Sphingosylphosphorylcholine blocks ovariectomy‐induced bone loss by suppressing Ca2+/calmodulin‐mediated osteoclast differentiation

Osteoporosis is a disease in which bone mineral density decreases due to abnormal activity of osteoclasts, and is commonly found in post‐menopausal women who have decreased levels of female hormones. Sphingosylphosphorylcholine (SPC) is an important biological lipid that can be converted to sphingosine‐1‐phosphate (S1P) by autotaxin. S1P is known to be involved in osteoclast activation by stimulating osteoblasts, but bone regulation by SPC is not well understood. In this study, we found that SPC strongly inhibits RANKL‐induced osteoclast differentiation. SPC‐induced inhibitory effects on osteoclast differentiation were not affected by several antagonists of S1P receptors or pertussis toxin, suggesting cell surface receptor independency. However, SPC inhibited RANKL‐induced calcineurin activation and subsequent NFATc1 activity, leading to decrease of the expression of Trap and Ctsk. Moreover, we found that bone loss in an experimental osteoporosis mouse model was recovered by SPC injection. SPC also blocked ovariectomy‐induced body weight increase and Nfatc1 gene expression in mice. We also found that SPC inhibits RANKL‐induced osteoclast differentiation in human macrophages. Since currently available treatments for osteoporosis, such as administration of female hormones or hormone receptor modulators, show serious side effects, SPC has potential as a new agent for osteoporosis treatment.

resorption occurs abnormally due to the imbalance between bone cells, resulting in weakened and easily fractured bones. 3 Osteoporosis can be caused by certain diseases, drugs or natural ageing, and, in many cases, is found in post-menopausal women. 4,5 Therefore, oestrogen is used to treat osteoporosis, 6 but causes serious side effects. [7][8][9] Osteoclasts are involved in bone resorption and are differentiated from bone marrow-derived macrophages (BMDMs) by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL) stimulation. 10,11 RANKL activates TNF receptor associated factor 6 and mitogen-activated protein kinase (MAPK) signals through receptor activator of nuclear factor kappa-Β (RANK) and stimulates transcription factors such as nuclear factor-κB (NF-κB), activator protein 1 (AP-1) and c-fos to regulate the expression of proteins important for OC differentiation. [12][13][14] RANKL also regulates nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) activity through calmodulin, which mediates Ca 2+ signal activation. [13][14][15] NFATc1 is a master regulator of OC differentiation, which is autoamplified and regulates the expression of several OC-related proteins such as ATPase H+ transporting V0 subunit D2 (ATP6v0d2), dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), tartrate-resistant acid phosphatase (TRAP) or cathepsin K (CTSK). 15,16 Ceramides can be metabolized to sphingosine, which can be converted to sphingosine 1-phosphate (S1P) via a specific enzyme. 17 Extracellular S1P stimulates OBs to produce RANKL, which is reported to be involved in promoting OC differentiation. 18 A previous report demonstrated that sphingosine kinase 1 expression and activity are increased, resulting in increased S1P during RANKLinduced OC differentiation. 18 However, intracellular S1P produced by RANKL in OCs has been reported to inhibit OC differentiation. 18 As such, S1P is known to be involved in the OC and OB balance. 18 Sphingosylphosphorylcholine (SPC) is a membrane lipid that is converted to S1P by autotoxin. 17 SPC is a bioactive lipid mediator that acts in a variety of biological processes, including intracellular calcium increase, cell migration, cell growth, proliferation and differentiation. 17 SPC is known to share receptors with S1P and lysophosphatidic acid (LPA), and to activate intracellular signal transduction through pertussis toxin (PTX)-sensitive G i protein. 19,20 In addition to membrane receptors, SPC is known to act on the ryanodine receptor or bind to calmodulin to regulate intracellular Ca 2+ signalling. 21,22 Ca 2+ -binding calmodulin is involved in OC formation by regulating cAMP-response element binding protein (CREB) phosphorylation through Ca 2+ /calmodulin-dependent protein kinase (CaMK) activity or by regulating NFATc1 nuclear translocation via calcineurin (CaN) activity. 23 SPC has been reported to bind to calmodulin and regulates calmodulin downstream signals. 22 A previous report also demonstrated that calmodulin inhibitors block OC formation. 24 However, it remains to be elucidated whether SPC has an effect on OC generation by RANKL.
In this study, we find that SPC blocks OC differentiation by inhibiting CaN activity and shows beneficial effects in an ovariectomized (OVX)-osteoporosis mouse model. Based on these results, we propose SPC as a useful agent for the development of new therapeutics to control osteoporosis.
All animal experiments were performed in accordance with the guidelines of the Korean Food and Drug Administration. All experiments involving animals received the approval of the Institutional Review Committee for Animal Care and Use at Sungkyunkwan University (Suwon, Korea). Mouse BMDMs were differentiated into OCs using 30 ng/mL M-CSF and 100 ng/mL RANKL, as previously described. 25 Peripheral blood was collected from healthy donors. These experiments were approved by Ajou University Hospital's Institutional Review Board for ethics. Human monocytes were isolated from peripheral blood as previously described. 26 The human monocytes were differentiated into macrophages by adding 30 ng/ mL M-CSF for 3 days. Differentiated human macrophages were further differentiated into OCs by adding 30 ng/mL M-CSF and 100 ng/mL RANKL for 17 days. The differentiated OCs were stained for TRAP using an acid phosphatase leukocyte diagnostic kit, as described previously. 27 The TRAP + multinuclear cells (>3 nuclei) with a value > 3 were considered to be OCs.

