Title

Insulin-like growth factor stimulation of myoblast proliferation is specified by a serum activity, mitogenic competence factor

Date of Award

1998

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

James R. Florini

Second Advisor

Richard Levy

Keywords

Rapamycin, Pertussis, Mitogenic, Insulin-like growth factor, Myoblast proliferation

Subject Categories

Biology

Abstract

The stimulation of both myoblast proliferation and differentiation by Insulin-like growth factors (IGF) is well documented, but how each response is specified remains unclear. IGF stimulation of myoblast proliferation is transient, only lasting 36 hours. Cessation of proliferation does not require cell contact and does not involve a loss in the mitogenic activity of the IGF in the extracellular medium. IGF stimulation of proliferation can be restored by the addition of horse serum. Thus, a serum component serves a competence function for IGF stimulation of proliferation. The mitogenic competence activity of serum cannot be replaced by PDGF, bFGF, TGF-$\beta,$ HGF, transferrin, or fetuin and is not one of the known IGF binding proteins. Fractionation of horse serum by ammonium sulfate precipitation reveals that the proliferation competence and differentiation suppressing effects are both recovered in the 60-80% ammonium sulfate fraction. The 60-80% fraction has little or no mitogenic activity alone and thus functions as a competence factor or cofactor for IGF in stimulating myablast proliferation. I have named this activity Mitogenic Competence Factor (MCF).

To explore how MCF specifies IGF stimulation of myoblast proliferation, the effect of IGF on mediators of cell cycle progression and myogenesis in the absence and presence of MCF was tested. IGF and MCF were both required for phosphorylation of the retinoblastoma (Rb) protein, but IGF alone was sufficient to maintain levels of cyclin D and decrease levels the the cyclin dependent kinase (cdk) inhibitor p27$\rm\sp{Kip1}.$ The Extracellular Regulated Kinases (ERKs), and p70 S6 kinase (p70 S6K), have been implicated in mediating stimulation of proliferation by several extracellular factors. IGF and MCF were co-required to increase the activities of ERK1 and ERK2 kinases and p70 S6 kinase. Proliferation stimulated by IGF-I and MCF was inhibited in the presence of PD098059 and rapamycin, inhibitors of ERK and p70 S6 kinase pathways, respectively. Proliferation was also inhibited by the G protein inhibitor pertussis toxin and the PI-3 kinase inhibitor LY294002. IGF and MCF stimulation of Rb phosphorylation was almost completely inhibited by PD098059 and partially inhibited by pertussis toxin. Rapamycin and LY294002 inhibited Rb phosphorylation very slightly. Although all four compounds inhibited proliferation, myoblasts treated with PD098059 and pertussis toxin fused into myotubes, although to different extents. Thus, both the ERK or p70 S6K pathways appear to be involved in L6A1 myoblast proliferation stimulated by IGF-I and MCF but the ERK pathway may be important in dictating the choice between proliferation and differentiation, which may be related to its involvement in Rb phosphorylation.

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