Role of glypican-1 in the trophic activity on PC12 cells induced by cultured sciatic nerve conditioned medium: identification of a glypican-1-neuregulin complex.

Glypican-1 is an extracellular matrix component found by microsequencing in a medium conditioned by cultured rat-sciatic nerves (CM). This CM was concentrated by ultrafiltration and fractionated by quaternary ammonium chromatography, followed by Hi-Trap blue affinity chromatography to obtain the active fraction B1.2. Previously, we have reported a 54 kDa neuregulin (NRG) in the same B1.2 fraction [Villegas et al., Brain Res. 852 (2001) 304]. The effect of Glypican-1 on the neuron-like differentiation of PC12 cells was investigated by immunoprecipitation, Western blot and cellular image analysis. Removal of glypican-1 by immunoprecipitation with increasing concentrations of specific antibodies revealed a gradual decrease of the differentiation activity of fraction B1.2, which paralleled the results obtained by removal of the 54 kDa NRG protein. Colorless native electrophoresis and Western blot analysis was used to identify a glypican-1-NRG protein complex, which could be afterwards separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis into its individual components. Additionally, it was demonstrated that glypican-1, in cooperation with the 54 kDa NRG, is involved in the neuronal-like differentiation of PC12 cells and could play an important role on the regeneration responses of peripheral nerves.