P2Y2 Receptor Regulates the Osteocytic Mechanosensitivity During Fluid Flow

Document Type

Conference Proceeding

Publication Date

2-1-2022

Publication Title

J Bone Miner Res

Abstract

Osteocytes sensitive to mechanical loading play a crucial role in maintaining bone mass. Mechanical stimulus in the form of oscillatory fluid flow (OFF) activates many cell-signaling pathways. For example, in osteocytes Ca2+ influx in response to loading induces purinergic signaling through release of ATP. Previous studies have demonstrated that OFF induced ATP activates the P2X7 receptor, which in turn contributes to the anabolic response to loading. However, the role of other purinergic receptors in mechanotransduction has not been analyzed in detail. Our previous work in osteoblasts demonstrated, ATP released during fluid flow activates the P2Y2 receptor (P2Y2R) and suppresses mechanosensitivity. Therefore, the purpose of this study is to examine the function of P2Y2R in osteocytes for response to loading. P2Y2R was knocked out in MLO-Y4 cells (P2Y2KO) using Crisper/ Cas9 that resulted in a frame shift mutation at the Exon-1 of the P2ry2 gene. Growth rate of P2Y2KO cells was reduced by 50% compared to WT MLO-Y4 cells. P2Y2KO cells had shorter canaliculi and cells appeared more circular than asteroidal. To check if P2Y2 impacts the down-stream response to loading, WT (with or without AR-C118925) and P2Y2KO cells were exposed to the OFF with a peak shear stress of 2 Pa at 1Hz. Downstream markers of osteogenesis such as PGE2 and pERK were significantly upregulated in WT MLO-Y4 cells upon OFF. Interestingly, P2Y2KO cells exhibited an even greater increase in PGE2 and pERK that is attributed to enhanced Ca2+ signaling observed when blocking P2Y2 with AR-C118925. Similarly, expression of other osteogenic markers such as RANKL, OPG, Wnt10, Lef1, Axin and Dkk1 was tested. Purinergic signaling is accompanied with actin stress fiber formation (ASFF). Upon shear stress, P2Y2KO cells did not form notable actin stress fibers compared to WT, suggesting KO cells were protected from ASFF. The absence of ASFF was associated with a lack of RhoA activation and phosphorylation of Cofilin. This study demonstrates the importance of P2Y2 activation as part of osteocytes' mechanotransduction pathway. In particular, P2Y2 appears to negatively impact the anabolic response due to increased ASFF. These findings position P2Y2R as a potential target to maintain osteocyte function, improve bone formation and bone quality.

Volume

37

First Page

288

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