| Cell viability assay
The cell viability assay was performed according to the manufacturer's instructions using the Cell Counting Kit-8 solution.

| RNA isolation and real-time qPCR
Total RNA from mouse and human OC was isolated using TRIzol reagent (Invitrogen), and cDNA was synthesized using the RT Premix Kit (iNtRON). Mouse femurs and tibiae were rapidly frozen in liquid nitrogen, ground in a mortar and used for RNA isolation using TRIzol reagent. The sequences of primers used include mouse Nfatc1-

| Nuclear/cytoplasmic fractionation
Mouse BMDMs were differentiated into preOCs (pOCs) using 30 ng/mL M-CSF and 100 ng/mL RANKL for 2 days. After changing culture medium to serum-free alpha-MEM for 4 hours, the cells were stimulated with 30 ng/mL M-CSF and 100 ng/mL RANKL for 2 hours in the absence or presence of SPC (10 μmol/L). Cells were fractionated using Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacturer's protocol.

| Measurement of CaN activity
Cellular CaN phosphatase activity was measured in cell extracts using a CaN cellular activity assay kit (Enzo Life Sciences). In brief, cells were lysed in a lysis buffer containing protease inhibitors and centrifuged. Proteins contained in the supernatant were used for the CaN activity assay using equal amounts (5 μg). Colorimetric measurements were performed at 620 nm.

| Isothermal titration calorimetry (ITC) analysis
Isothermal titration calorimetry studies were performed to determine the binding affinities between RANK and RANKL in the presence of SPC using a VP-ITC microcalorimeter (MicroCal). PBS

| Ovariectomy (OVX) mouse model and SPC injection
The OVX model is useful to see the effects of ovarian hormone action in menopause-associated osteoporosis. 28 For this purpose, control mice, which produce ovarian hormone normally, are needed. Eight-week-old C57BL/6 mice (female) were used for OVX, as previously reported. 28,29 Mice anaesthetized by inhalation anaesthesia had bilateral ovaries removed through the dorsal approach. SPC was injected three times a week via subcutaneous injection (s.c.) from day 1 post-operation. After 8 weeks, the mice were killed and the femurs separated and used in subsequent experiments.

| Microcomputed tomography (μCT) analysis of the distal femur
All mice were killed 8 weeks after surgery, and their bones were fixed in 4% paraformaldehyde solution. Bones were analysed using the SkyScan 1172 μCT scanner (70 kV, 141 μA, 6.92 pixel size; SkyScan). The data were analysed in 1-mm-thick areas of distal femurs, starting from 1 mm below the growth plate at thresholds of 75 minimum and 255 maximum. Bone parameters were calculated by a CT Analyzer program (version 1.7; SkyScan), and three-dimensional images were obtained by CT-volume software (version 1.11; SkyScan).

| Bone histology in OVX mice
All mice were killed 8 weeks after surgery, and their bones were fixed in 4% paraformaldehyde solution for 2 days and embedded in paraffin. For TRAP staining, bones were decalcified in 10% EDTA solution for 5 weeks, with changes of solution 3 times a week before embedding in paraffin. Bones were sectioned into 4 μm slices by a microtome and stained with haematoxylin and eosin for bone histopathological changes or for TRAP to determine osteoclast formation.

