Bone-protective Functions of Netrin 1 Protein*

Netrin 1 was initially identified as an axon guidance factor, and recent studies indicate that it inhibits chemokine-directed monocyte migration. Despite its importance as a neuroimmune guidance cue, the role of netrin 1 in osteoclasts is largely unknown. Here we detected high netrin 1 levels in the synovial fluid of rheumatoid arthritis patients. Netrin 1 is potently expressed in osteoblasts and synovial fibroblasts, and IL-17 robustly enhances netrin 1 expression in these cells. The binding of netrin 1 to its receptor UNC5b on osteoclasts resulted in activation of SHP1, which inhibited VAV3 phosphorylation and RAC1 activation. This significantly impaired the actin polymerization and fusion, but not the differentiation of osteoclast. Strikingly, netrin 1 treatment prevented bone erosion in an autoimmune arthritis model and age-related bone destruction. Therefore, the netrin 1-UNC5b axis is a novel therapeutic target for bone-destructive diseases.

administered Denosumab, including infection, hypocalcemia, and osteonecrosis of the jaw (14). Regulating the inhibition of osteoclast multinucleation may circumvent these adverse effects. Although the movement of osteoclast precursors, facilitated by increased actin polymerization, is a critical factor in the multinucleation step of osteoclast formation (15), the soluble factor that regulates this process has yet to be identified.
Netrin 1 is an axon guidance cue that mediates the attraction and repulsion of neural axons by regulating cytoskeletal rearrangements (16 -19). The determinant of these variable functions is the expression of netrin 1 surface receptor UNC5b on neural cells. Generally, the expression of this receptor mediates the repulsion of neural axons, whereas expression of another netrin 1 receptor, DCC, results in neuronal attraction (20). It has been reported that netrin 1 also inhibits monocyte migra-tion (21,22) by modifying the cytoskeleton of myeloid cell types. Furthermore, a previous study indicated that netrin 1 is expressed in human osteoblastic cells (23). These observations prompted us to explore whether netrin 1 also regulates osteoclast multinucleation.

Netrin 1 Is Expressed in Osteoblasts and Synovial Fibro-
blasts-To identify the netrin 1-producing cell population in the osteoimmune system, we examined netrin 1 expression in various hematopoietic and mesenchymal cell types. In mammals, three secreted netrins, netrin 1, netrin 3, and netrin 4, and two glycosylphosphatidylinositol-linked netrins, netrin G1 and netrin G2, have been reported (Fig. 1A). We found that netrin 1 is highly expressed in osteoblasts and synovial fibroblasts (Fig. 1B) but is barely detectable in T cells, B cells, neutrophils, dendritic cells, macrophages, and osteoclasts (Fig. 1C). When osteoblasts were stimulated with various cytokines, IL-17 was the most potent inducer of netrin 1 expression among the cytokines typically elevated in RA (Fig. 1, D and E). Furthermore, IL-17-induced netrin 1 expression in osteoblasts was NF-Bdependent (Fig. 1F).
To extend our findings to human RA, we examined the synovial fluid of RA patients (Fig. 2, A and B) and found that, when compared with the synovial fluid from osteoarthritis (OA) patients, it contained significantly higher levels of IL-17 and netrin 1 (Fig. 2B). We also found a significant correlation between the concentrations of IL-17 and IFN-␥ in the synovial fluid of RA but not OA patients (Fig. 2, C and D). In RA synovial fluid, there was correlation between netrin 1 and the IL-17 concentration (Fig. 2D). These data suggest that netrin 1 is induced in RA joints in an IL-17-dependent manner.
Netrin 1 Inhibits Osteoclast Multinucleation-In RA patients with high netrin 1 concentrations, the levels of the bone destruction marker human type 1 collagen cross-linked C-terminal telopeptide (CTXI) were relatively low (Fig. 2E). In the synovial fluid of OA patients, the netrin 1 concentration correlated negatively with that of CTXI (Fig. 2F), whereas in the synovial fluid of these patients and those with RA, there was no correlation between CTXI and IL-17 concentration (Fig. 2G). These results prompted us to explore the in vivo role of netrin 1 during osteoclast formation. First, because netrin 1 deficiency is lethal, we analyzed the bones of netrin 1 gene knock-out mice beginning on embryonic day 19. Histological analysis of the metaphyseal portion of the tibia showed that these netrin 1 gene-deficient mice had decreased trabecular bone accompanied by marked increases in osteoclast number and nuclei (Fig.  3, A-C). In contrast, adult mice heterozygous for netrin 1 gene exhibited normal bone volumes and osteoclast parameters (Fig.  3, D-F). We then examined the in vitro effect of netrin 1 on osteoclastogenesis and found that, in the presence of RANKL, netrin 1 inhibited osteoclast multinucleation (Fig. 3, G-I).
