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.