Osteocyte Apoptosis and Regulation of Bone Resorption with Aging

描述（由申请人提供）：骨骼老化与小鼠和人类骨细胞凋亡的发生率增加有关。该应用的工作表明，随着衰老而发生的骨骼综合症是通过间隙连接蛋白连接蛋白（Cx）43的骨细胞特异性缺失来模拟的。骨细胞中缺乏Cx43的小鼠表现出骨细胞凋亡增加和空腔积聚，以及OPG水平降低，并且Cx43缺陷的骨细胞表达较低水平的生存相关microRNA（miR）miR21。此外，年老小鼠的骨骼表现出 Cx43、OPG 和 miR21 表达减少。我们在体外证明，凋亡的骨细胞释放高迁移率族蛋白 1 (HMGB1)，该蛋白可降低 OPG 表达，并通过激活晚期糖基化终末产物 (RAGE) 受体来增加破骨细胞的募集和分化。 HMGB1 还激活 Toll 样受体 4 (TLR4)。然而，HMGB1 受体 RAGE 或 TLR4 的激活是否对 Cx43 缺陷或老年小鼠的骨骼表型有影响尚不清楚。凋亡细胞释放 HMGB1 的机制尚不清楚。一种潜在的机制可能涉及在凋亡细胞中打开 Pannexin1 (Pax1) 通道。这种机制是否在经历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针对骨细胞凋亡和吸收来改善对衰老对骨骼的不利影响的管理。本申请的具体目标是确定衰老过程中骨细胞凋亡和骨吸收升高所涉及的信号通路。我们认为，随着衰老而发生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所致。我们假设低 OPG 和随之而来的 RANKL/OPG 比率增加是破骨细胞发育的许可事件；而低miR21会减少抗凋亡激酶信号传导并激活半胱天冬酶，随后Panx1通道打开并释放HMGB1，将破骨细胞引导至凋亡骨细胞聚集的区域。为了检验这一假设，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以及已建立的原代骨细胞。目标 1 将检查 Cx43/miR21 对衰老过程中骨细胞凋亡的作用。目标 2 将研究 HMGB1 在 Cx43 缺陷和衰老过程中破骨细胞募集和分化中的作用。目标 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缺乏 Cx43 和老年动物中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和目标骨吸收之间的分子联系。此外，它们将为治疗提供基础，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。

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.