| Statistical analysis
The results were analysed with GraphPad prism software (GraphPad Software, Inc). Statistical analysis was performed using Student's t test. All results are expressed as the mean ± SE. A P-value < .05 was considered statistically significant.

| SPC inhibits RANKL-induced OC formation
Mouse BMDMs were differentiated into OCs by treatment with 30 ng/mL M-CSF and 100 ng/mL RANKL for 4 days, and differentiated OCs were identified by TRAP staining ( Figure 1A). To examine the effects of SPC on OC formation, we stimulated the BMDMs with SPC (10 μmol/L) in the presence of 30 ng/mL M-CSF and 100 ng/mL RANKL for 4 days. As shown in Figure 1A, SPC almost completely inhibited RANKL-induced OC formation. SPC-induced inhibitory effects on OC formation were concentration-dependent and showed significant effects at 1-10 μmol/L ( Figure 1B). We then investigated whether the inhibitory effects of SPC on RANKL-induced osteoclast formation were due to cell death in an in vitro culture system. As shown in Figure 1C, the viability of SPC-treated cells in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) was similar to that of vehicle (PBS)-treated cells in the presence of M-CSF and RANKL.
These results indicate that the inhibitory effects of SPC on RANKLinduced OC formation are not due to cell death.
The expression of many fusion-related genes is known to be increased during OC formation. 30,31 We investigated the effects of SPC on the expression of genes involved in cell fusion during OC formation. The expression of OC fusion-related genes (Atp6v0d2, Dc-stamp and Oc-stamp) was increased by RANKL treatment, and this increase was inhibited upon SPC treatment ( Figure 1D). These results suggest that SPC inhibits RANKL-induced OC formation through the regulation of RANKL-induced OC fusion-related gene expression.

| SPC-induced inhibitory effects on OC formation are not mediated by cell surface receptors
SPC is known to share receptors with S1P or LPA. [32][33][34] To investigate whether these receptors are involved in the inhibitory effects of SPC on RANKL-induced OC formation, several antagonists for S1P and LPA receptors were used. Mouse BMDMs were pre-incubated with these antagonists for 30 minutes according to previous reports, [35][36][37][38] prior to vehicle or SPC treatment in the

| SPC inhibits OC formation via inhibition of CaN activity
Since we found that the inhibitory effects of SPC on RANKLinduced OC formation are independent of cell surface receptors, we investigated whether SPC acts directly on intracellular modulators. According to previous reports, calmodulin is an endogenous intracellular SPC receptor. 22,41 Calmodulin is known to be involved in RANKL-induced OC formation through CREB phosphorylation or NFATc1 activation. 23  NFATc1 is known as a master regulator of osteoclastogenesis and is involved in OC-specific gene expression. 15,16,42 We investigated the effects of SPC on the expression of several OC-related genes (Nfatc1, Trap, and Ctsk) during RANKL-induced OC formation.
RANKL increased the expression of OC-specific genes; however, SPC strongly inhibited this increased gene expression ( Figure 3C).
These results suggest that SPC inhibits RANKL-induced OC formation by inhibiting OC-specific gene expression through inhibition of NFATc1 nuclear translocation and down-regulation of CaN activity.
In separate experiments, we also tested whether SPC inhibits RANKL-induced OC formation through direct binding with RANKL or RANK. Using ITC analysis, although we observed direct binding of RANK and RANKL, we found that SPC does not bind to RANK or RANKL directly ( Figure 3D).

| SPC administration shows beneficial effects on the OVX-induced osteoporosis mouse model
The OVX mouse model is often used as a post-menopausal osteoporosis mouse model. 43 Eight-week-old C57BL/6 female mice were divided into three groups: sham, OVX + vehicle and OVX + SPC (n = 8). Mice in the OVX group had their ovaries surgically removed and were injected with vehicle or SPC (2 mg/kg) three times a week for 8 weeks. As shown in Figure 4A, μCT analysis of mouse femurs showed that bone density of OVX mice was decreased compared to sham control mice, and this decrease was dramatically recovered upon SPC injection. Haematoxylin and eosin and TRAP staining analyses showed that OVX surgery elicited increased bone porosity and numbers of OCs in mouse femurs, which changes were markedly attenuated by SPC injection ( Figure 4A). OVX mice also showed decreased bone volume compared to trabecular volume (BV/TV), and also decreased trabecular numbers (Tb.N), which were recovered upon SPC injection ( Figure 4B). Trabecular separation (Tb.Sp) was also reduced by SPC injection ( Figure 4B). Ovariectomized mice have been reported to show increased body weight and decreased uterus size. 44 We also observed that OVX surgery causes an increase in body weight and decrease in uterus size ( Figure 4C). SPC administration blocked these effects ( Figure 4C). Femurs and tibiae isolated from sham, OVX and OVX + SPC mice were rapidly frozen with nitrogen, ground in a mortar and used for RNA isolation using TRIzol reagent. We found that the levels of Nfatc1 mRNA expression were increased in OVX mice, and these levels decreased upon SPC injection ( Figure 4D). These results suggest that SPC shows beneficial effects against the OVX-induced osteoporosis mouse model through regulation of OC differentiation and activation.

