The Roles of P2Y2 Purinergic Receptors in Osteoblasts and Mechanotransduction

Abstract

We previously demonstrated, using osteoblastic MC3T3-E1 cells, that P2Y2 purinergic receptors are involved in osteoblast mechanotransduction. In this study, our objective was to further investigate, using a knockout mouse model, the roles of P2Y2 receptors in bone mechanobiology. We first examined bone structure with micro-CT and measured bone mechanical properties with three point bending experiments in both wild type mice and P2Y2 knockout mice. We found that bones from P2Y2 knockout mice have significantly decreased bone volume, bone thickness, bone stiffness and bone ultimate breaking force at 17 week old age. In order to elucidate the mechanisms by which P2Y2 receptors contribute to bone biology, we examined differentiation and mineralization of bone marrow cells from wild type and P2Y2 knockout mice. We found that P2Y2 receptor deficiency reduces the differentiation and mineralization of bone marrow cells. Next, we compared the response of primary osteoblasts, from both wild type and P2Y2 knockout mice, to ATP and mechanical stimulation (oscillatory fluid flow), and found that osteoblasts from wild type mice have a stronger response, in terms of ERK1/2 phosphorylation, to both ATP and fluid flow, relative to P2Y2 knockout mice. However, we did not detect any difference in ATP release in response to fluid flow between wild type and P2Y2 knock out osteoblasts. Our findings suggest that P2Y2 receptors play important roles in bone marrow cell differentiation and mineralization as well as in bone cell mechanotransduction, leading to an osteopenic phenotype in P2Y2 knockout mice.

Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor

“…Corresponding changes in cortical bone appeared only after 17 weeks, while both trabecular and cortical BMD remained unchanged. Ultimate force and stiffness of cortical bone was decreased after 17 weeks, but not after 8 weeks [14]. The decrease in mechanical properties due to the lack of P2Y2R expression is in agreement with the increase in mechanical properties upon increased P2Y2R expression in our study.…”

“…Along with this, an increase in expression of BMP4 and BMP5, proteins involved in regulation of bone matrix formation, was seen upon stimulation with ATP and UTP [8]. Furthermore, Xing et al showed that primary osteoblasts from P2Y2R KO mice formed less mineralized nodules and had lower ALP activity compared to osteoblasts from Wt mice [14].…”

“…Orriss et al reported that 2-month-old male P2Y2R KO mice display a 17% increase in tibial BMC and a 9% increase in femoral BMC compared to Wt mice, supporting that P2Y2R activation inhibits mineralization [7], whereas Xing et al reported contrasting results on the bone phenotype in P2Y2R KO in a recent study [14]. They found that both 8-and 17-week-old male P2Y2 KO mice have decreased trabecular BV/TV and Tb.Th.…”

“…Whereas Orriss et al found higher bone mineral content (BMC) in P2Y2R KO mice compared to wild-type (Wt) mice [7], Xing et al found that P2Y2R KO mice had lower bone volume and bone strength than Wt mice [14].…”

GRK2 desensitizes flow-induced responses in osteoblasts

“…However, the mechanisms underlying the initiation and execution of these cellular responses have not been elucidated. Accumulating evidence suggests that oscillatory fluid flow is a very strong initiator for bone cell metabolic activities, including cell growth and differentiation, gene expression, and protein production (You et al, 2001(You et al, , 2002Ponik et al, 2007;Xing et al, 2014). In an earlier study, we demonstrated the role of oscillatory flow in the induction of cytosol Ca 2+ release from the ER, and enhancement of mRNA expression of osteopontin (You et al, 2001).…”