Other than netrin 1, osteoblasts produce osteoclast inhibitory axon guidance factors such as Sema3A (24), EphB4 (25), and netrin 4 (26). A comparison of these axon guidance factors showed that netrin 1 was the most potent inhibitor of osteoclastogenesis (Fig. 3J). Both apoptosis and TRAP activity were normal in netrin 1-stimulated osteoclasts (Fig. 3, K and L), but these cells had significantly fewer resorption pits than untreated cells (Fig. 3, M and N). The netrin 1-related decrease in the number of resorption pits in the netrin 1-treated osteoclasts correlated with the reduction in multinucleated cells, but the areas of the pits/osteoclast areas in these and the control cells were similar (Fig. 3, M and N). Thus, although netrin 1-treated osteoclasts are functionally normal, a fusion defect in these cells results in fewer resorption pits. Osteoblast-derived axon guidance factors such as Sema3A (24) and EphB4 (25) are local mediators of osteoclast and osteoblast functions and, as bidirectional regulators, may contribute to bone remodeling. We therefore explored whether netrin 1, as an osteoblast-derived axon guidance factor, had an effect on osteoblastogenesis (Fig. 4, A-E). In calvarial osteoblasts cultured with increasing netrin 1 concentrations, there was no effect on bone nodule formation or the expression of osteoblast differentiation markers such as ALP, Runx2, RANKL, and osteoprotegerin (OPG) (Fig. 4, A-E). We also examined the expression levels of osteoclast-derived osteoblast inducers ("clastokines") in netrin 1-treated osteoclasts and found an induction of bone morphogenetic protein (BMP)-2 expression (Fig. 4F). Thus, to test the hypothesis that netrin 1-induced BMP-2 released from osteoclasts influences osteoblastogenesis, calvarial cells were cultured in a concentrated culture supernatant from netrin 1-stimulated osteoclasts. This osteoclast medium (OM) significantly enhanced bone formation and mineralization when compared with OM from osteoclasts not treated with netrin 1 (Fig. 4, G and H). Additionally, BMP-2 neutralizing antibody attenuated the osteogenic effect of OM from netrin 1-treated osteoclasts (Fig. 4, G and H). To gain insight into the transcriptional regulation of BMP-2 by netrin 1, we examined the occupancy of the BMP-2 promoter by CREB, a pivotal BMP-2 transcription factor (27). Indeed, CREB binding to the BMP2 promoter was increased after netrin 1 stimulation (Fig. 4I). In CREB knockdown osteoclasts, however, netrin 1-induced BMP-2 expression was abolished (Fig. 4J). These data indicate that the netrin 1-CREB axis in osteoclasts induces BMP-2 expression, which in turn stimulates bone formation. Taken together, our findings suggest that netrin 1 does not affect osteoblast differentiation directly, but it may stimulate bone formation by a mechanism involving osteoclasts.
Netrin 1 Inhibits VAV3-RAC1 Signaling via the UNC5b Receptor-To gain further insight into the molecular mechanism of the netrin 1-induced inhibition of osteoclast formation, we analyzed netrin 1 signaling and its cross-talk with RANKL signaling. The possibility that the altered expression of RANK and Csf1R (colony-stimulating factor 1 receptor) is responsible for the inhibition of multinucleation in netrin 1-treated osteoclasts was excluded by the finding that their levels were similar after netrin 1 or PBS treatment (Fig. 5A). Because neural cells contain five netrin 1 surface receptors (UNC5a, UNC5b, DCC, Neo1, and Adora2b), we examined receptor expression on osteoclasts. Both UNC5b and Adora2b were detected at high levels ( Fig. 5B). UNC5b knockdown MDMs gave rise to significantly more and larger osteoclasts than control shRNA-transduced osteoclasts (Fig. 5, C and D), whereas Adora2b knockdown had no effect on netrin 1-induced osteoclast inhibition (data not shown). In co-cultures of UNC5b knockdown MDMs and calvarial osteoblasts, osteoclast numbers were slightly increased after 7 days (Fig. 5E). These data indicated that UNC5b is a bona fide netrin 1 receptor in osteoclasts.
