Figure 5. OSMR expression by osteoblasts is required for normal osteoclast and osteoblast activity; sclerostin is regulated by mOSM in Osmr^–/– mice.

(A) Tartrate-resistant acid phosphatase–positive multinucleated osteoclast-like cell (TRAP+ MNC) formation in response to RANKL from Osmr^–/– BM macrophages compared with WT BM. In the presence of Osmr^–/– osteoblasts, osteoclast formation from either Osmr^–/– or WT BM in response to mOSM was completely blocked, and osteoclast formation in response to PGE₂ and 1,25D₃ (PGE₂/D₃) was inhibited. (B) Rankl mRNA was increased by mOSM treatment in WT calvarial osteoblasts, but not in Osmr^–/– calvarial osteoblasts. Rankl mRNA levels were also significantly increased by PGE₂/D₃ in both WT and Osmr^–/– primary calvarial osteoblasts 8 hours after treatment was commenced. (C and D) Mineralization, measured by alizarin red staining after 21 days of culture in mineralizing medium, and ALP activity in cells grown in osteoblast differentiation medium were both significantly reduced in calvarial osteoblasts generated from Osmr^–/– mice compared with WT littermates. (E–H) Cebpb, Cebpd, Pparg, and Sost mRNA levels, relative to HPRT1 in WT and Osmr^–/– calvarial osteoblasts after treatment with 10 ng/ml mOSM. PPARγ and SOST are measured in cells differentiated for 17 days in osteoblast differentiation medium. All data in Figure 5 are shown as mean + SEM from 3–4 independent experiments, carried out in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 vs. untreated control at the same time point.