Phospholipase Cγ2 is required for basal but not oestrogen deficiency–induced bone resorption

Background Osteoclasts play a critical role in bone resorption under basal conditions, but they also contribute to pathological bone loss during diseases including postmenopausal osteoporosis. Phospholipase Cγ2 (PLCγ2) is an important signalling molecule in diverse haematopoietic lineages. Here, we tested the role of PLCγ2 in basal and ovariectomy-induced bone resorption, as well as in in vitro osteoclast cultures using PLCγ2-deficient (PLCγ2−/−) mice. Materials and methods The trabecular architecture of long bone metaphyses was tested by micro-CT and histomorphometric analyses. Postmenopausal osteoporosis was modelled by surgical ovariectomy. Osteoclast development and function, gene expression and PLCγ2 phosphorylation were tested on in vitro osteoclast and macrophage cultures. Results PLCγ2−/− mice had significantly higher trabecular bone mass under basal conditions than wild-type mice. PLCγ2 was required for in vitro development and resorptive function of osteoclasts, but not for upregulation of osteoclast-specific gene expression. PLCγ2 was phosphorylated in a Src-family-dependent manner upon macrophage adhesion but not upon stimulation by M-CSF or RANKL. Surprisingly, ovariectomy-induced bone resorption in PLCγ2−/− mice was similar to, or even more robust than, that in wild-type animals. Conclusions Our results indicate that PLCγ2 participates in bone resorption under basal conditions, likely because of its role in adhesion receptor signalling during osteoclast development. In contrast, PLCγ2 does not appear to play a major role in ovariectomy-induced bone loss. These results suggest that basal and oestrogen deficiency–induced bone resorption utilizes different signalling pathways and that PLCγ2 may not be a suitable therapeutic target in postmenopausal osteoporosis.


Introduction
While osteoclasts are important for normal bone turnover, they also contribute to pathological bone loss during osteoporosis, rheumatoid arthritis or osteolytic bone metastases [1][2][3]. However, it is incompletely understood how osteoclasts contribute to normal and pathological bone resorption and whether they utilize similar intracellular signalling machineries during the two processes.
Osteoclasts are highly specialized phagocytic cells of haematopoietic origin [4]. They develop by an initial macrophage-like differentiation, followed by reprogramming to the osteoclast lineage and fusion of preosteoclasts to mature multinucleated osteoclasts [1,4]. These processes are directed by the M-CSF and the osteoblast-derived RANK ligand (RANKL) cytokines.
A number of recent studies have indicated similar components of osteoclast biology and immune mechanisms, leading to the emergence of the new field of osteoimmunology [5]. Those similarities include activation by closely related cytokines [5][6][7], shared use of transcription factors [5,8,9] and the role of immunoreceptor-like signalling pathways (such as Syk activation by immunoreceptor-associated adapters) in osteoclast development [10][11][12][13][14]. The similarity between bone and immune cells is further supported by the similar components used by neutrophil, macrophage and osteoclast signalling and in vivo inflammatory processes [15][16][17][18][19].
PLCc2 is also activated downstream of the immunoreceptor signalling adapters DAP12 and the Fc-receptor c-chain (FcRc) in osteoclast precursors while the downstream activation of the NFATc1 transcription factor is mediated by Ca 2+ signalling through tyrosine phosphorylation pathways [11,31]. The overall similarity between immunoreceptor and PLCc2-mediated signalling pathways suggests a possible role for PLCc2 in osteoclast biology.
The above results prompted us to test the role of PLCc2 in osteoclast development and function, as well as in in vivo bone homeostasis under normal and pathological conditions. Our results indicate that PLCc2 plays an important role in basal bone resorption, likely due to its role in later phases of osteoclast development. Surprisingly, however, PLCc2 does not play a major role in ovariectomy-induced bone loss.