To evaluate the effects of netrin 1 on RANKL-induced osteoclast differentiation, the expression of various osteoclastogenic genes was examined by quantitative real-time PCR (qPCR) (Fig. 5F). At every time point, expression of the genes encoding NFATc1, c-Fos, Jdp2, TRAF6, TRAP, DC-STAMP, ␤3-integrin, and ATP6v0d2 was similar in netrin 1-treated and PBS-treated osteoclasts (Fig. 5F). In a study measuring the activation of NFATc1 in response to RANKL, we found that netrin 1 treatment did not alter the binding of NFATc1 to their target promoter regions (Fig. 5G). Netrin 1 also had no effect on calcium oscillations induced by RANKL (Fig. 5H). These observations suggest that osteoclast multinucleation, but not osteoclast differentiation, is suppressed by netrin 1.
Because the formation of actin belts (Fig. 6A) and actin rings (Fig. 6, B and C) was dramatically impaired in the netrin 1-treated osteoclasts, we next examined the role of netrin 1 in cytoskeletal rearrangement during osteoclast formation. Because actin dynamism is predominantly regulated by the Rho GTPase RAC1 and the adhesion kinase FAK, we first quantified the activated GTP-bound form of RAC1 and phosphorylated FAK (Fig. 6, D and E). The results showed a dramatic inhibition of the RANKL-induced activation of RAC1 (Fig. 6D) and FAK (Fig. 6E) by netrin 1 treatment. In addition, constitutively active RAC1 expression partially rescued the osteoclast multinucle-ation defect in netrin 1-treated cells (Fig. 6, F and G). Collectively, these data showed that impaired RAC1 activation is responsible for the fusion defect in netrin 1-treated osteoclasts. Next, given the role of VAV3, a Rho GTPase nucleotide exchanger, in osteoclast multinucleation and RAC1 activation (15), we measured the levels of phosphorylated VAV3 in netrin 1-treated osteoclasts (Fig. 6H). In response to RANKL, VAV3 phosphorylation was significantly impaired by netrin 1 (Fig.  6H). Furthermore, the RANKL-induced phosphorylation of SHP1, a phosphatase of the VAV family, was significantly enhanced in netrin 1-treated MDMs (Fig. 6I). Further, the knockdown of SHP1 in MDMs significantly rescued the osteoclast inhibitory effect of netrin 1 (Fig. 6, J and K).
Netrin 1 Prevents Bone Destruction in Arthritis-Based on our finding that netrin 1 is a potent inhibitor of osteoclast multinucleation, we explored the therapeutic potential of recombinant netrin 1 in CIA. Type II collagen was injected into 6-week-old mice, followed by a booster immunization 3 weeks later. The mice were then intravenously injected with netrin 1 or PBS (Fig. 7A). Netrin 1 failed to reduce the clinical scores, hind paw thicknesses, or serum proinflammatory cytokine levels of CIA mice (Fig. 7, B, C, and F), but it did significantly lower  the amount of bone erosion (Fig. 7D). The histological scores for inflammation in the hind paw were similar in netrin 1-treated and PBS-treated mice, but there were significantly fewer osteoclasts in the former (Fig. 7E). Finally, bone destruction was evaluated in a mouse model of CAIA using adult mice heterozygous for netrin 1 (Fig. 7, G-J). The clinical scores of netrin 1 heterozygous mice were normal (Fig. 7H), but the amount of bone erosion size was significantly higher than in the controls (Fig. 7, I and J). Although the histological scores for hind paw inflammation in the netrin 1 heterozygous mice were also normal, the number of osteoclasts in the calcaneus was increased (Fig. 7J). These data clearly demonstrated that netrin 1 protected the mice against autoimmune bone destruction in vivo.
