Chen Chen, Wu Xuan, Han Tiaotiao, Chen Jinnan, Bian Huiqin, Hei Ruoxuan, Tang Sitao, Li Zhenying, Lu Yaojuan, Gu Junxia, Qiao Longwei, Zheng Qiping
Department of Laboratory Medicine, School of Medicine, Jiangsu University Zhenjiang 212013, Jiangsu, China.
Department of Clinical Diagnose, Tangdu Hospital, Air Force Medical University Xi'an 710000, Shaanxi, China.
Am J Transl Res. 2023 Jun 15;15(6):4020-4032. eCollection 2023.
The type X collagen gene () is a signature gene of hypertrophic chondrocytes that are known as the main engine of long bone growth. Multiple transcription factors (TFs), including myocyte enhancer factor 2A (Mef2a), have previously been identified by analysis as potential gene regulators.
In this study, we aimed to investigate the correlation between Mef2a and Col10a1 expression and the possible effects on chondrocyte proliferation and hypertrophic differentiation .
First, Mef2a expression in proliferating and hypertrophic chondrocytes were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in two chondrocytic models, ATDC5 and MCT cells, as well as in mouse chondrocytes . Transfection with Mef2a small interfering fragments or Mef2a overexpression plasmids in the above chondrocytic models were performed to determine how Mef2a knockdown or overexpression may influence Col10a1 expression. The binding between Mef2a and its putative binding site within the 150 bp cis-enhancer which was evaluated by the dual luciferase reporter assay. The effect of Mef2a on chondrocyte differentiation was determined by examining the chondrogenic marker gene expression by qRT-PCR and by alcian blue, alkaline phosphatase (ALP), and alizarin red staining of the ATDC5 cells stably knocked down by Mef2a.
The expression of Mef2a in hypertrophic chondrocytes was significantly higher than that in proliferative chondrocytes in both chondrocytic models as well as in mouse chondrocytes . Interference with Mef2a caused decreased Col10a1 expression, while overexpression of Mef2a upregulated Col10a1. The result of the dual luciferase reporter assay showed that Mef2a enhanced Col10a1 gene enhancer activity via its putative Mef2a binding site. For the staining of ATDC5 stable cell lines, although no significant differences were seen in ALP staining, significantly weaker alcian blue staining intensity was noticed in Mef2a knockdown stable cell lines compared to the control cells at day 21, while slightly weaker alizarin red staining was seen in the stable cell lines at days 14 and 21. Correspondingly, we detected decreased runt-related transcription factor 2 (), increased SRY-box transcription factor 9 (), as well as differential expression of other chondrogenic markers in ATDC5 stable cell lines compared with the controls.
In conclusion, our results support that Mef2a upregulates Col10a1 expression possibly by interaction with its cis-enhancer. Altered levels of Mef2a affects the expression of chondrogenic marker genes, such as Runx2 and Sox9, but may only play an insignificant role during chondrocyte proliferation and maturation.
X型胶原蛋白基因（）是肥大软骨细胞的标志性基因，肥大软骨细胞是长骨生长的主要驱动力。先前通过分析已鉴定出多种转录因子（TFs），包括肌细胞增强因子2A（Mef2a），它们是潜在的基因调节因子。
在本研究中，我们旨在探讨Mef2a与Col10a1表达之间的相关性以及对软骨细胞增殖和肥大分化的可能影响。
首先，通过定量实时PCR（qRT-PCR）和蛋白质免疫印迹法在两种软骨细胞模型（ATDC5和MCT细胞）以及小鼠软骨细胞中检测增殖期和肥大期软骨细胞中Mef2a的表达。在上述软骨细胞模型中转染Mef2a小干扰片段或Mef2a过表达质粒，以确定Mef2a基因敲低或过表达如何影响Col10a1的表达。通过双荧光素酶报告基因检测评估Mef2a与其在150 bp顺式增强子内的假定结合位点之间的结合。通过qRT-PCR检测软骨生成标记基因的表达，并对Mef2a稳定敲低的ATDC5细胞进行阿尔辛蓝、碱性磷酸酶（ALP）和茜素红染色，以确定Mef2a对软骨细胞分化的影响。
在两种软骨细胞模型以及小鼠软骨细胞中，肥大软骨细胞中Mef2a的表达均显著高于增殖期软骨细胞。干扰Mef2a导致Col10a1表达降低，而Mef2a过表达则上调Col10a1。双荧光素酶报告基因检测结果表明，Mef2a通过其假定的Mef2a结合位点增强Col10a1基因增强子活性。对于ATDC5稳定细胞系的染色，虽然在第21天ALP染色未观察到显著差异，但与对照细胞相比，Mef2a敲低稳定细胞系在第21天的阿尔辛蓝染色强度明显较弱，而在第14天和第21天稳定细胞系的茜素红染色略弱。相应地，与对照相比，我们在ATDC5稳定细胞系中检测到矮小相关转录因子2（）表达降低、SRY盒转录因子9（）表达增加以及其他软骨生成标记物的差异表达。
总之，我们的结果支持Mef2a可能通过与其顺式增强子相互作用上调Col10a1表达。Mef2a水平的改变影响软骨生成标记基因如Runx2和Sox9的表达，但在软骨细胞增殖和成熟过程中可能仅起微不足道的作用。
