EXTRACELLULAR SIGNAL-REGULATED KINASE-1 AND KINASE-2 RESPOND DIFFERENTLY TO MITOGENIC AND DIFFERENTIATIVE SIGNALING PATHWAYS IN MYOBLASTS

In this report we show that extracellular signal-regulated kinase-1 an d -2 (ERK-1 and -2) respond differently to signals that elicit prolife ration and/or differentiation of myoblasts using the C2C12 cell line a nd nondifferentiating mutant NFB4 cells derived from them. Induction o f differentiation by withdrawal of serum rendered ERKs in C2C12 myobla sts relatively insensitive to restimulation by serum. Instead, myogeni c differentiation of C2C12 cells was associated with sustained activat ion of ERK-2 dependent on the insulin-like growth factor II(IGF-II) au tocrine loop. By contrast, mutant NFB4 cells cultured under the same c onditions remained proliferative and demonstrated robust activation of ERKs in response to serum. Similarly, a G(i)-dependent signaling path way induced activation of ERKs in NFB4 cells, but not in C2C12 cells, after stimulation by lysophosphatidic acid (LPA). In NFB4 cells partia lly rescued by prolonged IGF-I treatment, ERK activity remained respon sive to G(i)-dependent LPA stimulation, whereas rescue of NFB4 cells b y constitutive expression of myogenin or MyoD, associated with activat ion of the IGF-II autocrine loop, rendered the G(i)-signaling pathway refractory to LPA stimulation. Relatively high levels of G(alpha i2) w ere detected in NFB4 cells and IGF-I treated NFB4 cells, which correla ted with responsive Gi signaling. Activation of the IGF-II autocrine l oop in C2C12 and NFB4 myoblasts or treatment with IGF-II was associate d with loss of G and inhibition of G(i)-dependent signaling. Thus, IGF -I and IGF-II activate distinct signaling cascades, with IGF-II elicit ing 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 activat ion. Transient stimulation of NFB4 cells with IGF-I while blocking act ivation of G(i)-proteins is with pertussis toxin resulted in preferent ial activation of ERK-2 characteristic of differentiated C2C12 cells, suggesting that proliferation induced by IGF-I is G(i)-dependent and s eparable from the IGF-I-signaling pathway that leads to differentiatio n.