title: Raf-1 activation stimulates proliferation and inhibits IGF-stimulated differentiation in L6A1 myoblasts.
abstract: Our previous work has demonstrated that the insulin-like growth factors (IGFs), acting through a single receptor, stimulate both proliferation and differentiation of L6A1 myoblasts. This unique model system has enabled us to closely examine the switch that regulates these two opposing responses. We have previously shown, using specific inhibitors of the IGF-I signal transduction pathway, that the mitogenic response is mediated by the Ras/Raf/MAP kinase pathway and the myogenic response by the PI 3-kinase/p70s6k pathway (Coolican SA, Samuel DS, Ewton DZ, McWade FJ, Florini JR, J Biol Chem 1997; 272: 6653-62). In that study we found that PD098059, an inhibitor of MEK activation, inhibited the proliferative response, but dramatically enhanced IGF-stimulated differentiation which was associated with elevation of p70s6k activity. Since there have been reports of elevation of Raf-1 activity in PD098059-treated L6 myoblasts, and stimulation of p70s6k activity in cells expressing an activated Raf-1, it was important to determine whether or not Raf-1 elevation plays a role in the myogenic response. To test this, we have transfected L6A1 myoblasts with delta Raf-1:ER, an estradiol-regulated form of oncogenic Raf-1. We found that activation of Raf-1 by estradiol resulted in increased phosphorylation of p42 and p44 MAP kinases and stimulation of proliferation. In contrast, Raf-1 activation inhibited all measured aspects of the myogenic response: myogenin expression, creatine kinase elevation, and fusion of myoblasts to form myotubes. In addition, we found no elevation of p70s6k activity upon Raf-1 activation. These results indicate the following: (1) stimulation of myogenic differentiation by PD098059 treatment is not simply due to the elevation of Raf-1, (2) Raf-1 has a positive role in the MAP kinase pathway and myoblast proliferation, and (3) Raf-1 activation inhibits myogenesis, possibly by forcing cells to remain in the proliferative state.
authors.affiliation: Department of Biology, Syracuse University, NY, USA. dssamuel@mailbox.syr.edu
authors.forename: D S
authors.initials: DS
authors.lastname: Samuel
authors.forename: D Z
authors.initials: DZ
authors.lastname: Ewton
authors.forename: S A
authors.initials: SA
authors.lastname: Coolican
authors.forename: T D
authors.initials: TD
authors.lastname: Petley
authors.forename: F J
authors.initials: FJ
authors.lastname: McWade
authors.forename: J R
authors.initials: JR
authors.lastname: Florini
mesh_terms: D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines / Q000494:pharmacology; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D018482:Muscle, Skeletal / Q000166:cytology; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
chemical_list: D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009025:Morpholines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004958:Estradiol; D007334:Insulin-Like Growth Factor I; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one