Netrin 1 Increases Bone Mass-Age-related decreases in the serum netrin 1 concentration of the mice were determined (Fig.  8A). Netrin 1 expression was impaired in the bone marrow cells and mesenchymal stem cell (MSC)-derived osteoblasts of older versus younger mice (Fig. 8B). To gain insight into the mechanism underlying the age-dependent down-regulation of netrin 1, we measured the expression level of p53, a critical positive inducer of netrin 1 transcription (31), in the MSC-derived osteoblasts from mice of various ages. Both the expression and the binding of p53 to the netrin 1 promoter were impaired in MSC-derived osteoblasts from older versus younger mice (Fig.  8, C and D). Notably, netrin 1 was significantly lower in p53 knockdown osteoblasts (Fig. 8E), suggesting that abnormal p53 expression causes a decrease in netrin 1 expression in older mice.
To determine the impact of exogenous netrin 1 on the physiological condition of bone metabolism, 4-week-old (Fig. 8F) and 20-week-old (Fig. 8L) mice were intravenously injected with netrin 1 or PBS. Micro-computed tomography (CT) and histological analysis of these mice showed an increase in bone volume (Fig. 8, G, H, M, and N). In addition, histomorphometric analysis revealed significant reductions in osteoclast parameters and increase in bone formation parameters (Fig, 8, I, J, and O). The normal expression of TRAP in the bone marrow cells of netrin 1-treated mice was also confirmed, whereas BMP-2 expression was increased (Fig. 8K). These findings suggest that netrin 1 induces BMP-2 and stimulates bone formation in vivo.

Discussion
Accumulating evidence suggests that the proinflammatory cytokines such as IL-17 and TNF-␣ are also potent stimulators of osteoclastogenesis. Although the contribution of osteoblasts and synovial fibroblasts to the pathogenesis of inflammatory bone destruction has been extensively characterized, bone-protective factors produced from these cells during inflammation have been largely overlooked. In this study, we identified netrin 1 as an osteoblast-or synovial fibroblast-derived soluble pro- tein that limits osteoclast multinucleation. Among the netrin family proteins, only netrin 1 is strongly expressed by osteoblasts and synovial fibroblasts, and its expression is dramatically enhanced by IL-17. To our knowledge, our study is the first to highlight that osteoblasts and synovial fibroblasts rapidly release humoral factors that protect against excessive bone destruction during acute inflammation. Importantly, in the synovial fluid of OA patients, netrin 1 concentration correlated negatively with that of CTXI. This observation indicates that netrin 1 may also be a negative regulator of bone destruction in non-inflammatory conditions. Furthermore, netrin 1 treatment potently suppressed the bone erosion associated with experimental arthritis in mice, suggesting the prophylactic potential of netrin 1 in Th17-associated bone disease in humans. In this study, we also found that the osteoclast inhibitory function of netrin 1 is the strongest among the axon guidance cues released from osteoblasts. Previous studies showed 50% inhibition of osteoclast differentiation by 2 g of "clustered" EphB4/ml (25) and 1 g of Sema3A/ml, although when Sema3A is added after RANKL treatment, the inhibitory effect is not observed (24,32). By contrast, our results showed that 1 g of netrin 1/ml inhibited osteoclast multinucleation by Ͼ95%, even when it was added after RANKL treatment. Netrin 1 is a member of the laminin-related family of matrix-binding proteins, which contain domain VI, three EGF-like repeats, and a heparin-binding domain. Therefore, netrin 1 most likely binds the extracellular matrix, which in turn regulates its local concentration and tissue bioavailability. Consequently, the local concentration of netrin 1 is likely to be very high around osteoblasts and synovial cells, both of which secrete various NOVEMBER 11, 2016 • VOLUME 291 • NUMBER 46 extracellular matrix components. Nevertheless, further investigation is needed to understand the in vivo bioavailability of netrin 1.

Role of Netrin 1 in Bone Homeostasis
Our study is also the first to show that netrin 1 is a strong inhibitor of osteoclast fusion. The netrin 1 receptor UNC5b is potently expressed in osteoclasts. Netrin 1 binding to UNC5b causes the activation of SHP1, which, by suppressing VAV3 and RAC1 activation, leads to impaired osteoclast fusion. SHP1 is a phosphatase, and a reduction in its activity increases osteoclast multinucleation (33). In netrin 1-treated osteoclasts, the expression of ITIM-harboring receptors such as PIR-B and SIRP␣ was increased. Because this group of receptors activates SHP-1, it may be a target of UNC5b signaling. Further studies are required to understand the mechanism by which netrin 1 inhibits osteoclast fusion, but our data clearly indicate that VAV3-RAC1 signaling is inhibited by the netrin 1-UNC5b axis.
