CSF-1 Gene Expression in Osteoclast Biology

描述(由申请人提供)：骨质丢失和骨折是随年龄增长而发病的主要原因。年龄相关性骨病的发病机制尚未明确。我们的研究表明，骨细胞中CSF-1缺乏和NOX4氧化酶表达增加是氧化应激影响骨细胞生存/功能的关键决定因素，而氧化应激对骨重建至关重要。这项建议的长期目标是描述CSF-1/氧化应激调节骨细胞稳态的机制，并确定防止随着年龄增长而发生骨丢失的治疗靶点。CSF-1和CSF-1R主要由骨细胞表达。脑脊液-1或其异构体--可溶性的(S)和细胞表面的(Cs)的脑脊液-1调节骨细胞存活/功能的机制尚不清楚。随着年龄的增长，氧化应激会导致骨细胞死亡和骨质流失。我们的发现表明，随着年龄的增长，骨细胞中CSF-1的表达下降。我们建立了全局缺乏CSF-1(CSF-1KO)的小鼠，表现出骨化病，骨折增加，骨细胞缺陷，包括细胞凋亡，与NADPH氧化酶NOX4表达/活性增加和Cx43表达减少相关。与WT骨细胞相比，CSF-1降低了培养骨细胞的NADPH氧化酶活性，并显示了NOX4的升高和mTOR通路的激活，提示CSF-1对氧化应激具有保护作用。在骨细胞/晚期成骨细胞中条件基因敲除(CKO)的DMP1Cre-CSF-1cKO小鼠也表现出NOX4增加，骨细胞缺陷，破骨细胞和骨形成减少，容易随着年龄的增长而发生骨丢失和骨折。我们假设：a)CSF-1的骨细胞CKO增加了NOX4和氧化应激，损伤了骨细胞，加速了随年龄增长的骨缺损；b)缺失了CSF-1cKO骨细胞中的NOX4，降低了氧化应激，恢复了骨细胞功能/骨重建；c)在CSF-1cKO骨细胞中表达sCSF-1，比csCSF-1更大程度地促进了骨细胞的存活和适当的骨重建。我们将在以下特定目标中验证这些假说：1)确定骨细胞中的CSF-1cKO对衰老过程中的骨表型和氧化还原状态的影响。将检测WT和CSF-1cKO小鼠的骨表型，并评估骨细胞的凋亡、NOX4和基因表达谱；2)确定NOX4在CSF-1cKO小鼠骨细胞中的作用以及CSF-1调控骨细胞存活的机制。为了剖析CSF-1和NOX4之间的相互作用，我们将建立CSF-1和NOX4在骨细胞中的CKO小鼠，并在培养的骨细胞中分析CSF-1调控骨细胞存活的信号机制；3)确定CSF-1亚型修复CSF-1cKO小鼠骨缺损的能力。这将使用转基因方法在CSF-1cKO小鼠的骨细胞中靶向sCSF-1或csCSF-1。这些研究将为CSF-1控制骨细胞存活/功能提供新的机制见解，并可能导致新的治疗策略，以提高骨细胞的活性，对骨强度和寿命至关重要。

DESCRIPTION (provided by applicant): 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.