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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Method to Extend Research in Time (MERIT) Award (R37)
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Molecular Cytology Study Section (CTY)
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Syracuse University
Schools of Arts and Sciences
United States
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Samuel, D S; Ewton, D Z; Coolican, S A et al. (1999) Raf-1 activation stimulates proliferation and inhibits IGF-stimulated differentiation in L6A1 myoblasts. Horm Metab Res 31:55-64
Ewton, D Z; Coolican, S A; Mohan, S et al. (1998) Modulation of insulin-like growth factor actions in L6A1 myoblasts by insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5: a dual role for IGFBP-5. J Cell Physiol 177:47-57
Coolican, S A; Samuel, D S; Ewton, D Z et al. (1997) The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem 272:6653-62
Florini, J R; Ewton, D Z; Coolican, S A (1996) Growth hormone and the insulin-like growth factor system in myogenesis. Endocr Rev 17:481-517
Florini, J R; Samuel, D S; Ewton, D Z et al. (1996) Stimulation of myogenic differentiation by a neuregulin, glial growth factor 2. Are neuregulins the long-sought muscle trophic factors secreted by nerves? J Biol Chem 271:12699-702
Florini, J R; Ewton, D Z (1995) Actions of anabolic hormones and growth factors on cultured neonatal heart cells. Growth Regul 5:28-35
Ewton, D Z; Florini, J R (1995) IGF binding proteins-4, -5 and -6 may play specialized roles during L6 myoblast proliferation and differentiation. J Endocrinol 144:539-53
Bosche, W J; Ewton, D Z; Florini, J R (1995) Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis. J Cell Physiol 164:324-33
Ewton, D Z; Roof, S L; Magri, K A et al. (1994) IGF-II is more active than IGF-I in stimulating L6A1 myogenesis: greater mitogenic actions of IGF-I delay differentiation. J Cell Physiol 161:277-84
Florini, J R; Ewton, D Z; Magri, K A et al. (1993) IGFs and muscle differentiation. Adv Exp Med Biol 343:319-26

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