Different consequences of cellular aging in cortical versus cancellous bone- Resubmission

项目摘要/摘要 无论是女性还是男性，衰老都是造成骨折的主要原因。骨骼中的细胞变化 在老年老鼠身上观察到的情况与在老年人身上观察到的相似。在小鼠中，骨小梁丢失与低血压有关 骨改建，而皮质变薄和孔洞与高的骨改建有关。这些发现 提示不同的分子机制是导致这两个椎间骨质丢失的原因。蜂窝 随着年龄的增长，衰老会导致多个组织的功能衰退，DNA损伤是主要原因 衰老的迹象。DNA损伤通过激活P53和上调细胞周期抑制物导致衰老 P21和/或p16。DNA损伤还会导致转录因子GATA4的积累，从而促进 衰老相关分泌表型(SASP)。衰老细胞的系统性清除延迟了几个年龄- 相关的疾病和增加老年小鼠的骨量。我们已经证明，骨祖细胞的数量 在小鼠中，骨髓随着年龄的增长而下降，这些细胞具有更多的衰老标志。大脑皮层 在衰老的小鼠中，骨细胞也表现出更多的衰老标志物，这与 RANKL的生产。DNA损伤诱导骨器官培养细胞衰老足以增加 GATA4和RANKL生产。此外，在体外过表达GATA4足以增加RANKL 和SASP的其他组成部分。老年小鼠服用衰老药物可减弱衰老的标志物 在骨祖细胞和骨细胞中。值得注意的是，在骨细胞中缺乏RANKL的小鼠受到保护，不会丢失 随着年龄的增长，皮质骨而不是松质骨。我们假设骨祖细胞中p53/p21的激活 导致其衰老，从而减少成骨细胞数量和骨形成， 衰老的骨细胞在皮质而不是小梁中积累，增加了RANKL和骨 通过GATA4刺激的吸收。在目标1中，我们将确定成骨细胞系细胞的DNA损伤 足以诱导衰老和减少骨量。为了做到这一点，我们将产生氧化应激小鼠- 在整个成骨细胞谱系或仅在成熟的成骨细胞和骨细胞中诱导衰老。 使用抗衰老的PZ15227将揭示由于衰老而导致的表型的哪些成分。 在目标2中，我们将确定骨祖细胞中的p53/p21通路在衰老中对骨骼老化的贡献。 P53或p21功能丧失的小鼠。在目标3中，我们将研究衰老的不同贡献 骨细胞对GATA4衰老小鼠骨小梁与皮质骨随增龄增加骨吸收的影响 骨细胞功能丧失。我们还将量化皮质骨和松质骨中的骨细胞衰老，并 确定衰老的骨细胞是否表达更高水平的RANKL。成功完成这些任务 研究应该首次确定骨祖细胞和骨细胞的衰老是否有助于 随着年龄的增长，骨量的损失，有助于阐明皮质骨和松质骨不同的衰老机制。

Project Summary/Abstract Aging is responsible for the majority of fractures in both women and men. The cellular changes in the skeleton of aged mice are similar to those observed in aged humans. In mice, trabecular bone loss is associated with low bone remodeling, while cortical thinning and porosity are associated with high bone remodeling. These findings suggest that different molecular mechanisms underlie the bone loss in these two compartments. Cellular senescence contributes to the functional decline of multiple tissues with age and DNA damage is a major cause of senescence. DNA damage causes senescence via activation of p53 and up-regulation of the cell cycle inhibitor p21 and/or p16. DNA damage also causes accumulation of the transcription factor GATA4, which promotes the senescence associated secretory phenotype (SASP). Systemic clearance of senescent cells delays several age- associated disorders and increases bone mass in old mice. We have shown that the number of osteoprogenitors in murine bone marrow declines with age and that these cells have increased markers of senescence. Cortical osteocytes also exhibit increased markers of senescence in aged mice and this is associated with elevated production of RANKL. Induction of senescence in bone organ cultures by DNA damage is sufficient to increase GATA4 and RANKL production. Moreover, overexpression of GATA4 in vitro is sufficient to increase RANKL and other components of the SASP. Administration of senolytics to old mice attenuates markers of senescence in osteoprogenitors and osteocytes. Notably, mice lacking RANKL in osteocytes are protected from the loss of cortical but not trabecular bone with age. We hypothesize that activation of p53/p21 in osteoprogenitors causes their senescence and thereby decreases osteoblast number and bone formation and that accumulation of senescent osteocytes in cortical, but not trabecular, bone increases RANKL and bone resorption via GATA4 stimulation. In Aim 1 we will determine whether DNA damage in osteoblast lineage cells is sufficient to induce senescence and reduce bone mass. To do this, we will generate mice with oxidative stress- induced senescence in either the entire osteoblast lineage or only in mature osteoblasts and osteocytes. Administration of the senolytic PZ15227 will reveal what components of the phenotype are due to senescence. In Aim 2 we will determine the contribution of the p53/p21 pathway in osteoprogenitors to skeletal aging by aging mice with p53 or p21 loss-of-function. In Aim 3 we will investigate the differential contribution of senescent osteocytes to increased bone resorption in trabecular versus cortical bone with age by aging mice with GATA4 loss-of-function in osteocytes. We will also quantify osteocyte senescence in cortical versus trabecular bone and determine whether senescent osteocytes express higher levels of RANKL. Successful completion of these studies should establish for the first time whether senescence of osteoprogenitors and osteocytes contributes to the loss of bone mass with age, and help clarify different aging mechanisms in cortical versus trabecular bone.