Abstract
In many cell types including myoblasts, growth factors control proliferation and differentiation, in part, via the mitogen-activated protein kinase (MAPK) pathway (also known as the extracellular regulated kinase (Erk) pathway). In C2C12 myoblast cells, insulin-like growth factor-1 and basic fibroblast growth factor (bFGF) activate MAPK/Erk, and both growth factors promote myoblast proliferation. However, these factors have opposing roles with respect to differentiation; insulin-like growth factor-1 enhances muscle cell differentiation, whereas bFGF inhibits the expression of the muscle-specific transcription factors MyoD and myogenin. Cells treated with bFGF and PD98059, a specific inhibitor of the MAPK pathway, show enhanced expression of the muscle-specific transcription factors MyoD and myogenin as compared with cells not exposed to this inhibitor. Inhibiting MAPK activity also enhances myoblast fusion and the expression of the late differentiation marker myosin heavy chain. Basic FGF mediated repression of muscle-specific genes does not result from continued cell proliferation, since bFGF-treated cells progress through only one round of cell division. We have identified a critical boundary 16 to 20 h after plating during which bFGF induced MAPK activity is able to repress myogenic gene expression and differentiation. Thus, the targets of MAPK that regulate myogenesis are functional at this time and their identification is in progress.
Highlights
The mitogen-activated protein kinase (MAPK)1 signaling cascade has been implicated in the regulation of numerous cellular processes including cell growth and differentiation
The present study addresses the role of basic fibroblast growth factor (bFGF) induced p42/p44 MAPK activation in C2C12 differentiation
Quiescent C2C12 cells were stimulated with the indicated concentrations of bFGF or insulinlike growth factor-1 (IGF-1) and MAPK phosphorylation was measured after 5 min by Western blotting with a MAPK antibody that recognizes the activated form of MAPK (Fig. 1)
Summary
Mouse myoblast cells (C2C12) [22, 23] were maintained as subconfluent monolayers in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/liter glucose and L-glutamine supplemented with 20% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 g/ml streptomycin. Cells were rendered quiescent by suspension in methylcellulose [24, 25]. Subconfluent cells were trypsinized and ϳ2 ϫ 107 cells were suspended in 100 ml of 4% methylcellulose (Sigma) in DMEM supplemented with 20% FBS and penicillin/streptomycin. Cells were replated in DMEM supplemented with penicillin/streptomycin and 0.4% calf serum for 1 h in the presence of the MEK1 specific inhibitor, PD98059 (New England Biolabs, Beverly, MA) or dimethyl sulfoxide carrier prior to addition of the indicated concentration of bFGF or IGF-1 (Peprotech, Rocky Hill, NJ). Phase-contrast images of the cells were taken 96 h after treatment using an Olympus IX70 Inverted Research Microscope in conjunction with a Kodak EOS DCS5A digital camera (Eastman Kodak, New Haven, CT)
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