Results and Discussion MIP-1d Enhances Osteoclastogenesis in vitro Previously, we reported the first evidence that MIP-1d enhances RANKL-mediated OCL differentiation in vitro using murine bone marrow mononuclear cells (BM-MNC) [12]. Since BM- MNC is a heterogeneous population containing a minor fraction of OCL progenitors, it was unclear whether MIP-1d affected OCL differentiation via a direct effect on OCL progenitors or through indirect effects on other cells within the population (eg. marrow stromal cells). Thus, we examined the ability of MIP-1d to promote OCL differentiation in vitro using macrophage colony- stimulating factor (M-CSF)-dependent bone marrow macrophages (BMM), a refined population of committed OCL precursors. Consistent with our previous findings [12], while insufficient to stimulate OCL differentiation alone, MIP-1d significantly en- hanced OCL differentiation in response to RANKL, evidenced by increased numbers of TRAP-positive multinucleated cells (Fig. 1A). In addition, OCL formed in the presence of MIP-1d exhibited a significantly higher fusion index (Fig. 1B) compared to those treated with RANKL alone, highlighted by a greater than 7-fold increase in OCL containing more than 11 nuclei per cell (Fig. 1C, D). Notably, similar results were observed using the macrophage cell line RAW 264.7 (Fig. 1A, B, D), indicating that our findings using BMM were not likely affected by any minor stromal elements remaining within the BMM population. Taken together, these data suggest that MIP-1d enhances OCL formation via a direct effect on OCL precursors. MIP-1d Activates Key Pathways Regulating Osteoclastogenesis Prior studies identified several signaling pathways through which OCL differentiation is promoted, including ERK [13], JNK [14], PLCc2 [15], and NF-kB [16]. To investigate the mechanism whereby MIP-1d enhances OCL differentiation, we first deter- mined its ability to activate each of these signaling pathways in OCL precursors. Although exposure of BMM to MIP-1d did not induce phosphorylation of ERK or JNK, increased phosphoryla- tion of PLCc2 was observed, indicating activation of PLCc2 signaling (Fig. 2A). In addition, MIP-1d demonstrated an effect on both the canonical and non-canonical NF-kB signaling pathways. While activation of the canonical NF-kB pathway depends on phosphorylation and subsequent degradation of IkBs, largely IkBa, the non-canonical pathway depends on processing of p100 to p52 [17]. In addition, p50 traditionally partners with RelA or c- Rel in canonical signaling, while p52 partners with RelB in non- canonical signaling. Following exposure of BMM to MIP-1d, we observed phosphorylation and degradation of IkBa (Fig. 2A). Further, analysis of nuclear extracts revealed the translocation of p50 and its binding partners RelA and c-rel to the nucleus, indicating activation of canonical NF-kB signaling (Fig. 2B). It is also interesting to note that the kinetics of p50 and c-Rel nuclear translocation following stimulation with MIP-1d appear shorter than that of RelA. While this apparent shift in kinetics may be due to a number of events (eg. differences in NF-kB subunit degradation kinetics, nuclear translocation of p50 homodimers, etc.), this data suggests that MIP-1d may have a unique effect on NF-kB signaling, which may be of interest in subsequent studies. Examination of non-canonical NF-kB signaling revealed process- ing of p100 to p52 following stimulation with MIP-1d, however, nuclear translocation of the p52 binding partner RelB was not detected (Fig. 2C), suggesting that MIP-1d may favor activation of the canonical pathway. Lastly, using an oligonucleotide probe containing an NF-kB binding site, we observed increased NF-kB MIP-1d Activates NFATc1 via PLCc2 and NFkB activity in response to MIP-1d stimulation of BMM by EMSA (Fig. 2D). Collectively, these data demonstrate the ability of MIP- 1d to activate both PLCc2 and NF-kB signaling in OCL precursors, each having the potential to contribute to the effects of MIP-1d on OCL formation. MIP-1d Induces Critical Regulators of Osteoclastogenesis via activation of PLCc2 and NF-kB Recent studies suggest that the importance of the PLCc2 and NF-kB signaling pathways to osteoclastogenesis lies in their ability to stimulate expression and nuclear translocation of the transcrip- tion factor NFATc1, considered the ‘‘master regulator of OCL differentiation’’ [18,19]. NFATc1 auto-regulates its promoter together with c-fos, a component of the dimeric transcription factor AP-1, and controls expression of genes critical to OCL differentiation and function [20]. Because MIP-1d activated both the PLCc2 and NF-kB signaling pathways, we next examined its effect on expression and activity of c-fos and NFATc1. Treatment of BMM with MIP-1d significantly increased expression of c-fos (Fig. 3A) and, correspondingly, increased activation of AP-1 as determined by EMSA analysis (Fig. 3B). Moreover, similar to its effect on c-fos expression, MIP-1d significantly increased expres- sion (Fig. 3A) and translocation of NFATc1 to the nucleus (Fig. 3C). Correspondingly, increased mRNA expression of the NFATc1 downstream targets cathepsin K (CTSK), tartrate- resistant acid phosphatase (TRAP), and dendritic cell-specific transmembrane protein (DC-STAMP) was also observed (Fig. 3D). To determine the contribution of the NF-kB and PLCc2 signaling pathways to MIP-1d-induced expression of c-fos and NFATc1 we examined the effect of MIP-1d in the presence of chemical inhibitors of each pathway. Inhibition of both canonical and non- canonical NF-kB signaling using an IKK-2 inhibitor prevented c- fos and NFATc1 induction by MIP-1d (Fig. 3E). Similarly, exposure of BMM to the PLC inhibitor U73122 also inhibited the ability of MIP-1d to induce c-fos and NFATc1 expression (Fig. 3F). Taken together, these data indicate that MIP-1d induces the expression and activation of c-fos and NFATc1, critical for OCL differentiation, involving stimulation of both the PLCc2 and NF- kB pathways. It is interesting to note that although able to induce NFATc1 expression, MIP-1d was unable to induce OCL formation in the absence of RANKL ([12], data not shown). While seemingly incongruous, NFATc1 is reported to be necessary for OCL differentiation, yet insufficient in the absence of RANKL [21], indicating an additional effect of RANKL beyond the induction of NFATc1. Intriguingly, while both MIP-1d and RANKL activate NF-kB, MIP-1d appears to incompletely stimulate the non-canonical pathway, evidenced by the lack of RelB nuclear translocation (Fig. 2C). Given the importance of the non- canonical pathway for OCL differentiation [22], it is tempting to speculate that the limited ability of MIP-1d to stimulate this pathway contributes to its failure to induce OCL differentiation independently of RANKL. Despite a limited capacity to stimulate non-canonical NF-kB signaling, MIP-1d robustly activated the canonical pathway as evidenced by the nuclear translocation of RelA and c-Rel (Fig. 2B). Notably, canonical NF-kB signaling through induction of RelA has been reported to promote survival of OCL precursors, translating to increased OCL formation [23]. Thus, although unable to induce OCL differentiation alone, MIP-1d may enhance OCL formation not only by promoting differentiation via NFATc1 induction, but also by promoting survival of OCL progenitors via activation of RelA. PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40799