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

项目概要/摘要 衰老是造成大多数女性和男性骨折的原因。骨骼中的细胞变化 老年小鼠的死亡率与在老年人中观察到的相似。在小鼠中，骨小梁丢失与低 骨重建，而皮质变薄和多孔性与高骨重建相关。这些发现 表明这两个区室中骨丢失的分子机制不同。蜂窝 衰老导致多种组织的功能随年龄增长而下降，DNA损伤是主要原因 衰老DNA损伤通过激活p53和上调细胞周期抑制剂引起衰老 p21和/或p16。DNA损伤也会导致转录因子GATA 4的积累，这会促进DNA的降解。 衰老相关分泌表型（SASP）。全身清除衰老细胞延迟几个年龄- 相关疾病和增加老年小鼠的骨量。我们已经证明了骨祖细胞的数量 在小鼠骨髓中，这些细胞随着年龄的增长而下降，并且这些细胞具有增加的衰老标记。皮质 骨细胞在老年小鼠中也表现出增加的衰老标志物，这与骨细胞的衰老标志物的升高有关。 生产RANKL。通过DNA损伤诱导骨器官培养物的衰老足以增加 GATA 4和RANKL生产。此外，体外GATA 4的过表达足以增加RANKL 以及SASP的其他组成部分。向老年小鼠施用衰老抑制剂减弱衰老标志物 在骨祖细胞和骨细胞中。值得注意的是，骨细胞中缺乏RANKL的小鼠受到保护，免于骨细胞中RANKL的丢失。 皮质骨而非松质骨随年龄增长而变化。我们假设在骨祖细胞中p53/p21的激活 导致其衰老，从而减少成骨细胞数量和骨形成， 衰老骨细胞在皮质骨（而非小梁骨）中的蓄积增加了RANKL和骨 通过GATA 4刺激的吸收。在目标1中，我们将确定成骨细胞系细胞的DNA损伤是否 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的小鼠- 在整个成骨细胞谱系中或仅在成熟成骨细胞和骨细胞中诱导衰老。 衰老清除剂PZ 15227的施用将揭示表型的哪些组分是由于衰老。 在目标2中，我们将确定p53/p21通路在骨祖细胞中对骨骼衰老的贡献， p53或p21功能丧失的小鼠。在目标3中，我们将研究衰老的差异贡献， 骨细胞对GATA 4老化小鼠随年龄增长的骨吸收增加的影响 骨细胞功能丧失。我们还将量化皮质骨与小梁骨中的骨细胞衰老， 确定衰老骨细胞是否表达更高水平的RANKL。成功完成这些 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，并帮助澄清不同的老化机制，在皮质骨与松质骨。

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.