Timing-dependent actions of NGF required for cell differentiation.
BACKGROUND
Continuous NGF stimulation induces PC12 cell differentiation. However, why continuous NGF stimulation is required for differentiation is unclear. In this study, we investigated the underlying mechanisms of the timing-dependent requirement of NGF action for cell differentiation.


RESULTS
To address the timing-dependency of the NGF action, we performed a discontinuous stimulation assay consisting of a first transient stimulation followed by an interval and then a second sustained stimulation and quantified the neurite extension level. Consequently, we observed a timing-dependent action of NGF on cell differentiation, and discontinuous NGF stimulation similarly induced differentiation. The first stimulation did not induce neurite extension, whereas the second stimulation induced fast neurite extension; therefore, the first stimulation is likely required as a prerequisite condition. These observations indicate that the action of NGF can be divided into two processes: an initial stimulation-driven latent process and a second stimulation-driven extension process. The latent process appears to require the activities of ERK and transcription, but not PI3K, whereas the extension-process requires the activities of ERK and PI3K, but not transcription. We also found that during the first stimulation, the activity of NGF can be replaced by PACAP, but not by insulin, EGF, bFGF or forskolin; during the second stimulation, however, the activity of NGF 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, using a microarray.


CONCLUSIONS
These results demonstrate that NGF induces the differentiation of PC12 cells via mechanically distinct processes: an ERK-driven and transcription-dependent latent process, and an ERK- and PI3K-driven and transcription-independent extension process.
Chung
Jaehoon
J
Department of Biophysics and Biochemistry, Graduate School of Science, CREST, Japan Science and Technology Agency, University of Tokyo, Tokyo, Japan.
Kubota
Hiroyuki
H
Ozaki
Yu-ichi
Y
Uda
Shinsuke
S
Kuroda
Shinya
S
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D015151:Immunoblotting; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016501:Neurites / Q000187:drug effects* / Q000502:physiology; D020411:Oligonucleotide Array Sequence Analysis; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D020932:Nerve Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D048049:Extracellular Signal-Regulated MAP Kinases