Role of Mitochondrial Deacetylase Sirt3 in Skeletal Homeostasis

线粒体是氧化磷酸化过程中产生的细胞三磷酸腺苷的主要来源。最近的研究表明，线粒体还参与其他功能，如细胞死亡、活性氧(ROS)的产生、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。尽管仍未完全了解，但线粒体蛋白质网络的这种翻译后修饰似乎对维持健康、正常运作的线粒体很重要。毫不奇怪，在人类疾病和小鼠模型中，线粒体功能障碍与衰老的许多方面有关，包括神经退行性变、心脏病、胰岛素抵抗和骨质丢失。然而，随着年龄的增长，骨量的减少是由于破骨细胞中线粒体功能的改变(S)还是其他类型的细胞所致，目前尚不清楚。本申请中提出的工作的主要目标是确定线粒体功能在骨细胞生理学和疾病中的作用。为了研究线粒体的功能，我们将操纵Sirtuin 3(SIRT3)，这是一种对线粒体功能和能量平衡都具有关键作用的分子，但在破骨细胞和成骨细胞中尚未得到明确的研究。SIRT3是一种依赖NAD+的脱乙酰酶，主要定位于线粒体，在代谢酶和复合体I亚基的激活中具有独特的作用。SIRT3去乙酰化并激活Ndufa9，Ndufa9是一个重要的复合I亚单位，导致细胞内ATP水平的提高和氧化磷酸化。SIRT3还可以与乙酰辅酶A合成酶相互作用，影响Bcl2缺失引起的G1期停滞，从而调节细胞死亡。所有这些作用的丧失可能会导致线粒体功能障碍和ROS水平异常升高，很可能是通过下调复合体I的活性。我们的初步数据表明，雌激素的抗破骨作用与早期破骨细胞前体细胞复合体I活性和ATP产生减少有关，而SIRT3在破骨细胞形成过程中表达上调。此外，在小鼠中，SIRT3的全局缺失可以防止与年龄相关的骨丢失，并伴随着骨吸收的减少。从体外培养的成骨细胞前体细胞中去除SIRT3也会减少成骨细胞的分化和矿化。这些观察结果奠定了假设的基础，即SIRT3通过调节破骨细胞和成骨细胞中的线粒体功能在骨骼动态平衡中发挥重要作用(S)。为了验证这些假说，我们将研究SIRT3在破骨细胞(AIM 1)和成骨细胞(AIM 2)中的缺失是否可以防止因年龄增长或雌激素缺乏而导致的骨量损失。此外，我们将进行体外研究，以确定破骨细胞和成骨细胞中与其对分化和功能的影响有关的SIRT3靶蛋白，方法是对具有或不具有SIRT3基因的原代骨髓来源的巨噬细胞的整体蛋白质组和赖氨酸乙酰组进行定量分析(AIM 3)。这项工作的成功完成将有助于揭示导致骨质疏松症的新机制，并将促进对骨骼如何应对 线粒体功能的改变。

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.