Gα12 regulates osteoclastogenesis by modulating NFATc1 expression

Abstract The G12 family of G protein alpha subunits has been shown to participate in the regulation of various physiological processes. However, the role of Gα12 in bone physiology has not been well described. Here, by micro‐CT analysis, we discovered that Gα12‐knockout mice have an osteopetrotic phenotype. Histological examination showed lower osteoclast number in femoral tissue of Gα12‐knockout mice compared to wild‐type mice. Additionally, in vitro osteoclastic differentiation of precursor cells with receptor activator of nuclear factor‐κB ligand (RANKL) showed that Gα12 deficiency decreased the number of osteoclast generated and the bone resorption activity. The induction of nuclear factor of activated T‐cell c1 (NFATc1), the key transcription factor of osteoclastogenesis, and the activation of RhoA by RANKL was also significantly suppressed by Gα12 deficiency. We further found that the RANKL induction of NFATc1 was not dependent on RhoA signalling, while osteoclast precursor migration and bone resorption required RhoA in the Gα12‐mediated regulation of osteoclasts. Therefore, Gα12 plays a role in differentiation through NFATc1 and in cell migration and resorption activity through RhoA during osteoclastogenesis.


Introduction
Bone homeostasis is exquisitely maintained by a harmony of boneforming osteoblasts and bone-resorbing osteoclasts. Abnormal increases in the number or excessive stimulation of the activity of osteoclasts are often related to erosive bone diseases, such as osteoporosis, or inflammatory bone lysis. Osteoclasts are multinuclear giant cells that are differentiated from the monocyte/macrophage lineage of hematopoietic cells. For osteoclastogenesis, two major cytokines are required: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor jB ligand (RANKL). M-CSF supports the survival and proliferation of precursor cells, and RANKL induces their differentiation into osteoclasts [1]. The binding of RANKL to the RANK receptor recruits tumour necrosis factor receptor-associated factor 6 (TRAF6) and brings together the signalling molecules required for the activation of mitogen-activated protein kinase (MAPK) or NF-jB [2,3]. RANKL also induces and activates the c-Fos transcription factor and activates calcium signalling in cooperation with additional cell surface immune receptors. Eventually, these signalling pathways activate nuclear factor of activated T-cell c1 (NFATc1), the transcription factor regarded as a master regulator of osteoclastogenesis, and drives the expression of the osteoclast marker genes such as tartrate-resistant acid phosphatase (TRAP), cathepsin K and dendritic cell-specific transmembrane protein (DC-STAMP) [4]. When osteoclasts become mature and functionally activated, they resorb the bone by secreting bonedegrading enzymes in resorption lacunae.
Heterotrimeric G proteins are classified into four subfamilies based on their alpha subunits: Gs, Gi, Gq and G12 [5,6]. The most recently identified G12 subfamily consists of Ga12 and Ga13, which are ubiquitously expressed in various cells and tissues [7]. The first identified function of Ga12 was oncogenic transformation [8]; since then, Ga12 has been reported to regulate multiple cellular responses including growth, polarity, migration and apoptosis in various physiological processes, such as embryonic development, angiogenesis, platelet activation, and immune and neuronal actions [9][10][11]. The Ga12-mediated signalling response has mainly been described for small GTPase Rho activation through RhoGEF, which has been linked to the regulation of actin stress fibres and the induction of transcriptional activity [12][13][14]. Other than RhoGEF, more than 20 proteins have been revealed as effector molecules for Ga12, such as cadherin, Hsp90 and BTK [15][16][17]. This implies that Ga12 could function through diverse mechanisms in different tissues.
Ga12 shows a 67% amino acid identity with Ga13, and they share many of the same binding G protein-coupled receptors (GPCRs) and downstream effector molecules. However, the presence of significant differences between Ga12 and Ga13 has been demonstrated by knockout mice studies. Ga12-deficient mice are viable, fertile and show no noticeable gross abnormalities [18,19]. In striking contrast, Ga13-deficient mice are embryonic lethal due to angiogenic defects. The skeletal phenotypes of Ga12-knockout mice have not been investigated in detail.
In this study, we found increased trabecular bone volume in Ga12-knockout mice, and this result prompted us to investigate the function of Ga12 in osteoclastogenesis. Here, we report for the first time the bone phenotype of Ga12-knockout mice and suggest that Ga12 has a crucial role in RANKL-induced osteoclastogenesis.

