Anales RANF

P.99 ROLE OF UDP-GLUCOSE RECEPTOR P2Y14 IN OSTEOBLASTS Nicholas Mikolajewicz, Svetlana V. Komarova Shriners Hospital for Children – Canada, Montreal, Quebec, Canada; McGill University, Montreal, Quebec, Canada. Purinergic receptor P2Y14 is uniquely responsive to UDP-glucose (UDPG). P2Y14 expression was reported in all bone-residing cells, including mesenchymal stem cells and osteoblasts, however its role in bone is unknown. We confirmed gene and protein P2Y14 expression in C2C12-BMP2 osteoblastic cell line (C2-OB) and primary murine osteoblasts (P-OB). To study P2Y14 function, we generated CRISPR-Cas9-mediated P2Y14 knockout C2-OB clones (KO), or used P2Y14-antagonist PPTN. In P-OB, UDPG induced changes in the cytosolic free calcium concentration [Ca 2+ ] i , which were potentiated by PPTN. While C2-OB did not respond to UDPG with an increase in [Ca 2+ ] i , KO cells demonstrated a response that was similar to PPTN-treated P-OB. UDPG also stimulated PPTN-sensitive stress-fiber formation in P-OB cells, and PPTN- or KO-sensitive ERK1/2 phosphorylation in C2-OB cells. P2Y14 knockout or inhibition reduced proliferation in C2-OB and CB-OB cells, and was associated with higher AMPKα phosphor ylation. Osteogenic differentiation was induced by treating P-OB with ascorbic acid and β -glycerol phosphate in the presence of vehicle or PPTN for 28 days. Alkaline phosphatase activity, collagen deposition and mineralization were reduced in PPTN-treated cells at day 14 of differentiation, however normalized by day 28. Expression of OSX, RUNX2, COLA1, DMP1 and SOST significantly and similarly increased with osteogenic differentiation of vehicle-and PPTN-treated cells for the first 14 days, however at day 28 expression of these genes was significantly higher in PPTN- treated cultures compared to vehicle-treated. Of interest, while P2Y14 -/- mice from the International Mouse Phenotyping Consortium were similar to wild-type in bone mineral density, their tibia length was significantly increased. Taken together, these findings position P2Y14 as a modulator of osteoblast differentiation and further our understanding of the function of purinergic system in bone.