M-CSF Augments Resorption by Human Osteoclasts Figure 3. M-CSF pre-treatment facilitates RANKL-induced activation of c-fos but not of NFkB. NFATc1 is not activated by either RANKL or M-CSF. Mature human OCs cultured in 6-well plates for 21 d were serum-starved for 18 h in the absence or presence of M-CSF (37.5 ng/mL) followed by stimulation with RANKL (125 ng/mL) for 30 min. A. Relative activation of transcription factors quantified using ELISA-based transcription factor activation assays: NFkB(p65 subunit) and c-fos (Pierce Biotechnology, USA); NFATc1 (Active Motif, USA); representative data from 2 independent experiments. Results expressed as mean 6 SEM (n = 3 wells/group). Groups with different annotations are significantly different; p = 0.001; one-way ANOVA; Fishers multiple comparison test. B. Western analysis of c-fos protein; mature OCs cultured for 18 h in media alone, with or without M-CSF (25 ng/mL). doi:10.1371/journal.pone.0021462.g003 induced resorbing activity. Furthermore, c-fos induction by ERK is well characterised [25,26] and in the current study we have shown that c-fos is activated by M-CSF. In serum-starved OCs we found no detectable levels of constitutively phosphorylated ERK1/ 2. Treatment with M-CSF activated phosphorylation, with a peak increase of 477% at 5 min, dissipating to 70% at 10 min (Fig. 4A). Treatment with RANKL produced only a weak phosphorylation of ERK1/2 at 5 min, in comparison to M-CSF, with negligible levels detected at 10 min. Co-treatment with RANKL and M-CSF increased ERK1/2 phosphorylation at 5 min (44% increase) and 10 min (+19%), compared to M-CSF alone. Levels of total ERK1/2 were unchanged over the time-course investigated. M-CSF alone decreased phosphorylated IkBa by 42% at 10 min, whereas RANKL exposure stimulated phosphorylation by 278%. There was no additional effect in combination with M- CSF. Total IkBa was unchanged over the time-course (Fig. 4B). Blockade of MEK/ERK signalling inhibits M-CSF-induced stimulation of resorption The results above indicated that ERK1/2 phosphorylation was enhanced by co-treatment with M-CSF and RANKL, suggesting that MEK/ERK signalling may be involved in M-CSF enhance- ment of RANKL-induced resorption activity. To test this possibility, we examined the effects of PD98059, a specific inhibitor of the ERK1/2 activator MEK-1, on this effect. Mature OCs cultured on dentine slices were pre-treated for 1 h with PD98059 (5 mM), or vehicle, prior to treatment with RANKL alone or together with M-CSF for 72 h. Pre-treatment with PD98059 had no effect on OC survival, size or RANKL-induced resorption (Fig. 5). In cultures pre-treated with vehicle, treatment with RANKL and M-CSF produced a 330% increase in resorption compared to RANKL alone. This effect was attenuated in the presence of PD98059. In the experiment shown in Fig. 5, resorption in M-CSF group is not significantly different (ANOVA) to that of the RANKL-only control in the presence of PD98059. However, in 3 independent experiments PD98059 reduced the resorption-stimulating effect of M-CSF by 52.366.2% but resorption in the M-CSF group remained significantly greater than the control (p = 0.023, paired T-test). Discussion Utilising a human, mature OC activation assay, we have demonstrated that M-CSF effectively activates OC resorption, in PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21462