and reverse transcription
Total cellular RNA was isolated from freshly isolated bone marrow tissue, confluent mouse bone marrow cultures, bone marrow stromal cell cultures, RAW264.7 cells, and calvarial osteoblast cultures using TRI Reagent (Sigma, Sydney, Australia) according to the manufacturer's instructions. The RNA was quantified spectrophotometrically, and the integrity of the RNA preparations was examined by agarose gel electrophoresis. First-strand cDNA was synthesized from 1 μg RNA with Moloney murine leukemia virus RT using oligo(dT) primer (Ready-To-Go You-Prime First-Strand Beads; Pharmacia Biotech). The first-strand cDNA product was kept at −20°C until used for PCR.
Semiquantitative PCR was performed using 5 μl of the first-strand reaction in a 50 μl PCR reaction containing 5 μl PCR buffer, 0.8 mM dNTPs, 2.2 mM MgCl2, 0.25 U Hotstart Taq polymerase (Promega), and 1 μM forward and reverse primers (sequences of primers are shown in Table 1).(31–35) Amplification was performed using the following profile: hotstart at 95°C for 3 minutes, amplification of PCR products for between 20 and 36 cycles of denaturation at 94°C for 1 minute, annealing at 43–52°C for 1 minute, and extension at 72°C. After amplification, reactions were incubated at 72°C for 3 minutes. PCR products were analyzed on 1% agarose gels containing 0.5 mg/ml ethidium bromide and visualized under UV illumination. Digital images of the gels were captured by Pro Finder software, and the intensity of each band was measured by computerized densitometry using ImageQuant software. For each primer pair, a range of cycle numbers were performed (20-26 cycles for GAPDH and 30-36 cycles for all other primers). The optical intensity of the PCR products was plotted against cycle number, and the linear range of product amplification was determined. From this plot, a cycle number from within the linear range was chosen, and the optical intensity at this cycle number was collected and normalized to the optical intensity of GAPDH for each sample. Values presented in Figs. 5 and 6 and Table 2 represent the mean normalized optical intensity ± SE of 5 wells/treatment (N) from one experiment. For each gene product of interest, duplicate experiments were conducted and yielded similar results.
Reaction products were unaffected by pretreatment with DNase I, and no products were obtained when RT was omitted from the reaction or when the RNA was pretreated with RNase A. The identities of the amplified PCR products were confirmed by direct sequencing.
Data were analyzed using GraphPad Prism Version 3, and results are expressed as the mean ± SE. Results were analyzed using Student's t-test.
Expression of PAR-2 in mouse bone marrow and human macrophage RAW264.7 osteoclastogenic cells
RT-PCR studies were conducted to determine whether PAR-2 is expressed by whole bone marrow, bone marrow stromal cells, and the mouse macrophage RAW 264.7 osteoclastogenic cell line. Using PAR-2 primers, a band of the appropriate size was detected in RNA extracted from primary mouse calvarial osteoblast cultures, bone marrow stromal cell cultures, and freshly isolated bone marrow tissue (Fig. 1A, lanes 2–4). Bands of appropriate size were also detected in RNA extracted from the mouse macrophage RAW264.7 cell line grown in the absence of RANKL (Fig. 1B, lane 1) or in the presence of RANKL for 3, 5, and 7 days (Fig. 1B, lanes 2-4). No PCR product was detected in the negative control reaction (Fig. 1A, lane 1).
Effect of PAR-2 activation on osteoclast differentiation in mouse bone marrow cultures
The effect of PAR-2 activation on osteoclast differentiation was investigated in mouse bone marrow cultures in the presence or absence of three factors capable of stimulating osteoclast differentiation in this system. The cultures generated from C57BL6/J mice were incubated in the absence or presence of PTH, 1,25(OH)2D3, IL-11, RAP, or RAP in combination with PTH, 1,25(OH)2D3, or IL-11. The concentration of RAP used in these experiments (100 μM) was chosen as the concentration required for maximum calcium mobilization in primary osteoblast cultures.(22) Osteoclast differentiation was quantified at the end of the culture period by counting of multinucleate TRACP+ cells or by measuring resorption pit area formed on bone slices included in the bone marrow cultures. After 8 days of culture, low levels of osteoclast differentiation were seen in control cultures, and PTH, 1,25(OH)2D3, or IL-11 caused a substantial stimulation of osteoclast differentiation (Figs. 2A-2C). RAP alone did not stimulate osteoclast differentiation; it had no effect on the number of TRACP+ multinucleate cells, but caused a significant reduction in resorption pit area. Moreover, when it was administered simultaneously with PTH, 1,25(OH)2D3, or IL-11, RAP caused a significant decrease in osteoclast differentiation as assessed by both parameters (Figs. 2A-2C).
