Skeletal muscle neural cell adhesion molecule (N-CAM): changes in protein and mRNA species during myogenesis of muscle cell lines.

Abstract

Abstract. Qualitative and quantitative changes in neural cell adhesion molecule (N-CAM) protein and mRNA forms were measured during myogenesis in G8-1 and C2 cell lines. Indirect immunofluorescence assay showed that N-CAM was constitutively expressed by myoblasts in culture and that myotubes appeared to be stained more strongly. These changes were quantified using a dot blot assay. N-CAM levels increased almost 4-fold in G8-1 cells and 15-fold in C2 cells during myogenesis. The kinetics of accumulation of N-CAM were not coordinate with other muscle markers such as creatine kinase or acetylcholine receptor levels, since N-CAM accumulated significantly ahead of these markers. Immunoblotting showed that myogenesis was not associated with changes in the extent of sialylation of N-CAM. However, distinct changes in desialo forms were observed after neuraminidase treatment. Myogenesis was accompanied by increases in 125-and 155-kD desialo forms with minor changes in 120-and 145-kD forms. Biosynthetic labeling studies showed that myoblasts specifically expressed a transmembrane isoform of 145 kD that was phosphorylated and was down-regulated in myotubes. Pulse-chase analysis of myotubes showed that the 120-kD isoform and an isoform of 145 kD that co-migrated with, but was distinct from, the 145 kD transmembrane isoform of myoblasts were precursors of the 125-and 155-kD isoforms, respectively, that accumulated in myotubes. The 125-and 155-kD isoforms in myotubes are linked to the cell membrane via phosphatidylinositol linkage and can be released by phospholipase C. Indirect immunofluorescence analysis showed that phosphatidylinositol specific phospholipase C specifically released N-CAM from the myotube membrane generating N-CAM-free myotubes, while myoblasts were unaffected by this treatment. Three N-CAM mRNA species were observed in mouse muscle cell lines. Myoblasts were characterized by their expression of 6.7-and 5.2-kb transcripts while myotubes express 5.2-and 2.9-kb transcripts. Thus, myogenesis is qualitatively associated with a down regulation of the 6.7-kb transcript and an up regulation of the 5.2-and 2.9-kb transcript.

Transcriptional profile of GTP-mediated differentiation of C2C12 skeletal muscle cells

“…We found Vcam1 and Ncam-180 up-regulated, both of which code for cell adhesion molecules and which contribute to myotube formation through fusion of myoblasts [39,40]. Some studies have shown that for C2C12 myoblasts [67], Ncam expression is associated with the early stages of differentiation, and is concurrent with expression of T-type Ca 2+ channels and inward rectifier K + channels [68]. Our data again confirm the activation of these pathways during several phases of myogenesis.…”

Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation

“…Upon serum withdrawal, CD56 + /desmin + cells readily completed terminal differentiation to form large multinucleated myotubes expressing MHC (Fig. 2M), confirming CD56 as a useful marker for human primary myogenic cells as suggested by others (Cashman et al, 1987;Moore et al, 1987;Schubert et al, 1989;Illa et al, 1992;Mackey et al, 2009). By contrast, the CD56 2 cells obtained from sorting expressed the fibroblast specific antigen TE-7 in culture and were incapable of myogenic differentiation (Fig.…”

Neural cell adhesion molecule (NCAM) marks adult myogenic cells committed to differentiation

“…NCAM is an immunoglobulin superfamily adhesion molecule; its expression on human satellite cells was first observed in monoclonal antibody studies [53] while later work demonstrated that, in human [54] and rat [55], NCAM is expressed on quiescent satellite cells and is maintained during proliferation and differentiation, through the formation of regenerated myofibers. It is also detected on the widely-used myoblast cell line C2C12 [56] where its expression is associated with the early stages of differentiation, concurrent with expression of T-type Ca 2+ channels and inward rectifier K + channels [57]. However, these studies and others [58] also established that NCAM is not expressed by quiescent mouse satellite cells, and that in the mouse its expression on satellite cells is heterogeneous during regeneration in vivo [59,60].…”