Identification and characterization of multiple EGF-repeat containing protein 10 and its role in regulating satellite cell function during muscle regeneration

The regeneration of adult skeletal muscle is mediated by a small population of cells known as satellite cells. While a great deal is known about the events that occur following satellite cell activation, relatively little is known about the mechanisms of activation or maintenance of this population of cells. Previous work from our lab has demonstrated that primary myoblasts lacking MyoD, represent a primitive form of activated myogenic precursor, closely related to quiescent satellite cells. In an attempt to gain insight into the activation and maintenance of quiescent satellite cells we performed representational difference analysis to identify genes that function in these processes.;Investigation of several key signaling pathways that directly affect myogenic progression revealed that ectopic expression of Megf10 resulted in increased Rac1 activity and MEK1 phosphorylation providing a possible explanation for the inhibition of differentiation observed in Megf10 overexpressing cells. These results indicate that Megf10 impinges upon these signaling pathways supporting a role for Megf10 in regulating myogenic progression.;Our results clearly demonstrate that Megf10 is expressed in quiescent satellite cells in adult mouse skeletal muscle and indicate that Megf10 is capable of regulating the proliferation and differentiation of myogenic cells. Furthermore, our preliminary results implicate Megf10 as being a modulator of Rac1 and MEK1 signaling in myoblasts.;This screen led to the isolation a large number of candidates for examination. One of the clones, identified as mouse Megf10, is a multiple EGF-repeat transmembrane protein that is expressed in adult skeletal muscle, specifically in quiescent satellite cells. Retroviral expression of Megf10 in C2C12 myoblasts resulted in enhanced proliferation and inhibition of terminal differentiation. Furthermore, following serum withdrawal, Megf10 overexpressing cells entered a state of quiescence rather than undergoing terminal differentiation and remained capable of re-entering the cell cycle following serum restimulation. While no distinct effects on cell cycle regulation were observed, ectopic expression of Megf10 resulted in transcriptional upregulation of Myf5 and downregulation of Pax7 as well as post-transcriptional downregulation of MyoD. Importantly, ectopic expression of a truncated version of Megf10, lacking the cytoplasmic domain, had no effect on proliferation, differentiation or expression of transcriptional regulatory factors.