The effect of PAR-2 activation on osteoclast differentiation was also investigated in mouse bone marrow cultures generated from PAR-2-null and the corresponding wildtype mice. In cultures generated from PAR-2-null mice, RAP did not inhibit osteoclast differentiation when administered alone or in the presence of PTH, 1,25(OH)2D3, or IL-11 (Fig. 3A). In parallel cultures prepared from wildtype mice, RAP caused a significant decrease in osteoclast differentiation when administered simultaneously with PTH, 1,25(OH)2D3, or IL-11 (Fig. 3B). These results confirm that inhibition of osteoclast differentiation in response to RAP is specifically mediated by PAR-2.
The timing of the requirement for RAP for inhibition of PTH-, 1,25(OH)2D3-, and IL-11-induced osteoclast differentiation was investigated using C57BL6/J mice. Osteoclast differentiation assays were performed in which bone marrow cultures were treated continuously for 7 days with PTH, 1,25(OH)2D3, or IL-11; RAP was included simultaneously either for the entire culture period or for days 1–3, 3-5, or 5-7. Osteoclasts were quantified as TRACP+ cells at the end of the culture period.
In the presence of PTH or IL-11, RAP was effective at inhibiting osteoclast differentiation when administered at days 1–3, 3-5, 5-7, and the entire 7-day culture period (Figs. 4A and 4C). However, RAP was only 63% effective at inhibiting osteoclast differentiation when added at days 5-7 in PTH-treated cultures compared with the response obtained with continuous RAP. In the presence of 1,25(OH)2D3, RAP was equally effective at inhibiting osteoclast differentiation when added at days 1-3, 3-5, or the entire 7-day culture period, but ineffective when administered only for days 5-7 (Fig. 4B).
Effect of PAR-2 activation on expression of RANKL and OPG in mouse bone marrow and calvarial osteoblast cultures
Studies were carried out to determine whether PAR-2 activation regulates expression of RANKL and OPG in bone marrow cultures and osteoblast cultures treated with osteoclastogenic hormones and cytokines. Bone marrow cells were cultured for 3 or 7 days, and osteoblasts were cultured for 24 h in the absence or presence of PTH, 1,25(OH)2D3, IL-11, RAP, PTH and RAP, 1,25(OH)2D3 and RAP, or IL-11 and RAP. The level of RANKL and OPG expression was investigated using semiquantitative RT-PCR as described in the Materials and Methods section.
There was a significant increase in the ratio of RANKL:OPG expression at both days 3 and 7 in the presence of all the osteoclastogenic factors. When administered concomitantly with PTH, 1,25(OH)2D3, or IL-11, RAP caused a significant decrease in RANKL expression. Although it also decreased OPG expression at day 3 in 1,25(OH)2D3-treated cultures, RAP caused a significant lowering of the ratio of RANKL:OPG in the presence of all three osteoclastogenic factors at both time points investigated (Fig. 5).
It has been found in calvarial osteoblast cultures that 1,25(OH)2D3, PTH, and IL-11 increase RANKL expression while reducing OPG expression, resulting in an increase in the ratio of RANKL:OPG at 24 h.(31) The results shown in Fig. 6 confirm this pattern of expression in mouse calvarial osteoblast cultures. RAP caused a significant decrease in RANKL expression and increase in OPG expression, resulting in a highly significant decrease in the ratio of RANKL:OPG when it was administered together with PTH, 1,25(OH)2D3, or IL-11.
