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Notch Signaling in Cartilage Development

软骨发育中的Notch信号传导

基本信息

批准号：
8256561
负责人：
Matthew J. Hilton
金额：
$ 33.37万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-02 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8256561
关键词：
Address Binding Binding Sites Biological Assay Bromodeoxyuridine Cartilage Cartilage Diseases Cartilage injury Cell Differentiation process Cell Maintenance Cell Nucleus Cell surface Cells Chondrocytes Chondrogenesis Clinical Treatment Co-Immunoprecipitations Complex Data Degenerative polyarthritis Development Differentiation Antigens Disease Embryo Family Fracture Gel Gene Targeting Genes Genetic Goals Histology Immunohistochemistry In Situ Hybridization In Situ Nick-End Labeling In Vitro Injury Ligands Limb structure Luciferases Maintenance Mediating Membrane Mesenchymal Mesenchymal Stem Cells Mesenchyme Methodology Methods Mus Mutate Notch Signaling Pathway Pathway interactions Preparation Process Proteins Regulation Reverse Transcriptase Polymerase Chain Reaction Rheumatoid Arthritis Role Rosa Signal Pathway Signal Transduction Signaling Molecule Staging Staining method Stains Testing Time Tissue Engineering Tissues base beta catenin cartilage development cartilage repair gain of function gamma secretase in vitro Assay in vivo loss of function mouse model mutant mutant mouse model notch protein novel progenitor promoter protein complex public health relevance research study skeletal therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Signaling pathway components such as Ihh/Pthrp, TGF¿, BMPs, Wnt/¿-catenin, FGFs, and Sox-related proteins represent important regulators of cartilage formation and development. These same signaling pathways and related molecules are being targeted for clinical treatment of cartilage injuries and diseases (osteoarthritis, rheumatoid arthritis, and fracture repair) and cartilage tissue engineering applications (maintenance and manipulation of mesenchymal progenitor cells (MPCs)). We provide original, unpublished data indicating that the Notch signaling pathway is another important regulator of MPC differentiation and chondrocyte maturation, leading to questions regarding the mechanisms by which Notch controls these processes. To begin addressing these questions, we propose to test the novel hypothesis that RBPj?-dependent Notch signaling suppresses MPC differentiation and chondrogenesis by interacting with stabilized ¿-catenin to regulate Hes1 and ultimately Sox9, and is later required to promote chondrocyte maturation via cartilage specific regulation of Hes1 and Runx2 activities. To test these hypotheses, we will address two Specific Aims. Experiments in Specific Aim 1 will investigate whether Hes1 is required for MPC differentiation and chondrogenesis or the Notch-mediated suppression of MPC differentiation using limb mesenchyme specific conditional loss-of-function and genetic rescue mouse models. Secondly, we will determine whether the Wnt/beta-catenin signaling pathway is necessary and sufficient for Notch mediated induction of Hes1 and suppression of MPC differentiation. Finally, we will determine whether Hes1 suppresses MPC differentiation by directly regulating Sox9 expression. Specific Aim 2 will first examine the potential roles for both RBPJ?- dependent and -independent Notch signaling in promoting chondrocyte maturation using various tissue specific Notch gain- and loss-of-function mouse models. Additionally, we will perform in vitro experiments using primary chondrocyte cultures to identify RBPJ?-dependent Notch targets that regulate chondrocyte maturation and examine whether Notch mediates Wnt/beta-catenin or BMP signaling during this process. Secondly, we will use two different conditional Hes1 mutant mouse models to determine whether Hes1 is the primary RBPJk-dependent Notch regulator of chondrocyte maturation. Finally, in vitro studies will be performed to determine whether Hes1 promotes Runx2 activity and chondrocyte maturation via competitive interactions with the mutual co-repressor, Groucho related gene (Grg1). Completion of these aims will identify the Notch signaling mechanisms important in regulating MPC maintenance and expansion, as well as, chondrocyte maturation. These molecules will likely serve as therapeutic targets for cartilage injuries or diseases and provide us with potential tools for use in cartilage tissue engineering applications. PUBLIC HEALTH RELEVANCE: We have identified the RBPJ?-dependent Notch pathway as an important regulator of MPC differentiation and chondrocyte maturation. Our proposal will determine the exact Notch signaling mechanisms responsible for suppressing chondrogenic commitment from mesenchymal progenitor cells (MPCs) and for promoting chondrocyte maturation. Data generated by this proposal will likely implicate specific Notch signaling molecules as potential therapeutic targets for cartilage related injuries and diseases, as well as, provide potential tools in MPC maintenance and expansion for use in tissue engineering applications.

描述(申请人提供)：信号通路组件，如IHH/PTHrP、转化生长因子β、BMPs、Wnt/β-catenin、FGFs和SOX相关蛋白是软骨形成和发育的重要调节因子。这些信号通路和相关分子正被用于软骨损伤和疾病的临床治疗(骨关节炎、类风湿性关节炎和骨折修复)和软骨组织工程应用(间充质祖细胞的维护和操作)。我们提供了原始的、未发表的数据，表明Notch信号通路是MPC分化和软骨细胞成熟的另一个重要调节因子，这导致了关于Notch控制这些过程的机制的问题。为了开始解决这些问题，我们建议测试这一新的假设，即依赖RBPj的Notch信号通过与稳定的连接素相互作用来调节Hes1和最终的Sox9，从而抑制MPC的分化和软骨形成，并在以后需要通过软骨特异性调节Hes1和Runx2的活性来促进软骨细胞的成熟。为了检验这些假设，我们将解决两个具体目标。特定目的1的实验将利用肢体间充质特异性条件性功能丧失和遗传拯救小鼠模型，研究Hes1是MPC分化和软骨形成所必需的，还是Notch介导的抑制MPC分化的机制。其次，我们将确定Wnt/β-catenin信号通路对于Notch介导的Hes1诱导和抑制MPC分化是否必要和充分。最后，我们将确定Hes1是否通过直接调节Sox9的表达来抑制MPC的分化。具体目标2将首先利用不同组织特异性Notch获得和丧失功能的小鼠模型，研究RBPJ依赖和非依赖的Notch信号在促进软骨细胞成熟中的潜在作用。此外，我们将使用原代软骨细胞培养进行体外实验，以确定调控软骨细胞成熟的RBPJ？依赖的Notch靶标，并检查Notch在这一过程中是否介导Wnt/β-catenin或BMP信号。其次，我们将使用两种不同的条件性Hes1突变小鼠模型来确定Hes1是否是软骨细胞成熟的主要RBPJk依赖的Notch调节因子。最后，将进行体外研究，以确定Hes1是否通过与相互共抑制因子Groucho相关基因(Grg1)的竞争性相互作用来促进Runx2活性和软骨细胞成熟。这些目标的完成将确定Notch信号机制在调控MPC的维持和扩张以及软骨细胞成熟方面的重要作用。这些分子很可能成为软骨损伤或疾病的治疗靶点，并为软骨组织工程应用提供潜在的工具。公共卫生相关性：我们已经确定依赖RBPJ？的Notch通路是MPC分化和软骨细胞成熟的重要调节因子。我们的建议将确定确切的Notch信号机制，负责抑制间充质祖细胞(MPC)的成软骨承诺和促进软骨细胞成熟。这项提案产生的数据可能会暗示特定的Notch信号分子可能成为软骨相关损伤和疾病的潜在治疗靶点，并为组织工程应用中的MPC维护和扩增提供潜在的工具。