A recent study showed that netrin 1 is expressed in murine osteoclasts and stimulates their differentiation in an autocrine fashion (34). However, when we compared these results with osteoblasts and synovial fibroblasts, we found that netrin 1 is barely expressed in osteoclasts at both the mRNA and the protein levels. Significantly, osteoblasts and synovial fibroblasts exhibited more than 200-fold higher expression of netrin 1. Importantly, this comparison of netrin 1 expression between different cell types has not been done previously. Thus, we believe that the major source of netrin 1 inhibiting multinucleation of osteoclasts derives from osteoblasts. A previous study also showed enhanced in vitro osteoclastogenesis in netrin 1 knockdown osteoclasts (34). The lack of netrin 1 in osteoclasts may affect the signaling that regulates the intracellular differentiation of these cells (34). However, no enhancement of RANKL-induced osteoclastogenesis by netrin 1 knockdown in MDMs was observed in our experiment (data not shown). Strikingly, these previous studies also showed that 250 ng/ml recombinant netrin 1 (R&D Systems) significantly enhances (1.2-fold) the RANKL-induced osteoclast numbers. Discrepancies between our findings and their study may be explained by the differences between culture conditions. To generate osteoclasts, they changed media every 3 days, and after 7 days, they analyzed the number of osteoclasts. We did not change the culture media and performed analysis at days 3-5. Under our protocol, most of the osteoclasts underwent apoptosis at day 6. Because netrin 1 injection has a bone-protective function in vivo, we believe that the in vitro culture methods in Ref. 34 do not reflect the in vivo prophylactic potential of netrin 1. A previous study also showed a slight increase in the bone volume of wild-type mice engrafted with netrin 1 knock-out fetal liver cells (34). Because netrin 1 mediates adhesion of immune cells and promotes chemotaxis of CXCL12 (35), such results may reflect the difference of the stem cell trafficking to the bone marrow cavity. Generation of osteoclast/osteoblast-specific conditional netrin 1 knock-out mice will contribute to clarifying the role of netrin 1 in osteoclasts.
Previous work also suggested that the administration of netrin 1 inhibits inflammation in several models of animal disease (36,37), but in our CIA mice, an anti-inflammatory effect of netrin 1 was not detected. Indeed, netrin 1 may not always exhibit anti-inflammatory activity, and contradictory results have been reported. For instance, plasma IL-6 levels were 50% lower in netrin 1 transgenic mice than in wild-type mice (38).
Another study (39) demonstrated that although interference with netrin 1 levels does not affect inflammation, it does prevent tumor progression in a mouse model of inflammatory bowel disease-associated colorectal cancer. However, in two studies, netrin 1 promoted atherosclerosis and inflammation by inhibiting macrophage movement (40,41). In our models of bone destruction (CIA and CAIA), netrin 1 did not significantly modulate inflammation. Thus, our data collectively suggest that netrin 1 does not modulate arthritic inflammation; rather, it may modulate inflammation only under certain conditions, but not in the context of bone-destructive diseases. The potential anti-inflammatory function of netrin 1 remains to be evaluated in further experiments.
We also found that serum netrin 1 concentrations decline during aging and that the administration of netrin 1 to healthy mice inhibited osteoclast fusion, thus increasing their bone volume. Although netrin 1 had no effect on osteoblastogenesis in vitro, it increased the bone formation rate in vivo. Bone remodeling in DC-STAMP (10) and ATP6v0d2 (9) gene knock-out mice is uncoupled, as osteoclast parameters are reduced, whereas bone formation rates are increased. In cells from these mice, in vitro osteoclast fusion was significantly impaired, whereas in vitro osteoblastogenesis was normal, suggesting that the former stimulates bone formation in vivo. Our in vitro study indicated that netrin 1 released by osteoblasts induces the CREB-dependent expression of BMP-2 from osteoclasts, which stimulates osteoblastogenesis. This mechanism was confirmed in vivo, because BMP-2 expression was increased in the femurs of netrin 1-injected mice. According to these findings, the administration of netrin 1 stimulates bone formation via osteoclast-derived BMP-2. These observations demonstrate the potential of netrin 1 as a prophylactic agent in osteoporosis and in a broad range of other bone-destructive diseases.
