CSF-1 Gene Expression in Osteoclast Biology

描述（申请人提供）：骨质流失和骨折是老年发病的主要原因。老年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的关键决定因素，氧化应激影响骨细胞的存活/功能，这对骨重塑至关重要。该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途径，并确定治疗靶点以防止随年龄增长的骨质流失。CSF-1和CSF-1R由骨细胞表达。CSF-1或其异构体（可溶性CSF-1和细胞表面CSF-1）调节骨细胞存活/功能的机制尚未探索。氧化应激有助于骨细胞死亡和骨质流失随着年龄的增长。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。我们产生了具有全局CSF-1缺乏症（CSF-1KO）的小鼠，这些小鼠表现出骨质疏松，骨折增加，骨细胞缺陷包括凋亡，与NADPH氧化酶Nox4表达/活性增加和Cx43表达减少相关。CSF-1降低培养骨细胞的NADPH氧化酶活性，与WT骨细胞相比，CSF-1KO骨细胞显示Nox4升高和mTOR通路激活，表明CSF-1保护氧化应激。骨细胞/晚期成骨细胞中CSF-1条件敲除（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，随着年龄的增长易发生骨质流失和骨折。我们假设：a)随着年龄的增长，CSF-1的骨细胞cKO增加Nox4和氧化应激，损害骨细胞，加速骨缺损；b) CSF-1cKO骨细胞中Nox4的缺失减少氧化应激，随着年龄的增长恢复骨细胞功能/骨重塑；c)在衰老过程中，CSF-1cKO骨细胞中表达sCSF-1比csCSF-1更大程度地促进骨细胞存活和骨重塑。我们将在以下几个方面验证这些假设：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。将检测WT和CSF-1cKO小鼠的骨表型，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及CSF-1调控骨细胞存活的机制。为了解剖CSF-1和Nox4之间的相互作用，我们将在培养的骨细胞中产生CSF-1和Nox4缺失的小鼠，并分析CSF-1调节骨细胞存活的信号机制；3)测定CSF-1同种异构体对CSF-1cKO小鼠骨缺损的修复能力。这将通过转基因方法在CSF-1cKO小鼠的骨细胞中靶向sCSF-1或csCSF-1来实现。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，以提高对骨强度和寿命至关重要的骨细胞活力。

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.