Figure 1.

RANK mediates OPGL-induced osteoclastogenesis. (A) Expression of RANK in osteoclast precursor cells. A RANK-specific probe was used in the Northern blot analysis of 4 μg of total RNA prepared from the negative OPGL-binding population, positive OPGL-binding population, or 32-D cells. (B) Cross-linking of OPGL–RANK complexes on the surface of osteoclast precursors. OPGL positive and negative binding cells were prepared by FACS, incubated with ¹²⁵I-labeled OPGL, and then subjected to cross-linking with 1 mM disuccinimidyl tartrate. One-tenth of the cell lysates were directly fractionated by SDS/PAGE (lanes 1 and 2). The remainder of the cell lysates were immunoprecipitated with either pre-immune serum (lanes 3 and 5) or anti-RANK Ab (lanes 4 and 6). (C) Anti-RANK Abs stimulate osteoclastogenesis. Mouse bone marrow cells were cultured with CSF-1 (30 ng/ml) with and without 5 ng/ml murine OPGL(158–316) in the presence of various concentrations of either anti-RANK Ab or rabbit IgG. Osteoclastogenesis was quantified by TRAP solution assay. A405 represents absorbance at 405 nm and is presented as the mean ± SD (n = 3). TRAP cytochemical staining was shown at the bottom. (Bar = 200 μm.)