CCN PROTEINS IN CARTILAGE FORMATION AND MAINTENANCE

描述（由申请人提供）：生长板中软骨细胞增殖、存活和ECM产生需要基质细胞蛋白CCN家族的几个成员。然而，CCN介导这些活动的机制尚不清楚。类似地，尽管一些CCN蛋白在成人关节软骨和椎间盘（IVD）中表达，但它们在这些组织中的功能尚不清楚。因此，关于CCN蛋白在健康成人软骨和IVD中的作用以及它们在退行性疾病如骨关节炎（OA）和退行性椎间盘疾病（DDD）中的作用的知识存在很大差距，目前没有治疗方法。本提案中的研究涉及这些基本问题。核心假设是CCN 1、2和4在软骨形成和关节软骨和髓核（NP）的出生后维持中具有重叠的功能并且是所需的，并且它们部分地通过调节HIF 1a和HIF 2a介导它们的作用，部分地通过充当ECM蛋白的护送/伴侣。这一假设将在三个具体目标中得到检验。在目标1中，将确定CCN 1、2和4在生长板软骨发生和关节软骨中的作用。已经产生了携带Ccn 1和2的floxed等位基因的小鼠，如Ccn 4-/-小鼠，并且将用于在产前和产后切除Ccn基因。初步研究支持CCN 1和2在出生后软骨中具有重叠功能的假设。在此目的的其他研究建立在初步数据的基础上，这些数据表明Ccn 2的缺失导致NF κ B（RelA）的核定位减少，以及HIF 1a水平降低，这两者都在软骨中具有促生存功能。验证了CCN通过激活NFkB/RelA诱导HIF 1a和HIF 2a的假设。在目的2中，通过产生诱导切除CCN的小鼠来测试CCN在IVD的髓核（NP）的形成和维持中的作用。这一目的是基于初步数据，表明Ccn 2突变体在NP形成中具有主要缺陷，并且这些缺陷在Ccn 1/2双突变体中加剧。在这个目标的其他实验测试的假设，CCN介导的ECM生产和细胞存活的影响，部分通过维持HIF 1a和HIF 2a的水平，如在生长板软骨。在目的3中，测试了CCN 1和2直接充当"伴侣"以促进ECM组装的能力。这种作用模式的先例来自其他分泌蛋白具有这种活性的观察，以及来自CCN蛋白含有这种分子伴侣特征的CXXC基序的发现。这些研究是创新的，因为它们利用新的小鼠模型来测试CCN的两种新的作用模式：NFkB和HIF对细胞活力和基质产生的调节，以及通过分子伴侣样活性在ECM组装中的直接作用。这项研究意义重大，因为它有望首次证明CCN对关节软骨和IVD的维持至关重要。这些知识有可能导致新的治疗方法来治疗OA和DDD。 公共卫生相关性：骨关节炎（OA）是关节炎的最常见形式，也是美国长期残疾的主要原因。退行性椎间盘疾病（DDD）是最常见的腰痛形式，与OA一样，没有有效的治疗方法。CCN蛋白是软骨形成的关键调节因子;然而，它们在成人关节软骨和椎间盘中的作用尚不清楚。本提案中的研究调查了这些功能，并可能导致开发急需的治疗OA和DDD的疗法。

DESCRIPTION (provided by applicant): Several members of the CCN family of matricellular proteins are required for chondrocyte proliferation, survival, and ECM production in the growth plate. However, the mechanisms by which CCNs mediate these activities are unknown. Similarly, although some CCN proteins are expressed in adult articular cartilage and intervertebral discs (IVDs), their functions in these tissues are unknown. Thus there is a large gap in knowledge regarding the roles of CCN proteins in healthy adult cartilage and IVDs, and their roles in degenerative diseases such as osteoarthritis (OA) and degenerative disc disease (DDD), for which there are currently no therapies. The studies in this proposal address these fundamental issues. The central hypothesis is that CCNs 1, 2, and 4 have overlapping functions in and are required for chondrogenesis and postnatal maintenance of articular cartilage and nucleus pulposus (NP), and that they mediate their effects in part through regulation of HIF1a and HIF2a, and in part by acting as escorts/chaperones for ECM proteins. This hypothesis will be tested in three specific aims. In Aim 1, the roles of CCNs 1, 2, and 4 in growth plate chondrogenesis and articular cartilage will be determined. Mice carrying floxed alleles of Ccns 1 and 2 have been generated, as have Ccn4-/- mice, and will be used to excise Ccn genes prenatally and postnatally. Preliminary studies support the hypothesis that CCNs 1 and 2 have overlapping functions in postnatal cartilage. Other studies in this aim build on preliminary data showing that loss of Ccn2 leads to decreased nuclear localization of NFkB (RelA), and decreased levels of HIF1a, both of which have pro-survival functions in cartilage. The hypothesis that CCNs induce HIF1a and HIF2a by activating NFkB /RelA is tested. In Aim 2, the roles of CCNs in the formation and maintenance of the nucleus pulposus (NP) of the IVD are tested by generating mice in which excision of CCNs is induced. This aim is based on preliminary data showing that Ccn2 mutants have primary defects in formation of the NP, and these are exacerbated in Ccn1/2 double mutants. Other experiments in this aim test the hypothesis that CCNs mediate their effects on ECM production and cell survival in part through maintaining levels of HIF1a and HIF2a, as in growth plate cartilage. In Aim 3, the ability of CCNs 1 and 2 to act directly as "chaperones" to facilitate ECM assembly is tested. Precedent for this mode of action comes from observations that other secreted proteins have this activity, and from the finding that CCN proteins contain CXXC motifs characteristic of such chaperones. The studies are innovative, because they utilize new mouse models to test two novel modes of action for CCNs: regulation of cell viability and matrix production by NFkB and HIFs, and direct roles in ECM assembly via chaperone-like activity. The proposed research is significant, because it is expected to demonstrate for the first time that CCNs are essential for the maintenance of articular cartilage and IVD. This knowledge has the potential to lead to new therapeutic approaches to the treatment of OA and DDD. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is the most common form of arthritis and the leading cause of long-term disability in the US. Degenerative disc disease (DDD) is the most common form of low back pain, and as with OA, there are no effective therapies. CCN proteins are key regulators of cartilage formation; however, their roles in adult articular cartilage and intervertebral discs are unknown. The studies in this proposal investigate these functions and could lead to the development of sorely needed therapies for the treatment of OA and DDD.