Coolican S A, Samuel D S, Ewton D Z, McWade F J, Florini J R
Biology Department, Syracuse University, Syracuse, New York 13244, USA.
J Biol Chem. 1997 Mar 7;272(10):6653-62. doi: 10.1074/jbc.272.10.6653.
It is well established that mitogens inhibit differentiation of skeletal muscle cells, but the insulin-like growth factors (IGFs), acting through a single receptor, stimulate both proliferation and differentiation of myoblasts. Although the IGF-I mitogenic signaling pathway has been extensively studied in other cell types, little is known about the signaling pathway leading to differentiation in skeletal muscle. By using specific inhibitors of the IGF signal transduction pathway, we have begun to define the signaling intermediates mediating the two responses to IGFs. We found that PD098059, an inhibitor of mitogen-activated protein (MAP) kinase kinase activation, inhibited IGF-stimulated proliferation of L6A1 myoblasts and the events associated with it, such as phosphorylation of the MAP kinases and elevation of c-fos mRNA and cyclin D protein. Surprisingly, PD098059 caused a dramatic enhancement of differentiation, evident both at a morphological (fusion of myoblasts into myotubes) and biochemical level (elevation of myogenin and p21 cyclin-dependent kinase inhibitor expression, as well as creatine kinase activity). In sharp contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase, and rapamycin, an inhibitor of the activation of p70 S6 kinase (p70(S6k)), completely abolished IGF stimulation of L6A1 differentiation. We found that p70(S6k) activity increased substantially during differentiation, and this increase was further enhanced by PD098059. Our results demonstrate that the MAP kinase pathway plays a primary role in the mitogenic response and is inhibitory to the myogenic response in L6A1 myoblasts, while activation of the phosphatidylinositol 3-kinase/p70(S6k) pathway is essential for IGF-stimulated differentiation. Thus, it appears that signaling from the IGF-I receptor utilizes two distinct pathways leading either to proliferation or differentiation.
丝裂原抑制骨骼肌细胞分化已得到充分证实，但胰岛素样生长因子（IGFs）通过单一受体发挥作用，可刺激成肌细胞的增殖和分化。尽管IGF-I的促有丝分裂信号通路在其他细胞类型中已得到广泛研究，但对于导致骨骼肌分化的信号通路却知之甚少。通过使用IGF信号转导通路的特异性抑制剂，我们已开始确定介导对IGFs两种反应的信号中间体。我们发现，PD098059，一种丝裂原活化蛋白（MAP）激酶激酶激活的抑制剂，可抑制IGF刺激的L6A1成肌细胞增殖及其相关事件，如MAP激酶的磷酸化以及c-fos mRNA和细胞周期蛋白D蛋白的升高。令人惊讶的是，PD098059导致分化显著增强，这在形态学（成肌细胞融合为肌管）和生化水平（肌细胞生成素和p21细胞周期蛋白依赖性激酶抑制剂表达升高以及肌酸激酶活性升高）均很明显。形成鲜明对比的是，磷脂酰肌醇3-激酶抑制剂LY294002和p70 S6激酶（p70(S6k)）激活的抑制剂雷帕霉素完全消除了IGF对L6A1分化的刺激。我们发现，在分化过程中p70(S6k)活性大幅增加，并且PDdPDPD098059可进一步增强这种增加。我们的结果表明，MAP激酶通路在促有丝分裂反应中起主要作用，并且对L6A1成肌细胞的生肌反应具有抑制作用，而磷脂酰肌醇3-激酶/p70(S6k)通路的激活对于IGF刺激的分化至关重要。因此，似乎来自IGF-I受体的信号利用两条不同的途径分别导致增殖或分化。
