Role of BCP crystals in regulating the chondrocytic phenotype in osteoarthritis

碱性磷酸钙（BCP）软骨钙化是骨关节炎（OA）期间常见的发现，并与疾病的严重程度直接相关。我在之前的研究中发现BCP钙化与软骨细胞的肥厚分化有直接关系。发育过程中调节软骨细胞分化的一种形态因子是WNT。我在严重钙化的ttw/ttw小鼠中发现了典型的OA软骨变化迹象，这是一种天然的OA小鼠模型。在ttw/ttw小鼠中，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磺化模式。免疫组化染色显示，随着骨性关节炎严重程度的增加，软骨细胞周围积聚了WNT3a，可以用肝素酶去除，表明WNT3a与硫酸肝素蛋白多糖（HSPG）结合。我发现人类OA软骨中6-O硫转移酶的表达有所增加。与此发现一致，小鼠和人软骨中β-catenin染色随着OA等级的增加而增加。为了解释这一现象，我发现BCP晶体能够结合Wnt3a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dkk1阻断。在研究BCP晶体对软骨细胞表型的影响时，我发现随着胶原X和mmp13的增加，以及sox9和聚集蛋白表达的下调，软骨细胞向肥大转变。关节软骨的钙化似乎与典型WNT信号的激活和随后的软骨细胞肥厚分化有关。我假设BCP晶体将Wnt3a浓缩在细胞外基质中，从而增加了Wnt3a的可用性。随着骨性关节炎严重程度的增加，由于HSPGs的6-O硫酸化增加，软骨细胞更容易诱导典型Wnt信号。细胞外基质BCP矿化不是一种附带现象，而是骨关节炎中进一步延续软骨细胞分化的一个积极步骤。个别问题是：1)BCP晶体如何在受体水平上与软骨细胞相互作用？是LRP受体还是HSPGs重要？HSPGs的负电荷对矿化的生长是否重要？在软骨内骨形成过程中，HSPGs阴性是否也会发生同样的变化？BCP晶体激活了哪些细胞内通路？非典型WNT/Ca2+/ CamKII通路是BCP效应的原因吗？BCP诱导的软骨细胞分化是否涉及其他Ca2+依赖通路？BCP晶体如何诱导hspg的硫化模式改变？HS6ST1的诱导是否依赖于Ca2+？HS6ST1的变化是否依赖于年龄？蛋白质与BCP晶体表面的结合是如何介导的？是因为分泌蛋白脂化吗？BCP晶体的溶解或HSPG磺化的改变能否消除Wnt3a效应？

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?