Materials and methods Animals
Heterozygous mice carrying a deleted allele of the PLCc2encoding gene (Plcg2 tm1Jni , referred to as PLCc2 ) ) [23] were obtained from James N. Ihle (St. Jude Children's Research Hospital, Memphis, TN, USA) and has been backcrossed to the C57BL ⁄ 6 genetic background for more than 10 generations. Because of the limited fertility of homozygous PLCc2 ) ⁄ ) mice, the mutation was maintained in heterozygous form as described [26].
For in vivo experiments, PLCc2 + ⁄ + or PLCc2 + ⁄ ) mice of identical age and sex (mostly littermates) from the same colony were used as controls. For in vitro experiments, either PLCc2sufficient mice from the PLCc2 breeding colony or C57BL ⁄ 6 mice purchased from the Hungarian National Institute of Oncology (Budapest, Hungary) were used as controls. Because of the limited availability of PLCc2 ) ⁄ ) animals, some of the in vitro experiments were performed on cells from PLCc2 ) ⁄ ) (and appropriate control) bone marrow chimeras generated and tested as described [26]. No difference between the different sources of mice or bone marrow cells has been observed (not shown).
Mice were kept in individually sterile ventilated cages (Tecniplast, Buguggiate, Italy) in a conventional facility. All animal experiments were approved by the Semmelweis University Animal Experimentation Review Board.

Ovariectomy
To test oestrogen deficiency-induced bone loss, wild-type and PLCc2 ) ⁄ ) females at 8 weeks of age were anesthetized with ketamine and medetomidine and subjected to surgical ovariectomy or sham operation. Six weeks after the operation, the mice were sacrificed and their femurs or tibias were analysed.

Micro-CT and histomorphometry
Bone architecture under basal conditions was tested on agematched wild-type and PLCc2 ) ⁄ ) male mice at 8-10 weeks of age. Ovariectomy-induced bone loss was tested at 14 weeks of age on wild-type and PLCc2 ) ⁄ ) females.
Micro-CT studies were performed on the distal metaphysis of the femurs stored in PBS containing 0AE1% Na-azide. Samples were scanned on a SkyScan 1172 (SkyScan, Kontich, Belgium) micro-CT apparatus using a 50 kV and 200 lA X-ray source with 0AE5-mm aluminium filter, and a rotation step of 0AE5°with frame averaging turned on, resulting in an isometric voxel size of 4AE5 lm. Three-dimensional images were reconstituted and analysed using the NRecon and CT-Analyser software (both from SkyScan). For quantitative analysis, 400 horizontal sections starting 50 sections above the distal growth plate were selected, and the boundaries of trabecular area were selected manually a few voxels away from the endocortical surface [32]. The density threshold for bone tissue was set manually by an experienced investigator. For graphical presentation, the twodimensional representation of a horizontal section 250 sections above the distal growth plate, as well as the three-dimensional reconstitution of an axial cylinder of 700 lm diameter, expanding from 150 to 450 sections above the distal growth plate has been prepared.
Histomorphometry studies were performed on the proximal metaphysis of the tibias. After sacrificing the mice, the bones were placed in 70% ethanol, then fixed overnight in 4% formalin and embedded undecalcified in methylmetacrylate (Technovit; Heraeus Kulzer, Wehrheim, Germany). After polymerization, 3-to 4-lm sections were cut with a Jung micrometer (Jung, Heidelberg, Germany) and deplastinated in methoxymethylmetacrylate (Merck, Darmstadt, Germany). Sections were stained with von Kossa and Goldner stains. Bone histomorphometry was performed using a microscope (Nikon, Tokyo, Japan) equipped with a video camera and digital analysis system (OsteoMeasure; OsteoMetrics, Decatur, GA, USA).
In vitro cultures, resorption assays and flow cytometry Macrophages were generated by culturing osteoclast ⁄ macrophage precursors in the presence of M-CSF but not RANKL. M-CSF was supplied in the form of purified protein (parallel macrophage and osteoclast studies) or as a 10% conditioned medium from CMG14-12 cells [35] (biochemical studies). Expression of the F4 ⁄ 80 macrophage differentiation antigen was tested as described [19].