Bioinformatic analysis
Microarray data were downloaded from Gene Expression Omnibus (GSE57468). Differentially expressed genes (DEGs) upon osteoclast differentiation were identified independently using Student's t-test: DEGs were selected as the genes having P-values < 0.005 at every timepoints compared with control group. Statistically enriched signalling pathways of clustered DEGs were ranked and categorized according to Reactome pathways using DAVID 6.7 software (National Institutes of Health, Bethesda, MD). Hierarchical clustering with Pearson's correlation of the genes encoding Ga proteins was performed using R software with pvclust package (Bioconductor, http://www.r-project.org/). H3K27ac ChIP-seq data were collected from ENCODE. ChIP-seq data were analysed using Integrative Genomics Viewer (IGV) software (Broad Institute, Cambridge, MA).

Mice and cell culture
Ga12 À/À mice have been described previously [18]. Whole bone marrow cells were flushed from the tibias and femurs of 5-week-old ICR mice or 7-week-old Ga12 À/À mice. After the elimination of erythrocytes with hypotonic buffer, cells were incubated overnight in alpha-modified Eagle's medium (a-MEM) with 10% foetal bovine serum in culture dishes. Adherent cells were discarded, and floating cells were further cultured with 30 ng/ml M-CSF on Petri dishes. After three days, adherent cells were scraped and regarded as bone marrow-derived macrophages (BMMs). For the differentiation of osteoclasts, BMMs were cultured with 30 ng/ml M-CSF and 120 ng/ml RANKL.

siRNA transfection
BMMs were transfected with 40 nM control or Ga12-specific siRNAs (Santa Cruz) with HiPerFect transfection reagent (Qiagen, Hilden, Germany). After overnight incubation, the culture medium was replaced with a fresh medium.

Plasmid transfection and retroviral gene transfer
BMMs were transfected with pcDNA3-EGFP or pcDNA3-RhoA-CA (constitutively active form) using PolyFect (Qiagen). Retroviral packaging was performed by transfecting Plat E cells with pMX-puro or pMXpuro-hNFATc1CA using PolyFect. At 24 hrs after transfection, culture medium containing viral particles was collected and filtered through a 0.45-lm syringe filter. For retroviral infection, BMMs were incubated in the virus-containing medium with 4 lg/ml polybrene and 30 ng/ml M-CSF for 24 hrs.
lCT analysis Femurs from 9-week-old WT and Ga12 À/À male mice were analysed with a SkyScan 1172 (SkyScan, Aartselaar, Belgium; 70 Kv, 141 lA, 6.92 pixel size). Trabecular bone was measured in the 1-mm-thick region starting 1 mm below the growth plate at thresholds of 89 minimum and 255 maximum. Bone parameters were calculated by a CTanalyzer program (version 1.7; SkyScan), and three-dimensional images were obtained by CT-volume software (version 1.11; SkyScan).

TRAP staining
Cells were fixed with 3.7% formaldehyde and permeabilized in 0.1% Triton X-100 for 1 min. TRAP staining was performed for 5-15 min. using a commercial kit (Sigma-Aldrich, Cat. No. 387A-1KT) according to the manufacturer's instructions.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 3 lg RNA was used to synthesize cDNA using Superscript II reverse transcriptase (Invitrogen). Real-time PCR was performed using a KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA) and ABI7300 real-time system (Applied Biosystems, Walmington, MA, USA). Thermocycling conditions were as follows: an initial activation step at 95°C for 3 min., followed by 40 cycles of denaturation at 95°C for 3 sec. and amplification at 60°C for 33 sec. The primer sequences used are listed in Table 1. All reactions were run in triplicate, and gene expression levels were determined as fold changes using the cycle threshold comparison method. The housekeeping gene HPRT was used for normalization.