Effect of PAR-2 activation on expression of PGHS-1, PGHS-2, and IL-6 in mouse bone marrow cultures
Semiquantitative RT-PCR studies were carried out to determine whether PAR-2 activation regulates expression of the constitutive PGHS-1, the inducible PGHS-2, and IL-6 in bone marrow cultures treated with osteoclastogenic hormones. None of the resorptive agents stimulated expression of PGHS-1, but RAP caused a significant decrease in PGHS-1 levels in PTH- and 1,25(OH)2D3-treated cultures at day 3 (Table 2). PTH and IL-11 stimulated PGHS-2 expression at day 3 (p < 0.05 and p < 0.00001, respectively), but not at day 7, and 1,25(OH)2D3 did not affect PGHS-2 expression at either time point. Nevertheless, RAP decreased PGHS-2 levels in PTH-treated cultures at days 3 and 7 and in 1,25(OH)2D3- and IL-11-treated cultures at day 3 (Table 2). IL-6 was significantly increased by PTH or 1,25(OH)2D3 treatment in 3-day bone marrow cultures but not in 7-day cultures (Table 2). RAP inhibited expression of IL-6 in PTH-treated cultures at days 3 and 7 and in 1,25(OH)2D3-treated cultures at day 3 (Table 2).
Effect of PAR-2 activation on osteoclastic differentiation in the mouse macrophage RAW264.7 osteoclastogenic cell line
The ability of PAR-2 activation to inhibit osteoclast differentiation through direct effects on osteoclast precursors was investigated using the osteoclastogenic macrophage cell line, RAW264.7. RAP had no effect on the number of TRACP+ multinucleate cells generated in these cultures in response to treatment with RANKL (Fig. 7).
We have previously demonstrated expression of PAR-2 by osteoblasts,(22) but have not been able to identify any physiological responses to activation of PAR-2 in osteoblasts, other than elevation of [Ca2+]i.(22) Activation of PAR-2 in some cell types leads to secretion of IL-6 and prostanoids, both of which are known to stimulate osteoblast-mediated osteoclast differentiation. In addition, known activators of PAR-2 include proteases present during inflammation, which themselves are associated with increased bone resorption. These observations led us to hypothesize that activation of PAR-2 in osteoblastic cells stimulates osteoblast-mediated osteoclast differentiation. We used the mouse bone marrow culture system to investigate this hypothesis. Initially, we used RT-PCR to confirm that PAR-2 is expressed by bone marrow, as well as by bone marrow stromal cell-enriched cultures, which contain osteoblast precursors. Surprisingly, RAP alone had no effect on TRACP+ multinucleate cell number in bone marrow cultures, and it even caused a decrease in resorptive pit area. Moreover, when administered concomitantly with PTH, 1,25(OH)2D3, or IL-11, RAP caused a profound inhibition of osteoclast formation as assessed by both TRACP staining and resorption pit assays. In bone marrow cultures derived from PAR-2-null mice, RAP had no effect on osteoclast differentiation, indicating that inhibition of osteoclast differentiation in response to RAP is specifically mediated by PAR-2. These results clearly show that activation of PAR-2 by RAP inhibits PTH-, 1,25(OH)2D3-, and IL-11-induced osteoclast differentiation in bone marrow cultures. The fact that basal resorption pit area was decreased by RAP in the absence of an effect on TRACP+ multinucleate cell number suggests that RAP may influence osteoclast activity as well as differentiation.
Time course studies were used to investigate the timing of the requirement for RAP to inhibit PTH-, 1,25(OH)2D3-, and IL-11-induced osteoclast differentiation. RAP was added to PTH-, 1,25(OH)2D3-, and IL-11-treated cultures for 3-day periods at different stages of the culture period. In the presence of PTH, RAP caused maximal inhibition of osteoclast differentiation when administered at 0–3 or 3-5 days, but only partial inhibition when present at days 5-7. In the presence of 1,25(OH)2D3, RAP caused maximal inhibition of osteoclast differentiation when administered at 0-3 or 3-5 days, but was completely ineffective when administered only at days 5-7. In the presence of IL-11, RAP caused maximal inhibition of osteoclast differentiation when administered during any of the time periods. It is perhaps not surprising that RAP treatment shows different effects in these time course studies for PTH, 1,25(OH)2D3, and IL-11, because these hormones act through quite different receptor signaling systems.
In the mouse bone marrow culture system, osteoclast progenitors primarily proliferate during the first 4 days of culture and thereafter differentiate during the final 2–4 days of culture.(23) Hattersley and Chambers(36) demonstrated that addition of 1,25(OH)2D3 to mouse marrow cultures for 2 days after an initial 7-day hormone-free incubation period is sufficient to induce maximal formation of osteoclasts. Thus, 1,25(OH)2D3 can induce terminal differentiation and fusion of the osteoclast progenitors present in cultures. In contrast, we have previously demonstrated that PTH needs to be present throughout the entire culture period for maximal osteoclast differentiation to occur, although a small stimulation of osteoclast differentiation occurs in cultures treated with PTH for the last 3 days alone.(29) The current time course results indicate that RAP in the presence of 1,25(OH)2D3 does affect proliferation of osteoclast precursors but not fusion to form multinucleate osteoclasts. In contrast, when in the presence of PTH or IL-11, RAP seems to affect both the proliferation and fusion of osteoclasts (although the effect on fusion is not as great for PTH).
