ROS activation of Apoptosis Signal-regulated Kinase1 (Ask1) in chondrocytes

描述(申请人提供)：在软骨中，活性氧(ROS)信号在调控软骨细胞的增殖、分化和成熟中发挥重要作用。为了确保正常的细胞功能，ROS由抗氧化剂的产生来平衡；因为不受调控的ROS可以导致基因表达、转录因子信号和细胞周期的功能障碍。在衰老、全身性疾病、环境毒素暴露、损伤和炎症中，ROS的产生可以压倒抗氧化能力，触发异常信号，导致形成和永久软骨的细胞死亡、基质降解和病理损伤。细胞凋亡信号调节蛋白1(ASK1)是一种MAP激酶，是一种与病理性ROS信号转导相关的蛋白。在我们的初步数据中，我们发现Ask1存在于生长板和关节软骨细胞中。我们还发现，缺氧诱导因子2(HIF-2)是一种转录调节因子，控制软骨细胞中抗氧化剂的表达。综上所述，我们认为在软骨中，细胞的氧化状态是由HIF-2的表达控制的；而Ask1的激活是氧化应激的功能测量。我们将研究HIF-2控制的抗氧化剂的表达与ROS激活Ask1的关系。通过调控这两个分子的表达，我们将探索ROS在软骨细胞分化和停滞中的正常和病理作用。在具体目标1中，利用siRNA沉默Ask1基因的表达，我们将探讨Ask1作为软骨细胞氧化应激指标的作用机制及其在ROS诱导的软骨病理中的作用。在特定的目标2中，我们将研究HIF-2在通过抗氧化调节控制细胞氧化状态中的表达。通过操纵HIF-2的表达，用siRNA沉默HIF-2基因的表达，用低氧非依赖的质粒过表达，我们将检测细胞的氧化状态，ROS介导的Ask1的激活，软骨细胞的分解代谢活性和存活。通过仔细分析氧化应激改变软骨细胞的信号机制，我们提出的研究将对理解氧化应激的分子机制产生巨大影响，并为开发新的治疗干预靶点提供重要信息。 公共卫生相关性：这项建议将研究环境氧化剂和软骨细胞中抗氧化剂表达之间的关系；这种平衡如何影响软骨细胞功能和软骨退化。研究将包括对抗氧化功能受损或改善的小鼠的组织和分离细胞以及人类骨关节组织进行分析，以确定这些关系。我们将进行分析以确定改变软骨细胞功能的基因是否增加，从而导致软骨功能障碍的发展。

DESCRIPTION (provided by applicant): In cartilage, reactive oxygen species (ROS) signaling plays significant role in regulating chondrocyte proliferation, differentiation and maturation. To assure proper cellular function ROS is balanced by production of antioxidants; as unregulated ROS can cause dysfunction in gene expression, transcription factor signaling, and cell cycle. In aging, systemic disease, environmental toxin exposure, injury and inflammation, ROS production can overwhelm the antioxidant capacity triggering aberrant signaling leading to cell death, matrix degradation and pathological damage in both forming and permanent cartilages. Apoptosis signal-regulating kinase 1 (ASK1) a MAP kinase kinase kinase, is a well characterized protein implicated in pathological ROS signaling. In our preliminary data, we show Ask1 is present in growth plate and articular chondrocytes. We also show that hypoxia inducible factor 2 (HIF-2) is a transcriptional regulator controlling antioxidant expression in chondrocytes. Taken together, we propose that in cartilage the oxidative status of the cell is controlled by expression of HIF-2; and activation of Ask1 is a functional measurement of oxidative stress. We will investigate the relationship between HIF-2 controlled expression of antioxidants and activation of Ask1 by ROS. By manipulating the expression of these two molecules we will explore the normal and pathological role of ROS in chondrocyte differentiation and stasis. In Specific Aim 1, using siRNA to silence Ask1 gene expression we will investigate the mechanism of action of Ask1 as an indicator of oxidative stress in chondrocytes and its role in ROS induced cartilage pathology. In Specific Aim 2, we will investigate the expression of HIF-2 in controlling cellular oxidative status by antioxidant regulation. By manipulating the expression HIF-2, using siRNA to silence HIF-2 gene expression and a hypoxia independent plasmid for overexpression, we will determine cellular oxidative status, ROS mediated activation of Ask1, chondrocyte catabolic activity and survival. By carefully analyzing the signaling mechanisms altered by oxidative stress in chondrocytes, our proposed studies will have an enormous impact on understanding molecular mechanisms of oxidative stress, and provide important information for developing novel targets for therapeutic intervention. Public Health Relevance: This proposal will examine the relationship between environmental oxidants and antioxidant expression in cartilage cells; how this balance affects cartilage cell function and cartilage degeneration. Studies will include analysis of tissue and isolated cells from mice with impaired or improved antioxidant function and human osteoarthris tissue to determine these relationships. We will perform analyses to determine if genes that alter cartilage cell function are increased leading to the development of cartilage dysfunction.