FIGURE 2.

Breast cancer cells activate Hh signaling and promote osteoclast differentiation. A, DM supports differentiation of RAW264.7 into osteoclasts. Supplementing DM with recombinant human OPN (100 ng/ml) or SHH (100 nm) significantly (* indicates p < 0.005) increases the numbers of multinucleate (more than three nuclei) TRAP-positive cells. B, conditioned serum-free medium from breast cancer cells, MDA-MB-231, SUM159, and SUM1315, significantly (*, p < 0.01) increases the numbers of multinucleate, TRAP-positive cells. The addition of Hh ligand-neutralizing antibody, 5E1, to differentiation conditions notably (^, p < 0.05) reduces the efficiency of breast cancer cell-conditioned medium to elicit osteoclast differentiation. C, breast cancer cells (MCF10CA clone d, MDA-MB-231, SUM159, and SUM1315) express IHH and SHH ligands. Shown is an immunoblot of the lysate from the breast cancer cells. β-Tubulin serves as a loading control. D, images represent photomicrographs showing the TRAP-stained osteoclasts formed in response to various differentiation conditions. The bar represents 100 μm. Panel a, growth medium (GM); panels b and e, DM; panels c and d, DM supplemented with recombinant OPN (100 ng/ml) (panel c) and SHH (100 nm) (panel d); panel f, DM + 5E1 (2.5 μg/ml); panels g, i, and k, DM supplemented with conditioned medium from MDA-MB-231 cells (panel g), SUM159 cells (panel i), and SUM1315 cells (panel k); panels h, j, and l, 5E1 (2.5 μg/ml) added to DM supplemented with conditioned medium from MDA-MB-231 (panel h), SUM159 (panel j), and SUM1315 cells (panel l). The osteoclasts are marked within circles in panel c. The data were recorded at 10× magnification using the Nikon Eclipse TS 100 microscope and are represented as a percentage of multinucleate TRAP-positive cells relative to the total number of cells in the field. The data were verified by two independent experiments.