The overall objective of this research is to identify agents and mechanisms that are important in stimulating the growth and repair of damaged skeletal and cardiac muscle. After extensive work in this area, I have concluded that the somatomedins/Insulin- like Growth Factors (IGFs) are the most active anabolic hormones and that they play a major role in the growth, development, and maintenance of muscle tissue. The recent availability of relatively large quantities of these (and other) growth factors produced by recombinant DNA technology has made it feasible to do experiments that were previously impossible. This fact, coupled with our characterization of agents stimulating and inhibiting muscle differentiation, now allows a sharply focussed study of the control of this important aspect of development. During the current grant period, we have developed a system in which differentiation of cloned L6 myoblasts (in serum-free medium) can be stimulated by addition of 10 ng/ml (1.3nM) IGF-I and inhibited by 0.5 ng/m1 (20 pM) transforming growth factor- beta (TGF-beta). We (and others) have demonstratd that TGF- beta inhibits all measured aspects of differentiation, acting at an early stage of the process. Using incubations of other inhibitors followed by TGF-beta, we have confirmed our expectation that both RNA and protein synthesis are required for the commitment to terminal differentiation. We now propose to exploit this well- defined system by investigating molecular events that occur during commitment to myogenesis, as has recently been done in studies of the initial events that occur as quiescent cells reenter the cell cycle. Our experiments are designed to evaluate the following hypothesis: commitment of myoblasts to terminal differentiation involves the expression of specific genes during a period in which there is little or no formation of muscle-specific proteins. We will optimize the system by seeking conditions that synchronize onset of differentiation, and determining the most selective time to measure commitment. On the basis of these results, we will screen a lambda gt11 cDNA library prepared from IGF-I stimulated cells at the selective time. The initial screen will be based on comparisons of poly(A)+RNA populations from IGF- stimulated cells incubated in the presence or absence of TGF- beta. Selected clones will be further screened with probes corresponding to proteins expressed later in differentiation in order to focus attention on early controlling events. We will look for clones corresponding to the time course of commitment, and then characterize the selected clones, the corresponding mRNAs, and their protein products. We will also take advantage of the inhibition of differentiation (but not of other actions of IGF-I) by TGF-beta to investigate the role of receptor phosphorylation and tyrosine kinase activities in mediating the various actions of IGF-I on myoblasts.
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