Osteocyte Apoptosis and Regulation of Bone Resorption with Aging

描述（由申请人提供）：在小鼠和人类中，骨骼的老化与骨细胞凋亡的患病率增加有关。导致这一应用的工作表明，骨细胞间隙连接蛋白（Cx） 43的特异性缺失模仿了随着衰老而发生的骨骼综合征。骨细胞中缺乏Cx43的小鼠表现出骨细胞凋亡增加和空腔隙积累，OPG水平降低，而缺乏Cx43的骨细胞表达较低水平的存活相关microRNA (miR) miR21。此外，老龄小鼠的骨骼表现出Cx43、OPG和miR21表达的降低。我们在体外发现，凋亡的骨细胞释放高迁移率的组框蛋白1 (HMGB1)，通过激活晚期糖基化终产物受体（RAGE），降低OPG的表达，增加破骨细胞的募集和分化。HMGB1也激活toll样受体4 （TLR4）。然而，HMGB1受体RAGE或TLR4的激活是否在cx43缺陷或老年小鼠的骨骼表型中起作用尚不清楚。凋亡细胞释放HMGB1的机制尚不清楚。一个潜在的机制可能涉及凋亡细胞中Pannexin1 （Pax1）通道的打开。这种机制是否在发生凋亡的骨细胞中起作用尚不清楚。我们研究的长期目标是通过靶向骨细胞凋亡和再吸收来改善衰老对骨骼不利影响的管理。本应用的具体目的是确定衰老过程中骨细胞凋亡和骨吸收升高的信号通路。我们认为，随着年龄增长而出现的骨脆性综合征是由于骨细胞Cx43、OPG和miR21表达减少导致骨细胞凋亡增加。我们假设低OPG和随之而来的RANKL/OPG比值的增加是破骨细胞发育的许可事件；而低miR21降低抗凋亡激酶信号并激活caspase，导致Panx1通道打开和HMGB1释放，将破骨细胞引导到累积凋亡的骨细胞区域。为了验证这一假设，我们将进行体内和体外研究的结合，包括使用新型转基因小鼠，建立和原代骨细胞。目的1将研究Cx43/miR21在衰老过程中骨细胞凋亡中的作用。目的2将探讨HMGB1在Cx43缺乏和衰老时破骨细胞募集和分化中的作用。目的3将探讨骨细胞Panx1去除对HMGB1释放和破骨细胞募集的影响。这些研究的成功完成将为老年动物在缺乏Cx43的情况下骨细胞凋亡增加提供分子基础。此外，这些研究将建立死亡骨细胞与靶向骨吸收之间的分子联系。此外，它们将为通过靶向HMGB1-RAGE/TLR4系统来抵消骨细胞凋亡增加对破骨细胞募集的影响的治疗提供基础。

DESCRIPTION (provided by applicant): Aging of the skeleton is associated with increased prevalence of osteocyte apoptosis in mice and humans. Work leading to this application shows that the skeletal syndrome that ensues with aging is mimicked by osteocyte specific deletion of the gap junction protein connexin (Cx) 43. Mice lacking Cx43 in osteocytes exhibit increased osteocyte apoptosis and accumulation of empty lacunae, and reduced OPG levels, and Cx43- deficient osteocytes express lower levels of the survival-associated microRNA (miR) miR21. In addition, bones from old mice exhibit reduced Cx43, OPG, and miR21 expression. We showed in vitro that apoptotic osteocytes release high mobility group box protein1 (HMGB1), which reduces OPG expression and increases osteoclast recruitment and differentiation through activation of receptors for advanced glycation end products (RAGE). HMGB1 also activated toll-like receptor 4 (TLR4). However, whether activation of HMGB1 receptors RAGE or TLR4 has a role in the skeletal phenotype of Cx43-deficient or old mice is not known. The mechanism by which HMGB1 is released by apoptotic cells is not known. A potential mechanism could involve opening of Pannexin1 (Pax1) channels in cells undergoing apoptosis. Whether this mechanism operates in osteocytes undergoing apoptosis is not known. The long-term goal of our studies is to improve the management of the adverse skeletal effects of aging by targeting osteocyte apoptosis and resorption. The specific objective of this application is to define the signaling pathways involved in the elevated osteocyte apoptosis and bone resorption in aging. We propose that the bone fragility syndrome that ensues with aging is due to increased osteocyte apoptosis as a consequence of reduced expression of osteocytic Cx43, OPG, and miR21. We hypothesize that low OPG and the consequent increase in the RANKL/OPG ratio acts as a permissive event for osteoclast development; whereas low miR21 reduces anti-apoptotic kinase signaling and activates caspases, with the consequent Panx1 channel opening and HMGB1 release, directing osteoclasts to areas with accumulated apoptotic osteocytes. To test this hypothesis, we will pursue a combination of in vivo and in vitro studies that include the use of novel genetically modified mice, and established and primary osteocytic cells. Aim 1 will examine the role of Cx43/miR21 on osteocyte apoptosis with aging. Aim 2 will investigate the role of HMGB1 in osteoclast recruitment and differentiation in Cx43 deficiency and aging. Aim 3 will explore the consequence of osteocytic Panx1 removal on HMGB1 release and osteoclast recruitment. Successful completion of these studies will provide the molecular basis for the in- creased osteocyte apoptosis in the absence of Cx43 and in old animals. In addition, these studies will establish the molecular link between dying osteocytes and targeted bone resorption. Moreover, they will offer the basis for treatments in which the effect of increased osteocyte apoptosis on osteoclast recruitment are counteracted by targeting the HMGB1-RAGE/TLR4 system.