Mechanism of Tgfbr2 in Chondroprotection

项目摘要。 骨关节炎（OA）是工业化国家残疾的主要原因，但没有批准的疾病 治疗OA的药物我的实验室的长期目标是了解介导 关节软骨的发育和维持，以便可以预防或治疗OA的特定靶点， 鉴定TGF-β是一种多功能肽，已显示其调节骨骼发育， 组织特异性基因表达。我们是第一个鉴定TGF-β作为软骨保护因子的人。的 TGF-β 1在人骨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TGF-β下游靶点 软骨中的蛋白质特别是3-初级-磷酸腺苷5-初级-磷酸硫酸合酶2 （Papss2），与人类和小鼠的骨关节炎有关，并且是适当硫酸化 软骨中的糖胺聚糖。转录因子Sox 9在软骨中具有与TGF-β相似的作用， 在上一个资助期，我们发现TGF-β调节Sox 9的翻译后修饰， 增加了Sox 9蛋白的半衰期。此外，我们还证明了Sox 9是足够的，并且是必需的。 TGF-β 1介导的Papss2转录调控定义了软骨中TGF-β 1的一种新的信号通路。 我们还发现，使用生物反应器模型，Sox 9在TGF-β信号传导缺陷的软骨中的表达， 足以恢复软骨的蛋白多糖染色。在这段拨款期内，我们建议继续 以确定TGF-β 1后修饰Sox 9蛋白的机制，并确定TGF-β 1对Sox 9蛋白的影响。 修改的生物学后果。我们还将确定TGF-β的分子机制 PAPSS通过一种新的Sox9依赖性机制调节Papss2的表达。详细的分子信息 关于TGF-β 1在关节软骨中作用的机制，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：1A）确定TGF-β如何调节 Sox 9在丝氨酸181上的磷酸化; 1B）确定丝氨酸211在TGF-β介导的细胞凋亡中的作用。 Sox 9蛋白的稳定化; 1C）确定TGF-β 1处理导致类小泛素化的机制 3）鉴定TGF-β 1/Sox 9 Papss2基因中的响应性DNA元件;和4）确定TGF-β 1的下游靶点是否可以预防 软骨退化和OA。在此项目期间获得的信息将提供软骨独特的 用于预防或治疗骨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.