Figure 4. Activation of ERK1/2 and IkBa by RANKL and M-CSF in mature OCs. Mature OCs were serum starved for 18 h then acutely treated with RANKL (125 ng/mL) or M-CSF (25 ng/mL) alone, or in combination. Cytoplasmic protein extracts were harvested and subjected to SDS-PAGE followed by western analysis utilising antibodies against total and phosphorylated ERK1/2 and IkBa. A. Augmentation of phospho-ERK1/2 in the presence of RANKL and M-CSF. B. M-CSF has no effect on phospho-IkBa; representative data from 2 independent experiments. doi:10.1371/journal.pone.0021462.g004 addition to its roles in survival and motility. It is established that RANKL rapidly activates resorption in isolated mouse OCs [27], increasing actin-ring formation within 30 minutes. Furthermore, treatment of mice with intravenous RANKL increases ionized calcium at 1 h, consistent with acute activation of mature OCs [27]. In our system, exposure to RANKL alone initiates, within 4 h, the formation of resorption pits that progressively enlarge with time, while the addition of M-CSF results in a rapid increase in the relative number of pits, which are not larger or longer, indicating a fundamental difference in the mechanism. The rapidity of this effect suggests that it is not due to an effect on OC differentiation but, rather, activation of mature cells. Pits progressively accumu- late so the increased number could be due to an increase in the proportion of OCs activated to form pits, or an increase in the activation frequency among the same proportion of the OCs. However, since the presence of an actin ring indicates current OC activation [28], the observed increase in sealing zone number confirms that the increased pit number is due to a greater proportion of activated OCs. Functional RANK is expressed on mature OCs and RANKL stimulates their activation [27,29,30]. Since resorption is RANKL-dependent, we surmised that augmentation of RANK signalling by M-CSF was a possible mechanism of its stimulation of OC activation. In OC precursors, a number of signalling cascades are known to be activated downstream of RANK leading to activation of NFkB, AP-1, p38, ERK, Src and NFAT (reviewed by Boyle et al. [12]). However, most of this knowledge is based on investigations done in rodent osteoclast generation models and the signalling pathways involved in the activation of mature OCs have not been well characterised. Likewise, signalling downstream of c-fms, the tyrosine kinase receptor of M-CSF is not well characterised in mature OCs although the important roles of Src kinase in resorption [31,32], PI-3-kinase in motility [33] and PI-3-kinase as well as MEK 1 in survival [34] are well established. Therefore, we investigated the effects of acute treatment of OCs with RANKL on activation of NFkB, c- M-CSF Augments Resorption by Human Osteoclasts fos and NFATc1, and the modulation of this by M-CSF. In fully differentiated OCs under these experimental conditions, RANKL activation of NFkB is independent of M-CSF. However, RANKL activation of c-fos is dependent on the presence of M-CSF. NFATc1 appears to have no role in RANKL-induced activation of differentiated OCs. These results are consistent with the results of our resorption assays where RANKL initiates sub-maximal resorption activity independent of M-CSF while M-CSF augments RANKL-induced resorption, the former mediated by NFkB and the latter by c-fos. Yao et al. [35] have previously shown that M-CSF treatment of mature mouse OCs results in increased c-fos expression and transcriptional activation [35]. The key role of c-fos in OC differentiation is well established [36,37]. Our results indicate that in mature OCs c-fos activation is not essential for the initiation of resorption but that its activation by M-CSF greatly augments RANKL-induced activation of resorption. Moreover, although NFATc1 is critical in osteoclast differentiation it appears to play no part in resorption activation of fully differentiated OCs. Lee and Kim [26] have previously highlighted that existing data show that signalling pathways stimulated by RANKL are different in OC precursors and mature OCs [26]. Miyazaki et al. [24] used adenoviral gene transfer methods to activate and inhibit ERK and NFkB in semi-pure mouse OC cultures. Activation of ERK increased OC survival and inhibition of ERK decreased survival but neither had any effect on their bone- resorbing activity. In contrast, activation and inhibition of NFkB increased and decreased OC resorbing activity, respectively, but had no effect on survival [24]. We have shown that in fully differentiated human OCs, functional MEK/ERK pathways are not essential for the pro-survival and pro-resorption effects of RANKL but these pathways are involved in M-CSF augmentation of RANKL-induced resorption activation. The involvement of ERK is consistent with the involvement of c-fos activation that we have demonstrated. The results do not exclude the possibility that other MAPK parallel cascades, such as JNK and p38 are also involved [25]. Our novel finding that M-CSF regulates the rate of osteoclastic bone resorption in vitro, independent of OC formation, is supported by data from two murine in vivo studies. The first investigated the capacity of cell-surface M-CSF (csCSF-1) to restore in vivo M-CSF function in the CSF-1-deficient osteopetrotic (Csf1op/Csf1op) back- ground [6]. Transgenic expression of csCSF-1 corrected the gross defects of these mice, including growth retardation and failure of tooth eruption. However, residual osteopetrosis and significantly delayed trabecular bone resorption in the subepiphyseal region of the long bones, along with incomplete correction of hematologic abnormalities were found. Complete restoration was achieved by transgenic expression of the full length CSF-1 gene, which encodes both circulating CSF-1 and some csCSF-1 [38]. Only partial correction of osteopetrosis in csCSF-1 transgenic mice was observed, even though there was an equivalent number and size of OCs to wild-type controls. This finding prompted the authors to conclude that while local csCSF-1 expression was sufficient to support osteoclastogenesis, the rate of bone resorption in these animals lacking circulating M-CSF was slower [6]. These investigators subsequently demonstrated [7] that the osteopetrosis phenotype of CSF-1- and CSF1R-deficient mice could be replicated by post-natal administration of a neutralising anti-CSF-1 antibody. The number of OCs was dramatically reduced at 15.5 but not at 36.5 or 64.5 days post-natal, and increased trabecular bone density remained. Very low to undetectable levels of circulating antibody were present in the older mice, suggesting that some M-CSF was present (although not measured in this study) but at sub-optimal levels for OC resorption to proceed [7]. PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21462