Molecular Mechanisms in Joint Development

描述(由申请人提供)：骨性关节炎(OA)影响着4600万美国人，造成了重大的社会经济负担。目前，还没有治疗骨性关节炎的有效药物。尽管它具有临床意义，但我们对关节发育过程的了解仍然有限。滑膜关节发育是由带间层的形成启动的，其中关节形成细胞出现在分段的软骨性胚基内。这项建议的总体目标是确定导致关节带间发展的机制，其概念是，对关节形成的更多了解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，转化生长因子-2 II型受体(T2RII)信号对关节带间细胞的出现是必不可少的。发育中的肢体缺乏T2RII信号，导致指间关节缺失和分化的软骨细胞异常持续存在，以及Notch配体Noggin和Jagge-1的缺失。在缺乏肢体T2RII信号的小鼠中，逐渐释放Jagge-1的数字植入物导致软骨细胞分裂和表达关节标记的区间细胞的出现，包括Noggin。Noggin植入可导致软骨细胞分裂，但不能恢复关节标志物的表达。结合RNA激光捕获显微镜和基因图谱分析，我们发现带间细胞表达特定的细胞膜相关标记，并在细胞因子(如CCL-12)和血管生成因子(如VEGF-A)的受控环境中发育，这与邻近的生长板软骨细胞环境明显不同。基于T2RII在带间细胞中的独特表达模式及其在带间发育中的作用，我们从胚胎自体足间充质中分离出一个表达联合特异性基因、缺乏软骨细胞分化且在形态上与缺乏这种标记的细胞群不同而分化为软骨细胞的带间细胞群。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通路的激活是否是T2RII信号决定关节带间形成的机制；2)确定转化生长因子-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)确定关节带间细胞在关节发育过程中的个体发育；4)表征CCL-12和VEGF-A在关节带间形成中的功能作用以及T2RII信号在控制其表达方面的要求。为了实现这些目标，我们将结合使用小鼠模型、生物化学和新型纳米颗粒递送系统技术。随着这些研究的完成，我们将更好地了解T2RII信号如何通过作用于关键的关节形态发生基因的上游启动关节间隙的形成，从而调节关节间隙细胞的存活和软骨细胞的分割。

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.