Mechanism of Tgfbr2 in Chondroprotection

Tgfbr2 软骨保护机制

• 批准号: 10394844
• 负责人: Rosa A. Serra
• 金额: $ 32.34万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10394844
• 关键词: Arthritis; Binding; Binding Proteins; Biological; Biological Assay; Biological Process; Bioreactors; CRISPR/Cas technology; Cartilage; Cells; Centers for Disease Control and Prevention (U.S.); Chondrocytes; Cyclic AMP-Dependent Protein Kinases; DNA; Degenerative polyarthritis; Development; Disease; Dominant-Negative Mutation; Drug Targeting; Drug usage; Elements; Engineering; Extracellular Matrix Proteins; Funding; Future; Gel; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Half-Life; Homeostasis; Human; In Vitro; Industrialization; Knock-in; Knock-out; Knowledge; Laboratories; Luciferases; Maintenance; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Modification; Molecular; Mus; Mutation; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphorylation; Phosphorylation Site; Polysaccharides; Post-Translational Modification Site; Post-Translational Protein Processing; Post-Translational Regulation; Proteoglycan; Regulation; Reporter; Role; SOX9 protein; Serine; Signal Pathway; Signal Transduction; Site; Skeletal Development; Stains; Stream; Sulfate; Sumoylation Pathway; TGFBR2 gene; Tissue-Specific Gene Expression; Tissues; Transforming Growth Factor beta; Transgenic Mice; Western Blotting; articular cartilage; cartilage degradation; chromatin immunoprecipitation; disability; drug discovery; experimental study; genetic manipulation; high throughput screening; joint function; loss of function; mutant; novel; p38 Mitogen Activated Protein Kinase; parathyroid hormone-related protein; polysulfated glycosaminoglycan; prevent; response; sex; skeletal disorder; skeletal tissue; stable cell line; transcription factor

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.

项目摘要。 骨关节炎 (OA) 是工业化国家导致残疾的主要原因，但目前还没有经过批准的疾病 改良治疗 OA 的药物。我实验室的长期目标是了解介导这一现象的因素 关节软骨的发育和维护，以便可以实现预防或治疗 OA 的特定目标 确定。 TGF-ß 是一种多功能肽，已被证明可以调节骨骼发育和 组织特异性基因表达。我们是第一个将 TGF-β 确定为软骨保护因子的人。这 TGF-β 在人类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 TGF-β 的下游靶标，调节主要细胞外基质的翻译后加工 软骨中的蛋白质。特别是 3-Prime-磷酸腺苷 5-Prime-磷酸硫酸合酶 2 (Papss2)，与人类和小鼠的骨关节炎有关，并且是适当硫酸化所必需的 软骨中的糖胺聚糖。转录因子 Sox9 在软骨中的作用与 TGF-ß 相似，并且在 上一个资助期我们表明 TGF-ß 调节 Sox9 和 延长 Sox9 蛋白的半衰期。此外，我们证明 Sox9 是足够的并且是 TGF-β 介导的 Papss2 转录调节定义了软骨中 TGF-β 的新信号通路。 我们还使用生物反应器模型表明，TGF-β 信号传导缺陷的软骨中 Sox9 的表达 足以恢复该软骨的蛋白多糖染色。在此资助期内，我们建议继续 确定 TGF-ß 翻译后修饰 Sox9 蛋白的机制并确定 修改的生物学后果。我们还将确定 TGF-β 的分子机制。 ß 通过一种新颖的 Sox9 依赖性机制调节 Papss2 表达。详细的分子信息 关于 TGF-β 在关节软骨中的作用机制将使我们能够开发高通量检测方法 未来的药物发现屏幕。我们提出以下具体目标： 1A) 确定 TGF-ß 如何调节 Sox9 在丝氨酸 181 上的磷酸化； 1B) 确定丝氨酸 211 在 TGF-ß 介导中的作用 Sox9 蛋白的稳定性； 1C) 确定 TGF-ß 治疗导致苏酰化的机制 Sox9 的； 2) 确定翻译后修饰的 Sox9 的生物学功能 3) 识别 TGF-ß /Sox9 Papss2 基因中的响应 DNA 元件； 4) 确定 TGF-ß 的下游靶点是否可以预防 软骨退变和 OA。在该项目期间获得的信息将提供软骨独特的信息 预防或治疗骨关节炎的药物发现目标。