Further studies were performed to elucidate the mechanism by which RAP inhibits osteoclast differentiation. Because the ability of PTH, 1,25(OH)2D3, and IL-11 to stimulate osteoclast differentiation is dependent on their ability to increase the ratio of RANKL:OPG expression, the effect of RAP on this response was investigated. When RAP was administered together with PTH, 1,25(OH)2D3, or IL-11, there was a significant decrease in the RANKL:OPG ratio in mouse marrow cultures at days 3 and 7, which correlated with the decrease in osteoclast numbers in bone marrow cultures treated in the same way. RAP exerted similar effects on the RANKL:OPG ratio in osteoblasts treated with the three factors as were seen in bone marrow cultures. These results are consistent with a conclusion that inhibition of hormone-induced osteoclast differentiation by PAR-2 activation is at least in part dependent on its effects on RANKL and OPG expression in cells of the osteoblast lineage. When administered alone, RAP had no effect on the RANKL:OPG ratio, although it did reduce resorption pit area, suggesting that the apparent effect of RAP on osteoclast activity may be independent of RANKL and OPG expression, and may even act directly through PAR-2 on osteoclasts.
PAR-2 was not only found to be expressed by bone marrow stromal cells, but also by RAW264.7 cells, which represent osteoclast precursors. RAP was shown to have no effect on RANKL-induced osteoclast differentiation in RAW264.7 cells. Results obtained with a transformed cell line should always be interpreted with caution, but these observations seem to indicate that activation of PAR-2 on osteoclast precursors is unlikely to make a major contribution to the effect of RAP on osteoclast differentiation in bone marrow cultures. Thus, the inhibition of osteoclast differentiation by PAR-2 activation seems to be mediated largely by cells of the osteoblast lineage.
Our original hypothesis that PAR-2 activation stimulates osteoclast differentiation was based on the expectation that PAR-2 activation would stimulate prostanoid and IL-6 production in cells of the osteoblast lineage. Once our hypothesis had been disproved, it seemed nevertheless appropriate to investigate whether PAR-2 activation had any effect on expression of IL-6 and the two enzymes that play a pivotal role in prostanoid synthesis, PGHS-1 and −2. The results of these experiments were in keeping with PAR-2's inhibition of osteoclast differentiation in the presence of osteoclastogenic factors, indicating that this could partly be mediated by a downregulation of IL-6, PGHS-1, and PGHS-2. The fact that PAR-2 caused a suppression of expression of the supposedly constitutive PGHS-1 in 3-day bone marrow cultures was not entirely surprising, because this enzyme has previously been found to be regulated in ST2 stromal cells.(37)
What is the physiological significance of our observation that PAR-2 activation results in inhibition of PTH- and 1,25(OH)2D3-induced osteoclast differentiation? The PAR-2-null mice used in this study had grossly normal skeletons,(27) and PAR-2-null mice from other groups are also described as being grossly normal,(38, 39) although no detailed study of bone development and structure has yet been carried out. Any role for PAR-2, must however, depend on the availability of PAR-2 activators, and it is likely that these are only present in pathological conditions. A number of PAR-2 activators, including mast cell tryptase, factor Xa, neutrophil protease 3, and gingipain-R, are likely to be present in the bone environment in inflammatory conditions affecting bone. The role of PAR-2 may be to protect bone from uncontrolled resorption resulting from exposure to inflammatory mediators. Indeed, a cytoprotective role has already been ascribed to PAR-2 in the gastrointestinal and respiratory tracts.(40) Our identification of a novel pathway leading to inhibition of osteoclast differentiation may have important implications for the development of antiresorptive agents.
The authors thank A Lie and the late MR de Niese for excellent technical assistance. This work was supported by National Health and Medical Research Council of Australia Grant 208960, the University of Melbourne Research Development Grants Scheme, and a Monash University Small Grant.
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