Figure 5.

OPGL and anti-RANK agonistic Abs stimulate osteoclast differentiation of the murine myeloid RAW 264.7 cell line. (A) Northern blots of RNA from various sources hybridized with an RANK probe. (B) Approximately 10⁶ RAW cells were exposed to 100 ng/ml OPGL for the indicated length of time. The cell lysates were immunoprecipitated with monoclonal anti-JNK Ab. Immunoprecipitates were assayed for kinase activity by using GST-JUN as substrate and resolved by SDS/PAGE. (C) RAW cells were plated on bone slices and treated for 7 days with OPGL. The Upper and Lower panels show the results of TRAP cytochemistry and toluidine blue staining, respectively. Note that OPGL induces the formation of TRAP positive cells (Upper) that form resorption lacunae (Lower). (Bars = 50 μm.) (D) Total cell RNA was extracted from RAW cells treated with media alone (lane 1) or media supplemented with either 100 ng/ml OPGL (lane 2) or 1,750 ng/ml anti-RANK antibodies (lane 3). RNA from OPGL plus CSF-1-treated bone marrow cultures (lane C) served as the positive control. RNase protection assays were performed by using probes for the calcitonin receptor (CTR), β3 integrin (B3), c-fms, cathepsin k (cathK), TRAP, c-src, PU.1, and cyclophilin (cyclo). Lanes P and Y represent the undigested probe and yeast hybridization controls, respectively.