FIGURE 9.

HS-OPG interactions lower the inhibitory threshold of OPG against RANKL. A, inhibition of osteoclastogenesis in osteoblasts/bone marrow macrophage co-culture by WT OPG dimer or triple mutant K359A/R370A/R379A at 1, 3, 10, 30, and 100 ng/ml. The extent of osteoclastogenesis is assessed by an enzymatic assay of TRAP activity in whole cell lysate. n = 3. Error bars represent S.D. * represents p < 0.01 by Student's t test. Data are representative of at least three separate assays. B–E, representative images of differently treated co-culture. Mature osteoclasts are visualized by TRAP staining. The pink multinucleated cells are osteoclasts and the round clear cells are undifferentiated bone marrow macrophages. F, inhibition of osteoclastogenesis in bone marrow macrophage monoculture by WT OPG dimer or triple mutant OPG at 50, 100, 200, and 500 ng/ml. Osteoclastogenesis is induced by recombinant soluble RANKL (50 ng/ml) and M-CSF (20 ng/ml).