Molecular basis of mechanotransduction in bone cells

描述（由申请人提供）： 骨质疏松症是美国的一个重大健康问题，是一种骨形成和骨吸收过程不平衡的疾病。众所周知，机械负载会影响这些过程。我们的长期目标是阐明骨细胞对机械载荷反应的分子机制。在过去的几年中，大量证据表明，细胞外核苷酸（例如 ATP）通过 P2 嘌呤能受体发出信号，在骨行为的调节中发挥着重要作用。我们和其他人证明，机械负荷诱导的流体流动导致骨细胞中 ATP 释放，并且 ATP 随后通过 P2Y 嘌呤能受体激活钙信号传导途径。 P2Y 受体是一种 G 蛋白偶联受体 (GPCR)，在正常生物过程中普遍至关重要。然而，P2Y 受体在骨生物学，特别是骨力转导中的作用尚不清楚。我们的初步数据表明，P2Y2（P2Y 受体的一种亚型）由 ATP 激活，参与响应流体流动的基因表达变化。此外，GPCR 的脱敏最近已被证明是骨骼中机械传感装置的重要组成部分。我们的结果表明，G 蛋白偶联受体激酶 2 (GRK2) 参与响应流体流动的 P2Y 激活脱敏。有趣的是，我们的初步结果表明 P2Y2 缺陷小鼠表现出中等的骨表型。更重要的是，我们的数据表明 P2Y2 缺陷小鼠对机械负荷的成骨反应受到抑制。因此，我们的中心假设是生物物理信号，例如流体流动，通过涉及 P2Y 嘌呤能受体的机制调节骨细胞行为，P2Y 嘌呤能受体被 G 蛋白偶联受体激酶脱敏。为了检验这一假设，我们将进行一系列体外和体内实验，以检查 P2Y 受体在骨细胞机械转导中的作用（目标 1）、GRK（目标 2）和甲状旁腺激素 (PTH)（目标 3）对 P2Y 受体的调节，以及 P2Y 和 GRK 缺陷小鼠完整骨骼的机械负荷效应（目标 4）。公共卫生相关性。骨质疏松症是一个严重的健康问题，影响着超过 4400 万美国人。拟议的项目是确定通过 P2Y 受体负责骨骼中机械转导的分子机制。该项目的完成将最终为具有机械成分的骨疾病（例如骨质疏松症）提供药物干预的新目标。

DESCRIPTION (provided by applicant): Osteoporosis, a significant health problem in US, is a disease with imbalance between the processes of bone formation and bone resorption. It is known that mechanical loading can influence these processes. Our long-term goal is to elucidate the molecular mechanisms of bone cell response to mechanical loading. Over the past several years, substantial evidence has indicated that extracellular nucleotides, such as ATP, signaling through P2 purinergic receptors, play an important role in the regulation of bone behavior. We and others demonstrated that mechanical loading induced fluid flow causes ATP release in bone cells, and ATP subsequently activates calcium signaling pathways via P2Y purinergic receptors. P2Y receptor is a G protein-coupled receptor (GPCR), which is universally critical in normal biological processes. However, the role of P2Y receptors in bone biology, particularly in bone mechanotransduction, is unknown. Our preliminary data suggest that P2Y2, one subtype of P2Y receptors, activated by ATP is involved in changes in gene expression in response to fluid flow. In addition, desensitization of a GPCR's has recently been shown to be an important component of the mechanosensing apparatus in bone. Our results suggest that G-protein coupled receptor kinase 2 (GRK2) is involved in the desensitization of P2Y activation in response to fluid flow. Interestingly, our preliminary results suggest that P2Y2 deficient mice exhibit moderate bone phenotype. More importantly, our data suggest that the osteogenic response to mechanical loading in P2Y2 deficient mice is suppressed. Thus, our central hypothesis is that biophysical signals, such as fluid flow, regulate bone cell behavior via a mechanism involving P2Y purinergic receptors which are desensitized by G-protein coupled receptor kinases. To test this hypothesis we will conduct a series of in vitro and in vivo experiments to examine the role of P2Y receptors in bone cell mechanotransduction (aim 1), the regulation of P2Y receptors by GRK (aim 2) and parathyroid hormone (PTH) (aim 3), and mechanical loading effects on intact bone from mice deficient in P2Y and GRK (aim 4). PUBLIC HEALTH RELVANCE. Osteoporosis is a significant health problem that affects over 44 million Americans. The proposed project is to determine the molecular mechanism responsible for mechanotransduction in bone via P2Y receptors. Completion of this project will ultimately lead to novel targets for pharmacological intervention in bone diseases that have a mechanical component, such as osteoporosis.