Histological analysis
Histological measurements were performed as previously described [20]. Briefly, femurs were fixed in 4% paraformaldehyde and decalcified with 12% ethylenediaminetetraacetic acid (EDTA) for four weeks. Decalcified femurs were dehydrated and embedded in paraffin. Then, 5-lmthick sections were stained for TRAP and with haematoxylin and were analysed using Osteomeasure software (Osteometrics, Decatur, CA, USA).

Actin ring analysis
BMMs were cultured on 15-mm glass coverslips and then fixed with 3.7% formaldehyde. Cells were permeabilized with 0.1% Triton X-100, and non-specific binding sites were blocked with 1% bovine serum albumin in phosphate-buffered saline (PBS) for 90 min. After the cells were washed with PBS, they were stained with actin using rhodaminephalloidin for 2 hrs and mounted in the presence of 4 0 ,6-diamidino-2phenylindole (DAPI). Images were obtained using a Zeiss LSM700 confocal microscope (Carl Zeiss).

Transwell migration assay
BMMs were transfected with pcDNA3-EGFP or RhoCA (the constitutively active form of RhoA) constructs with a polyfect transfection reagent (Qiagen). Transfected BMMs (1 9 10 5 ) in serum-free a-MEM were transferred to the upper chamber of transwell plates with 8-lm pores (Corning, Corning, NY, USA). The lower chambers were filled with serum-free a-MEM containing 240 ng/ml RANKL as an attractant. After 16 hrs of incubation in a CO 2 incubator at 37°C, cells were fixed and stained with crystal violet.

RhoA activity assay
Rho activity was measured using a Rho Activation Assay Kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer's protocol. Briefly, after 3 hrs of serum starvation, BMMs were stimulated with 360 ng/ml RANKL and harvested at 0 and 15 min. Cells were lysed with lysis buffer and spun down, and then the supernatant was incubated with GTP-Rho-specific binding beads (containing Rhotekin Rho-binding domain) at 4°C for 45 min. After the beads were washed three times, the samples were boiled with sample buffer. The supernatants of the boiled samples were loaded on 12°C sodium dodecyl sulphate (SDS)polyacrylamide gels, and immunoblotting was performed using anti-Rho antibodies.

BrdU assay
BrdU incorporation into cells was measured using a BrdU Cell Proliferation Assay Kit (Calbiochem, La Jolla, CA, USA) following the manufacturer's protocol.

Western blotting
Cells were lysed with RIPA buffer [120 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na 2 EDTA, 1 mM EGTA, 0.5% NP-40, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na 3 VO 4 , 1 mM NaF and a protease inhibitor cocktail (Roche, Mannheim, Germany)]. Protein concentrations of the cell lysates were measured using a DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA), and the same amounts of protein were loaded onto 10% SDS-polyacrylamide gels for electrophoresis. After protein transfer onto nitrocellulose membranes (Amersham Pharmacia, Uppsala, Sweden), non-specific binding sites on the membranes were blocked with 5% skim milk for 1 hr, and the membranes were incubated with primary antibodies overnight at 4°C. The membranes were then washed several times with Tris-buffered saline with Tween-20 and incubated with secondary antibodies conjugated with horseradish peroxidase in 2% skim milk for 1 hr.

Statistical analysis
Statistical differences between the two groups were determined using Student's t-test. P-values <0.05 were regarded as significant.

Decrease in Ga12 transcript level during osteoclast differentiation
To explore the putative signalling pathway responsible for the regulation of bone homeostasis, we first analysed a microarray data set obtained using the mouse bone marrow-derived macrophages (BMDMs), primary cells of osteoclast precursor. We found that expression of genes involved in several G protein-coupled receptor pathways changed during osteoclast differentiation triggered by M-CSF and RANKL treatment (Fig. 1A). Along with Ga12/13 and Gaq signalling events, PAR and P2Y1 signalling pathways shown to link to Ga12/13 or Gaq showed significant changes. Next, we analysed Ga transcript levels; Ga12 transcript levels were significantly down-regulated along with those of Gai2 and Ga15, whereas Ga13, Gaq or Gat3 transcript levels were rather increased (Fig. 1B). We then analysed the profile of histone modification on each gene and the surrounding regions in BMDMs and other tissues using H3K27ac signals and found that Gna12, Gnai2, Gna15, Gna13 and Gnaq DNA regions were transcriptionally active in BMMs, whereas Gnat3 DNA regions were not (Fig. 1C, and Fig. S1). The H3K27ac signals on the Gna12 DNA regions were strongest in BMMs among the tissues examined, suggestive of the tissue-specific role of Ga12 in osteoclast differentiation. Consistently, Ga12 mRNA levels were decreased by M-CSF and RANKL treatment in our real-time PCR analyses (Fig. 1D). In contrast, Ga12 mRNA levels were not changed by M-CSF treatment alone. Increases in NFATc1 mRNA levels confirmed successful osteoclast differentiation (Fig. 1D). These results led us to further explore the role of Ga12 in osteoclast differentiation and bone homeostasis.

