Abstract
Satellite cells are muscle-resident stem cells, which locate beneath the basement membrane of myofibers. Because the number of satellite cells is normally constant, there must be tight regulation of satellite cell activation and self-renewal. However, molecular mechanisms in satellite cell maintenance are largely unknown, and thus has become subject to extensive study in these days. Although RNA interference with a small interfering RNA has been widely used to investigate the role of specific gene products, inefficient knockdown of Grb2 expression occurred in quiescent reserve cells, a model for quiescent satellite cells, by ordinary transfection protocol. We report in this study that pretreatment with trypsin greatly enhanced siRNA delivery into quiescent reserve cells, resulting in efficient silencing of Grb2 expression. By applying combination of Grb2-silencing and protein kinase C inhibitors, we demonstrated that extracellular signal-regulated kinase (ERK) phosphorylation induced with fibroblast growth factor 2 (FGF2) was dependent on both Grb2 and protein kinase C (PKC) with different kinetics. We conclude that PKC-mediated pathway contributes to rapid initiation and termination of ERK phosphorylation while Grb2-mediated pathway contributes delayed and sustained ERK phosphorylation.
