CSF-1 Gene Expression in Osteoclast Biology

摘要 骨丢失和骨折是随着年龄增长而发病的主要原因。年龄相关骨的机制 疾病尚未被定义。我们的研究表明，CSF-1缺乏和Nox 4氧化酶增加， 在骨细胞中的表达是氧化应激的关键决定因素，其影响骨细胞存活/功能， 对骨骼重塑至关重要这项提案的长期目标是描绘出 通过CSF-1/氧化应激调节骨细胞稳态并鉴定治疗靶点以预防 随着年龄的增长骨质流失。CSF-1和CSF-1 R由骨细胞表达。CSF-1或 其同种型，可溶性和细胞表面CSF-1，调节骨细胞存活/功能， 探讨了随着年龄的增长，氧化应激导致骨细胞死亡和骨丢失。我们的研究结果表明 随着年龄的增长，骨细胞中CSF-1的表达下降。我们制造了CSF-1缺乏的小鼠， （CSF-1 KO），其显示具有增加的骨折和包括凋亡的骨细胞缺陷的骨硬化症， 与NADPH氧化酶Nox 4表达/活性增加和Cx43表达减少相关。CSF-1 降低培养的骨细胞中NADPH氧化酶的活性，并且CSF-1 KO骨细胞显示升高的 与WT骨细胞相比，Nox 4和mTOR通路的激活，表明CSF-1保护了骨细胞的增殖。 氧化应激骨细胞中CSF-1条件性敲除（cKO）的DMP 1Cre-CSF-1cKO小鼠/晚期 成骨细胞还显示出增加的Nox 4、骨细胞缺陷、减少的破骨细胞和骨形成， 随着年龄的增长易导致骨质流失和骨折。我们假设：a）CSF-1的骨细胞cKO增加 Nox 4和氧化应激损伤骨细胞并加速骨缺损，B）Nox 4的缺失， CSF-1cKO骨细胞降低氧化应激，恢复骨细胞功能/随年龄的骨重塑，c） CSF-1cKO骨细胞中sCSF-1的表达促进骨细胞存活和适当的骨重建， 比csCSF-1在衰老过程中的作用更大。我们将在以下具体目标中测试这些假设：1） 确定骨细胞中CSF-1cKO对衰老过程中骨表型和氧化还原状态的影响。WT和 将检查CSF-1cKO小鼠的骨表型，并评估骨细胞的凋亡、Nox 4 2）确定Nox 4在CSF-1cKO小鼠骨细胞中的作用， CSF-1调节骨细胞存活的机制。为了分析CSF-1和 Nox 4，将产生具有CSF-1和Nox 4在骨细胞中的cKO的小鼠，并且通过 将在培养的骨细胞中分析CSF-1调节骨细胞存活的能力; 3）确定CSF-1调节骨细胞存活的能力。 CSF-1同种型在CSF-1cKO小鼠中挽救骨缺损。这将通过使用转基因 靶向CSF-1cKO小鼠骨细胞中sCSF-1或csCSF-1的方法。这些研究将提供新的 CSF-1控制骨细胞存活/功能的机制见解，并可能导致新的治疗方法 提高骨细胞活力的策略对骨强度和寿命至关重要。

ABSTRACT Bone loss and fracture are leading causes of morbidity with aging. The mechanisms of age-related bone disease have not been defined. Our studies suggest that CSF-1 deficiency and increased Nox4 oxidase expression in osteocytes are key determinants of oxidant stress that impact osteocyte survival/function that is essential for bone remodeling. The long-term goal of this proposal is to delineate mechanistic pathways by which CSF-1/oxidative stress regulate osteocyte homeostasis and identify therapeutic targets to prevent bone loss with age. CSF-1 and CSF-1R are expressed by osteocytes. The mechanisms by which CSF-1 or its isoforms, soluble (s) and cell-surface (cs) CSF-1, regulate osteocyte survival/function have not been explored. Oxidative stress contributes to osteocyte demise and bone loss with aging. Our findings indicate that, with aging, CSF-1 expression declines in osteocytes. We generated mice with global CSF-1 deficiency (CSF-1KO) that show osteopetrosis with increased fractures and osteocyte defects including apoptosis, associated with increased NADPH oxidase Nox4 expression/activity and reduced Cx43 expression. CSF-1 decreases NADPH oxidase activity in cultured osteocytes and CSF-1KO bone osteocytes show elevated Nox4 and activation of the mTOR pathway compared to WT osteocytes, suggesting that CSF-1 protects from oxidant stress. DMP1Cre-CSF-1cKO mice with conditional knockout (cKO) of CSF-1 in osteocytes/late osteoblasts also show increased Nox4, osteocyte defects, reduced osteoclasts and bone formation, predisposing to bone loss and fracture with age. We hypothesize that: a) osteocyte cKO of CSF-1 increases Nox4 and oxidative stress, impairs osteocytes and accelerates bone defects with age, b) deletion of Nox4 in CSF-1cKO osteocytes decreases oxidative stress, restores osteocyte function/bone remodeling with age, c) expression of sCSF-1 in CSF-1cKO osteocytes promotes osteocyte survival and proper bone remodeling to a greater extent than csCSF-1 during aging. We will test these hypotheses in the following specific aims: 1) Determine the effect of CSF-1cKO in osteocytes on bone phenotype and redox state during aging. WT and CSF-1cKO mice will be examined for bone phenotype and osteocytes will be assessed for apoptosis, Nox4 and gene expression profile; 2) Determine the role of Nox4 in osteocytes of CSF-1cKO mice and mechanisms by which CSF-1 regulates osteocyte survival. To dissect the interplay between CSF-1 and Nox4, mice with cKO of CSF-1 and Nox4 in osteocytes will be generated and signaling mechanisms by which CSF-1 regulates osteocyte survival will be analyzed in cultured osteocytes; 3) Determine the ability of CSF-1 isoforms to rescue bone defects in CSF-1cKO mice. This will be accomplished using a transgenic approach to target sCSF-1 or csCSF-1 in osteocytes of CSF-1cKO mice. These studies will provide new mechanistic insights by which CSF-1 controls osteocyte survival/function and may lead to novel therapeutic strategies for improving osteocyte viability crucial for bone strength and longevity.