Gene expression and secretion of insulin-like growth factor-binding proteins during myoblast differentiation.
Numerous cell types have been reported to secrete insulin-like growth factor-binding proteins (IGFBPs) in vitro. We have observed such IGFBPs in culture medium conditioned by the mouse myoblast cell line C2C12 and have identified IGFBP-2 among the secreted IGFBPs. Since C2C12 cells fuse in culture, these cells were used to examine IGFBP-2 mRNA expression and protein secretion during myogenic differentiation. Cells were harvested at approximately 80% of confluent density. Additional cultures were rinsed, fed differentiation medium, and harvested when approximately 15%, 60%, and 85% differentiated (fused). Northern and dot blot analyses were performed using total cellular RNA and a labeled cDNA specific for rat IGFBP-2. A single mRNA transcript of approximately 1.8 kilobases was observed. The level of expression of IGFBP-2 mRNA was highest in proliferating cells and decreased to 35%, 20%, and less than 10% of initial levels as differentiation progressed. Serum-free medium was conditioned for 24 h at each time point and collected from similar cultures. Three IGFBP species of 32,000, 30,000, and 24,000 mol wt (Mr) were detected in conditioned medium by probing Western blots with [125I]IGF-I (ligand blot analysis). The intensity of the 32,000 Mr band decreased with differentiation. These same blots were probed with an antibody raised against the 34,000 Mr bovine IGFBP-2. This antibody specifically bound to only the 32,000 Mr IGFBP. The level of antibody binding decreased by 50%, 90%, and nearly 100% as differentiation progressed. It, therefore, appears that IGFBP-2 is expressed and secreted in a differentiation-dependent manner by C2C12 myoblasts and may, thus, be involved in the process of myoblast differentiation.
Department of Animal Science, Ohio State University, Columbus 43210.
C W
CW
Ernst
R H
RH
McCusker
M E
ME
White
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D002999:Clone Cells; D015870:Gene Expression; D018971:Insulin-Like Growth Factor Binding Protein 2; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D051379:Mice; D009132:Muscles / Q000166:cytology / Q000502:physiology*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification
D002352:Carrier Proteins; D018971:Insulin-Like Growth Factor Binding Protein 2; D012313:RNA; D007334:Insulin-Like Growth Factor I