Fig. 3.

c-FMS proteolysis positively regulates osteoclastogenesis. a Schematic showing mutations in the TACE cleavage sites of c-FMS. TACE cleavage sites of c-FMS¹⁰ were replaced by the addition of 14 amino acids from insulin receptor sequences (FMS^mut). b 293 T cells did not express c-FMS and were transduced with lentiviral particles encoding control, FMS^wt or FMS^mut. The cells were then stimulated with M-CSF for the indicated times. Protein expression of phospho-ERK, phospho-JNK, phospho-p38, and α-tubulin was determined by immunoblot analysis. c–f BMDMs from c-FMS^f/+ Mx1-Cre mice were transduced with lentivirus encoding control, wild-type FMS (FMS^wt), or the TACE-uncleavable mutant FMS (FMS^mut). The transduced BMDMs were stimulated with LPS (10 ng·mL⁻¹) for 3 h (c) and 24 h (d). c The mRNA expression of TNFα and IL6 was measured by q-PCR. d TNFα and IL6 protein levels in the culture media were measured by Luminex multiplex cytokine assays. e Osteoclastogenesis assay. The left panel shows representative images of TRAP-stained cells. The right panel shows the percentage of TRAP-positive multinuclear cells (MNCs: more than three nuclei) per control (n = 6). Black scale bar: 100 μm. f Resorption pit assay. Bone resorption activity analysis of FMS^con, FMS^wt, or FMS^mut cells. The left panel shows representative images, and the right panel shows the percentage of the resorbed pit area per total area. Red scale bar: 200 μm. All data are shown as the mean ± SEM. ns not significant, ND not detected. *P < 0.05 by one-way ANOVA with a post hoc Tukey test (c–f). The data represent at least three experiments (b–d, f).