Mechanism of Tgfbr2 in chondroprotection

描述(申请人提供)：骨性关节炎是工业化世界中导致残疾的主要原因，但人们对与骨性关节炎相关的软骨破坏机制知之甚少。老鼠基因操作的最新进展导致了新的动物模型和新的概念，这些模型和概念与理解人类的骨关节炎有关。我的实验室的长期目标是了解影响关节软骨发育、持续和修复的因素，并确定预防和治疗骨性关节炎的具体靶点。转化生长因子β是一种多功能多肽，已被证明可调节细胞分化和组织特异性基因表达。此前，我们培育了表达关节软骨中转化生长因子II型受体(TGFBR2)显性负突变的转基因小鼠。对转化生长因子-β的反应改变导致了一种进行性骨骼疾病，类似于人类的骨关节炎。我们最近发现了几个调节关节软骨主要细胞外基质蛋白翻译后加工的转化生长因子-β下游靶点。其中一个特别是3-Prime-Phohoadosine 5-Prime-Phososulate Synthase 2(Papss2)，它与人类的脊柱干骨干端发育不良有关，是软骨中蛋白多糖正确硫酸盐化所必需的。除了转化生长因子β，转录因子Sox9也与成熟关节软骨的维持有关。我们的初步研究表明，转化生长因子β提高了软骨细胞中Sox9蛋白的水平，而不是依赖于mRNA的变化。初步数据表明，用转化生长因子β处理会导致Sox9的苏莫化。苏莫化已被证明可以调节蛋白质的稳定性、活性和细胞定位。我们假设转化生长因子-β维持 永久软骨中分化的软骨细胞表型，如关节软骨，通过蛋白总甲基化调节Sox9水平和活性。我们建议测试这一模型的具体目的如下：1)确定Sox9上哪些位点被转化生长因子β激活，并确定其在转化生长因子β介导的Sox9水平、定位和活性中的作用；1)确定转化生长因子β介导的Sox9总甲基化的机制；2)确定转化生长因子β介导的PAPSS2表达的机制；3)确定当转化生长因子β信号转导中断时，PAPSS2活性是否能够减轻软骨退变，并确定激活转化生长因子β的软骨保护信号是否能够恢复骨关节炎软骨的生化和生物力学特性。这些研究将确定可用作骨关节炎治疗靶点的软骨保护机制。

DESCRIPTION (provided by applicant): Osteoarthritis is a leading cause of disability in the industrialized world but little is known about mechanisms of cartilage destruction associated with osteoarthritis. Recent advances in the genetic manipulation of mice have lead to new animal models and new concepts that are relevant to understanding osteoarthritis in humans. The long-term objective of my laboratory is to understand the factors that mediate the development, persistence, and repair of articular cartilage and to identify specific targets for prevention and treatment strategies for osteoarthritis. TGF-¿ is a multifunctional peptide that has been shown to regulate cellular differentiation and tissue-specific gene expression. Previously, we generated transgenic mice that express a dominant-negative mutation of the TGF-¿ type II receptor (Tgfbr2) in articular cartilage. Altered responsiveness to TGF-¿ resulted in a progressive skeletal disease that resembled osteoarthritis in humans. We recently identified several down-stream targets of TGF-¿ that regulate post-translational processing of the major extracellular matrix proteins in articular cartilage. One in particular, 3-Prime-Phoshoadenosine 5-Prime-Phosphosulfate Synthase 2 (Papss2), has been associated with Spondyloepimetaphyseal Dysplasias in humans and is required for proper sulfation of proteoglycans in cartilage. In addition to TGF-¿, the transcription factor Sox9 is also associated with the maintenance of mature articular cartilage. Our preliminary studies indicate that TGF-¿ enhances the level of Sox9 protein in chondrocytes independently of changes in mRNA. The preliminary data suggest that treatment with TGF-¿ results in sumoylation of Sox9. Sumoylation has been shown to regulate protein stability, activity and cellular localization. We hypothesize that TGF-¿ maintains the differentiated chondrocyte phenotype in permanent cartilages, like articular cartilage, by regulating Sox9 levels and activity via protein sumoylation. We propose to test this model with the following specific aims: 1a) to determine which sites on Sox9 are sumoylated in response to TGF-¿ and determine the role of sumoylation in TGF-¿-mediated Sox9 levels, localization, and activity; 1b) to determine the mechanism of TGF-¿-mediated sumoylation of Sox9; 2) to determine the mechanism of TGF-¿ mediated expression of Papss2 and 3) to determine if Papss2 activity can alleviate cartilage degeneration when TGF-¿ signaling is disrupted and determine if activation of TGF-¿'s chondroprotective signals can restore biochemical and biomechanical properties to OA cartilage. These studies will identify mechanisms of chondroprotection that can be used as targets for therapies in osteoarthritis.