Molecular Mechanisms in Joint Development

描述（由申请人提供）：骨关节炎（OA）影响超过4600万美国人，造成重大的社会经济负担。目前，没有有效的药物治疗OA。尽管其临床意义，我们的知识的联合开发过程仍然有限。滑膜关节的发育是由关节间层的形成开始的，其中关节形成细胞出现在分节的软骨形成芽基内。该提案的总体目标是确定导致关节间区发育的机制，其概念是更好地理解关节形成将为理解软骨如何退化和开发治疗OA的新疗法提供基础。我们实验室的研究表明，TGF-2 II型受体（T2 RII）信号传导对于关节间区细胞的出现至关重要。在发育中的肢体中缺乏T2 RII信号传导，导致指间关节的缺乏和分化的软骨细胞的异常持久性，以及Noggin和Jagged-1（一种Notch配体）的缺乏。在缺乏肢体T2 RII信号传导的小鼠中，逐渐释放Jagged-1的数字植入物导致软骨细胞分割和表达关节标志物（包括Noggin）的区间细胞的出现。头蛋白植入导致软骨细胞分割，但没有恢复关节标记物的表达。结合RNA激光捕获显微镜与基因分析，我们发现，区间细胞表达特定的细胞膜相关的标志物，并在细胞因子，如CCL-12和血管生成因子，如VEGF-A，这是明显不同于相邻的生长板软骨细胞环境的受控环境中发展。基于T2 RII在间区细胞中的独特表达模式及其在间区发育中的功能作用，我们已经从表达关节特异性基因、缺乏软骨细胞分化且在形态学上不同于缺乏此类标记物并分化成软骨细胞的细胞群的胚胎自体足间充质中分离出间区细胞群。基于我们令人兴奋的初步数据，我们提出了以下具体目标：1）确定Notch途径的激活是否是T2 RII信号传导决定关节间区形成的机制; 2）确定TGF-2诱导的Notch激活和Noggin在抑制关节软骨细胞分化中的相互作用; 3）确定关节发育过程中关节间区细胞的个体发生; 4）研究CCL-12和VEGF-A在细胞间区形成中的功能作用，以及T2 RII信号在调控其表达中的作用。为了实现这些目标，我们将联合收割机结合使用小鼠模型，生物化学和新的纳米颗粒输送系统技术。在拟议的研究完成后，我们将有一个更好的了解T2 RII信号如何启动关节间区形成的关键关节形态发生基因的上游操作，因此，调节关节间区细胞存活和软骨细胞分割。 公共卫生相关性：骨关节炎（OA）是工业化国家最常见的残疾原因，影响超过4600万美国人。目前所有的OA治疗都是针对症状，没有一种可以改变疾病的退行性过程。该提案的总体目标是了解对联合开发至关重要的机制和分子。从我们的研究中获得的信息具有重要的医学相关性和意义。我们的研究将为导致OA的事件链提供重要的见解，并将为研究方向指明新的治疗方法，以恢复OA的关节功能。

DESCRIPTION (provided by applicant): Osteoarthritis (OA) affects over 46 million Americans imposing a major socio-economic burden. Currently, there are no effective medical treatments for OA. Despite its clinical significance, our knowledge of the joint development process remains limited. Synovial joint development is initiated by the formation of an interzone layer wherein joint-forming cells emerge within the segmenting chondrogenic blastema. The overall goal of this proposal is to determine the mechanisms that lead to joint interzone development, with the concept that a greater understanding of joint formation will provide the foundations to comprehend how cartilage degenerates and to develop novel therapies to treat OA. Investigations from our laboratory have revealed that TGF-2 type II receptor (T2RII) signaling is essential for the emergence of joint interzone cells. Lack of T2RII signaling in developing limbs, resulted in a lack of interphalangeal joints and an aberrant persistence of differentiated chondrocytes, as well as the absence of Noggin and Jagged-1, a Notch ligand. In mice lacking limb T2RII signaling, digital implants that gradually released Jagged-1 led to chondrocyte segmentation and emergence of interzone cells expressing joint markers, including Noggin. Noggin implants led to chondrocyte segmentation, but did not restore joint marker expression. Combining RNA laser-capture microscopy with gene-profiling analysis we have found that interzone cells express specific cell membrane-associated markers and develop in a controlled environment of cytokines, such as CCL-12, and angiogenic factors like VEGF-A, that is clearly distinct from the adjacent growth plate chondrocyte milieu. Based on the unique expression pattern of T2RII in the interzone cells and its functional role in the interzone development, we have isolated an interzone cell population from embryonic autopod mesenchyme that expresses joint specific genes, lack chondrocytic differentiation and is morphologically distinct from the cell population that lacks such markers and differentiates into chondrocytes. Based on our exciting Preliminary Data we propose the following Specific Aims: 1) to define whether activation of the Notch pathway is a mechanism by which T2RII signaling determines joint interzone formation; 2) to determine the interplay between TGF-2-induced Notch activation and Noggin in repressing joint chondrocyte differentiation; 3) to define the ontogeny of joint interzone cells throughout joint development; 4) to characterize the functional roles for CCL-12 and VEGF-A in interzone formation and the requirement for T2RII signaling in controlling their expression. To achieve these Aims, we will combine the use of mouse models, biochemistry and novel nanoparticle delivery system techniques. At the completion of the proposed studies, we will have a better understanding of how T2RII signaling initiates joint interzone formation by operating up-stream of key joint morphogenic genes and, therefore, regulating joint interzone cell survival and chondrocyte segmentation. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is the most common cause of disability in industrialized countries and affects more than 46 million Americans. All present therapies for OA are targeted at symptoms, and none exists that can alter the degenerative course of the disease. The overall goal of this proposal is to understand the mechanisms and molecules that are critical for joint development. The information derived from our studies has major medical relevance and implications. Our studies will provide critical insights into the chain of events that lead to OA and will point the research's direction to novel therapies to restore joint function in OA.