Louis Magali, Zanou Nadège, Van Schoor Monique, Gailly Philippe
Université catholique de Louvain, Institute of Neuroscience, Laboratory of Cell Physiology, 55/40 avenue Hippocrate, 1200 Brussels, Belgium.
J Cell Sci. 2008 Dec 1;121(Pt 23):3951-9. doi: 10.1242/jcs.037218. Epub 2008 Nov 11.
Myoblast migration is a key step in myogenesis and regeneration. It allows myoblast alignment and their fusion into myotubes. The process has been shown to involve m-calpain or mu-calpain, two Ca(2+)-dependent cysteine proteases. Here we measure calpain activity in cultured cells and show a peak of activity at the beginning of the differentiation process. We also observed a concomitant and transient increase of the influx of Ca(2+) and expression of TRPC1 protein. Calpains are specifically activated by a store-operated entry of Ca(2+) in adult skeletal muscle fibres. We therefore repressed the expression of TRPC1 in myoblasts and studied the effects on Ca(2+) fluxes and on differentiation. TRPC1-depleted myoblasts presented a largely reduced store-operated entry of Ca(2+) and a significantly diminished transient influx of Ca(2+) at the beginning of differentiation. The concomitant peak of calpain activity was abolished. TRPC1-knockdown myoblasts also accumulated myristoylated alanine-rich C-kinase substrate (MARCKS), an actin-binding protein and substrate of calpain. Their fusion into myotubes was significantly slowed down as a result of the reduced speed of cell migration. Accordingly, migration of control myoblasts was inhibited by 2-5 microM GsMTx4 toxin, an inhibitor of TRP channels or by 50 microM Z-Leu-Leu, an inhibitor of calpain. By contrast, stimulation of control myoblasts with IGF-1 increased the basal influx of Ca(2+), activated calpain and accelerated migration. These effects were not observed in TRPC1-knockdown cells. We therefore suggest that entry of Ca(2+) through TRPC1 channels induces a transient activation of calpain and subsequent proteolysis of MARCKS, which allows in turn, myoblast migration and fusion.
成肌细胞迁移是肌生成和再生的关键步骤。它使成肌细胞排列并融合形成肌管。已表明该过程涉及m-钙蛋白酶或μ-钙蛋白酶，这两种钙（2+）依赖性半胱氨酸蛋白酶。在这里，我们测量了培养细胞中的钙蛋白酶活性，并显示在分化过程开始时活性达到峰值。我们还观察到钙（2+）内流和TRPC1蛋白表达同时出现短暂增加。钙蛋白酶在成年骨骼肌纤维中通过钙库操纵的钙（2+）内流被特异性激活。因此，我们抑制了成肌细胞中TRPC1的表达，并研究了对钙（2+）通量和分化的影响。TRPC1缺失的成肌细胞在分化开始时钙库操纵的钙（2+）内流大幅减少，钙（2+）的瞬时内流也显著减少。钙蛋白酶活性的伴随峰值消失。TRPC1敲低的成肌细胞还积累了肉豆蔻酰化富含丙氨酸的蛋白激酶C底物（MARCKS），一种肌动蛋白结合蛋白和钙蛋白酶的底物。由于细胞迁移速度降低，它们融合形成肌管的过程明显减慢。相应地，对照成肌细胞的迁移受到TRP通道抑制剂2-5 microM GsMTx4毒素或钙蛋白酶抑制剂50 microM Z-Leu-Leu的抑制。相比之下，用IGF-1刺激对照成肌细胞会增加钙（2+）的基础内流，激活钙蛋白酶并加速迁移。在TRPC1敲低的细胞中未观察到这些效应。因此，我们认为通过TRPC1通道的钙（2+）内流诱导钙蛋白酶的瞬时激活以及随后MARCKS的蛋白水解，这反过来又允许成肌细胞迁移和融合。