Ga12 knockout mice show osteopetrotic phenotype
To evaluate the potential role of Ga12 in bone metabolism, femurs from 9-week-old Ga12-knockout (Ga12 À/À ) mice were analysed by micro-computed tomography (lCT). The trabecular bone volume (BV) of the Ga12 À/À mice was about threefold higher than that of the wild-type (WT) mice ( Fig. 2A and B). The trabecular number (Tb.N) and trabecular thickness (Tb.Th) were also higher, while trabecular separation (Tb.Sp) was lower in the Ga12 À/À mice compared to the WT mice (Fig. 2B). To investigate whether the increment in bone mass was caused by changes in the population of bone cells, we assessed the numbers of osteoclasts and osteoblasts by histological methods. With increased trabecular bone area, we observed decreased numbers of osteoclasts in the Ga12 À/À bone tissue sections ( Fig. 2C and D). However, the numbers of osteoblasts were not significantly different between the WT and Ga12 À/À mice (Fig. 2D). The lCT analyses of 15-week-old mice also revealed a similar osteopetrotic phenotype in Ga12 À/À femurs (Fig. S2). These observations suggest that Ga12 plays a role in bone metabolism, perhaps by regulating osteoclastogenesis.

Ga12 depletion impairs osteoclast differentiation and bone resorptive function
After the lCT and histologic analyses, we assessed the function of Ga12 in osteoclast differentiation. BMMs were prepared from WT and Ga12 À/À mice, and the cells were cultured with osteoclastogenic medium containing M-CSF and RANKL. On day 3 after culturing, WT cells formed a considerable number of multinucleated cells that were positive for the osteoclast marker TRAP. However, <50% of multinucleated osteoclasts were generated in the Ga12 À/À group (Fig. 3A). A similar pattern was observed with the BMMs, in which Ga12 expression was reduced with a siRNA-mediated knockdown system (Fig. 3B).
Next, we examined the bone resorptive function of Ga12 À/À osteoclasts. Osteoclasts were cultured on dentin slices, and the area and depth of the resorption pits were measured. Compared to the WT osteoclasts, the Ga12 À/À osteoclasts generated smaller and shallower pits (Fig. 3C). To specify the reduced resorption is due to impaired resorption activity or decreased osteoclast differentiation, we tested resorption activity of mature osteoclasts. Ga12-specific siRNAs were introduced to osteoclasts generated by culturing BMMs for 4 days with RANKL (Fig. 3D). Ga12 knockdowned osteoclasts showed significantly diminished resorption area and resorption depth (Fig. 3E) when the degree of osteoclast differentiation was similar to that of the control cells (Fig. 3D). It implies that Ga12 is involved not only in the osteoclastic differentiation but also in the resorption activity of mature osteoclasts.
We also assessed actin ring formation, because the actin ring is a functionally important cytoskeletal structure for bone resorption and a typical morphological feature of active mature osteoclasts. As expected, we observed a reduced number of osteoclasts containing actin rings in Ga12 À/À group compared to the WT group (Fig. 3F). However, the actin ring morphology in mature osteoclasts did not show evident differences between the WT and Ga12 À/À mice.
Taken together, these results indicate that Ga12 depletion impaired RANKL-induced osteoclast differentiation and bone resorption.

Ga12 regulates osteoclastogenesis via NFATc1
Ga12 has been implicated in oncogenic transformation and proliferation in other cell types, mainly fibroblast cell line [8,21]. To examine whether Ga12 promotes osteoclast differentiation by increasing the proliferation of osteoclast precursors, we performed a bromodeoxyuridine (BrdU) cell proliferation assay. However, there was no difference in proliferation between WT and Ga12 À/À BMMs during osteoclast differentiation (Fig. 4A).