Analysis of gene expression
Osteoclast-specific gene expression was tested using quantitative real-time PCR analysis [36] from wild-type or PLCc2 ) ⁄ ) cultures generated in the indicated periods of time using the indicated cytokine concentrations. Total RNA was then isolated from the cells with Trizol reagent (Invitrogen). Reverse transcription was performed at 37°C for 120 min from 100 ng total RNA using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). Quantitative realtime PCRs were performed in triplicates with a control reaction containing no reverse transcriptase on an ABI PRISM 7900 (Applied Biosystems) equipment with 40 cycles at 94°C for 12 s and 60°C for 60 s using Applied Biosystems Taqman Gene Expression Assay kits. We tested the expression of the mouse Acp5 (TRAP; Taqman Mm00475698_m1), Calcr (Calcitonin receptor; Mm00432271_m1), Ctsk (cathepsin K; Mm00484039_m1), Fos (c-Fos; Mm00487425_m1), Nfatc1 (NFATc1; Mm00479445_m1), Oscar (OSCAR; Mm00558665_ m1) and Tm7sf4 (DC-STAMP; Mm04209235_m1) genes and normalized it to the expression of the housekeeping gene Gapdh (GAPDH; Mm99999915_g1). The comparative C t method was used to quantify transcripts.

Biochemical and signalling studies
PLCc2 expression was determined from Triton X-100-soluble whole osteoclast or macrophage lysates as described [26].

Statistical analysis
Experiments were performed at the indicated times with comparable results. Statistical analyses were performed using Student's unpaired two-population t-test with unequal variance or by two-way ANOVA. Analysis of the interaction between the effects of genotypes and surgical treatments was performed using Tukey's post hoc test. P values below 0AE05 were considered statistically significant.

micro-CT and histomorphometric analysis of wild-type and PLCc2 ) ⁄ ) animals
We first analysed the composition of trabecular bone of wildtype and PLCc2 ) ⁄ ) male mice using micro-CT analysis of the distal metaphysis of the femurs. Significantly more trabeculae were visible in PLCc2 ) ⁄ ) animals than in the wild-type mice both in raw micro-CT slices (Fig. 1a) and in three-dimensional reconstitution images of an axial cylindrical region (Fig. 1b). Quantification of the entire three-dimensional reconstitution image (Fig. 1c) revealed a significant increase in the per cent bone volume (BV ⁄ TV) of PLCc2 ) ⁄ ) animals (P = 0AE011; n = 5), which was primarily because of increased trabecular number rather than increased thickness of the individual trabeculae (Fig. 1c).
We also performed histomorphometric analysis of the trabecular bone of the proximal tibia of male mice. Those studies confirmed an increased relative bone volume (BV ⁄ TV; P = 0AE0012; n = 4) and trabecular number, but not trabecular thickness is PLCc2 ) ⁄ ) animals (Fig. 1d). In addition, a significantly lower number of osteoclasts was seen in PLCc2 ) ⁄ ) bones while the number of osteoblasts was not affected (Fig. 1e). Taken together, PLCc2 ) ⁄ ) animals have increased trabecular bone volume likely due to an osteoclast defect.

PLCc2 is required for in vitro osteoclast development
To test the role of PLCc2 in osteoclasts, we have cultured wildtype and PLCc2 ) ⁄ ) bone marrow cells under osteoclastogenic conditions in vitro. As shown in the TRAP-stained images in Fig. 2a and their quantification in Fig. 2b, 20 ng ⁄ mL M-CSF and 20 ng ⁄ mL RANKL induced significant osteoclast development from wild-type but not from PLCc2 ) ⁄ ) bone marrow cells. This defect could not be overcome by increasing the concentration of M-CSF, RANKL or both to 50 ng ⁄ mL (Figs 2a,b). However, both wild-type and PLCc2 ) ⁄ ) cultures consistently stained positive for TRAP (Fig. 2c), and the percentage of TRAP-positive cells among all mononuclear cells was very similar in the two to the trabecular bone surface. Data were obtained from five (a-c) or four (d,e) mice per group at 8-10 weeks of age. The analyses were performed on the distal metaphysis of the femurs (a-c) or the proximal metaphysis of the tibias (d,e). Error bars represent SEM. *P < 0AE05; **P < 0AE01; ***P < 0AE002; n.s., not significant; BV ⁄ TV, per cent bone volume (bone volume ⁄ total volume).
genotypes (30-35% at 20 ng ⁄ mL M-CSF + 20 ng ⁄ mL RANKL and 40-45% at 50 ng ⁄ mL M-CSF and 50 ng ⁄ mL RANKL, irrespective of the genotype of the cells). Taken together, PLCc2 is required for the in vitro development of mature multinucleated osteoclasts in the presence of M-CSF and RANKL but likely not for the initial steps of preosteoclast differentiation.

