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.
Highlights
Purinergic (P2) receptors are a primitive family of extracellular nucleotide-sensing receptors that emerged early in our evolutionary history [1]
We found that compact bone-derived murine osteoblasts (CB-Ob) expressed P2Y14 (Figure 1a)
We examined cAMP signalling and observed that forskolin (FK)-stimulated cAMP production was not reversed by UDPG-mediated P2Y14 stimulation, inhibiting P2Y14 led to significant increases in basal cAMP levels (p < 0.01) (Figure 1c)
Summary
Purinergic (P2) receptors are a primitive family of extracellular nucleotide-sensing receptors that emerged early in our evolutionary history [1]. The P2Y14 receptor is unique among the P2 receptor family because it is the only member that selectively responds to UDP-sugars including UDP-glucose (UDPG) and UDP-galactose [9]. Just as adenine nucleotides were first described for their intracellular role in cellular energetics [10], UDP-sugars are primarily known as sugar donors for glycosylation reactions in the endoplasmic reticulum and Golgi apparatus [11,12]. Among these UDP-glucose serves as an intermediate in several biosynthetic pathways, including synthesis of polysaccharides such as glycogen, lipopolysaccharides and glycosphingolipids [13]. The extracellular function of UDP-sugars was shown to follow their release through Ca2+-regulated exocytosis into the extracellular space, where they are enzymatically stable and capable of stimulating the P2Y14 receptor [14,15,16,17,18]
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