Morishima Nobuhiro, Ito Yoshihiro
Nano Medical Engineering Laboratory, Cluster for Pioneering Research, RIKEN, Wako, Japan.
Nano Medical Engineering Laboratory, Cluster for Pioneering Research, RIKEN, Wako, Japan; Emergent Bioengineering Materials Research Team, Center for Emergent Matter Science, RIKEN, Wako, Japan.
J Biol Chem. 2024 Nov;300(11):107842. doi: 10.1016/j.jbc.2024.107842. Epub 2024 Sep 30.
Intracellular calcium dynamics is key to regulating various physiological events. Myotube formation by myoblast fusion is controlled by the release of Ca from the endoplasmic reticulum (ER), and the calpain (CAPN) family is postulated to be an executioner of the process. However, the activation of a specific member of the family or its physiological substrates is unclear. In this study, we explore the involvement of a CAPN in myoblast differentiation. Time-course experiments showed that the reduction in potential substrates of calpains, c-Myc and STAT3 (signal transducer and activator of transcription 3) and generation of STAT3 fragments occurred multiple times at an early stage of myoblast differentiation. Inhibition of the ER Ca release suppressed these phenomena, suggesting that the reduction was dependent on the cleavage by a CAPN. CAPN5 knockdown suppressed the reduction. In vitro reconstitution assay showed Ca- and CAPN5-dependent degradation of c-Myc and STAT3. These results suggest the activation of CAPN5 in differentiating myoblasts. Fusion of the Capn5 knockdown myoblast efficiently occurred; however, the upregulation of muscle-specific proteins (myosin and actinin) was suppressed. Myofibrils were poorly formed in the fused cells with a bulge where nuclei formed a cluster, suggesting that the myonuclear positioning was abnormal. STAT3 was hyperactivated in those fused cells, possibly inhibiting the upregulation of muscle-specific proteins necessary for the maturation of myotubes. These results suggest that the CAPN5 activity is essential in myoblast differentiation.
细胞内钙动力学是调节各种生理事件的关键。成肌细胞融合形成肌管受内质网（ER）钙释放的控制，钙蛋白酶（CAPN）家族被认为是该过程的执行者。然而，该家族特定成员的激活或其生理底物尚不清楚。在本研究中，我们探讨了一种钙蛋白酶在成肌细胞分化中的作用。时间进程实验表明，在成肌细胞分化早期，钙蛋白酶的潜在底物c-Myc和信号转导及转录激活因子3（STAT3）减少以及STAT3片段的产生多次发生。内质网钙释放的抑制抑制了这些现象，表明这种减少依赖于钙蛋白酶的切割作用。钙蛋白酶5（CAPN5）基因敲低抑制了这种减少。体外重组实验表明c-Myc和STAT3的降解依赖于钙和CAPN5。这些结果表明分化的成肌细胞中CAPN5被激活。钙蛋白酶5基因敲低的成肌细胞融合有效发生；然而，肌肉特异性蛋白（肌球蛋白和辅肌动蛋白）的上调受到抑制。在融合细胞中肌原纤维形成不良，有一个细胞核聚集的凸起，表明肌核定位异常。在这些融合细胞中STAT3过度激活，可能抑制了肌管成熟所需的肌肉特异性蛋白的上调。这些结果表明CAPN5活性在成肌细胞分化中至关重要。
