Method: Bone Marrow Macrophages (BMMs) were isolated from mouse femur and tibia and differentiated into mature osteoclasts by culturing in the presence of RANKL. Whole-cell and nuclear extracts were harvested and analyzed by western blot. mRNA was extracted from osteoclasts, reverse transcribed, and subjected to real-time PCR analysis.
Result: Differentiating BMMs in the presence of BMP2 leads to and increase in p38 phosphorylation at days 1 and 2 compared to cells differentiated with RANKL alone, whereas Smad1/5/8 phosphorylation is increased on days 2 through 5. On day 3 of differentiation, stimulation of starved osteoclasts with BMP2 rapidly induces phosphorylation of p38. Following this rapid induction levels of P-p38 are quickly reduced, corresponding with an increase in the phosphorylated form of MAPK phosphatase, MKP-1. BMP2 also reduces MKP-1 mRNA expression in differentiating osteoclasts. Dorsomorphin, a SMAD inhibitor, effectively inhibits osteoclast maturation when administered day 3 and beyond. Future studies are exploring the role of MKP-1 in modulating BMP2 signaling pathways in osteoclasts.
Conclusion: BMP2 modulates osteoclast differentiation utilizing the p38 MAPK pathway at early stages of differentiation. Around the time of fusion of osteoclast precursors, a signaling switch occurs and BMP2 signals through the SMAD pathway. BMP2 regulates expression of MKP-1 RNA and phosphorylation of MKP-1 protein.