Genetic dissection of the role of chondroitin sulfate in cartilage

描述（由申请人提供）：硫酸软骨素（CS）是一种硫酸化糖胺聚糖，是软骨中最丰富的成分之一，占组织的30%。由于其高度负电荷和绝对丰富，CS长期以来一直被认为是决定关节软骨独特生物力学特性的关键组分。然而，我们对CS在体内的生理作用及其在人类疾病中的参与的理解是有限的，主要是由于缺乏通用的小鼠遗传模型。为了将CS研究推进到一个新的水平，我们已经创建了一个条件无效等位基因Chsy 1，基因编码硫酸软骨素合酶1。值得注意的是，使用Prx 1-Cre靶向肢体骨骼系统的条件性Chsy 1敲除小鼠表现出与经典人类骨关节炎（OA）的关节病理学惊人相似的关节病理学。这些观察结果，连同先前在体外观察到的CS对软骨细胞具有保护作用，使我们假设CS合成能力降低是人类OA发病机制的基础。为了验证这一假设，我们提出了以下研究：在目标1中，我们将确定CS缺陷软骨变性的信号基础，重点是经典的Notch通路。在目标2中，我们将采用体外机械应力模型来表征CS缺陷软骨细胞对机械应力的细胞死亡和抗氧化反应。在目标3中，我们将转向额外的条件敲除实验，以进一步剖析软骨细胞特异性和成骨细胞特异性CS在OA发展的背景下的作用。在目标4中，我们将与人类相关研究专家合作，在人类CHSY 1和其他CS合成基因中寻找OA相关的功能性SNP。通过这些研究，我们希望获得新的见解软骨退行性变的分子机制在OA，并将我们在小鼠中的观察转化为识别新的易感基因为人类OA。

DESCRIPTION (provided by applicant): Chondroitin sulfate (CS), a sulfated glycosaminoglycan, is one of the most abundant components of cartilage, comprising as much as 30% of the tissue. Because of its highly negative charge and sheer abundance, CS has long been assumed to be a key component to determine the unique biomechanical property of articular cartilage. Nevertheless, our understanding of the physiological role of CS in vivo and it involvement in human diseases is limited, mainly due to the lack of versatile mouse genetic models. To advance CS research to the next level, we have created a conditional null allele of Chsy1, the gene encoding chondroitin sulfate synthase 1. Remarkably, conditional Chsy1 knockout mice targeted to the limb skeletal system using Prx1-Cre exhibit joint pathologies strikingly similar to those of classical human osteoarthritis (OA). These observations, together with the previous in vitro observation that CS is protective to chondrocytes, led us to hypothesize that a decreased capability in CS synthesis underlie the pathogenesis of human OA. To examine this hypothesis, we propose the following studies: In Aim 1, we will determine the signaling basis of the degeneration of CS-deficient cartilage, focusing on the canonical Notch pathway. In Aim 2, we will employ an in vitro mechanical stress model to characterize cell death and antioxidant responses of CS-deficient chondrocytes to mechanical stress. In Aim 3, we will turn to additional conditional knockout experiments to further dissect chondrocyte-specific and osteoblast-specific roles of CS in the context of OA development. In Aim 4, collaborating with an expert in human association study, we will search for OA-associated, functional SNPs in human CHSY1 and other CS synthesizing genes. By these studies, we wish to gain novel insight into the molecular mechanisms of cartilage degeneration in OA, and to translate our observations in mice into the identification of novel susceptibility genes for human OA.