Although to date, studies of netrin 1 have focused on its neurobiological role, our results clearly show that netrin 1 suppresses osteoclast fusion, both in vivo and in vitro. Further studies of netrin 1, including netrin 1-UNC5b signaling in osteoclasts, together with the generation of conditional netrin 1 gene knock-out mice, will contribute to clarifying the role of netrin 1 in bone metabolism.

Experimental Procedures
Mice-Female C57BL/6 mice and female DBA/1J mice were purchased from CLEA Japan and Japan SLC, respectively. ICR or C57BL/6 background female mice heterozygous for a netrin 1 mutation (16) were crossed to obtain homozygous embryos. All animal experiments were performed with the approval of the Animal Research Committee of the Research Institute for Microbial Diseases, Osaka University.
Cells and Reagents-Synovial fibroblasts were harvested as described previously (42). Briefly, the articular capsules of the ankle joints isolated from the hind paw of mice with CIA were treated with 1 mg of collagenase/ml. The resulting suspension was filtered through a 40-m cell strainer. The filtered cells were cultured in DMEM supplemented with 10% FCS and 50 mg of L-glutamine/ml for 24 h and then washed with PBS. Adherent cells from passage 3 were used as synovial fibroblasts. B cells and T cells were collected from the spleen by positive selection using anti-B220 and anti-Thy-1.2 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. Splenic CD11b ϩ macrophages were sorted using a FACSAria flow cytometer (BD Biosciences). Dendritic cells were isolated by positive selection using anti-CD11c magnetic beads (Miltenyi Biotec). Th1, Th2, and Th17 cells were generated from CD4 ϩ cells isolated from splenocytes using anti-CD4 magnetic beads (Miltenyi Biotec), as described previously (43). Recombinant murine M-CSF, IL-17A, IL-4, IL-6, IL-12, and IFN-␥ were purchased from PeproTech. Recombinant RANKL, netrin 1, netrin 4, Sema3A-Fc, and EphB4-Fc were purchased from R&D Systems. TNF and IL-6 ELISA kits were purchased from R&D Systems. ELISA kits were used for analyses of mouse CTXI (USCN Life Science), human CTXI (MBS454442, MyBio-Source), mouse netrin 1 (E91827Mu (USCN Life Science), human netrin 1 (E91827Hu, USCN Life Science), human IL-17A (Diaclone SAS), and human IFN-␥ (KHC4021, Invitrogen). The ALP quantification kit (LabAssay) was purchased from Wako (Tokyo, Japan). Nuclear extracts were prepared as described (44). The DNA binding activities of NFATc1 was quantified using a TransAM transcription factor assay (Active Motif, Carlsbad, CA). The histone-DNA fragment in the culture supernatant was quantified using a cell death detection ELISA (Roche Applied Science). The NF-B inhibitor BAY117085 was from R&D Systems. OM was prepared from MDMs cultured with 40 ng of netrin 1/ml and/or 100 ng of RANKL/ml for 4 days, after which the culture medium was concentrated 10-fold using an Amicon Centricon Plus-70 concentrator. Antibodies for FACS analysis were from BD Biosciences. Data were collected using a FACSCalibur (BD Biosciences) and analyzed by FlowJo (Ashland, OR).
Osteoclast/Osteoblast Culture-MDMs were prepared as described (45). Osteoclasts were generated by incubating the cells with 25 ng of M-CSF/ml and various concentrations of RANKL. TRAP staining was performed as described (46). For the pit assay, MDMs were plated on dentine slices with RANKL. After 5 days, the cells were immersed for 3 h in 1 M NH 4 OH, after which pit numbers were counted. Calvariae from 2-day-old mice were collected as described previously (46). Mesenchymal stem cells from compact bone were prepared as described previously (47). Osteoblastogenesis was induced by culturing the cells with an osteoblast-inducing reagent (Takara). ALP and calcified nodules were stained using TRAP/ ALP (Wako) and calcified nodule (AK-21; Primary Cell Co. Ltd., Hokkaido, Japan) staining kits, respectively. The calcium concentration was quantified using the Metallo Assay LS-MPR kit (AKJ Global Technology). For osteoclast/osteoblast co-culture, MDMs (6 ϫ 10 5 ) and calvarial cells (5 ϫ 10 5 ) were cultured in 24-well plates containing ␣-minimum essential medium supplemented with 30 nM 1␣,25(OH) 2 D 3 plus prostaglandin E 2 (PGE 2 ).