PLCc2 is required for in vitro bone resorption
We next tested the effect of PLCc2 deficiency on osteoclastmediated bone resorption by culturing bone marrow cells on an artificial hydroxyapatite layer. As shown in Fig. 3, wild-type cells cultured in the presence of 20 ng ⁄ mL M-CSF and 20 ng ⁄ mL RANKL had a moderate resorptive capacity that was strongly increased by increasing the concentration of both cytokines to 50 ng ⁄ mL. In contrast, practically, no resorption could be observed in PLCc2 ) ⁄ ) cultures at either cytokine concentration. Therefore, PLCc2 is also required for osteoclastmediated bone resorption, likely reflecting the previously mentioned osteoclast developmental defect (Fig. 2).
PLCc2 is not required for macrophage differentiation or expression of osteoclast-specific genes Our next aim was to address whether PLCc2 is involved in an earlier or a later phase of osteoclast differentiation. Because we were able to obtain normal numbers of apparently normal macrophages from PLCc2 ) ⁄ ) bone marrow cells (Fig. 2c and data not shown) and those macrophages expressed normal levels of the macrophage differentiation marker F4 ⁄ 80 (Fig. 4a), it is unlikely that PLCc2 is required for the first steps of general myeloid cell differentiation.
We next tested the time course of osteoclast-specific gene expression in in vitro cultures by quantitative RT-PCR. As shown in Fig. 4b, the expression of the Acp5 (encoding for TRAP), Calcr (calcitonin receptor), Ctsk (cathepsin K), Fos (c-Fos), Nfatc1 (NFATc1), Oscar (OSCAR) and Tm7sf4 (DC-STAMP) genes was strongly increased during osteoclast differentiation, but none of these genes showed increased expression in parallel macrophage samples. The genetic deficiency of PLCc2 did not induce any major reduction in osteoclast-specific gene expression, although some partial decrease in expression could be observed, particularly in the case of Calcr (Fig. 4b). Most importantly, the expressions of the genes encoding for the early maturation marker TRAP (Acp5), the most plausible PLCc2 effector NFATc1 (Nfatc1) [11,31] and of DC-STAMP (Tm7sf4), a critical player of the preosteoclast fusion machinery [37,38], were all upregulated normally in PLCc2 ) ⁄ ) cultures (Fig. 4b).
These results indicate that PLCc2 is mostly dispensable for initiation of osteoclast-specific gene expression.
Biochemical characterization of the PLCc2-mediated osteoclast signalling pathway Next, we aimed at the biochemical characterization of PLCc2 activation in osteoclasts. We first tested the presence of PLCc2 in parallel macrophage and osteoclast cultures and found that PLCc2 was expressed at comparable levels in wild-type macrophages and osteoclasts but, as expected, not in PLCc2 ) ⁄ ) cells (Fig. 5a). Osteoclast development is triggered by three major extracellular signals: M-CSF, RANKL and adhesive interactions with the environment (e.g. with tissue culture plastic surface). We next tested which of these three signals trigger PLCc2 activation, using wild-type macrophages stimulated with M-CSF or RANKL in suspension (which was required to avoid parallel engagement of adhesion receptors), or plated on a tissue culture plastic surface. Both an immunoprecipitation approach followed by immunoblotting with anti-phosphotyrosine antibodies (Fig. 5b) and a direct immunoblotting using phospho-specific PLCc2 antibodies (Fig. 5c) revealed PLCc2 phosphorylation upon adhesion of macrophages but not upon M-CSF or RANKL stimulation in suspension. Additional attempts with M-CSF or RANKL stimulation for various periods of time or using various cytokine concentrations ranging from 10 to 100 ng ⁄ mL did not reveal a consistent PLCc2 phosphorylation in suspension either (not shown). On the other hand, M-CSF-induced ERK phosphorylation and RANKLinduced p38 MAP kinase phosphorylation and NFjB activation (degradation of IjBa) could readily be observed under these conditions (Fig. 5c), indicating intact basic M-CSF and RANKL signalling in suspension. Therefore, PLCc2 appears to be activated by adhesive interactions rather than by stimulation with M-CSF or RANKL cytokines.
We have also tested the role of Src-family kinases in PLCc2 phosphorylation. As shown in Fig. 5d, pretreatment of macrophages with the Src-family inhibitor PP2 completely abrogated the PLCc2 phosphorylation response, indicating that the adhesion-induced PLCc2 activation requires members of the Src kinase family.

