Zimmermann Peter, Boeuf Stéphane, Dickhut Andrea, Boehmer Sandra, Olek Sven, Richter Wiltrud
Orthopaedic University Hospital of Heidelberg, Heidelberg, Germany.
Arthritis Rheum. 2008 Sep;58(9):2743-53. doi: 10.1002/art.23736.
Human articular chondrocytes do not express COL10A1 and do not undergo hypertrophy except in close vicinity to subchondral bone. In contrast, chondrocytes produced in vitro from mesenchymal stem cells (MSCs) show premature COL10A1 expression and cannot form stable ectopic cartilage transplants, which indicates that they may be phenotypically unstable and not suitable for treatment of articular cartilage lesions. CpG methylation established during natural development may play a role in suppression of COL10A1 expression and hypertrophy in human articular chondrocytes. This study was undertaken to compare gene methylation patterns and expression of COL10A1 and COL2A1 in chondrocyte and MSC populations, in order to determine whether failed genomic methylation patterns correlate with an unstable chondrocyte phenotype after chondrogenesis of MSCs.
COL10A1 and COL2A1 regulatory gene regions were computationally searched for CpG-rich regions. CpG methylation of genomic DNA from human articular chondrocytes, MSCs, and MSC-derived chondrocytes was analyzed by Combined Bisulfite Restriction Analysis and by sequencing of polymerase chain reaction fragments amplified from bisulfite-treated genomic DNA.
The CpG island around the transcription start site of COL2A1 was unmethylated in all cell groups independent of COL2A1 expression, while 9 tested CpG sites in the sparse CpG promoter of COL10A1 were consistently methylated in human articular chondrocytes. Induction of COL10A1 expression during chondrogenesis of MSCs correlated with demethylation of 2 CpG sites in the COL10A1 promoter.
Our findings indicate that methylation-based COL10A1 gene silencing is established in cartilage tissue and human articular chondrocytes. Altered methylation levels at 2 CpG sites of COL10A1 in MSCs and their demethylation during chondrogenesis may facilitate induction of COL10A1 as observed during in vitro chondrogenesis of MSCs.
人类关节软骨细胞不表达COL10A1，也不会发生肥大，除非在软骨下骨附近。相比之下，由间充质干细胞（MSC）体外产生的软骨细胞会过早表达COL10A1，且无法形成稳定的异位软骨移植物，这表明它们在表型上可能不稳定，不适合用于治疗关节软骨损伤。自然发育过程中建立的CpG甲基化可能在抑制人类关节软骨细胞中COL10A1的表达和肥大方面发挥作用。本研究旨在比较软骨细胞和MSC群体中基因甲基化模式以及COL10A1和COL2A1的表达，以确定基因组甲基化模式异常是否与MSC软骨形成后软骨细胞表型不稳定相关。
通过计算机搜索COL10A1和COL2A1调控基因区域中富含CpG的区域。采用亚硫酸氢盐联合限制性分析以及对经亚硫酸氢盐处理的基因组DNA扩增的聚合酶链反应片段进行测序，分析来自人类关节软骨细胞、MSC和MSC来源的软骨细胞的基因组DNA的CpG甲基化情况。
COL2A1转录起始位点周围的CpG岛在所有细胞组中均未甲基化，与COL2A1的表达无关，而COL10A1稀疏CpG启动子中的9个测试CpG位点在人类关节软骨细胞中始终处于甲基化状态。MSC软骨形成过程中COL10A1表达的诱导与COL10A1启动子中2个CpG位点的去甲基化相关。
我们的研究结果表明，基于甲基化的COL10A1基因沉默在软骨组织和人类关节软骨细胞中得以建立。MSC中COL10A1的2个CpG位点甲基化水平改变以及它们在软骨形成过程中的去甲基化可能有助于诱导COL10A1表达，如在MSC体外软骨形成过程中所观察到的那样。
