Jahn L, Sadoshima J, Izumo S
Molecular Medicine Division, Beth Israel Hospital, Boston, Massachusetts.
Exp Cell Res. 1994 Jun;212(2):297-307. doi: 10.1006/excr.1994.1147.
Differentiation of skeletal myoblasts into contractile myotubes is associated with permanent withdrawal from the cell cycle. Little is known about the expression of cell cycle regulating genes during terminal differentiation of muscle cells. We investigated the expression pattern, biological activity, and cellular localization of cyclins and cyclin-dependent kinases during terminal differentiation of the mouse skeletal myogenic cell line C2C12. After induction of differentiation by serum deprivation, cdc2 mRNA levels transiently increased, followed by a down-regulation to undetectable levels within 42 h. In contrast, cdk2 mRNA stayed constant during this period. During differentiation cyclin A, B, and C were down-regulated within 24 h to undetectable levels. Interestingly, cyclin D1/CYL1 mRNA was up-regulated by twofold at 9-12 h after serum deprivation followed by a down-regulation to undetectable levels within 42 h, while cyclin D3/CYL3 mRNA levels remained constant. Restimulation of the differentiated myotube culture with serum reinduced cdc2 as well as cyclin D1/CYL1 mRNA close to the levels observed in dividing myoblasts. At the protein level p34cdc2 was detected in nuclei of proliferating myoblasts and nascent myotubes, but not in mature myotubes. Restimulation with serum-induced p34cdc2 protein in a small minority of unfused myoblasts, but never in myotubes. Histone H1 kinase activity of p34cdc2 decreased during differentiation while p33cdk2 activity did not change. These findings suggest that terminal differentiation of skeletal muscle cells is associated with a differential regulation of cyclins and their associated kinases. Inability to accumulate p34cdc2 protein in response to serum stimulation, despite the induction of its mRNA, in differentiated myotubes may play an important role in maintaining the postmitotic state of skeletal muscle in the presence of high concentrations of growth factors.
骨骼肌成肌细胞向收缩性肌管的分化与细胞周期的永久性退出相关。关于肌肉细胞终末分化过程中细胞周期调控基因的表达了解甚少。我们研究了细胞周期蛋白和细胞周期蛋白依赖性激酶在小鼠骨骼肌成肌细胞系C2C12终末分化过程中的表达模式、生物学活性和细胞定位。血清剥夺诱导分化后，cdc2 mRNA水平短暂升高，随后在42小时内下调至无法检测的水平。相比之下，cdk2 mRNA在此期间保持恒定。在分化过程中，细胞周期蛋白A、B和C在24小时内下调至无法检测的水平。有趣的是，血清剥夺后9 - 12小时，细胞周期蛋白D1/CYL1 mRNA上调两倍，随后在42小时内下调至无法检测的水平，而细胞周期蛋白D3/CYL3 mRNA水平保持恒定。用血清重新刺激分化的肌管培养物可重新诱导cdc2以及细胞周期蛋白D1/CYL1 mRNA接近在增殖成肌细胞中观察到的水平。在蛋白质水平上，p34cdc2在增殖的成肌细胞和新生肌管的细胞核中被检测到，但在成熟肌管中未检测到。用血清重新刺激在少数未融合的成肌细胞中诱导出p34cdc2蛋白，但在肌管中从未诱导出。p34cdc2的组蛋白H1激酶活性在分化过程中降低，而p33cdk2活性未改变。这些发现表明骨骼肌细胞的终末分化与细胞周期蛋白及其相关激酶的差异调节有关。尽管在分化的肌管中诱导了其mRNA，但无法响应血清刺激而积累p34cdc2蛋白可能在高浓度生长因子存在的情况下维持骨骼肌的有丝分裂后状态中起重要作用。
