Role of p21 positive senescent cells in radiation-induced skeletal injury and repair

项目摘要/摘要 老年癌症幸存者接受治疗后骨骼退化和相关骨折风险加剧 放射治疗(RTX)，影响独立生活，降低生活质量。细胞衰老，其中之一 RTX诱导DNA损伤后诱导的主要途径以促炎为特征 衰老相关分泌表型(SASP，由趋化因子、细胞因子、生长因子、基质组成 降解酶等)，主要受细胞周期蛋白依赖性激酶抑制物(CDKi‘s)p16INK4a和 P21Cip1。直到最近，人们了解到p16INK4a和p21Cip1在所有衰老细胞中共同表达并受到调控 它们的功能可以互换。然而，这一理论与p16INK4a和p16INK4a的表达模式不匹配 P21Cip1在RTX后或老化中。利用基因表达和RNA原位杂交研究，我们最近 研究表明，在骨髓细胞、成骨细胞和骨细胞中，细胞表达p21或p16INK4a。 不依赖于两个衰老标记的表达。只有一小部分细胞同时表达p16INK4a 和p21Cip1。此外，我们有令人信服的初步数据使用骨细胞的质量细胞学，这是 使我们能够可视化这些独立的、独特的p16INK4a和p21Cip1表达细胞群体，而没有任何 SASP的共表达，提示这些CDK具有生理功能。有趣的是，在一项平行分析中 在受辐射的骨骼中，我们发现CD11b+p21+髓系细胞群水平升高，并伴随着 通过表达几个SASP因子，从而使我们能够将这些p21+细胞定性为p21+衰老 (p21+SEN)细胞。在最近的一项开创性发现中，使用携带转基因的转基因小鼠能够选择性地 P21-ATTAC中p16INK4a和p21Cip1表达细胞的消除及p21+SEN细胞的消除 [通过靶向激活caspase实现细胞凋亡]小鼠，但不能消除p16+SEN细胞中的p16-ink- ATTAC小鼠，可以减轻幼鼠骨骼中大部分与RTX相关的不良事件。这是不是 清除p21+SEN细胞的方法将减轻RTX相关的老年小鼠的骨退化，这是 有没有预先存在的高负担衰老细胞，还有待观察。基于我们令人信服的初步调查 数据，我们将检验我们的中心假说：“p21+SEN细胞的急性生成介导了RTX相关的骨骼 恶化和BMSC功能障碍，但有针对性的早期清除p21+SEN细胞可以缓解RTX相关 慢性骨骼退化和促进骨折愈合“。为了检验我们的中心假设，我们的目标是：(Aim 1)确定早期清除RTX相关骨骼退化的关键机制 年轻和老年小鼠的p21+SEN细胞，(目标2)：评估细胞特异性后骨结构的变化 清除(使用我们的新型Cre-loxP小鼠，p21-LOX-ATTAC)p21+CD11b+SEN髓系细胞和(Aim3) 评估衰老细胞的预先清除是通过药物清除还是通过p21+SEN细胞的遗传清除 促进骨折愈合。该项目将解决与衰老的基本生物学相关的问题以及p21在 并为支持早期干预可能被证明有效对抗 RTX的不良变化，并缓解慢性骨骼退化和降低潜在的风险 骨折。

Project Summary/Abstract Skeletal deterioration and related fracture risk is exacerbated in the elderly cancer survivors receiving radiation treatment (RTx), affecting independent living, and reducing quality of life. Cellular senescence, one of the major pathways induced following RTx-induced DNA damage, is characterized by a pro-inflammatory senescence associated secretory phenotype (SASP, consisting of chemokines, cytokines, growth factors, matrix degrading enzyme, etc), and is mainly regulated by cyclin dependent kinase inhibitors (CDKi’s) p16Ink4a and p21Cip1. Till recently, it was understood that p16Ink4a and p21Cip1 co-expressed in all senescent cells and regulated their function interchangeably. This theory however did not match with the expression pattern of p16Ink4a and p21Cip1 post-RTx or in aging. Using gene expression and RNA in situ hybridization studies, we have recently shown that cells express p21 or p16Ink4a in unique populations of bone marrow cells, osteoblasts, and osteocytes independent of the expression of either senescence marker. Only a small proportion of cells express both p16Ink4a and p21Cip1. Furthermore, we have compelling preliminary data using mass cytometry of bone cells, which allowed us to visualize these independent unique populations of p16Ink4a and p21Cip1 expressing cells without any coexpression of SASP, suggesting a physiological function of these CDKis. Interestingly, in a parallel analysis in radiated bones, we identified elevated levels of Cd11b+p21+ myeloid cell population, which was accompanied by expression of several SASP factors, thus allowing us to characterize these p21+ cells as p21+ senescent (p21+SEN) cells. In a recent seminal finding, using transgenic mice harboring transgenes that enable the selective elimination of either p16Ink4a or p21Cip1 expressing cells, the elimination of p21+SEN cells in the p21-ATTAC [apoptosis through targeted activation of caspase] mice, but not the elimination of p16+SEN cells in the p16-INK- ATTAC mice, could mitigate most of the RTx-related adverse events in bone in young mice. Whether this approach of clearance of p21+SEN cells will work to alleviate RTx-related bone deterioration in old mice, which have a pre-existing high burden of senescent cells, remains to be seen. Based on our compelling preliminary data, we will test our central hypothesis that: “Acute generation of p21+SEN cells mediate RTx-related skeletal deterioration and BMSC dysfunction, but targeted early clearance of p21+SEN cells can alleviate RTx-related chronic skeletal deterioration and promote fracture healing”. To test our central hypothesis, our aims are: (aim 1)To identify key mechanisms that are involved in RTx-related skeletal deterioration following early clearance of p21+SEN cells in young and aged mice, (aim 2): To assess bone architectural changes following cell specific clearance (using our novel Cre-LoxP mice, p21-LOX-ATTAC) of p21+Cd11b+SEN myeloid cells and (aim3): To assess if prior clearance of senescent cells pharmacologically or by genetic clearance of p21+SEN cells will promote fracture healing. The project will address questions related to basic biology of aging and role of p21 in skeletal cells and lay the groundwork to support the idea that an early intervention could prove effective to counter adverse changes from RTx, and to alleviate chronic skeletal deterioration and reduce the risk of potential fractures.