Osteoporotic fractures are common, increasing in incidence, and have a high associated economic burden. This significant clinical problem is further compounded by a lack of therapeutic strategies to increase bone formation and improve tissue strength. Bone formation is a function of osteocytes? response to mechanical loading during physical activity and exercise. Osteocytes? purinergic signaling through the release of nucleotides plays a key role in regulating bone adaptation in response to loading. In particular we have found the P2Y2 receptor downregulates osteocytes? sensitivity to loading and that the loss in mechanosensitivity is accompanied by an increase in actin-stress fiber formation (ASFF) through cofilin phosphorylation. These findings are significant because they suggest targeting P2Y2 signaling as a potential strategy to enhance osteocyte mechanotransduction and increase bone formation. However, the extent to which P2Y2 influences bone formation by regulating osteocytes? sensitivity to loading through ASFF remains unknown. These gaps in knowledge limit our development of new therapeutic strategies that increase bone formation and reduce fracture risk in an aging population. Our long-term goal is to prevent osteoporosis and reduce fracture risk in an aging population. The objective of this study is to determine the role of purinergic signaling through the P2Y2 receptor in regulating the anabolic response to loading. The premise for this study is that blocking P2Y2 signaling has therapeutic potential to prevent age-related bone loss and reduce fracture risk. The central hypothesis states that blocking P2Y2 signaling will increase osteocytes? sensitivity to loading, allowing greater gains in bone mass and tissue strength in response to loading. The central hypothesis will be tested under three specific aims.
Aim 1 will determine the extent to which P2Y2 signaling in-vitro influences osteocytes? sensitivity and overall response to loading by regulating ASFF through cofilin phosphorylation. Our approach in aim 1 utilizes osteocyte knockout cell lines generated using CRISPR/Cas9 to examine in-vitro their response to fluid flow.
Aim 2 will determine the extent to which P2Y2 expression in-vivo contributes to bone formation in response to loading and unloading. Our approach in aim 2 will prescribe treadmill exercise and hindlimb immobilization to conditional knockout mice that target osteocytes? P2Y2 expression.
Aim 3 will examine the efficacy of AR-C118925, a selective P2Y2R inhibitor, to increase the anabolic response to loading in aged mice as well as prevent age-related bone loss. Our approach in aim 3 will treat wild-type as well as P2Y2- knockout mice with AR-C118925 to identify off-target effects that are not specific to osteocytes. This study is innovative because it 1) evaluates a novel therapeutic agent (AR-C118925) for increasing bone formation, and 2) uses new cell-lines and animal models to establish P2Y2 signaling as a unique mechanism to increase bone mass. Overall, this study is significant because we expect it to demonstrate the therapeutic potential of targeting P2Y2R signaling to increase bone formation and reduce fracture risk.
The proposed study is relevant to public health because understanding the underlying mechanisms that regulate bone formation in response to loading during normal physical activity and exercise will form the basis for developing novel therapeutic targets to prevent bone loss and reduce fracture risk in our aging population. As a result, this study aligns well with the NIH mission of developing strategies for the prevention of osteoporotic fractures and improving the health and well-being of older adults.