Autocrine regulation of neurite outgrowth from PC12 cells by nerve growth factor

Abstract

The PC12 cell line may be used as a model of NGF-induced neuronal differentiation. Exposure to NGF is accompanied by extension of neurites, cessation of growth and differentiation into cells resembling sympathetic neurons. In this study neurite outgrowth from PC12 cells was induced in serum-free, NGF-free medium conditions. Neurite outgrowth in serum-free conditions was abolished by exposure to anti-NGF antisera. Reverse transcription combined with polymerase chain reaction (RT-PCR) and in situ hybridization of PC12 cells in serum-free medium conditions revealed NGF transcripts. Western blot analysis of these cells revealed tyrosine phosphorylation of the high affinity NGF receptor (TrkA/gp140) and activation of a downstream signal cascade element, ERK-1/MAP kinase. NGF was also detected by a specific enzyme-linked immunoabsorbant assay (ELISA) revealing picogram levels of protein in conditioned medium and cell lysates. Survival of embryonic rat dorsal root ganglion neurons was maintained in cultures grown in this serum-free conditioned medium. This demonstrated that NGF may act as an autocrine or paracrine growth factor for PC12 cell differentiation.