Department of Orthopaedic Surgery, Otto-von-Guericke University, Magdeburg, Germany
Division of Mol Medicine of Musculoskeletal Tissue, University Munster, Munster, Germany.
Ann Rheum Dis. 2020 Jul;79(7):975-984. doi: 10.1136/annrheumdis-2019-216648. Epub 2020 May 5.
Calcification of cartilage with basic calcium phosphate (BCP) crystals is a common phenomenon during osteoarthritis (OA). It is directly linked to the severity of the disease and known to be associated to hypertrophic differentiation of chondrocytes. One morphogen regulating hypertrophic chondrocyte differentiation is Wnt3a.
Calcification and sulfation of extracellular matrix of the cartilage was analysed over a time course from 6 to 22 weeks in mice and different OA grades of human cartilage. Wnt3a and ß-catenin was stained in human and murine cartilage. Expression of sulfation modulating enzymes (HS2St1, HS6St1) was analysed using quantitative reverse transcription PCR (RT-PCR). The influence of BCP crystals on the chondrocyte phenotype was investigated using quantitative RT-PCR for the marker genes Axin2, Sox9, Col2, MMP13, ColX and Aggrecan. Using western blot for β-catenin and pLRP6 we investigated the activation of Wnt signalling. The binding capacity of BCP for Wnt3a was analysed using immunohistochemical staining and western blot.
Here, we report that pericellular matrix sulfation is increased in human and murine OA. Wnt3a co-localised with heparan sulfate proteoglycans in the pericellular matrix of chondrocytes in OA cartilage, in which canonical Wnt signalling was activated. In vitro, BCP crystals physically bound to Wnt3a. Interestingly, BCP crystals were sufficient to induce canonical Wnt signalling as assessed by phosphorylation of LRP6 and stabilisation of β-catenin, and to induce a hypertrophic shift of the chondrocyte phenotype.
Consequently, our data identify BCP crystals as a concentrating factor for Wnt3a in the pericellular matrix and an inducer of chondrocyte hypertrophy.
软骨中以碱性磷酸钙（BCP）晶体形式存在的钙化是骨关节炎（OA）的一种常见现象。它与疾病的严重程度直接相关，并且已知与软骨细胞的肥大分化有关。一种调节肥大软骨细胞分化的形态发生素是 Wnt3a。
在从小鼠到人类不同 OA 等级的软骨中，分析了从 6 周到 22 周的时间范围内软骨细胞外基质的钙化和硫酸化。在人和鼠软骨中对 Wnt3a 和β-连环蛋白进行染色。使用定量逆转录 PCR（RT-PCR）分析硫酸化调节酶（HS2St1、HS6St1）的表达。使用定量 RT-PCR 分析 Axin2、Sox9、Col2、MMP13、ColX 和 Aggrecan 等标记基因，研究 BCP 晶体对软骨细胞表型的影响。通过对β-连环蛋白和 pLRP6 的 Western blot 分析，研究 Wnt 信号通路的激活情况。使用免疫组织化学染色和 Western blot 分析 BCP 对 Wnt3a 的结合能力。
在这里，我们报告在人和鼠 OA 中细胞周基质硫酸化增加。在 OA 软骨中，Wnt3a 与软骨细胞细胞周基质中的硫酸乙酰肝素蛋白聚糖共定位，其中经典 Wnt 信号通路被激活。在体外，BCP 晶体与 Wnt3a 物理结合。有趣的是，BCP 晶体足以诱导经典 Wnt 信号通路，如 LRP6 的磷酸化和β-连环蛋白的稳定，并诱导软骨细胞表型向肥大转化。
因此，我们的数据将 BCP 晶体鉴定为细胞周基质中 Wnt3a 的浓缩因子，也是软骨细胞肥大的诱导剂。