Fig. 3 Ga12 depletion impairs osteoclast differentiation and bone resorptive function. (A) Bone marrow-derived macrophages (BMMs) of WT or
Ga12 À/À mice were cultured with 30 ng/ml M-CSF and 120 ng/ml RANKL for three days and stained for TRAP. Images were captured using a light microscope (magnification 100 9), and TRAP-positive multinucleated cells (MNCs) were counted. (B) BMMs were transfected with 40 nM of negative control or Ga12-specific siRNA and cultured with M-CSF and RANKL for three days. Images of TRAP-stained cells are shown, and knockdown was confirmed by RT-PCR (right upper) and Western blotting (right lower). (C) BMMs were cultured on dentin slices for nine days with RANKL and M-CSF. The dentin slices were observed using a confocal microscope. Bone resorption area and depth were measured with image analysis software. (D) BMMs were seeded on dentin slices and cultured with M-CSF and RANKL for 4 days. After siRNA transfection at day 4, the cells were further cultured for additional 2 days. After removing the cells, dentin surface was analysed using a confocal microscope. (E) Bone resorption area and depth were measured with image analysis software. (F) BMMs were cultured with M-CSF and RANKL for 4 days, and actin was labelled with rhodamine-phalloidin (red). Nuclei were counter-stained with DAPI. Images were captured using a confocal microscope (magnification 200 9), and actin ring-positive osteoclasts were counted. Data are presented as means AE S.D. *P < 0.05; **P < 0.005.  NFATc1 is a key transcription factor in osteoclastogenesis and mediates the expression of many essential genes for osteoclast differentiation [4]. To investigate whether Ga12 regulates NFATc1 expression during RANKL-induced osteoclastogenesis, we measured the expression level of NFATc1 in Ga12 À/À BMMs. NFATc1 protein level was lower in Ga12 À/À BMMs compared to that in WT BMMs at 24 and 48 hrs after RANKL stimulation (Fig. 4B). Sequentially, the induction of osteoclast-specific genes that are direct targets of NFATc1, such as Acp5, Ctsk, Dcstamp and Atp6v0d2, was also much less prominent in the Ga12 À/À BMMs than in the WT BMMs during RANKL-induced osteoclast differentiation (Fig. 4C). However, c-Fos, which is one of the transcription factors inducing NFATc1, did not differ between the two groups at protein level (Fig. 4B). To clarify that Ga12 regulates osteoclast differentiation through NFATc1, we overexpressed constitutive active (CA) NFATc1 in Ga12 À/À BMMs using a retroviral system. NFATc1CA overexpression partially recovered the generation of osteoclasts from Ga12 À/À BMMs (Fig. 4D) and the expression of osteoclast marker genes (Fig. 4E). These results indicate that Ga12 regulates osteoclastogenesis by modulating NFATc1 expression and, consequently, the expression of NFATc1driven genes.

