Neurotrophic Factors Prevent Ceramide-Induced Apoptosis Downstream of c-Jun N-Terminal Kinase Activation in PC12 Cells


Address correspondence and reprint requests to Dr. P. J. Hartfield at School of Biological Sciences, Flinders University of South Australia, G.P.O. Box 2100, Adelaide 5001, South Australia, Australia.


Abstract: Neurotrophic factors prevent apoptosis of PC12 cells in serum-free medium. The present study determines whether neurotrophic factors can prevent ceramide-induced apoptosis in PC12 cells and investigates the role that c-Jun N-terminal kinase (JNK) activation may play in this system. Ceramide-induced apoptosis was inhibited by nerve growth factor, basic fibroblast growth factor, pituitary adenylyl cyclase-activating peptide, 4-(8-chlorophenylthio)cyclic AMP, and the caspase inhibitor benzyloxycarbonyl-Val-Ala-dl-Asp fluoromethyl ketone (zVAD-FMK). It was surprising that inhibition of extracellular signal-regulated kinase and/or phosphatidylinositol 3-kinase did not markedly block the protective effects exerted by neurotrophic factors against ceramide-induced apoptosis, suggesting that neurotrophic factors can promote survival independently of these signaling pathways. Treatment of PC12 cells with ceramide resulted in a time-dependent increase in JNK activity. However, neither neurotrophic factors nor zVAD-FMK attenuated ceramide-stimulated JNK activation. