Identification of genes which are responsible for induction of the initial differentiation process of PC12 cells

Continuous NGF stimulation induces PC12 cell differentiation; however, it is unclear whether NGF is continuously required for differentiation. We found that discontinuous NGF stimulation, consisting of the first transient stimulation followed by an interval and the second sustained stimulation, similarly induces differentiation. The first stimulation did not induce neurite extension, whereas the second stimulation induced fast neurite extension; therefore, the first stimulation is required as prerequisite conditions. This indicates that the action of NGF can be divided into two processes: the first stimulation-driven latent process and the second stimulation-driven extension process. The latent process appears to require ERK and transcription, but not PI3K, activities, whereas the extension-process requires ERK and PI3K, but not transcription, activities. We also found that NGF in the first stimulation can be replaced by PACAP, but not by insulin, EGF, bFGF or forskolin, whereas NGF in the second stimulation cannot be replaced by any of these stimulants. These findings allowed us to identify potential genes specifically involved in the latent process, rather than in other processes. These results demonstrate that NGF induces differentiation of PC12 cells via mechanically distinct processes: the ERK-driven and transcription-dependent latent process; and the ERK- and PI3K-driven, and transcription-independent extension process. Gene expression in PC12 cells was measured at 3 hours after following stimulations, complete medium (n = 4), continuous NGF (n = 2), 1 hour of NGF (n = 4), 1 hour of PACAP (n = 2), 1 hour of Insulin (n = 2), 1 hour of NGF in presence of U0126 (n = 2) or 1 hour of NGF in presence of LY294002 (n = 2).