Modifying the mechanotransduction of bone by targeting purinergic receptors

通过靶向嘌呤能受体来改变骨的机械传导

• 批准号: 10242712
• 负责人: Joseph Daniel Gardinier
• 金额: $ 32.12万
• 依托单位: HENRY FORD HEALTH SYSTEM
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10242712
• 关键词: Actins; Adult; Age-Related Bone Loss; Animal Model; Bone Tissue; CRISPR/Cas technology; Calcium Signaling; Cell Line; Cells; Clinical; Development; Economic Burden; Elderly; Engineering; Exercise; Gene Expression; Goals; Health; Health Care Costs; Hindlimb Suspension; In Vitro; Incidence; Knock-out; Knockout Mice; Knowledge; Malignant neoplasm of lung; Malignant neoplasm of prostate; Mission; Modeling; Mus; Muscle; Nucleotides; Osteocytes; Osteogenesis; Osteoporosis; Osteoporosis prevention; Outcome; P2Y2 receptor; Paralysed; Personal Satisfaction; Phosphorylation; Physical Exercise; Physical activity; Play; Prevalence; Prevention strategy; Public Health; Purinoceptor; Role; Series; Signal Transduction; Stress Fibers; Testing; Therapeutic; Therapeutic Agents; Tissues; United States National Institutes of Health; aged; aging population; base; bone; bone loss; bone mass; cofilin; conditional knockout; dentin matrix protein 1; efficacy evaluation; experimental study; fluid flow; fracture risk; improved; in vivo; inhibitor/antagonist; innovation; malignant breast neoplasm; mechanical load; mechanical properties; mechanotransduction; new therapeutic target; novel therapeutic intervention; novel therapeutics; osteoporosis with pathological fracture; overexpression; prevent; protein expression; response; therapy design; treadmill

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.

骨质疏松性骨折是常见的，发病率增加，并有很高的相关经济负担。 由于缺乏增加骨密度的治疗策略， 形成并提高组织强度。骨形成是骨细胞对机械力的反应的函数， 在体力活动和锻炼期间的负荷。骨细胞嘌呤能信号通过释放 核苷酸在调节骨对负荷的适应中起关键作用。特别是我们发现 P2 Y2受体下调骨细胞对负荷的敏感性， 伴随着通过cofilin磷酸化的肌动蛋白应激纤维形成（ASFF）的增加。这些 研究结果是重要的，因为他们建议靶向P2 Y2信号传导作为一种潜在的策略，以提高 骨细胞机械传导和增加骨形成。然而，P2 Y2影响的程度 通过ASFF调节骨细胞对负荷的敏感性来形成骨尚不清楚。这些差距在 知识限制了我们开发新的治疗策略， 老年人骨折的风险。我们的长期目标是预防骨质疏松症，降低骨折风险， 人口老龄化本研究的目的是确定嘌呤能信号通过P2 Y2的作用， 受体在调节合成代谢反应负荷。本研究的前提是阻断P2 Y2 信号传导具有预防年龄相关的骨丢失和降低骨折风险的治疗潜力。中央 一种假说认为，阻断P2 Y2信号传导将增加骨细胞对负荷的敏感性， 增加骨量和组织强度以响应负荷。中心假设将在以下条件下进行检验： 三个具体目标。目的1将确定P2 Y2信号在体外影响骨细胞增殖的程度。 通过cofilin磷酸化调节ASFF来提高对负荷的敏感性和总体响应。我们的方法在 目的1利用使用CRISPR/Cas9产生的骨细胞敲除细胞系来检查它们对CRISPR/Cas9的体外应答。 流体流动目的2将确定体内P2 Y2表达在多大程度上有助于骨形成。 响应加载和卸载。我们在目标2中的方法将规定跑步机锻炼和后肢锻炼 固定到靶向骨细胞P2 Y2表达的条件敲除小鼠。目标3将审查 选择性P2 Y2 R抑制剂AR-C118925增加老年人对负荷的合成代谢反应的疗效 以及预防与年龄相关的骨质流失。我们在目标3中的方法将治疗野生型以及P2 Y2- 用AR-C118925敲除小鼠，以鉴定非骨细胞特异性的脱靶效应。本研究 创新之处在于：1）评价了一种新型治疗剂（AR-C118925）用于增加骨形成， 2)使用新的细胞系和动物模型来建立P2 Y2信号传导作为增加骨骼的独特机制。 马萨诸塞州总的来说，这项研究意义重大，因为我们希望它能证明 靶向P2 Y2 R信号传导以增加骨形成并降低骨折风险。