Role of UDP-Sugar Receptor P2Y 14 in Murine Osteoblasts.

The purinergic (P2) receptor P2Y ₁₄ is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y ₁₄ expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca ²⁺ ] _i ) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y ₁₄ function, we used the P2Y ₁₄ inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y ₁₄ knockout C2-Ob clones (Y14 _KO ). P2Y ₁₄ inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y ₁₄ inhibition augmented Ca ²⁺ signalling in response to ATP, ADP and mechanical stimulation. P2Y ₁₄ knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y ₁₄ inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14 ^-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y ₁₄ in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.