due to increased activation of resorption in OCs. We also demonstrate that M-CSF potentiates RANKL-induced c-fos activation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, and that the resorption-stimulating effect of M- CSF is blocked by the mitogen-activated protein kinase (MEK) inhibitor PD98059. This is the first study to definitively identify M- CSF as a potent enhancer of resorption activation of mature OCs, a paradigm-shift for its role in physiology and pathophysiology. Materials and Methods Materials Eagle’s MEM, penicillin/streptomycin, paraformaldehyde, Sig- ma Cell Dissociation Solution (1X) non-enzymatic (Cat. No. C5914), Fast Garnett GBC, MEK1/2 inhibitor PD98059 and naphthol AS-BI-phosphate were purchased from Sigma. Non- essential amino acids (100X) and FBS were purchased from Bovogen (Melbourne, Australia). Ficoll-Paque was purchased from Pharmacia Biotech. MethoCult GF H4534 (Iscove’s MDM containing 1% methylcellulose, 30% FBS, 1% BSA, 1024 M 2- mercaptoethanol, 2 mM L-glutamine, 10 ng/ml recombinant human GM-CSF, 10 ng/ml IL-3, and 50 ng/ml stem cell factor) was purchased from Stem-Cell Technologies. Human M-CSF and neutralising polyclonal antibody against M-CSF was purchased from Chemicon. Primary rabbit monoclonal antibodies raised against b-actin, c-fos and total or phosphorylated forms of IkBa and ERK1/2 and HRP-conjugated anti-rabbit polyclonal anti- body were from Cell Signaling Technology (MA, USA). Soluble RANKL coupled to GST fusion protein (RANKL) was generously provided by Drs Matthew Gillespie and Julian Quinn (Prince Henry’s Institute, Monash Medical Centre, Melbourne, Australia). Transcription factor activation kits incorporating detection antibody for the p65 subunit of Nuclear Factor kappa B (NFkB) and c-fos component of AP-1 were purchased from Pierce Biotechnology (Illinois, USA). TransAM assay kits for Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) were from Active Motif (California, USA). Ethics statement Human umbilical cord blood was obtained with written informed consent from healthy donors under a protocol approved by Barwon Health Human Research and Ethics Committee. Mature human osteoclasts Collection of human umbilical cord blood, isolation of a mononuclear cell fraction, expansion of CFU-GM-derived OC precursors and differentiation of mature human OCs have been previously described [19]. Specifically, a mononuclear cell fraction of cord blood (CBMC) was isolated by Ficoll-Paque density gradient centrifugation. CBMCs (36106 cells/culture) were suspended in 3.0 ml Methocult GF H4534 in 6-well plates and incubated at 37C in humidified atmosphere of 5% CO2-air for 10 days. Pooled colonies (CFU-GM-derived OC precursors) were harvested into PBS. Precursors (6.56106 cells/175 cm 2 ) were then seeded into 175 cm2 tissue culture flasks. The cells were cultured in 20 mL of MEM containing 10% FBS, nonessential amino acids, penicillin 50 U/ml, streptomycin 50 mg/ml, 2 mM L-glutamine, M-CSF (25 ng/ml), and RANKL (125 ng/ml) for 14–21 days. The cultures were refreshed weekly by replacing additives in one half volume of media. Mature OCs were used in situ for signalling and transcription factor assays or dissociated and re-settled on dentine slices for survival and resorption assays. M-CSF Augments Resorption by Human Osteoclasts Mature OC survival and resorption assay Mature OC cultures were treated with dissociation buffer (5 mL/75 cm2 flask) for 30 min at 37C when most were detached by agitation. Remaining adherent cells were removed with a cell scraper. Detached OCs were washed, pelleted by centrifugation (200 g; 2 min), resuspended in MEM/10% FBS and settled onto round dentine slices (6 mm diameter) in 96-well plates (n = 500– 1000 OCs/well) and cultured for 4–72 h in 200 mL of media. Cells were fixed in 1% formalin and reacted for TRAP as previously described [19]. The formation of OCs was assessed by transmission light microscopy; quantified using microcomputer image analysis software (MCID - Imaging Research Inc. Ontario, Canada). F-actin stained sealing zones were quantified by confocal microscopy using rhodamine-conjugated phalloidin [20]. Cells were removed from dentine slices by brief sonication in chloroform:methanol 2:1. Xylene-free black ink was applied to the surface of each slice and residual ink removed by wiping on absorbent paper. Resorption was assessed by reflective light microscopy and the percentage area resorbed was quantified using MCID software. This method was validated by comparison with scanning electron microscopy [21]. NFkB, c-fos and NFATc1 transcription factor activity Mature OC cultures in 6-well plates were rinsed, pre-treated with or without M-CSF (37.5 ng/mL) for 18 h in MEM/ 0.2%FBS and then treated with RANKL (125 ng/mL) or vehicle for 30 min. Nuclear extracts (Ne-PER; Pierce, IL, USA) were assessed for activation of NFkB(p65), c-fos and NFATc1 using ELISA-based transcription factor assay kits. Briefly, an oligonu- cleotide containing the consensus binding sequence for the relevant transcription factor is immobilised to a 96-well plate. Transcription factor contained in nuclear extracts binds specifi- cally to this oligonucleotide and is detected through use of an antibody directed against either p65, in the case of NFkB, c-fos or NFATc1. Addition of a secondary antibody conjugated to horseradish peroxidise provides sensitive chemiluminescent or colorimetric readout that is quantified by spectrophotometry. ERK1/2, IkBa signalling and c-fos Western Blot Mature OCs in 25 cm2 flasks were serum-starved for 18 h when media was replaced with MEM/10%FBS with vehicle, RANKL (125 ng/mL), M-CSF (25 ng/mL) or both. Cytoplasmic extracts (M-PER, Pierce, IL, USA) were collected at 0, 5 min and 10 min. For c-fos expression, mature OCs were treated with or without M- CSF for 18 h before cell lysis. Total protein was subjected to SDS- PAGE on 4–20% gradient gels (NuSep, Georgia, USA). Protein gels were transferred to nylon membranes (Amersham Biosciences, Buckinghamshire, UK) overnight at 4C (50 V; 25 mA) and blocked in PBS containing 5%BSA; 0.2% Tween 20 for 1 h. Immunodetection used primary rabbit monoclonal antibodies that recognised either total or phosphorylated forms of IkBa, ERK1/2, c-fos, or b-actin (Cell Signaling Technology, MA, USA). Secondary antibody was HRP-conjugated anti-rabbit polyclonal antibody (Cell Signaling Technology, MA, USA) A chemilumi- nescent substrate followed by autoradiography was used for detection (GE Healthcare Bio-Sciences, NJ, USA). MCID software was used for densitometric analysis. Statistical analyses Data are expressed as the mean 6 SEM where applicable. Differences between groups where determined using one-way ANOVA followed by Fisher’s multiple comparison test, two-way ANOVA – general linear model (GLM) followed by Tukey’s PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21462