Chondrocyte Metabolic Stress in the Development of Osteoarthritis

描述(申请人提供)：肥胖是发生骨关节炎(OA)的最重要和可预防的危险因素之一。努力查明造成这种情况的原因 传统上，风险集中在肥胖导致的关节压力触发因素上，如机械因素和炎症因素。然而，对于肥胖如何损害软骨细胞的细胞防御机制，从而导致对关节压力的不适当或不充分的反应，我们的理解存在着根本的差距。申请者的长期目标是开发策略，要么上调软骨保护通路，要么下调因肥胖和衰老而变得失调的细胞分解代谢通路。这里的目的是确定肥胖如何增加线粒体蛋白的乙酰化，最终调节老化和炎症条件下的软骨分解代谢。这一重点来源于申请人令人兴奋的初步数据，将线粒体脱乙酰酶SIRT3对线粒体蛋白乙酰化的调节与年龄相关的下降与抗氧化剂防御和骨关节炎的损害联系起来。中心假设是，肥胖加剧了与年龄相关的慢性线粒体超乙酰化增加，导致软骨细胞氧化还原压力和软骨分解代谢。据认为，这种失衡是由SIRT3表达随年龄增长而下降，以及肥胖导致乙酰辅酶A产生和炎症增加所致。初步数据显示，线粒体抗氧化剂SOD2是软骨细胞超乙酰化的关键靶点。在这些和其他初步数据的指导下，该假说将通过三个具体目标进行检验：1)确定肥胖如何诱导促进线粒体蛋白乙酰化的代谢变化；2)确定积极或消极地操纵SIRT3表达对软骨细胞氧化还原动态平衡和软骨分解代谢的衰老和肥胖依赖效应；以及3)确定SIRT3调节促炎挑战后线粒体氧化还原动态平衡和软骨分解代谢途径激活的机制。成熟的饮食诱导肥胖和骨关节炎小鼠模型将与转基因小鼠结合使用，转基因小鼠允许有条件地删除或过度表达软骨中的SIRT3。AIMS 1和AIMS 2有针对性的基因组、蛋白质和代谢物检测方法，以确定促进线粒体乙酰化、改变抗氧化能力和诱发骨性关节炎的因素。目的3将使用体外白细胞介素1刺激试验来确定SIRT3敏感的软骨分解代谢途径。软骨特异性SOD2基因缺失的小鼠将为评估SOD2超乙酰化对软骨细胞氧化应激和下游分解代谢通路激活的影响提供参考。这种方法是创新的，因为它将肥胖研究的重点从细胞应激转移到应激易感性。这项拟议的研究具有重要意义，因为它将启动一项系统研究，即如何对线粒体蛋白质的可逆翻译后赖氨酸乙酰化进行积极或消极的操纵，以促进衰老和肥胖时的软骨保护。

DESCRIPTION (provided by applicant): Obesity is among the most significant and preventable risk factors for developing osteoarthritis (OA). Efforts to identify the causes of this risk have traditionally focused on obesity-induced triggers of joint stress, such as mechanical and inflammatory factors. However, there is a fundamental gap in our understanding about how obesity impairs chondrocyte cellular defense mechanisms resulting in inappropriate or insufficient responses to joint stresses. The applicant's long-term goal is to develop strategies t either up-regulate chondroprotective pathways or down-regulate cell catabolic pathways that become dysregulated with obesity and aging. The objective here is to determine how obesity increases the acetylation of mitochondrial proteins that ultimately regulate cartilage catabolism under aging and inflammatory conditions. This focus is derived from the applicant's exciting preliminary data linking an age-related decline in the regulation of mitochondrial protein acetylation by the mitochondrial deacetylase SIRT3 to impaired antioxidant defense and OA. The central hypothesis is that obesity exacerbates an age-related increase in chronic mitochondrial hyper-acetylation resulting in chondrocyte redox stress and cartilage catabolism. It is proposed that this imbalance is driven by an aging-dependent decline in SIRT3 expression coupled with an obesity-driven increase in acetyl-CoA production and inflammation. Preliminary data show that the mitochondrial antioxidant, SOD2, is a key target of hyper-acetylation in chondrocytes. Guided by these and additional preliminary data, the hypothesis will be tested by three specific aims: 1) Determine how obesity induces metabolic changes that promote mitochondrial protein acetylation; 2) Determine the aging and obesity-dependent effects of manipulating SIRT3 expression, positively or negatively, on chondrocyte redox homeostasis and cartilage catabolism; and 3) Identify the mechanisms by which SIRT3 regulates mitochondrial redox homeostasis and activation of cartilage catabolic pathways following a pro-inflammatory challenge. Well-established mouse models of diet-induced obesity and OA will be used in combination with genetically modified mice that allow for the conditional deletion or over- expression of SIRT3 in cartilage. Targeted genomic, proteomic, and metabolite detection methods are in place for aims 1 and 2 to determine the factors that promote mitochondrial acetylation, alter antioxidant capacity, and induce OA. Aim 3 will use ex vivo interleukin-1 stimulation assays to identify SIRT3-sensitive cartilage catabolic pathways. Mice with cartilage-specific deletion of SOD2 will provide a reference for evaluating the effect of SOD2 hyper-acetylation on chondrocyte oxidative stress and activation of downstream catabolic pathways. This approach is innovative because it shifts the focus of obesity research on OA from cellular stress triggers to stress susceptibility. The proposed research is significant because it will initiate the systematic study of how reversible post-translational lysine acetylation of mitochondrial proteins may be manipulated, either positively or negatively, to promote chondroprotection with aging and obesity.