NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro

Aims/hypothesis. Using primary cultures of human pancreatic islets, purifie d human pancreatic beta cells and the mouse beta TC6-F7 cell line, we analy sed the expression of nerve growth factor, (NGF/NGF) receptors in beta cell s. To investigate whether NGF could sub-serve an autocrine antiapoptotic ro le in beta cells, we studied the effects of NGF withdrawal using a neutrali zing monoclonal anti-NGF antibody. Methods. The expression of NGF and NGF receptors (gp140(Trk-A) and p75(NTR) ) were analysed by RT-PCR and immunofluorescence. Pulse-chase experiments a nd beta cell/PC12 co-cultures were used to investigate NGF production and s ecretion from beta cells. Possible apoptosis induced by NGF withdrawal was monitored by phosphatidylserine translocation, nucleosomal formation, DNA l addering and FACS analysis. Involvement of transcription/translation mechan isms were investigated as well as the gp140(Trk-A) required. Finally, signa l transduction pathways typically involved in apoptotic mechanisms were ana lysed by western blot analysis. Results. We show that NGF and both NGF receptors, gp140(Trk-A) and p75(NTR) are expressed in beta cells where NGF is produced and secreted in a biolog ically active form. NGF-withdrawal induces beta-cell transcription/translat ion independent apoptosis but mediated by gp140Trk-A. Analysis of signal tr ansduction pathways revealed that NGF withdrawal inhibits the PI3-K, protei n kinase B (AKT), Bad survival pathway and activates c-Jun kinase (JNK) whe reas ERKs and p38 mitogen-activated protein kinase (MAPK) are not affected. Moreover, Bcl-XL, but not Bcl-2 protein expression are reduced. Conclusion/interpretation. We suggest that the integrity of the NGF/NGF rec eptor system and NGF bioavailability participate in controlling beta-cell s urvival in culture which represents a key issue for improving possibilities for transplantations in the treatment of diabetes.