Role of Mitochondrial Deacetylase Sirt3 in Skeletal Homeostasis

线粒体是通过氧化磷酸化过程产生的细胞ATP的主要来源。最近的见解表明，线粒体还参与其他任务，如细胞死亡，活性氧（ROS）产生，免疫和钙稳态。线粒体蛋白质赖氨酸乙酰化在这些代谢过程中起着关键作用。虽然还没有完全理解，但线粒体蛋白网络的这种翻译后修饰似乎对维持健康，正常功能的线粒体很重要。不出所料，在人类疾病以及小鼠模型中，线粒体功能障碍与衰老的许多方面有关，包括神经变性、心脏病、胰岛素抵抗和骨质流失。然而，随着年龄的增长，骨量减少是否是由于破骨细胞或其他细胞类型中线粒体功能的变化，这在很大程度上是未知的。本申请中提出的工作的主要目标是建立线粒体功能在骨细胞生理学和疾病中的作用。为了研究线粒体功能，我们将操纵Sirtuin 3（Sirt 3），这是一种对线粒体功能和能量稳态都具有关键作用的分子，在破骨细胞和成骨细胞中尚未明确研究。Sirt 3是一种主要定位于线粒体的NAD+依赖性脱乙酰酶，在代谢酶和复合物I亚基的活化中具有独特的作用。Sirt 3去乙酰化并激活重要的复合物I亚基Ndufa 9，导致细胞ATP水平和氧化磷酸化的增强。Sirt 3还可以与乙酰辅酶A合成酶相互作用，影响Bcl-2缺失诱导的G1期阻滞，从而调节细胞死亡。所有这些作用的丧失可能导致线粒体功能障碍和异常增强的ROS水平，最有可能是通过下调复合物I活性。我们的初步数据表明，雌激素的抗破骨细胞生成作用与早期破骨细胞前体中复合物I活性和ATP产生的降低有关，并且在破骨细胞生成过程中Sirt 3表达上调。此外，小鼠中Sirt 3的整体缺失防止了年龄相关的骨丢失，伴随着骨吸收的减少。体外从成骨细胞祖细胞中去除Sirt 3也会降低成骨细胞分化和矿化。这些观察结果形成了Sirt 3通过调节破骨细胞和成骨细胞中的线粒体功能在骨骼稳态中起重要作用的假设的基础。为了验证这些假设，我们将研究破骨细胞（Aim 1）和成骨细胞（Aim 2）中Sirt 3的缺失是否可以防止由于年龄增长或雌激素缺乏引起的骨量丢失。此外，我们将进行体外研究，以确定在破骨细胞和成骨细胞的Sirt 3靶蛋白，负责其对分化和功能的影响，使用定量分析的整体蛋白质组和赖氨酸乙酰组在原代骨髓衍生的巨噬细胞有或没有Sirt 3基因（目标3）。这项工作的成功完成将揭示新的机制，有助于骨质疏松症，并将推进知识的骨骼如何回应， 线粒体功能的变化。

Mitochondria are the major source of cellular ATP generated through the process of oxidative phosphorylation. Recent insights have revealed that mitochondria are also involved in other tasks, such as cell death, reactive oxygen species (ROS) generation, immunity, and calcium homeostasis. Mitochondrial protein lysine acetylation plays a key role in these metabolic processes. Although still not completely understood, this post-translational modification of mitochondrial protein networks seem to be important for maintenance of healthy, properly functioning mitochondria. Unsurprisingly, in human diseases as well as mouse models, mitochondrial dysfunction has been linked to numerous aspects of aging, including neurodegeneration, cardiopathologies, insulin resistance, and bone loss. However, it is largely unknown whether the reduced bone mass with advancing age is due to a change in mitochondrial function(s) in osteoclasts or in other cell types. The primary goal of the work proposed in this application is to establish the role of mitochondrial function in bone cell physiology and disease. In order to investigate mitochondrial function, we will manipulate Sirtuin 3 (Sirt3), a molecule with critical roles for both mitochondrial function and energy homeostasis that has not clearly been studied in osteoclasts and osteoblasts. Sirt3 is an NAD+- dependent deacetylase localized primarily to the mitochondria, with a unique role in the activation of metabolic enzymes and Complex I subunits. Sirt3 deacetylates and activates Ndufa9, an important Complex I subunit, leading to enhancement of cellular ATP levels and oxidative phosphorylation. Sirt3 can also interact with the acetyl-CoA synthetase, affect G1 arrest induced by loss of Bcl-2 and, thereby, regulate cell death. Loss of all of these roles can contribute to mitochondrial dysfunction and aberrantly enhanced ROS levels, most likely by down-regulating Complex I activity. Our preliminary data shows that the anti-osteoclastogenic actions of estrogens are associated with decreased Complex I activity and ATP production in early osteoclast precursors, and that Sirt3 expression is upregulated during osteoclastogenesis. Further, global deletion of Sirt3 in mice prevents age-related bone loss, accompanied by a decrease in bone resorption. Removal of Sirt3 from osteoblast progenitors in vitro also reduces osteoblast differentiation and mineralization. These observations form the foundation for the hypothesis that Sirt3 plays an essential role in skeletal homeostasis by regulating mitochondrial function(s) in osteoclasts and osteoblasts. To test these hypotheses, we will examine whether deletion of Sirt3 in osteoclasts (Aim 1) and osteoblasts (Aim 2) prevents the loss of bone mass caused by advancing age or estrogen deficiency. Further, we will perform in vitro studies to identify Sirt3 target proteins in osteoclasts and osteoblasts that are responsible for their effects on differentiation and function using quantitative analysis of global proteome and lysine acetylome in primary bone marrow-derived macrophages with or without Sirt3 genes (Aim 3). Successful completion of this work will shed light on novel mechanisms that contribute to ost oporosis and will advance knowledge of how the skeleton responds to changes in mitochondrial function.