Role of BCP crystals in regulating the chondrocytic phenotype in osteoarthritis

BCP晶体在调节骨关节炎软骨细胞表型中的作用

• 批准号: 430270172
• 负责人: Professorin Dr. Jessica Bertrand
• 金额: --
• 依托单位: Orthopädische Universitätsklinik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/430270172?language=en
• 关键词: Role; BCP; crystals; regulating; chondrocytic

Basic calcium phosphate (BCP) calcification of cartilage is a common finding during osteoarthritis (OA) and is directly linked to the severity of the disease. I have found in a previous study that BCP calcification is directly linked to hypertrophic differentiation of chondrocytes. One morphogen regulating chondrocyte differentiation during development is WNT. I found typical signs of OA cartilage changes in severely calcified ttw/ttw mice, which are a natural OA mouse model. OA in ttw/ttw mice was accompanied by an increased pericellular matrix sulfation. The same sulfation pattern was observed in human OA cartilage sections. Immunohistochemical staining showed an accumulation of WNT3a around chondrocytes with increasing OA severity, which could be removed using heparitinase, indicating a binding of Wnt3a to heparansulfate proteoglycans (HSPG). I found an increase in 6-O sulfotransferase expression in human OA cartilage. In line with this finding, β-catenin staining was increased with increasing OA grade in mouse and human cartilage. Explaining this phenomenon I found that BCP crystals were able to bind Wnt3a and induced canonical WNT signalling in monolayer chondrocytes. This effect could be blocked by the extracellular Wnt inhibitor DKK 1. Investigating the effect of BCP crystals on the chondrocyte phenotype, I found a shift towards hypertrophy with increased collagen X and MMP 13, as well as downregulated Sox 9 and aggrecan expression. The calcification of articular cartilage seems to be associated with activation of canonical WNT signalling and subsequent hypertrophic differentiation of chondrocytes. I hypothesize that BCP crystals concentrate Wnt3a in the pericellular matrix, thereby increasing the availability of Wnt3a. The chondrocytes are more prone to induce canonical Wnt signalling with increasing OA severity due to the increased 6-O sulfation of HSPGs. BCP mineralization of extracellular matrix is not an epiphenomenon, but an active step in further perpetuating chondrocyte differentiation in osteoarthritis. The individual questions are:1.) How do BCP crystals interact with chondrocytes at receptor level? Is the LRP receptor or the HSPGs important? Is the negative charge of HSPGs important for the growth of mineralisation? Can the same changes in negative HSPGs be observed during endochondral bone formation?2.) Which intracellular pathways are activated by BCP crystals? Is the non-canonical WNT/Ca2+/ CamKII pathway responsible for the BCP effect? Are other Ca2+ dependent pathways involved in BCP induced chondrocyte differentiation? How do BCP crystals induce changes in the sulfation pattern of HSPGs? Is the induction of HS6ST1 dependent on Ca2+? Are changes in HS6ST1 age dependent?3.) How is the binding of proteins to the surface of BCP crystals mediated? Is it due to lipidation of secreted proteins? Can the dissolution of BCP crystals or the change in HSPG sulfation eliminate the Wnt3a effect?

软骨的碱性磷酸钙（BCP）钙化是骨关节炎（OA）期间的常见发现，并与疾病的严重程度直接相关。我在以前的研究中发现BCP钙化与软骨细胞的肥大分化直接相关。在发育过程中调节软骨细胞分化的一种形态原是WNT。我在严重钙化的ttw/ttw小鼠中发现了OA软骨变化的典型迹象，这是一种自然的OA小鼠模型。ttw/ttw小鼠的OA伴随着细胞周围基质硫酸化的增加。在人OA软骨切片中观察到相同的硫酸化模式。免疫组织化学染色显示，随着OA严重程度的增加，WNT 3a在软骨细胞周围积聚，可以使用肝素酶去除，表明WNT 3a与硫酸乙酰肝素蛋白聚糖（HSPG）结合。我发现人OA软骨中6-O磺基转移酶表达增加。与这一发现一致，在小鼠和人软骨中，β-连环蛋白染色随着OA等级的增加而增加。为了解释这种现象，我发现BCP晶体能够结合Wnt 3a并在单层软骨细胞中诱导典型的WNT信号传导。这种作用可被细胞外Wnt抑制剂DKK 1阻断。研究BCP晶体对软骨细胞表型的影响，我发现随着胶原X和MMP 13的增加，以及Sox 9和聚集蛋白聚糖表达的下调，向肥大转变。关节软骨的钙化似乎与经典WNT信号的激活和随后的软骨细胞肥大分化有关。我假设BCP晶体将Wnt 3a集中在细胞周围基质中，从而增加了Wnt 3a的可用性。由于HSPG的6-O硫酸化增加，软骨细胞更倾向于随着OA严重程度的增加而诱导经典Wnt信号传导。BCP矿化细胞外基质不是一种附带现象，而是骨关节炎中软骨细胞分化进一步延续的一个积极步骤。具体问题如下：1）BCP晶体如何在受体水平与软骨细胞相互作用？LRP受体或HSPGs重要吗？HSPG的负电荷对矿化的生长重要吗？在软骨内成骨过程中是否也能观察到同样的HSPGs阴性变化？2.）的情况。BCP晶体激活了哪些细胞内途径？非经典的WNT/Ca 2 +/ CamKII通路是否是BCP效应的原因？BCP诱导的软骨细胞分化是否涉及其他钙依赖性途径？BCP晶体如何诱导HSPG硫酸化模式的变化？HS 6ST 1的诱导依赖于Ca ~（2+）吗？HS 6ST 1的变化是否与年龄有关？3.）第三章蛋白质与BCP晶体表面的结合是如何介导的？是由于分泌蛋白质的脂化吗？BCP晶体的溶解或HSPG硫酸化的变化能否消除Wnt 3a效应？