Role of UDP-Sugar Receptor P2Y₁₄ in Murine Osteoblasts

The purinergic (P2) receptor P2Y₁₄ is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y₁₄ 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 ([Ca²⁺]_i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y₁₄ function, we used the P2Y₁₄ inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y₁₄ knockout C2-Ob clones (Y14_KO). P2Y₁₄ inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y₁₄ inhibition augmented Ca²⁺ signalling in response to ATP, ADP and mechanical stimulation. P2Y₁₄ knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y₁₄ 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 P2Y₁₄ in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.