| SPC inhibits OC formation from human macrophages
To investigate the effects of SPC on human OC formation, we used human monocyte-derived macrophages. Human macrophages were obtained by treatment of primary human monocytes with 30 ng/ mL M-CSF for 3 days. Differentiated human macrophages were incubated with 30 ng/mL M-CSF and 100 ng/mL RANKL for 17 days to differentiate into OCs, which were identified by TRAP staining.
When 10 μmol/L SPC was administered under OC-forming conditions, RANKL-induced OC formation was inhibited by SPC treatment ( Figure 5A,B). RANKL-induced expression of OC-specific genes (NFATc1and CTSK) was also significantly inhibited by SPC treatment ( Figure 5C). These results indicate that SPC can inhibit OC formation in humans as well as in mice, suggesting a potential beneficial role of SPC on human osteoporosis.

| D ISCUSS I ON
SPC is an important bioactive lipid that mediates a variety of biological processes. 17 SPC can be converted to S1P, another important bioactive lipid, by autotoxin. 17 Previous studies have reported that S1P is involved in the regulation of bone formation and resorption in bone metabolism in addition to various cellular responses. 18 Although SPC has been reported to modulate the activity of pre-OB cell lines, 45 little has been reported about its effects on OCs. In this study, we investigated the effects of SPC on OC formation and the therapeutic effects of SPC in an osteoporosis mouse model. SPC inhibits RANKL-induced OC formation in a concentration-dependent manner (Figure 1). Although SPC is known to share receptors with S1P or LPA, antagonists for the S1P and LPA receptors showed no effects on the inhibitory effects of SPC on RANKL-induced OC formation (Figure 2A,C). SPC is also known to act through the PTX-sensitive G i protein, but PTX did not affect the SPC-induced inhibition of RANKL-induced OC formation ( Figure 2C). We also found  Osteoporosis is a bone disease commonly found in post-menopausal woman. Oestrogen is a representative female hormone mainly produced in ovaries and decreases with age or in post-menopausal woman. Oestrogen is known to induce the apoptosis of OCs involved in bone resorption, and thus, it has been reported that osteoporosis is alleviated by oestrogen treatment in an osteoporosis animal model. 46,47 For this reason, in many cases, ovarian resection is used as a model for post-menopause osteoporosis. Oestrogen is known to inhibit the accumulation of ceramides, and ceramide levels have been reported to be increased in older and post-menopausal woman. 48 Ceramides can be metabolized to S1P by ceramidase and S1P phosphatase, and S1P stimulates OBs to promote RANKL production and to promote OC differentiation. 17,18 OCs are known to produce autotoxin, 49 which can convert SPC to S1P. Based on previous literature, it is reasonable to assume that as autotoxin increases, the levels of SPC decrease, and eventually, osteoporosis will worsen as SPC decreases. In support of this hypothesis, in this study, we demonstrated that SPC shows beneficial effects against OVX-induced osteoporosis. SPC also reverses the effects of OVX on body weight and uterine size ( Figure 4C). To the best of our knowledge, there has been no report on the oestrogenic effects of SPC. Therefore, it is very difficult to explain how SPC reverses OVXinduced changes in body weight and uterine size. Detailed mechanisms of action of SPC on the OVX model should be examined in the future.
In conclusion, the results of this study show that SPC is effective in the treatment of mouse osteoporosis by inhibition of OC differentiation. Since SPC also showed inhibitory effects on human OC differentiation, we suggest that SPC can be used as a new therapeutic agent for the treatment of osteoporosis, which can avoid the side effects of currently used treatments such as oestrogen.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.  The data that support the findings of this study are available from the corresponding author upon reasonable request.