PLCc2 ) ⁄ ) mice show normal ovariectomy-induced bone resorption
Because osteoclast-mediated bone resorption contributes to postmenopausal osteoporosis [39], we hypothesized that PLCc2 may also play a role in oestrogen deficiency-induced bone loss. That possibility was tested by subjecting wild-type and PLCc2 ) ⁄ ) animals to surgical ovariectomy, followed by micro-CT and histomorphometric analysis 6 weeks later. Representative raw micro-CT sections (Fig. 6a), three-dimensional reconstitution images (Fig. 6b) and quantitative micro-CT  analyses (Fig. 6c) indicated that, similar to intact male animals ( Fig. 1), sham-operated PLCc2 ) ⁄ ) females also had increased trabecular bone density, which was reflected in a nearly twofold increase in relative bone volume (BV ⁄ TV; P = 0AE00031; n = 7 (wild type) vs. 4 (PLCc2 ) ⁄ ) )). As expected, surgical ovariectomy led to a significant reduction in the per cent bone volume (BV ⁄ TV) of wild-type mice (P = 0AE025; n = 7). Contrary to our expectations, however, the per cent bone volume of PLCc2 ) ⁄ ) animals was also significantly reduced (P = 0AE00023; n = 4) and that reduction was even higher in PLCc2 ) ⁄ ) mice than in wild-type animals both in terms of absolute reduction in BV ⁄ TV values (4AE1 vs. 1AE6 percentage points, respectively) and in percentage of the BV ⁄ TV values of the sham-operated control animals (50% vs. 36%, respectively). The difference of the effect of ovariectomy on wild-type and PLCc2 ) ⁄ ) animals (interaction of the genotypes and surgical procedures) proved to be statistically significant (P = 0AE0090). Importantly, while the BV ⁄ TV values of sham-operated wild-type and PLCc2 ) ⁄ ) animals were statistically highly significant (P = 0AE00025), there was no significant difference between the two genotypes after the ovariectomy procedure (P = 0AE25; n = 7 (wild type) vs. 4 (PLCc2 ) ⁄ ) )). Similar differences could be observed in the trabecular numbers, whereas the trabecular thickness remained unaffected by the different genotypes and surgical procedures (Fig. 6c). The above-mentioned findings were also confirmed by histomorphometric analysis of ovariectomy-induced bone loss in the proximal tibia. As shown in Fig. 6d, that analysis confirmed the increased per cent bone volume (BV ⁄ TV) in sham-operated PLCc2 ) ⁄ ) animals (P = 0AE0010; n = 3) and a reduction in per cent bone volume in ovariectomized wild-type mice (P = 0AE038; n = 3). Importantly, the ovariectomy procedure induced a significantly more pronounced reduction in per cent bone volume in PLCc2 ) ⁄ ) mice than in wild-type animals, both . Surgical operation was performed at 8 weeks of age followed by an additional 6 weeks before the mice were sacrificed and their bones were removed for analysis. Error bars represent SEM of the indicated number of animals. *P < 0AE05; **P < 0AE01; ***P < 0AE002; ****P < 0AE0004; n.s., not significant; BV ⁄ TV, per cent bone volume (bone volume ⁄ total volume).
in terms of absolute reduction in BV ⁄ TV values (9AE9 vs. 4AE3 percentage points, respectively) and in per cent of the BV ⁄ TV values of the sham-operated control animals (38% vs. 24%, respectively). The difference of the effect of ovariectomy on wild-type and PLCc2 ) ⁄ ) animals (interaction of the genotypes and surgical procedures) proved again to be statistically significant (P = 0AE013). Similar to the micro-CT data (Fig. 6a), while the BV ⁄ TV values of sham-operated wild-type and PLCc2 ) ⁄ ) animals were statistically highly significant (P = 0AE0010), that difference faded away after the ovariectomy procedure (P = 0AE24; n = 3). A similar picture was seen during the analysis of trabecular numbers, whereas the trabecular thickness remained unaffected by the different genotypes and surgical procedures (Fig. 6d).
Additional studies testing the number of osteoclasts and osteoblasts attached to the bone surface indicated that although the number of osteoclasts was significantly lower in the shamoperated PLCc2 ) ⁄ ) mice than in the wild-type ones (3AE0 ± 0AE1 vs. 1AE4 ± 0AE5 per mm, respectively; P = 0AE0036; n = 3), the number of osteoclasts was strongly increased and reached a comparable, though, still significantly different level (6AE3 ± 0AE4 vs. 5AE0 ± 0AE4 per mm, respectively; P = 0AE010; n = 3) in the two genotypes after the ovariectomy procedure (Fig. 6e). The difference of the effect of ovariectomy on wild-type and PLCc2 ) ⁄ ) animals (interaction of the genotypes and surgical procedures) did not prove to be statistically significant (P = 0AE56; n = 3), indicating that PLCc2 ) ⁄ ) animals were able to upregulate osteoclast numbers upon oestrogen deficiency normally. Analysis of the number of osteoblasts did not find any significant difference between any of the groups tested (Fig. 6e).
Taken together, these results suggest that ovariectomized PLCc2 ) ⁄ ) animals are capable of reducing their bone mass to levels comparable to those seen in similarly treated wild-type animals, likely because of similar oestrogen deficiency-induced increase in osteoclast numbers in the two genotypes.

