Department of Biochemistry & Molecular Biology, The University of Calgary, Calgary, Albeta, Canada.
J Cell Physiol. 2013 Oct;228(10):1989-95. doi: 10.1002/jcp.24365.
L6 rat myoblasts undergo differentiation and myotube formation when cultured in medium containing a low-concentration of serum, but the underlying mechanism is not well understood. The role of atrogin-1, an E3 ligase with well-characterized roles in muscle atrophy, has not been defined in muscle differentiation. Myocardin is a coactivator of serum response factor (SRF), which together promotes smooth muscle differentiation. Myocardin is transiently expressed in skeletal muscle progenitor cells with inhibitory effects on the expression of myogenin and muscle differentiation. It remains unknown whether myocardin, which undergoes ubiquitination degradation, plays a role in L6 cell differentiation. The current study aimed to investigate the potential roles of myocardin and atrogin-1 in differentiation of L6 cells. As reported by many others, shifting to medium containing 2% serum induced myotube formation of L6 cells. Differentiation was accompanied by up-regulation of atrogin-1 and down-regulation of myocardin, suggesting that both may be involved in muscle differentiation. As expected, over-expression of atrogin-1 stimulated the expression of troponin T and myogenin and differentiation of the L6 myoblasts. Co-expression of myocardin with atrogin-1 inhibited atrogin-1-induced myogenin expression. Over-expression of atrogin-1 decreased myocardin protein level, albeit without affecting its mRNA level. Small-interfering RNA-mediated knockdown of atrogin-1 increased myocardin protein. Consistently, ectopic expression of myocardin inhibited myogenic differentiation. Unexpectedly, myocardin decreased the expression of atrogin-1 without involving Foxo1. Taken together, our results have demonstrated that atrogin-1 plays a positive role in skeletal muscle differentiation through down-regulation of myocardin.
L6 大鼠成肌细胞在含低浓度血清的培养基中培养时会发生分化和肌管形成，但其中的机制尚不清楚。E3 连接酶 atrogin-1 在肌肉萎缩中具有明确的作用，但在肌肉分化中其作用尚未确定。肌球蛋白结合蛋白是血清反应因子（SRF）的共激活因子，共同促进平滑肌分化。肌球蛋白结合蛋白在骨骼肌祖细胞中短暂表达，对肌生成素和肌肉分化的表达具有抑制作用。尚不清楚经历泛素化降解的肌球蛋白结合蛋白是否在 L6 细胞分化中发挥作用。本研究旨在探讨肌球蛋白结合蛋白和 atrogin-1 在 L6 细胞分化中的潜在作用。如许多人所报道的，将培养基中的血清浓度从 20%切换到 2%会诱导 L6 细胞形成肌管。分化伴随着 atrogin-1 的上调和肌球蛋白结合蛋白的下调，表明两者可能都参与了肌肉分化。正如预期的那样，atrogin-1 的过表达刺激了肌钙蛋白 T 和肌生成素的表达，并促进了 L6 成肌细胞的分化。肌球蛋白结合蛋白与 atrogin-1 共表达抑制了 atrogin-1 诱导的肌生成素表达。atrogin-1 的过表达降低了肌球蛋白结合蛋白的蛋白水平，尽管不影响其 mRNA 水平。小干扰 RNA 介导的 atrogin-1 敲低增加了肌球蛋白结合蛋白的蛋白水平。一致地，异位表达肌球蛋白结合蛋白抑制了成肌分化。出乎意料的是，肌球蛋白结合蛋白抑制了 atrogin-1 的表达，而不涉及 Foxo1。综上所述，我们的结果表明，atrogin-1 通过下调肌球蛋白结合蛋白在骨骼肌分化中发挥正向作用。
