CCN PROTEINS IN CARTILAGE FORMATION AND MAINTENANCE

描述（由申请人提供）：基质细胞蛋白CCN家族的几个成员是生长板中软骨细胞增殖、存活和ECM产生所必需的。然而，ccn介导这些活动的机制尚不清楚。同样，尽管一些CCN蛋白在成人关节软骨和椎间盘（IVDs）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，目前尚无治疗方法，在知识方面存在很大差距。本提案中的研究涉及这些基本问题。核心假设是CCNs 1、2和4在关节软骨和髓核（NP）的软骨形成和出生后维持中具有重叠的功能，并且它们部分通过调节HIF1a和HIF2a来调节其作用，部分通过作为ECM蛋白的护送/伴侣来调节。这一假设将在三个具体目标中得到检验。在Aim 1中，将确定CCNs 1、2和4在生长板软骨形成和关节软骨中的作用。携带ccn1和ccn2的固定等位基因的小鼠，以及Ccn4-/-小鼠已经产生，并将用于在产前和产后切除Ccn基因。初步研究支持CCNs 1和CCNs 2在出生后软骨中具有重叠功能的假设。该目的的其他研究建立在初步数据的基础上，表明Ccn2的缺失导致NFkB （RelA）的核定位降低，HIF1a水平降低，这两者在软骨中都具有促进生存的功能。CCNs通过激活NFkB /RelA诱导HIF1a和HIF2a的假设得到验证。在目的2中，通过诱导CCNs切除的小鼠来测试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表明，Ccn2突变体在NP的形成上存在主要缺陷，而这些缺陷在Ccn1/2双突变体中加剧。该目的的其他实验验证了ccn部分通过维持HIF1a和HIF2a水平介导其对ECM产生和细胞存活的影响的假设，如在生长板软骨中。在Aim 3中，测试了CCNs 1和CCNs 2直接作为“伴侣”促进ECM组装的能力。这种作用模式的先例来自于对其他分泌蛋白具有这种活性的观察，以及发现CCN蛋白含有这种伴侣蛋白特征的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ccn的两种新作用模式：NFkB和hif对细胞活力和基质产生的调节，以及通过伴侣样活性在ECM组装中的直接作用。拟议的研究意义重大，因为它有望首次证明ccn对维持关节软骨和IVD至关重要。这一知识有可能导致OA和DDD治疗的新方法。

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.