Interaction of heparan sulfate and integrin signaling in maintaining the articular cartilage

硫酸乙酰肝素和整合素信号在维持关节软骨中的相互作用

• 批准号: 289229882
• 负责人: Professorin Dr. Andrea Vortkamp
• 金额: --
• 依托单位: Abteilung Entwicklungsbiologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/289229882?language=en
• 关键词: Interaction; heparan; sulfate; integrin; signaling

Heparan sulfate (HS) carrying proteoglycans are important components of the articular cartilage (AC) matrix. HS chains bind to many secreted proteins, such as signaling factors, cytokines and proteases, thereby regulating their distribution, activity and receptor binding. The specificity of these interactions is determined by the degree of HS sulfation and its pattern.During the first funding period, we could show that mice carrying clones of HS-deficient cells in the AC and mice with altered Ndst1-dependent sulfation show reduced OA progression during aging and after anterior crucial ligament transection (ACLT). We demonstrated reduced Aggrecan degradation by AdamTS and Mmp proteases and found a decreased gelatinase activity of Mmp2 in Ndst1 mutants. Biochemical analysis of the relative composition of HS and chondroitin sulfate (CS) in the ECM of chondrocytes with reduced HS levels (Extgt/gt) or lacking 2-O sulfation (Hs2st1-/-) revealed that an altered HS structure is compensated by increased CS levels resulting in a decreased cartilage stiffness in the growth plate cartilage of Extgt/gt mice (with SP1). To decipher how distinct alterations of the HS structure affect OA, we surgically destabilized the medial meniscus (DMM) in Hs2st1 mutants and in mice lacking HS epimerization (Glce-/-). We did not detect an acceleration of OA, but due to the mild OA induced by DMM we could not assess a possible protective function.We now plan to induce OA by the harsher ACLT method to identify a potential protective effect against OA in Hs2st1 mutants (WP1). To receive comprehensive insight into the relevance of the HS structure on AC homeostasis, we will complete the ongoing systematic analysis of GAG composition (RPIP-HPLC), cartilage stiffness (AFM; SP1) and subchondral bone structure (nCT; SP5) of Ext1, Ndst1 and Hs2st1 mutants (WP4). Furthermore, the initiated transcriptome analyses of the impact of HS on mechanical stress responses (Extgt/gt) will be completed (with SP3; WP3). To address the potential role of HS as a therapeutic target, we plan to inhibit HS function by the small molecule inhibitor Surfen in cartilage explants and in mice after ACLT surgery (WP2). We will analyze cartilage degradation, OA progression, osteocyte numbers (with SP4) and synovial inflammation (with SP6). To further investigate the role of integrin signaling in sensing the altered HS structure, we will treat cells with Surfen and corroborate preliminary findings of altered Actin cytoskeleton, focal adhesions and cell migration (WP5). Epistasis will be evaluated by co-activation or inhibition of integrin signaling, in vitro, and the results will be confirmed in primary HS-deficient cells (Ext1fl/fl;R26RmT/mG). To detect mechanisms that translate the information on ECM composition into gene expression changes, we will investigate signaling pathways affected by an altered cytoskeleton (YAP) or by HS deficiency (Bmp/Smad, Ihh, Wnt).

硫酸乙酰肝素（HS）携带的蛋白多糖是关节软骨（AC）基质的重要组成部分。HS链结合许多分泌蛋白，如信号传导因子、细胞因子和蛋白酶，从而调节它们的分布、活性和受体结合。这些相互作用的特异性由HS硫酸化程度及其模式决定。在第一个资助期内，我们可以证明，在AC中携带HS缺陷细胞克隆的小鼠和Ndst 1依赖性硫酸化改变的小鼠在衰老过程中和前交叉韧带横断（ACLT）后显示OA进展减少。我们证明了AdamTS和Mmp蛋白酶降低的聚集蛋白聚糖降解，并发现Ndst 1突变体中Mmp 2的明胶酶活性降低。HS水平降低（Extgt/gt）或缺乏2-O硫酸化（Hs 2st 1-/-）的软骨细胞ECM中HS和硫酸软骨素（CS）的相对组成的生物化学分析显示，改变的HS结构通过增加CS水平得到补偿，导致Extgt/gt小鼠（SP1）生长板软骨硬度降低。为了解释HS结构的不同改变如何影响OA，我们通过手术使Hs 2st 1突变体和缺乏HS差向异构化（Glce-/-）的小鼠的内侧半月板（DMM）不稳定。我们没有检测到OA的加速，但由于DMM诱导的轻度OA，我们无法评估可能的保护功能。我们现在计划通过更严厉的ACLT方法诱导OA，以确定Hs 2st 1突变体（WP 1）对OA的潜在保护作用。为了全面了解HS结构对AC稳态的相关性，我们将完成Ext 1、Ndst 1和Hs 2st 1突变体（WP 4）的GAG组成（RPIP-HPLC）、软骨硬度（AFM; SP1）和软骨下骨结构（nCT; SP 5）的持续系统分析。此外，将完成HS对机械应力反应（Extgt/gt）影响的启动转录组分析（使用SP3; WP 3）。为了解决HS作为治疗靶点的潜在作用，我们计划通过小分子抑制剂Surfen在软骨外植体和ACLT手术后的小鼠中抑制HS功能（WP 2）。我们将分析软骨降解、OA进展、骨细胞数量（SP 4）和滑膜炎症（SP 6）。为了进一步研究整合素信号传导在感知改变的HS结构中的作用，我们将用Surfen处理细胞，并证实改变的肌动蛋白细胞骨架、粘着斑和细胞迁移（WP 5）的初步发现。将通过体外共激活或抑制整联蛋白信号传导来评价上位性，并将在原代HS缺陷细胞（Ext 1fl/fl; R26 RmT/mG）中确认结果。为了检测将ECM组成信息转化为基因表达变化的机制，我们将研究受细胞骨架改变（雅普）或HS缺陷（Bmp/Smad，Ihh，Wnt）影响的信号传导途径。