Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, China.
Department of Histology and Embryology of Medical College, Nantong University, Nantong, Jiangsu 226001, China.
Gene. 2018 Aug 20;668:204-210. doi: 10.1016/j.gene.2018.05.056. Epub 2018 May 18.
Calpain 3 (Capn3), a skeletal muscle-specific member of the calpain family, executes some non-proteolytic functions besides its role as a Ca-regulated proteolytic enzyme. Previously, we found that changes in Capn3 expression were linearly correlated with the degree of muscular atrophy following reversible sciatic nerve injury and that knockdown of Capn3 gene expression promoted myoblast differentiation. While the regulation of capn3 gene expression is interesting, transcriptional regulation of Capn3 is still unclear. In the present study, we provided experimental data showing that the myogenic enhancer factor 2A (MEF2A) regulated Capn3 gene expression. Firstly, the luciferase reporter assay and EMSA were performed and showed that ectopic expression of the Mef2a gene could bind to the predicted site of the Capn3 promoter region. Furthermore, in the L6 myoblast differentiation model in vitro, Capn3 gene expression was shown to be positively associated with the level of Mef2a by qRT-PCR, western-blotting, and immunocytochemistry. The Capn3 protein level decreased as MEF2A decreased when induced by Mef2a siRNA transfection in L6 myoblasts. Finally, the results of ChIP indicated that MEF2A occupied the promoter region of the Capn3 gene in rat denervated gastrocnemius muscle tissue. Based on these results, we proposed that MEF2A is a transcriptional regulator for Capn3 gene expression.
钙蛋白酶 3（Capn3）是钙蛋白酶家族中骨骼肌特异性成员，除了作为 Ca 调节的蛋白水解酶外，还具有一些非蛋白水解功能。以前，我们发现 Capn3 表达的变化与可逆性坐骨神经损伤后肌肉萎缩的程度呈线性相关，并且 Capn3 基因表达的敲低促进了成肌细胞分化。虽然 Capn3 基因表达的调节很有趣，但 Capn3 的转录调节仍不清楚。在本研究中，我们提供了实验数据表明，肌细胞增强因子 2A（MEF2A）调节 Capn3 基因表达。首先，进行了荧光素酶报告基因测定和电泳迁移率变动分析（EMSA），结果表明外源性表达 Mef2a 基因可以与 Capn3 启动子区域的预测位点结合。此外，在体外 L6 成肌细胞分化模型中，通过 qRT-PCR、western-blotting 和免疫细胞化学检测到 Capn3 基因表达与 Mef2a 水平呈正相关。当用 Mef2a siRNA 转染诱导 L6 成肌细胞时，Capn3 蛋白水平随着 MEF2A 的减少而降低。最后，ChIP 的结果表明 MEF2A 占据了大鼠失神经腓肠肌组织中 Capn3 基因的启动子区域。基于这些结果，我们提出 MEF2A 是 Capn3 基因表达的转录调节剂。