PCR Analysis-RNA was extracted using TRIzol Reagent (Invitrogen Life Science Technologies). cDNA was generated using ReverTra Ace (Toyobo Co., Ltd, Japan). qPCR was performed with an ABI PRISM 7500 Real-Time PCR System using TaqMan Assays-on-Demand primers (NFATc1 (Mm00479445_ Viral Gene Transfer/Knockdown-Retroviral constructs for constitutively active RAC1 (pM-RAC1) (49) and the retroviral particles (50) were generated as described previously. Control virus particles were generated using the empty pMIEG3 plasmid. Cells were transfected with the retroviral gene as described elsewhere (51). The transfectants were resuspended in DME medium containing 10% FCS and supplemented with 25 ng of M-CSF/ml. The medium was refreshed after 3 days, and the cells were stimulated with 60 ng of RANKL/ml plus 25 ng of M-CSF/ml to induce osteoclastogenesis. shRNA lentivirus of netrin 1 (sc-42045-V), UNC5b (sc-61847-V), SHP1 (sc-29479-V), CREB (sc-35111), p53 (sc-29436), and control shRNA lentiviral particles (sc-108080) were purchased from Santa Cruz Biotechnology. For lentiviral gene transfer, the virus in 2 g of Polybrene/ml was added to cells. After 10 h, the cells were washed and cultured for 30 h with 2 g of puromycin/ml, and the puromycin-resistant cells were identified.
Analysis of the Bone Phenotype-The bone formation rate was quantified by double calcein labeling (25). The bones were fixed in 70% ethanol, and the hind paws and femurs were analyzed by three-dimensional CT using a Scan-Xmate RB080SS110 (Comscan Techno Co., Ltd, Sagamihara, Japan) and the TRI/3D-Bon software (Ratoc System Engineering Co., Ltd, Tokyo, Japan). Bone microarchitectural parameters in the trabecular regions were quantified in an area 0.1-1.5 mm from the chondro-osseous junction. Bone histomorphometric analysis was carried out in tibias stained with Villanueva bone stain (Wako) and embedded in methyl methacrylate. Serial longitudinal sections (6-mm-thick) were prepared using a microtome (RM2255, Leica) and analyzed with a Histometry RT camera (System Supply Co., Ltd, Nagano, Japan). Hind paw sections were stained with TRAP or H&E. Histological scores were graded from 0 to 4 based on a previously described scoring system (52).
Induction of Arthritis-Bovine type II collagen and complete Freund's adjuvant were purchased from Chondrex. To induce CIA in DBA/1J mice, the mice were immunized according to the manufacturer's protocol, using an emulsion of these two ingredients. To induce CAIA, 1.5 mg of Arthrogen-CIA arthritogenic monoclonal antibody (53040, Chondrex) and 25 mg of LPS were injected intraperitoneally into DBA/1J mice. The mice were then observed daily for signs of joint inflammation and scored for clinical signs as described previously (53). Briefly, joint inflammation was scored from 0 to 4, with 4 being the maximum arthritis score per paw, and 16 being the maximum score per mouse.
Synovial Fluid Collection-Synovial fluids from RA and OA patients (diagnosed according to the criteria for RA and OA of the American College of Rheumatology) were collected and analyzed by ELISA. All patients were Ͼ18 years of age. The inclusion criteria for patients with OA were the presence of osteophytes and joint space narrowing on an X-ray of Kellgren-Lawrence grade Ն2. For RA patients, they were: fulfillment of the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria for RA or the 1987 ACR criteria for RA. Juveniles were excluded from the study. The mean (ϮS.E.) ages of the OA and RA patients were 75.1 Ϯ 6.1 and 65 Ϯ 12.8 years, respectively. The experiments were performed with the approval of the Ethics Committee of the Kyoto University Graduate School of Medicine.
Statistical Analysis-Student's t test was used to evaluate the significance of the differences, indicated by a p level Ͻ 0.05.