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 C₂C₁₂and have identified IGFBP-2 among the secreted IGFBPS. Since C₂C₁₂cells 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 (M_r) were detected in conditioned medium by probing Western blots with [¹²⁵I]Igf-I (ligand blot analysis). The intensity of the 32, 000 M_rband 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 M_rIGFBP. 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 C₂C₁₂myoblasts and may, thus, be involved in the process of myoblast differentiation.