<br>软骨细胞肥大分化过程中新型 Col10a1 调节机制的鉴定和表征。,Cell Death & Disease

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软骨细胞肥大分化过程中新型 Col10a1 调节机制的鉴定和表征。
Cell Death & Disease ( IF 8.1 ) Pub Date : 2014-Oct-16 , DOI: 10.1038/cddis.2014.444
J Gu ₁ , Y Lu ₂ , F Li ₃ , L Qiao ₁ , Q Wang ₁ , N Li ₁ , J A Borgia ₄ , Y Deng ₅ , G Lei ₆ , Q Zheng ₂

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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软骨细胞，最终发生细胞凋亡并被骨取代。尽管处于终末分化阶段，肥大软骨细胞仍被认为是骨生长的主要引擎。软骨细胞异常肥大见于许多骨骼发育不良和骨关节炎。同时，作为肥大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COL10A1表达改变常常伴随软骨细胞异常肥大。然而，X型胶原蛋白基因在软骨细胞肥大过程中如何受到调控尚未完全阐明。我们最近证明，Runx2 与 150 bp 小鼠 Col10a1 顺式增强子的相互作用是必要的，但对于其在转基因小鼠中肥大软骨细胞特异性报告基因的表达来说还不够，这表明需要额外的 Col10a1 调节剂。在本研究中，我们报告了该 150 bp 增强子的计算机序列分析及其多个结合因子的鉴定，包括 AP1、MEF2、NFAT、Runx1 和 TBX5。使用这种增强子作为诱饵，我们进行了酵母单杂交测定，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-α、Fus、GdF7 和 Runx3 作为顺式增强子和丰富表达 Col10a1 的肥大 MCT（用大 T 抗原永生化的小鼠软骨细胞）细胞的核提取物形成的特定复合物的成分。值得注意的是，一些候选基因在肥大的 MCT 细胞中差异表达，并与软骨细胞肥大和 Runx2（一种不可或缺的 Col10a1 调节因子）相关。有趣的是，我们在肥大的 MCT 细胞中检测到高水平的 Cox-2 表达。电泳迁移率变动测定和染色质免疫沉淀测定证实了 Cox-2 和 Col10a1 顺式增强子之间的相互作用，支持其作为候选 Col10a1 调节剂的作用。总之，我们的数据支持软骨细胞肥大分化过程中包含 Cox-2、以 Runx2 为中心的 Col10a1 调节机制。

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

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.

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