Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation.
Identification and characterization of the novel Col10a1 regulatory mechanism during chondrocyte hypertrophic differentiation.
复制标题
软骨细胞肥大分化过程中新型 Col10a1 调控机制的鉴定和表征
DOI：
10.1038/cddis.2014.444
复制
发表时间：
2014-10-16
影响因子：
9
通讯作者：
Zheng Q
中科院分区：
文献类型：
--
作者：
Gu J;Lu Y;Li F;Qiao L;Wang Q;Li N;Borgia JA;Deng Y;Lei G;Zheng Q
大多数人类骨骼通过软骨内途径发育，其中软骨形成软骨细胞增殖并扩大成肥大软骨细胞，肥大软骨细胞最终经历凋亡并被骨取代。虽然处于终末分化阶段，但肥大软骨细胞已被认为是骨生长的主要引擎。在许多骨骼发育不良和骨关节炎中可以看到异常的软骨细胞肥大。同时，作为肥大软骨细胞的特异性标志物，X型胶原基因（COL 10A 1）对于软骨内骨形成也是至关重要的，因为在许多骨骼疾病中，COL 10A 1的突变和改变的表达常常伴随着软骨细胞的异常肥大。然而，X型胶原基因在软骨细胞肥大过程中是如何调控的还没有完全阐明。我们最近证明，Runx 2与150 bp的小鼠Col 10a 1顺式增强子的相互作用是必需的，但不足以在转基因小鼠中表达其肥大软骨细胞特异性报告基因，这表明需要额外的Col 10a 1调节剂。在这项研究中，我们报告了这个150-bp增强子的计算机序列分析及其多种结合因子的鉴定，包括AP 1，MEF 2，NFAT，Runx 1和TBX 5。使用这种增强子作为诱饵，我们进行了酵母单杂交试验，并确定了多个候选Col 10 a1相互作用的基因，包括环氧合酶1（考克斯-1）和考克斯-2。我们还进行了质谱分析，并检测EF 1-α，Fus，GdF 7和Runx 3作为由顺式增强子和肥大MCT（小鼠软骨细胞永生化与大T抗原）细胞的核提取物形成的特异性复合物的组分，该细胞大量表达Col 10a 1。值得注意的是，一些候选基因在肥大MCT细胞中差异表达，并与软骨细胞肥大和Runx 2（一种不可或缺的Col 10a 1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考克斯-2表达。电泳迁移率变动分析和染色质免疫沉淀分析证实了考克斯-2和Col 10 a1顺式增强子之间的相互作用，支持其作为候选Col 10 a1调节剂的作用。总之，我们的数据支持考克斯-2，Runx 2为中心的Col 10a 1的调节机制，在软骨细胞肥大分化。
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.
影响因子：
3.7
作者：
Miller AK;Print CG;Nielsen PM;Crampin EJ
通讯作者：
Crampin EJ