Bone Abstracts

The PTH type 1 receptor (PTH1R) is an important modulator of bone remodeling. In mice, PTH1R ablation in osteocytes produces trabecular bone reduction and impaired calcium homeostasis; meanwhile, its overexpression in these cells promotes periosteal and endocortical bone formation. Osteocytes can translate mechanical stimuli into bone-forming signals. Skeletal unloading induces osteocyte apoptosis and bone loss, whereas mechanical stimuli prevent osteocyte apoptosis through inducing β-catenin accumulation and ERK nuclear translocation. PTH1R activation stimulates the canonical Wnt/β-catenin pathway and promotes osteoblast survival. Here, we aimed to explore the possible involvement of PTH1R activation in cell protection conferred to osteocyte-like MLO-Y4 cells by mechanical stimulation. Cells were subjected to hypotonic shock (240 mOsm) for a short time (≤10 min) in the presence or absence of PTH1R antagonists or after transfection with a PTH1R siRNA. Cell viability was assessed by Trypan blue exclusion after incubation with the proapoptotic agent etoposide (50 μM) for 6 h. Changes in cell localization of β-catenin and nuclear ERK were examined by western blot. Calcium signaling measurement was assessed by confocal microscopy in cells preloaded with the fluorochrome Fluo-4 AM. Hypotonic shock stimulated a rapid (<1 min) and transient Ca_i²⁺ response in MLO-Y4 cells. In addition, this mechanical stimulus induced β-catenin stabilization, related to an increased nuclear and membrane localization, and increased nuclear ERK at 10 min. These changes were associated with an enhanced MLO-Y4 cell survival. These events were all inhibited by cell pre-treatment with two PTH1R antagonists, PTHrP (7–34) and JB4250 (1 μM).or transfection with PTH1R siRNA. Moreover, both the calcium antagonist verapamil (1 μM) and Rp-cAMPS (25 μM), which prevents protein kinase A activation (another PTH1R signaling pathway) also prevented these changes triggered by mechanical stimulus. Collectively, these findings indicate that a rapid activation of the PTH1R occurs after mechanical stimulus in osteocytes, leading to an increased survival.