CCN PROTEINS IN CARTILAGE FORMATION AND MAINTENANCE

描述(申请人提供)：在生长板中，软骨细胞的增殖、存活和细胞外基质的产生需要CCN家族的几个成员。然而，CCNS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，对于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中的作用，如骨关节炎(OA)和退行性间盘疾病(DDD)，目前还没有治疗方法，这方面的知识差距很大。本提案中的研究涉及这些基本问题。核心假设是CCN1、2和4在关节软骨和髓核(NP)的软骨形成和出生后的维持中具有重叠的功能和所需的功能，它们部分通过调节HIF1a和HIF2a，部分通过充当ECM蛋白的护卫/伴侣来调节它们的作用。这一假设将在三个具体目标上得到检验。在目标1中，将确定CCNS1、2和4在生长板软骨形成和关节软骨中的作用。已经产生了携带CCNS1和CCN2等位基因的小鼠，以及Ccn4-/-小鼠，并将用于产前和出生后切除CCN基因。初步研究支持CCNS1和CCN2在出生后软骨中功能重叠的假说。在这一目标的其他研究建立在初步数据的基础上，表明Ccn2的丢失导致NFkB(RelA)核定位减少，HIF1a水平降低，这两者在软骨中都具有促进生存的功能。验证了CCNS通过激活NFkB/relA诱导HIF1a和HIF2a的假说。目的2通过建立CCNS切除小鼠模型，研究CCNS在IVD髓核(NP)形成和维持中的作用。这一目的是基于初步数据显示，Ccn2突变体在NP的形成方面存在初级缺陷，这些缺陷在CCN1/2双重突变体中加剧。这一目的的其他实验验证了这一假设，即CCNS部分通过维持生长板软骨中HIF1a和HIF2a的水平来调节其对ECM产生和细胞存活的影响。在目标3中，测试了CCNS 1和2直接作为促进细胞外基质组装的“伴侣”的能力。这种作用模式的先例来自于观察到其他分泌的蛋白质具有这种活性，以及发现CCN蛋白含有这种伴侣蛋白特有的CXXC基序。这些研究具有创新性，因为他们利用新的小鼠模型来测试CCNS的两种新的作用模式：NFkB和HIFs对细胞活性和基质产生的调节，以及通过伴侣样活性在ECM组装中的直接作用。这项拟议的研究意义重大，因为它有望首次证明CCNS对关节软骨的维持和IVD是必不可少的。这一知识有可能导致治疗骨性关节炎和DDD的新的治疗方法。 公共卫生相关性：骨关节炎(OA)是美国最常见的关节炎形式，也是导致长期残疾的主要原因。退行性腰椎间盘疾病(DDD)是最常见的腰痛形式，与OA一样，目前还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。这项建议中的研究调查了这些功能，并可能导致开发治疗骨性关节炎和DDD的迫切需要的疗法。

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.