The Role of PGRN Growth Factor in Osteoarthritis. - Renewal - 1

项目摘要 骨关节炎是最常见的关节疾病，目前尚无有效的预防手段 或减缓关节退变。然而，生长因子和细胞因子强烈地与启动和 加重骨性关节炎的损害。因此，对这些分子之间相互作用的分子理解将提供 寻找新的骨性关节炎治疗靶点的宝贵信息。我们的全基因组差异筛选 在骨性关节炎中狂热表达的基因导致了作为骨性关节炎相关生长因子的前颗粒蛋白(PGRN)的分离，而对PGRN结合伙伴的遗传筛选导致了作为PGRN结合受体的TNFR的分离 (唐，《科学》，2011)。我们已经开发出一种名为Atsttrin的工程蛋白质，它由三个 在治疗炎性关节炎方面，PGRN和Atsttrin的TNFR结合域均优于PGRN。在.期间 在最初的资金阶段，我们成功地鉴定了14-3-3ε，一个重要的细胞内信号分子， 在蛋白质组学筛选中，响应PGRN刺激的TNFR2复合体的一个新成分。此外，软骨细胞中14-3-3ε的敲除也取消了PGRN的信号转导。此外，我们最近分离到了与TNFR1同源性最高的DR3，作为一个与PGRN/Atsttrin结合的新的TNFR成员，以及PGRN/Atsttrin 干扰DR3与肿瘤坏死因子样配体1A(TL1A)的相互作用。因此，这种竞争性的继续应用主要基于1)14-3-3ε作为PGRN/TNFR2途径的新成分的鉴定， 2)DR3作为一种新的额外的PGRN结合受体的分离。这种更新的中心假设是，PGRN通过其a)募集在OA的发病中发挥其软骨保护作用。 14-3-3ε转TnFR2和激活PgRn/TnFR2合成代谢途径；b)相互作用和抑制肿瘤坏死因子α/TnFr1和TL1a/DR3炎症/分解代谢途径。具体目标是：(1)什么是 PGRN和Atsttrin调节软骨细胞代谢的分子机制和信号通路？我们将确定PGRN、Atsttrin、肿瘤坏死因子α和TL1a对软骨细胞代谢、它们的信号通路、靶基因表达谱以及软骨细胞中相互作用的影响(SA#1a)； 软骨细胞(SA#1B)中的Atsttrin信号转导和靶基因表达依赖于14-3-3ε； PGRN/Atsttrin也通过调节TL-1A/DR3途径调节软骨细胞(SA#1C)。(2)确实如此 PGRN/Atsttrin在骨性关节炎中具有治疗和保护作用，其作用机制是什么？ 我们将确定类似于PGRN的重组Atsttrin是否能逆转PGRN-/-小鼠的易感性 对于OA的挑战，以及PGRN和爱特林是否改善现有的OA(SA#2a)；软骨细胞是否- 14-3-3ε的表达对PGRN/Atsttrin在骨性关节炎(SA#2B)中的作用以及是否也参与了PGRN/Atsttrin在骨性关节炎(SA#2C)中的保护作用具有重要意义。完成拟议的研究不仅将 阐明14-3-3ε和DR3在PGRN介导的软骨细胞和骨关节炎调节中的重要性，但可能 这也导致了治疗骨性关节炎和其他退行性疾病的新治疗策略的发展。

Project Summary Osteoarthritis (OA) is the most common joint disease, and currently there is no effective means of preventing or slowing joint degeneration. However, growth factors and cytokines are strongly implicated in initiating and aggravating OA lesions. Thus, a molecular understanding of the interplay among these molecules will provide invaluable information toward the search for novel therapeutic targets for OA. Our genome-wide screen for dif ferentially expressed genes in OA led to the isolation of progranulin (PGRN) as an OA-associated growth factor, and genetic screen for PGRN binding partners led to the isolation of TNFR as the PGRN-binding receptor (Tang, Science, 2011). We have developed an engineered protein named Atsttrin which is composed of three TNFR-binding domains of PGRN, and Atsttrin surpassed PGRN in treating inflammatory arthritis. During the initial funding period, we have successfully identified 14-3-3ε, an important intracellular signaling molecule, as a novel component of TNFR2 complexes in response to PGRN stimulation in a proteomics screen. In addition, knockout of 14-3-3ε in chondrocytes abolished PGRN's signaling. Further, we recently isolated DR3, the highest homology to TNFR1, as a novel additional TNFR member that binds to PGRN/Atsttrin, and PGRN/Atsttrin disturbed the interaction between DR3 and TNF-like ligand 1A (TL1A). Thus, this competitive continued application is primarily based on 1) the identification of 14-3-3ε as a novel component of PGRN/TNFR2 pathway, and 2) the isolation of DR3 as a novel additional PGRN-binding receptor. The central hypothesis of this renewal is that PGRN exerts its chondroprotective role in the pathogenesis of OA through its a) recruitment of 14-3-3ε to TNFR2 and activation of the PGRN/TNFR2 anabolic pathway; and b) interplay with and inhibition of TNFα/TNFR1 and TL1A/DR3 inflammatory/catabolic pathways. The Specific Aims are: (1) What are the molecular mechanisms and signaling pathways by which PGRN and Atsttrin regulate chondrocyte metabolism? We will determine the effects of PGRN, Atsttrin, TNFα and TL1A on chondrocyte metabolism, their signaling pathways, target gene expression profiling and interplay in chondrocytes (SA#1A); whether PGRN and Atsttrin signaling and target gene expressions depends on 14-3-3ε in chondrocytes (SA#1B); and whether the PGRN/Atsttrin regulates chondrocytes through modulating TL-1A/DR3 pathway as well (SA#1C). (2) Does PGRN/Atsttrin have therapeutic and protective role in OA, and what are the mechanisms of its action in OA? We will determine whether recombinant Atsttrin, similar to PGRN, reverses the susceptibility of PGRN-/- mice to OA challenge, and whether PGRN and Atsttrin ameliorate existing OA(SA#2A); whether chondrocyte- expressed 14-3-3ε is important for PGRN/Atsttrin's role in OA(SA#2B); and whether DR3 pathway also contributes to PGRN/Atsttrin's protective role in OA (SA#2C). Completion of the proposed research will not only elucidate the importance of 14-3-3ε and DR3 in PGRN-mediated regulation of chondrocytes and OA, but may also lead to the development of novel therapeutic strategies for treating OA and other degenerative diseases.