Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells.

Activation of the neurotrophin receptor TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events leading to neuronal differentiation in vitro and might play an important role in the differentiation of favorable neuroblastomas (NB) in vivo. To study TrkA signal transduction pathways and their effects on differentiation in NB, we stably expressed wild-type TrkA and five different TrkA mutants in the NGF unresponsive human NB cell line SH-SY5Y. Resulting clones were characterized by TrkA mRNA and protein expression, and by autophosphorylation of the receptor. Introduction of wild-type TrkA restored NGF responsiveness of SH-SY5Y cells, as demonstrated by morphological differentiation, activation of mitogen-activated protein kinases (MAPK) and induction of immediate-early genes. Expression of TrkA in the absence of NGF resulted in growth inhibition of transfectants compared to parental cells, whereas NGF-treatment increased their proliferation rate. Analysis of downstream signal transduction pathways indicated that NGF-induced differentiation was dependent on TrkA kinase activity. Our data suggest that several redundant pathways are present further downstream, but activation of the RAS/MAPK signaling pathway seems to be of major importance for NGF mediated differentiation of NB cells. Our results also show that the signaling effector SH2-B is a substrate of NGF-mediated Trk signaling in NB, whereas it is not activated by NGF in rat pheochromocytoma PC12 cells. This might explain the differences we observed in TrkA signaling between neuroblastoma and PC12 cells. Further insight into TrkA signaling may suggest new options for the treatment of NB.