Mechanism of Tgfbr2 in Chondroprotection

项目摘要。 在工业化世界中，骨关节炎(OA)是导致残疾的主要原因，但目前还没有得到批准的疾病 修改治疗骨性关节炎的药物。我的实验室的长期目标是了解影响 关节软骨的发展和维护，以便可以预防或治疗骨性关节炎的特定靶点 已确认身份。转化生长因子是一种多功能多肽，已被证明调节骨骼发育和 组织特异性基因表达。我们首次将转化生长因子确定为软骨保护因子。这个 转化生长因子在人类骨性关节炎中的重要性现已得到充分证实。我们之前确定了几个 调节主要细胞外基质翻译后加工的转化生长因子下游靶点 软骨中的蛋白质。其中一个特别的是，3-Prime-Phshoososine 5-Prime-Phososulate Synthase 2 在人类和小鼠中与骨关节炎有关，并且是适当的硫酸盐化所必需的。 软骨中的糖氨基葡聚糖。转录因子Sox9在软骨中的作用与转化生长因子类似，在 在上一个资助期，我们展示了转化生长因子调节Sox9和Sox9的翻译后修饰 增加Sox9蛋白的半衰期。此外，我们还证明了Sox9是充分和必要的 转化生长因子-β介导的PAPSS2转录调控为转化生长因子-β在软骨中定义了一条新的信号通路。 我们还利用生物反应器模型显示，Sox9在软骨中的表达缺乏转化生长因子-β信号 足以将蛋白多糖染色恢复到软骨上。在这一资助期内，我们建议继续 为了确定转化生长因子β对Sox9蛋白进行翻译后修饰的机制，并确定 这些改变的生物学后果。我们还将确定转化生长因子-1的分子机制。 通过一种新的Sox9依赖机制来调节Papss2的表达。详细的分子信息 关于转化生长因子在关节软骨中的作用机制将使我们能够开发高通量的检测方法 未来的药物发现筛选。我们提出了以下具体目标：1)确定转化生长因子是如何调节的 Sox9在丝氨酸181上的磷酸化；1B)决定丝氨酸211在转化生长因子介导的信号转导中的作用 Sox9蛋白的稳定化；1C)决定了转化生长因子-贝类处理导致总甲基化的机制 2)确定翻译后修饰的Sox9的生物学功能3)鉴定转化生长因子β/Sox9 Papss2基因中有反应的DNA元件；以及4)确定转化生长因子下游靶点是否可以阻止 软骨退变和骨性关节炎。在此项目期间获得的信息将为软骨提供独特的 预防或治疗骨性关节炎的药物发现的靶点。

Project Summary. Osteoarthritis (OA) is a leading cause of disability in the industrialized world but there are no approved disease modifying drugs for OA. The long-term objective of my laboratory is to understand the factors that mediate the development and maintenance of articular cartilage so that specific targets to prevent or treat OA can be identified. TGF-ß is a multifunctional peptide that has been shown to regulate skeletal development and tissue-specific gene expression. We were the first to identify TGF-ß as a chondroprotective factor. The importance of TGF-ß in human osteoarthritis has now been well established. We previously identified several down-stream targets of TGF-ß that regulate post-translational processing of the major extracellular matrix proteins in cartilage. One in particular, 3-Prime-Phoshoadenosine 5-Prime-Phosphosulfate Synthase 2 (Papss2), has been associated with osteoarthritis in humans and mice and is required for proper sulfation of glycosamino glycans in cartilage. The transcription factor Sox9 has similar roles in cartilage as TGF-ß and in the last funding period we showed that TGF-ß regulates post-translational modifications on Sox9 and increases the half-life of the Sox9 protein. Furthermore, we showed that Sox9 is sufficient and required for TGF-ß-mediated regulation of Papss2 transcription defining a novel signaling pathway for TGF-ß in cartilage. We also showed, using a bioreactor model, that expression of Sox9 in cartilage deficient for TGF-ß signaling was sufficient to restore proteoglycan staining to that cartilage. In this funding period, we propose to continue to determine the mechanisms by which TGF-ß post-translationally modifies the Sox9 protein and determine the biological consequences of the modifications. We will also determine the molecular mechanism whereby TGF- ß regulates Papss2 expression through a novel Sox9-dependent mechanism. Detailed molecular information about the mechanisms of TGF-ß action in articular cartilage will allow us to develop high throughput assays for future drug discovery screens. We propose the following specific aims: 1A) To determine how TGF-ß regulates the phosphorylation of Sox9 on Serine 181; 1B) Determine the role of Serine 211 in TGF-ß-mediated stabilization of Sox9 protein; 1C) Determine the mechanism whereby TGF-ß treatment results in sumoylation of Sox9; 2) Determine the biological functions of post translationally modified Sox9 3) Identify TGF-ß /Sox9 responsive DNA elements in the Papss2 gene; and 4) Determine if downstream targets of TGF-ß can prevent cartilage degeneration and OA. The information acquired during this project period will provide cartilage unique targets for drug discovery to prevent or treat osteoarthritis.