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 (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_r band decreased with differentiation. These same blots were probed with an antibody raised against the 34,000 M_r bovine IGFBP-2. This antibody specifically bound to only the 32,000 M_r 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.