Ga12 affects BMM migration and osteoclast resorption through Rho activation
To define the molecular mechanism involved in osteoclast regulation by Ga12, we tested signalling pathways that have been reported to be activated by RANKL. BMMs were stimulated with RANKL, and the phosphorylation of MAPK, IjB and Akt was assessed by Western blotting. In the Ga12 À/À and WT cells, the RANKL-mediated activation of ERK, JNK, and p38 MAPK was detected at similar levels, and the activity of canonical NF-jB signalling reflected as IjB phosphorylation, and degradation was also not significantly different between Ga12 À/À and WT BMMs (Fig. 5A). However, Akt phosphorylation was increased in Ga12 À/À BMMs (Fig. 5A).
Ga12 has been well described to have RhoGEF as its effector molecule and to regulate cellular migration and polarity through Rho signalling activation [22,23]. To test whether Ga12 affects Rho activation in osteoclasts, we performed Rho-GTP pull-down assays. By RANKL stimulation, the level of GTP-bound RhoA (the active form of RhoA) was substantially increased after 15 min. in WT BMMs. In contrast, the Ga12 À/À BMMs showed only a slight increase in GTP-RhoA (Fig. 5B). To demonstrate that Ga12 affects the migration of osteoclast precursor cells through RhoA activation, we evaluated the migrating ability of Ga12 À/À BMMs versus WT BMMs. Ga12 À/À BMMs showed a decreased migration level (32.4 AE 8.4%), and this decline was rescued by the overexpression of constitutively active RhoA (RhoCA) (Fig. 5C).
We next tested whether the overexpression of RhoCA could recover osteoclast formation from and NFATc1 expression in Ga12 À/À BMMs. As shown in Figure 5D, osteoclastogenesis was not influenced by RhoCA overexpression. Furthermore, NFATc1 expression was not elevated, rather inhibited at a later stage, by RhoCA overexpression (Fig. 5F). However, the impaired resorption capacity of Ga12 À/À osteoclasts was partially restored by RhoCA overexpression (Fig. 5E). Taken together, these results indicate that Ga12 regulates BMM migration and bone resorption through RhoA activation while influencing osteoclast differentiation through NFATc1 up-regulation.

