Figure 2. Lack of direct effect of PTH on cells of the osteoclast lineage.

(A) Non-adherent cells expanded in the presence or absence of Flt-3L (100 ng/ml) or PTH (10 nM), a combination of both, or vehicle (control), were counted and plated at equal numbers and allowed to differentiate in osteoclastogenic media, then induced via RANK-L (0–30 ng/ml) and M-CSF (50 ng/ml). Five days later, multi-nucleated TRAP⁺ cells were counted. Data are mean ± SEM of 2 experiments performed in duplicate. * p<0.05 versus respective vehicle or PTH alone ** p<0.05 versus all other groups in their respective RANK-L concentrations. The 0 ng/ml RANK-L resulted in no osteoclasts; therefore, the data was not plotted. (B) Cells were expanded in Flt-3L (5 ng/ml) media for 8 days with or without 10 nM PTH. Cells were counted and plated at equal numbers, then induced via RANK-L (30 ng/ml) and M-CSF (50 ng/ml) to differentiate with additional treatments of PTH, forskolin or tetrahydrofuryladenine (THFA). Multinucleated TRAP⁺ cells were enumerated 5 days later. Data are mean ± SEM of 2 experiments performed in duplicate. *p<0.05 and **p<0.01 versus vehicle. (C) Visualization of the cytoskeleton of actin, by confocal microscopy in mature osteoclasts seeded on coverslips or ACC and stained for actin and vinculin at different time points. All images are the same magnification. (D) Osteoclast transmigration assay: osteoclasts were seeded on MC3T3-E1 cell layers, treated with 0–10 nM PTH then fixed. Cells were stained with phalloidin to visualize actin under confocal microscopy. Data are mean ± SEM of number of osteoclasts that transmigrated compared to the total number of osteoclasts. Experiments were performed a minimum of 3 times. * p<0.05 versus vehicle or PTH. (E) Osteoclast functional assay: Cells were expanded in the presence of Flt-3L (100 ng/ml). At day 8, they were seeded onto ACC (TRAP staining) or osteologic disks (resorption pit assay) and induced to differentiate in the presence of 50 ng/ml M-CSF and 30 ng/ml RANK-L. When osteoclasts started to form, PTH, calcitonin or vehicle (control) were added to the medium. Data are mean ± SEM of the area of the pit divided by the total area. Experiments were performed a minimum of 3 times in duplicate. * p<0.05 versus vehicle.