Discussion
In the first part of this study, we showed that PLCc2 ) ⁄ ) mice have increased basal bone density, likely due to reduced in vivo osteoclast number reflecting the role of PLCc2 in a later phase of osteoclast development. These results raised the possibility that PLCc2 may also participate in pathological bone resorption, such as oestrogen deficiency-induced osteoporosis. Much to our surprise, however, PLCc2 ) ⁄ ) mice showed similar, or even more pronounced, ovariectomy-induced bone resorption than their wild-type counterparts. Therefore, PLCc2 does not appear to be required for oestrogen deficiency-induced bone loss.
The experiments presented in this paper (part of which were published in an abstract form before [40]) were initiated based on our prior experiments showing defective osteoclast develop-ment and in vivo bone resorption in mice lacking immunoreceptor signalling adapter molecules or the Syk tyrosine kinase [10], as well as the similarity between various Syk ) ⁄ ) and PLCc2 ) ⁄ ) phenotypes [15,19,[23][24][25][26]41]. However, two other groups have also independently reported in vitro osteoclast development and in vivo bone resorption defects in PLCc2 ) ⁄ ) mice [42,43]. Although all three reports conclude that PLCc2 is required for in vitro osteoclast development and basal bone resorption in vivo, they provide different explanations for those observations. Mao et al. [42] and Chen et al. [43] reported dramatically reduced expression of osteoclast-specific genes (such as those encoding TRAP, NFATc1, cathepsin K or the calcitonin receptor) in PLCc2 ) ⁄ ) cultures, suggesting that PLCc2 is required for an early step of osteoclast differentiation. A plausible explanation was that a PLCc2-induced intracellular Ca 2+ signal triggered activation of NFATc1, a Ca 2+ -sensitive master regulator of osteoclast-specific gene expression [31]. Surprisingly, our own more detailed analyses did not reveal any substantial defect of osteoclast-specific gene expression in PLCc2 ) ⁄ ) cultures, and the expression of NFATc1 was not at all affected by the PLCc2 mutation (Fig. 4b). Based on the time course of the expression of those genes and their low expression in parallel macrophage samples (Fig. 4b), it is unlikely that our results are attributed to non-specific amplification artefacts. It is also unlikely that our results are owing to inappropriate selection of the cytokine concentrations used because we did not observe reduced gene expression levels in PLCc2 ) ⁄ ) cultures even when the concentration of both M-CSF and RANKL was reduced to 20 ng ⁄ mL, whereas the PLCc2 ) ⁄ ) mutation caused severe osteoclast developmental defect even when the concentration of both cytokines was increased to 100 ng ⁄ mL (not shown). In addition, we consistently observed a large percentage of TRAP-positive mononuclear cells in PLCc2 ) ⁄ ) osteoclast cultures (Fig. 2c), and such cells were also present in the PLCc2 ) ⁄ ) osteoclast cultures shown by Mao et al. [42] and Chen et al. [43]. Taken together, osteoclast-specific gene expression is not (or not completely) blocked in PLCc2 ) ⁄ ) cultures, necessitating alternative explanations for the PLCc2 ) ⁄ ) osteoclast phenotype.
Another possible explanation for the PLCc2 ) ⁄ ) osteoclast phenotype could be the participation of PLCc2 in preosteoclast fusion, a process mediated in part by the DC-STAMP molecule [37,38]. While our gene expression studies (Fig. 4b) did not reveal any major role for PLCc2 in RANKL-induced upregulation of DC-STAMP, it has yet to be tested whether PLCc2 is involved in signal transduction by DC-STAMP or another preosteoclast fusion receptor.