Discussion
By lCT analyses, we found increased trabecular bone volume in Ga12 À/À mice compared to that in WT mice. This observation suggests that Ga12 has a significant role in bone homeostasis. Histological experiments with femoral tissues revealed that Ga12 deficiency selectively affected osteoclast numbers with little effect on osteoblasts. In addition to, in vitro cultures also showed Ga12 deficiency impairs osteoclast differentiation. These results seem to result from the decreased expression of NFATc1, the master transcription factor of osteoclastogenesis.
A well-established downstream effector of Ga12 is monomeric GTPase RhoA [22]. RhoA regulates cytoskeletal dynamics, cell movement and divergent cellular functions. In osteoclasts, it has been reported that RhoA is crucial for osteoclast podosome organization, motility and bone resorption [24,25]. In our experiments, Ga12 À/À BMMs showed decreased RhoA activity, which caused a reduction in osteoclast precursor migration and bone resorption ( Fig. 5C and E). However, overexpression of the constitutively active RhoA could not recue impaired osteoclast formation by Ga12 deficiency (Fig. 5D). It implies that RhoA is more crucial for the precursor migration and resorption activity than for the differentiation in Ga12-mediated osteoclast control. Nonetheless, indirect contribution of RhoA to acceleration of osteoclast formation by stimulating recruitment of precursors to bone surface in vivo is still conceivable.
In investigations on the RANKL-induced signalling, we found that Ga12 deficiency increased Akt phosphorylation (Fig. 5A). RhoA has been reported to inhibit Akt [26], and the Akt-dependent GSK3b-NFATc1 pathway has been suggested to positively regulate osteoclastogenesis [27]. Therefore, one may expect that RhoA activation by  Ga12 leads to attenuation of NFATc1 expression. Indeed, we detected slightly down-regulated NFATc1 expression by RhoCA overexpression (Fig. 5F). However, Ga12 À/À cells consistently displayed elevated osteoclastogenesis and NFATc1 expression (Figs 2-4). Based on our findings and results shown in previous reports, we propose that RhoA activation by Ga12 increases cell mobility and bone resorption activity while negatively acting on NFATc1 via Akt inhibition. However, the direct positive effect of Ga12 on NFATc1 overrides the NFAT attenuation by RhoA, leading to enhanced osteoclastogenesis (Fig. 5G). Further studies are required to reveal the molecular mechanism for NFATc1 up-regulation by Ga12 in osteoclasts.
Through Ga transcript analysis, we found Gna12 expression is down-regulated during osteoclastogenesis, and it was verified by our real-time PCR analyses. Although the molecular mechanism of Ga12 suppression during osteoclastogenesis was not investigated in this study, we could find, using a program for prediction of transcription factor binding sites, several transcription factors that were supposed to bind the Ga12 promoter region. Among them, Pax6, a transcriptional repressor of which expression is increased during osteoclastogenesis [28] and Foxo1 and ATF3, transcriptional activator of which expression is decreased during osteoclastogenesis [29,30] may be attributed to the down-regulation of Ga12 during RANKL-induced osteoclastogenesis.
Although it is apparent that Ga12 modulates RANKL-induced osteoclastogenesis, whether Ga12 is activated directly through RANKL stimulation is not clear. Considering that Ga12 is activated by its binding to GPCR, Ga12 might be activated through some GPCR ligands secreted from osteoclasts during RANKL-induced osteoclastogenesis. More than 30 GPCRs, including P2Y receptor and sphingosine-1-phaophate receptor, have been reported to couple to either Ga12, Ga13 or both, and these GPCRs have various ligands or agonists [31]. In RANKL-induced osteoclastogenesis, the identification of the GPCR that acts as the main upstream mediator of Ga12 requires further study. Furthermore, the Ga12-mediated signalling pathway functionally interacts with not only Ga13, but also Ga q/11 -mediated signalling systems [18,32]. This complexity makes it difficult to distinguish contribution of each pathway from that of the others to the regulation of osteoclastogenesis.
In our histological analysis, no difference in osteoblast numbers between Ga12 À/À and WT femurs was observed. There have been some reports that suggest Ga12 activation by parathyroid hormone (PTH) in osteoblastic cells can regulate osteoclastogenesis. In UMR-106 osteoblastic cells, Ga12 was shown to mediate PTH-stimulated phospholipase D activation through Rho [33], and in turn, phospholipase D activates osteoclastogenesis through IL-6 production [34]. In the coculture of UMR-106 and RAW264.7 cells, active Ga12 reduced the ratio of RANKL/osteoprotegerin production from UMR-106 cells in response to PTH and inhibited the TRAP activity of RAW264.7 cells [35]. These in vitro studies proposed both positive and negative roles of Ga12 in osteoblasts to affect osteoclastogenesis. However, the in vivo implication of these studies is not clear. Considering that the crosstalk between osteoblasts and osteoclasts is a very important feature in bone physiology, studies with Ga12 À/À mice under various conditions, including ovariectomized or hormone-stimulated conditions, will be helpful for exploring the function of Ga12 in bone metabolism. Meanwhile, the roles of Ga12 in various human diseases have been recognized [36]. Our results suggest the possibility of involvement of Ga12 in the pathophysiology of bone diseases like osteoporosis or rheumatoid arthritis.
Here, for the first time, we demonstrated that the loss of Ga12 expression results in abnormally augmented trabecular bone volume, coincident with a decrease in osteoclast numbers. We also revealed that Ga12 regulates osteoclastogenesis via NFATc1 along with modulating osteoclast migration and resorption through RhoA activation.    5 Ga12 regulates precursor migration and bone resorption through mediating Rho activation by RANKL. (A) Bone marrow-derived macrophages (BMMs) were serum-starved for 4 hrs and then stimulated with 600 ng/ml RANKL. Stimulated cells were harvested after 0, 5, 15 and 30 min., and protein lysates were prepared. Western blot analysis was performed using antibodies against the phospho forms or total forms of the indicated proteins. (B) BMMs were serum-starved for 3 hrs, and proteins were harvested at 0 and 15 min. after RANKL stimulation. After GTP-Rho was precipitated, immunoblotting was performed using anti-Rho antibody. For a loading control, the Rhotekin Rho-binding domain used for pull-down was stained with Ponceau S after transfer to membranes. (C) Transwell migration assays were performed for 16 hrs using pcDNA3-EGFP-or RhoCAtransfected WT or Ga12 À/À BMMs. Migrated cells were stained with crystal violet and counted. (D) BMMs transfected with pcDNA3-EGFP-or RhoCA were cultured with M-CSF and RANKL for 6 days and TRAP+ MNC were counted (magnification 40x). (E) Transfected BMMs were cultured with M-CSF and RANKL on calcium phosphate-coated plates for 6 days. Remained calcium phosphate was stained by vonKossa, and resorption area is quantified with ImageJ software. (F) Transfected BMMs were cultured with M-CSF and RANKL for 3 days, and NFATc1 mRNA levels were analysed by real-time PCR. (G) Schematic diagram on the role of Ga12 for osteoclast differentiation and bone resorption. NS, not significant. Data are presented as means AE S.D.. *P < 0.05; **P < 0.005.