Sarbassov D D, Jones L G, Peterson C A
Department of Medicine, University of Arkansas for Medical Sciences McClellan Veterans Hospital, Little Rock 72205, USA.
Mol Endocrinol. 1997 Dec;11(13):2038-47. doi: 10.1210/mend.11.13.0036.
In this report we show that extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) respond differently to signals that elicit proliferation and/or differentiation of myoblasts using the C2C12 cell line and nondifferentiating mutant NFB4 cells derived from them. Induction of differentiation by withdrawal of serum rendered ERKs in C2C12 myoblasts relatively insensitive to restimulation by serum. Instead, myogenic differentiation of C2C12 cells was associated with sustained activation of ERK-2 dependent on the insulin-like growth factor II (IGF-II) autocrine loop. By contrast, mutant NFB4 cells cultured under the same conditions remained proliferative and demonstrated robust activation of ERKs in response to serum. Similarly, a Gi-dependent signaling pathway induced activation of ERKs in NFB4 cells, but not in C2C12 cells, after stimulation by lysophosphatidic acid (LPA). In NFB4 cells partially rescued by prolonged IGF-I treatment, ERK activity remained responsive to Gi-dependent LPA stimulation, whereas rescue of NFB4 cells by constitutive expression of myogenin or MyoD, associated with activation of the IGF-II autocrine loop, rendered the Gi-signaling pathway refractory to LPA stimulation. Relatively high levels of G(alpha i2) were detected in NFB4 cells and IGF-I treated NFB4 cells, which correlated with responsive Gi signaling. Activation of the IGF-II autocrine loop in C2C12 and NFB4 myoblasts or treatment with IGF-II was associated with loss of G(alpha i2) and inhibition of Gi-dependent signaling. Thus, IGF-I and IGF-II activate distinct signaling cascades, with IGF-II eliciting a stronger differentiation effect correlated with down-regulation of G(alpha i2) protein. Short-term stimulation of NFB4 cells with IGF-I, a mitogenic signal for myoblasts, also induced ERK-1 and -2 activation. Transient stimulation of NFB4 cells with IGF-I while blocking activation of Gi-proteins is with pertussis toxin resulted in preferential activation of ERK-2 characteristic of differentiated C2C12 cells, suggesting that proliferation induced by IGF-I is Gi-dependent and separable from the IGF-I-signaling pathway that leads to differentiation.
在本报告中，我们表明，利用C2C12细胞系以及从中衍生出的未分化突变体NFB4细胞，细胞外信号调节激酶1和2（ERK-1和ERK-2）对引发成肌细胞增殖和/或分化的信号反应不同。血清撤除诱导分化使得C2C12成肌细胞中的ERK对血清再刺激相对不敏感。相反，C2C12细胞的肌源性分化与依赖胰岛素样生长因子II（IGF-II）自分泌环的ERK-2持续激活相关。相比之下，在相同条件下培养的突变体NFB4细胞仍具有增殖能力，并且在响应血清时表现出ERK的强烈激活。同样，溶血磷脂酸（LPA）刺激后，一种依赖Gi的信号通路在NFB4细胞中诱导了ERK的激活，但在C2C12细胞中未诱导。在用延长的IGF-I处理部分挽救的NFB4细胞中，ERK活性仍对依赖Gi的LPA刺激有反应，而通过组成型表达肌细胞生成素或MyoD对NFB4细胞进行挽救，这与IGF-II自分泌环的激活相关，使得Gi信号通路对LPA刺激无反应。在NFB4细胞和经IGF-I处理的NFB4细胞中检测到相对高水平的G(αi2)，这与有反应的Gi信号相关。C2C12和NFB4成肌细胞中IGF-II自分泌环的激活或用IGF-II处理与G(αi2)的丧失和Gi依赖信号的抑制相关。因此，IGF-I和IGF-II激活不同的信号级联，其中IGF-II引发更强的分化效应，这与G(αi2)蛋白的下调相关。用IGF-I对NFB4细胞进行短期刺激，IGF-I是成肌细胞的促有丝分裂信号，也诱导了ERK-1和ERK-2的激活。在用百日咳毒素阻断Gi蛋白激活的同时，用IGF-I对NFB4细胞进行短暂刺激，导致了分化的C2C12细胞特有的ERK-2优先激活，这表明IGF-I诱导的增殖是Gi依赖的，并且与导致分化的IGF-I信号通路可分离。