Osteoclast development requires a complex interplay between signals from M-CSF, RANKL and ligation of adhesion receptors [44]. Because RANKL stimulation of adherent bone marrow-derived macrophages triggered PLCc2 phosphorylation, Mao et al. [42] and Chen et al. [43] suggested that PLCc2 is activated downstream of RANK. That conclusion, however, is confounded by the ligation of both RANK and adhesion receptors in that assay. In contrast, we did not observe any PLCc2 phosphorylation upon stimulating wild-type macrophages with RANKL (or M-CSF) in suspension, whereas cellular adhesion consistently triggered robust phosphorylation of the protein in the absence of any cytokines (Fig. 5b,c). These results suggest that PLCc2 is primarily involved in adhesion receptor rather than in RANK signal transduction, a possibility consistent with the role of PLCc2 in integrin signalling of neutrophils [24,26] and its proposed modulatory effect on integrin signalling in preosteoclasts [45].
The analysis of ovariectomy-induced bone resorption is likely the most clinically relevant aspect of our study. While the increased basal bone volume ( Fig. 1) and the defective in vitro osteoclast development and function (Figs 2 and 3) in PLCc2 ) ⁄ ) mice suggested a role for PLCc2 in pathological bone resorption, ovariectomy-induced bone loss in PLCc2 ) ⁄ ) mice was unexpectedly normal or even more pronounced than in wild-type animals (Fig. 6). Because this was observed both in the distal femur and in the proximal tibia and by two independent approaches (micro-CT and histomorphometry), we believe that PLCc2 is not a major general component of oestrogen deficiency-induced bone resorption. However, we cannot exclude the possibility that ovariectomy-induced bone resorption at certain sites or under some specific conditions would be defective in PLCc2 ) ⁄ ) animals.
It is at present unclear to us how exactly PLCc2 ) ⁄ ) animals are able to reduce their bone mass during oestrogen deficiency. However, the fact that the ovariectomy-induced increase in the number of osteoclasts was similar in the two genotypes despite significantly reduced basal number of osteoclasts in PLCc2 ) ⁄ ) mice (Fig. 6e) suggests the existence of PLCc2-independent mechanisms triggering oestrogen deficiency-induced osteoclast development. Whether those are mediated by cell-cell interactions (e.g. with osteoblasts) not present in the in vitro cultures [10,11], by excessive release of cytokines overcoming osteoclast developmental defects [46] or by the amplification of PLCc2independent signal transduction pathways, should be the subject of future research.
It has been generally believed that osteoclasts use similar signal transduction pathways during basal and induced (e.g. oestrogen deficiency-induced) bone resorption. Therefore, it has been assumed that the identification of novel osteoclast signalling molecules may provide suitable targets for the therapeutic intervention in pathological bone loss, such as postmenopausal osteoporosis. Our results showing normal ovariectomy-induced bone loss in PLCc2 ) ⁄ ) animals indicate that this may not be the case. Interestingly, a prior study showed that ovariectomy-induced bone loss in the highly osteopetrotic DAP12 ) ⁄ ) FcRc ) ⁄ ) animals is comparable to, or even higher than, that in wild-type mice [47]. All these results suggest differential osteoclast signalling requirements for basal and oestrogen deficiency-induced bone resorption and indicate that care should be taken when extrapolating findings on basal bone resorption to pathological conditions. These results also indicate that limited clinical benefit can be expected from therapeutic targeting of PLCc2 in postmenopausal osteoporosis.