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Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation

机译：软骨细胞肥大分化过程中新型Col10a1调控机制的鉴定与表征

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The majority of human skeleton develops through the endochondral pathway, in which cartilage-forming chondrocytes proliferate and enlarge into hypertrophic chondrocytes that eventually undergo apoptosis and are replaced by bone. Although at a terminal differentiation stage, hypertrophic chondrocytes have been implicated as the principal engine of bone growth. Abnormal chondrocyte hypertrophy has been seen in many skeletal dysplasia and osteoarthritis. Meanwhile, as a specific marker of hypertrophic chondrocytes, the type X collagen gene ( COL10A1 ) is also critical for endochondral bone formation, as mutation and altered COL10A1 expression are often accompanied by abnormal chondrocyte hypertrophy in many skeletal diseases. However, how the type X collagen gene is regulated during chondrocyte hypertrophy has not been fully elucidated. We have recently demonstrated that Runx2 interaction with a 150-bp mouse Col10a1 cis-enhancer is required but not sufficient for its hypertrophic chondrocyte-specific reporter expression in transgenic mice, suggesting requirement of additional Col10a1 regulators. In this study, we report in silico sequence analysis of this 150-bp enhancer and identification of its multiple binding factors, including AP1, MEF2, NFAT, Runx1 and TBX5. Using this enhancer as bait, we performed yeast one-hybrid assay and identified multiple candidate Col10a1 -interacting genes, including cyclooxygenase 1 ( Cox-1 ) and Cox-2 . We have also performed mass spectrometry analysis and detected EF1-alpha, Fus, GdF7 and Runx3 as components of the specific complex formed by the cis-enhancer and nuclear extracts from hypertrophic MCT (mouse chondrocytes immortalized with large T antigen) cells that express Col10a1 abundantly. Notably, some of the candidate genes are differentially expressed in hypertrophic MCT cells and have been associated with chondrocyte hypertrophy and Runx2, an indispensible Col10a1 regulator. Intriguingly, we detected high-level Cox-2 expression in hypertrophic MCT cells. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays confirmed the interaction between Cox-2 and Col10a1 cis-enhancer, supporting its role as a candidate Col10a1 regulator. Together, our data support a Cox-2-containing, Runx2-centered Col10a1 regulatory mechanism, during chondrocyte hypertrophic differentiation.
机译：大多数人体骨骼通过软骨内途径发育，其中形成软骨的软骨细胞增殖并扩大为肥大的软骨细胞，这些软骨细胞最终会发生凋亡并被骨骼所取代。尽管处于晚期分化阶段，但是肥大的软骨细胞被认为是骨骼生长的主要引擎。在许多骨骼发育不良和骨关节炎中都发现了异常的软骨细胞肥大。同时，作为肥大性软骨细胞的特异性标志物，X型胶原基因（COL10A1）对于软骨内骨形成也至关重要，因为在许多骨骼疾病中突变和改变的COL10A1表达常伴有异常的软骨细胞肥大。然而，尚未充分阐明在软骨细胞肥大过程中如何调节X型胶原基因。我们最近证明，需要Runx2与150 bp小鼠Col10a1顺式增强子的相互作用，但不足以使其在转基因小鼠中表达肥大的软骨细胞特异性报告基因，提示需要其他Col10a1调节剂。在这项研究中，我们报告了该150 bp增强子的计算机序列分析，并鉴定了其多种结合因子，包括AP1，MEF2，NFAT，Runx1和TBX5。使用这种增强剂作为诱饵，我们进行了酵母一杂交测定，并鉴定了多个候选Col10a1相互作用基因，包括环氧合酶1（Cox-1）和Cox-2。我们还进行了质谱分析，并检测到EF1-alpha，Fus，GdF7和Runx3作为顺式增强子和肥大MCT细胞（富含大T抗原的永生的软骨细胞）的核提取物形成的特定复合物的成分，这些细胞大量表达Col10a1 。值得注意的是，某些候选基因在肥厚性MCT细胞中差异表达，并与软骨细胞肥大和Runx2（一种不可缺少的Col10a1调节剂）相关。有趣的是，我们在肥大性MCT细胞中检测到高水平的Cox-2表达。电泳迁移率迁移测定和染色质免疫沉淀测定证实了Cox-2和Col10a1顺式增强剂之间的相互作用，支持其作为候选Col10a1调节剂的作用。总之，我们的数据支持在软骨细胞肥大性分化过程中以Cox-2为中心，以Runx2为中心的Col10a1调节机制。

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