M-CSF Potently Augments RANKL-Induced Resorption Activation in Mature Human Osteoclasts Jason M. Hodge1,2*, Fiona M. Collier2, Nathan J. Pavlos3, Mark A. Kirkland1,2, Geoffrey C. Nicholson4 1 The Department of Clinical and Biomedical Sciences, Barwon Health, The University of Melbourne, Geelong, Victoria, Australia, 2 Barwon Biomedical Research, Barwon Health, Geelong, Victoria, Australia, 3 Centre for Orthopaedic Research, School of Surgery, The University of Western Australia, Nedlands, Western Australia, Australia, 4 Rural Clinical School, School of Medicine, The University of Queensland, Toowoomba, Queensland, Australia Abstract Macrophage-CSF (M-CSF) is critical for osteoclast (OC) differentiation and is reported to enhance mature OC survival and motility. However, its role in the regulation of bone resorption, the main function of OCs, has not been well characterised. To address this we analysed short-term cultures of fully differentiated OCs derived from human colony forming unit- granulocyte macrophages (CFU-GM). When cultured on dentine, OC survival was enhanced by M-CSF but more effectively by receptor activator of NFkB ligand (RANKL). Resorption was entirely dependent on the presence of RANKL. Co-treatment with M-CSF augmented RANKL-induced resorption in a concentration-dependent manner with a (200–300%) stimulation at 25 ng/mL, an effect observed within 4–6 h. M-CSF co-treatment also increased number of resorption pits and F-actin sealing zones, but not the number of OCs or pit size, indicating stimulation of the proportion of OCs activated. M-CSF facilitated RANKL-induced activation of c-fos and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, but not NFkB nor nuclear factor of activated T-cells, cytoplasmic-1 (NFATc1). The mitogen-activated protein kinase kinase (MEK) 1 inhibitor PD98059 partially blocked augmentation of resorption by M-CSF. Our results reveal a previously unidentified role of M-CSF as a potent stimulator of mature OC resorbing activity, possibly mediated via ERK upstream of c-fos. Citation: Hodge JM, Collier FM, Pavlos NJ, Kirkland MA, Nicholson GC (2011) M-CSF Potently Augments RANKL-Induced Resorption Activation in Mature Human Osteoclasts. PLoS ONE 6(6): e21462. doi:10.1371/journal.pone.0021462 Editor: Vincent Laudet, Ecole Normale Supe´rieure de Lyon, France Received February 21, 2011; Accepted May 31, 2011; Published June 29, 2011 Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jhodge@unimelb.edu.au Copyright: 2011 Hodge et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction The critical role of macrophage-colony stimulating factor (M- CSF) in osteoclast (OC) differentiation from hematopoietic precursors was demonstrated in mice with osteopetrosis due to disruption of the CSF-1 gene (Csf1op/Csf1op). Osteoclasts were completely absent in these mutant animals [1–5]. However, the role of M-CSF in the function of fully-differentiated OCs, in particular bone resorbing activity, is unclear. Two recent studies in mice suggest a role for M-CSF in OC activation. Mice rendered osteopetrotic, either by disruption of the CSF-1 gene (Csf1op/ Csf1op), or administration of a neutralizing anti-CSF-1 antibody, displayed this high bone mass phenotype due to total lack, or significant reduction in the number of OCs [6,7]. Partial correction of osteopetrosis was achieved by either co-expression of cell-surface M-CSF in the case of the Csf1op/Csf1op background, or cessation of neutralizing anti-CSF-1 antibody administration. In both cases there were equivalent levels of OCs to those seen in control animals, leading the authors to deduce that the rate of bone resorption in animals with sub-optimal levels of M-CSF was slower. In short-term (6 h) semi-pure cultures of isolated neonatal rat OCs, human M-CSF enhanced survival, motility and cytoplasmic spreading, but inhibited resorbing activity, in a concentration- dependent manner; maximum effect at 0.5 ng/mL [8]. In contrast, in 6–18 h cultures of isolated human fetal OCs, M- CSF had no apparent effect on resorbing activity but did enhance survival; maximum effect at 50 ng/mL [9]. Experiments in a human bone marrow osteoclastogenesis assay suggested that resorption per OC was enhanced in the presence of M-CSF 50 ng/mL [10]. Likewise, in an osteoclastogenesis assay employ- ing human colony forming unit-granulocyte macrophage (CFU- GM) precursors, we found that resorption per OC was increased in the presence of M-CSF 25 ng/mL [11]. However, investigation of resorbing activity in osteoclastogenesis assays is problematic because of the confounding effects of proliferation, differentiation and fusion. Furthermore, these findings were inconsistent with the widely held view [12–16] that, in fully differentiated OCs, the role of M-CSF is limited to regulation of survival and motility while receptor activator of NFkB ligand (RANKL) and perhaps interleukin-1 (IL-1) [17,18] are the key regulators of resorption activation. To address this knowledge gap, we established a mature OC model based on that of Fuller and colleagues [17] that would allow the characterization of the role of M-CSF in the short-term regulation of resorption activation and survival in these cells. We generated OCs en masse by treating CFU-GM-derived cells with M-CSF and RANKL for 14d [19]. The OCs were then either detached from the plastic and cultured on dentine slices for 4–72 h for resorption and survival assays, or used in situ for transcription factor activation and signalling phosphorylation assays. We demonstrate that although RANKL is necessary for resorption, M-CSF 10–25 ng/mL potently augments RANKL-induced re- sorption, an effect that is not due to enhanced survival but, rather, PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21462