Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts

Abstract

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 (Y14KO). 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.

Purinergic P2 Receptors: Novel Mediators of Mechanotransduction

“…Subsequently, VNUTmediated ATP release inhibited osteoblast differentiation through P2X7R and/or P2Y2R. A recent study also reported that P2Y14R, the only P2YR stimulated by uridine diphosphate sugars, contributed to the differentiation of osteoblasts under a mechanical stimulus, which was confirmed in primary murine osteoblasts and a C2C12-BMP2 osteoblastic cell line (Mikolajewicz and Komarova, 2020). Another study indicated that ATP release induced by low-intensity pulsed ultrasound promotes osteoblast proliferation and osteogenic responses via the activation of P2X7R (Manaka et al, 2015).…”

Purinergic Signaling Mediates PTH and Fluid Flow-Induced Osteoblast Proliferation

“…The results are reasonable since reduction of ATP and P2Y2 inhibited ERK1/2 and CREB phosphorylation based on our previous findings. Additionally, other researchers have demonstrated that purinergic receptors were closely related to cell proliferation [ 38 ].…”

In Search of a Role for Extracellular Purine Enzymes in Bone Function

“…recently pointed out that mechanically stimulated ATP release from murine bone cells could be regulated by a reversible membrane disruption, which did not cause cell death but a rapid repair [ 19 ]. This event would be mediated by P2Y 14 receptors (P2Y 14 R), the inhibition of which determined cell hypersensitivity to mechanical stimulation [ 20 ]. Interestingly, the same research group has developed a theoretical model to determine the amount of ATP released from cells and the propagation of the purinergic signal following a mechanical “injury” (that is, a rapidly reversible perturbation/damage of the membrane).…”

“…As well, increased levels of extracellular ATP, interacting with P2 × 7R present on fibroblasts and osteoblasts, induced the generation of the receptor activator of nuclear factor kB ligand (RANKL), which further activated osteoclastic alveolar bone resorption and bone loss in periodontitis [ 26 ]. Very recently, it was found that also the P2Y 14 R activated by UDP-sugar reduced, in addition to the responsiveness to mechanical stimuli and the differentiation of osteoblasts, while stimulating their proliferation [ 20 ]. Likewise, low concentrations of ATP and UTP strongly inhibited osteogenesis in osteoblasts via P2Y 2 R. Interestingly, the P2Y 2 R performed their activity by primarily regulating extracellular ATP levels in both osteoclasts and osteoblasts [ 27 ].…”