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.