IGF-II is more active than IGF-I in stimulating L6A1 myogenesis: greater mitogenic actions of IGF-I delay differentiation.

Mitogens are generally thought to inhibit myogenesis, and many cell biologists have found it hard to interpret observations that the insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of muscle cells in culture. Our previous studies suggested that the Type I IGF receptor mediates these actions. However, IGF-II and insulin treatment caused myoblasts to differentiate much more extensively, suggesting that more complex mechanisms may be involved. Here we present evidence that the greater mitogenic activity of IGF-I (compared to IGF-II and insulin) delays L6A1 myoblast differentiation. Under conditions in which the mitogenic actions of IGF-I are suppressed, the stimulation of myogenesis by IGF-I approached that by IGF-II: (1) in L6A1 cultures plated at a higher cell density; (2) in L6A1 cultures in which cell proliferation was inhibited by cytosine arabinoside or aphidicolin; and (3) in cultures of primary human muscle cells, which exhibit a smaller mitogenic response to IGF-I. Further evidence that the Type I receptor plays a major role in relaying the signal for differentiation was obtained by using IGF-I and IGF-II analogs. Analogs which have reduced affinity for the Type I receptor showed a dramatic decrease in activity, while an analog with increased affinity for the Type II receptor was no more active than native IGF-I. Our results indicate that both mitogenic and myogenic actions of IGF-I are mediated by the Type I receptor. We conclude that IGF-I delays the onset of myogenesis as a result of its mitogenic actions, and only subsequently stimulates myogenesis. These observations reconcile the apparent conflict between our results with the IGFs and other investigators' reports of effects of other mitogens.

Paradoxical decrease in myf-5 messenger RNA levels during induction of myogenic differentiation by insulin-like growth factors.

Having previously demonstrated that the insulin-like growth factors (IGFs) induce expression of the myogenin gene, we have now extended our investigation of the induction of myogenesis by the IGFs to a second member of the MyoD family, myf-5. This is the only myogenesis gene other than myogenin expressed early in the differentiation of L6 myoblasts, so its regulation was of particular interest because of our observations on myogenin. In contrast to myogenin, myf-5 mRNA was detectable in proliferating myoblasts, but the steady state levels of myf-5 mRNA fell strikingly for 48 h after the cells were switched to low serum medium containing IGF-II in both murine cell lines and myoblasts cultured from human muscle. In spite of this decrease, translation of myf-5 mRNA appeared essential during the early stages of stimulation of myogenesis by the IGFs; an antisense oligodeoxynucleotide complementary to the first five codons of myf-5 blocked the increase in myogenin mRNA and inhibited morphological (cell fusion) and biochemical (creatine kinase elevation) aspects of myogenesis. We conclude that expression of myf-5 is essential for the initial induction of myogenin by the IGFs, but that subsequent elevation of myogenin expression is independent of myf-5, possibly resulting from autoinduction of the myogenin gene. The functional significance of the dramatic decrease in myf-5 mRNA levels during differentiation is not obvious.

Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression.

Stimulation of myogenic differentiation by the insulin-like growth factors (IGFs) has been established for many years, but our attempts to elucidate the mechanism of that stimulation have been successful only in eliminating some likely possibilities. The recent discovery of a family of muscle determination genes has opened a new approach to this question, allowing specific focus on those genes that might play central roles in controlling myogenesis. We now report that IGF-I stimulates terminal myogenic differentiation in L6A1 cells by inducing a large increase in expression of the myogenin gene. This conclusion is supported by the following observations. 1) Myogenin mRNA is elevated by IGF-I, with a concentration dependency that parallels the stimulation of differentiation, including a decrease in stimulation at higher concentrations. 2) The time course of elevation of myogenin mRNA is consistent with its acting as an intermediate in the signalling pathway between occupancy of the IGF-I receptor and induction of expression of muscle-specific genes. 3) Inhibitors of myogenesis also inhibit elevation of myogenin mRNA in response to IGF-I. 4) An antisense oligonucleotide to the N-terminus of myogenin prevents the stimulation of differentiation by IGF-I and IGF-II, but has no effect on other actions of IGF-I on myoblasts. MyoD has been reported not to be expressed in L6 cells, and the expression of myf-5 and herculin/myf-6/MRF4 is reportedly low or undetectable. Thus, the stimulation of differentiation by IGF-I can be attributed largely, if not entirely, to increased expression of the myogenin gene. However, the relatively long time period between addition of the IGFs and elevation of myogenin mRNA as well as the inhibition of this process by several inhibitors indicate that increased myogenin mRNA levels are not a simple direct result of occupation of the IGF-I receptor.