Bian Huiqin, Zhu Ting, Liang Yuting, Hei Ruoxuan, Zhang Xiaojing, Li Xiaochen, Chen Jinnan, Lu Yaojuan, Gu Junxia, Qiao Longwei, Zheng Qiping
Department of Hematology and Hematological Laboratory Science, Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang, China.
Laboratory of Clinical Medicine, Huai'an Women & Children Hospital, Affiliated to Yangzhou University, Huai'an, China.
Front Genet. 2021 Jul 2;12:683939. doi: 10.3389/fgene.2021.683939. eCollection 2021.
Hypertrophic chondrocytes and their specific marker, the type X collagen gene (), are critical components of endochondral bone formation during skeletal development. We previously found that Runx2 is an indispensable mouse gene regulator and identified many other transcription factors (TFs) that potentially interact with the 150-bp cis-enhancer. However, the roles of these candidate TFs in expression and chondrocyte hypertrophy have not been elucidated. Here, we focus on 32 candidate TFs recently identified by analyzing the 150-bp enhancer using the transcription factor affinity prediction (TRAP) program. We found that 12 TFs (Hoxa3, Lsx, Evx2, Dlx5, S8, Pax2, Egr2, Mef2a, Barhl2, GKlf, Sox17, and Crx) were significantly upregulated and four TFs (Lhx4, Tbx5, Mef2c, and Hb9) were significantly downregulated in hypertrophic MCT cells, which show upregulation of expression. Most of the differential expression pattern of these TFs conformed with the results obtained from ATDC5 cell model and primary mouse chondrocytes. Notably, was downregulated upon upregulation, overexpression of decreased expression, and knock-down of increased expression in hypertrophic chondrocytes, suggesting that Tbx5 is a negative regulator of . We further generated a stable -overexpressing ATDC5 cell line and transgenic mice driven by -specific enhancers and promoters. overexpression decreased expression in ATDC5 cells cultured as early as day 7 and in limb tissue on post-natal day 1. Slightly weaker alkaline phosphatase staining was also observed in cell culture on day 7 and in limb digits on embryonic day 17.5, indicating mildly delayed ossification. Further characterization of these candidate transcriptional regulators could help identify novel therapeutic targets for skeletal diseases associated with abnormal chondrocyte hypertrophy.
肥大软骨细胞及其特异性标志物X型胶原蛋白基因（）是骨骼发育过程中软骨内成骨的关键组成部分。我们之前发现Runx2是小鼠不可或缺的基因调节因子，并鉴定了许多其他可能与150 bp顺式增强子相互作用的转录因子（TFs）。然而，这些候选TFs在表达和软骨细胞肥大中的作用尚未阐明。在这里，我们聚焦于最近通过使用转录因子亲和力预测（TRAP）程序分析150 bp增强子而鉴定出的32个候选TFs。我们发现，在显示表达上调的肥大MCT细胞中，12个TFs（Hoxa3、Lsx、Evx2、Dlx5、S8、Pax2、Egr2、Mef2a、Barhl2、GKlf、Sox17和Crx）显著上调，4个TFs（Lhx4、Tbx5、Mef2c和Hb9）显著下调。这些TFs的大多数差异表达模式与从ATDC5细胞模型和原代小鼠软骨细胞获得的结果一致。值得注意的是，在肥大软骨细胞中，上调时下调，过表达时表达降低；敲低时表达增加，这表明Tbx5是表达的负调节因子。我们进一步构建了稳定过表达的ATDC5细胞系以及由特异性增强子和启动子驱动的转基因小鼠。过表达在培养至第7天的ATDC5细胞以及出生后第1天的肢体组织中降低了表达。在第7天的细胞培养物以及胚胎第17.5天的肢体指中也观察到碱性磷酸酶染色略弱，表明骨化略有延迟。对这些候选转录调节因子的进一步表征可能有助于识别与软骨细胞肥大异常相关的骨骼疾病的新治疗靶点。
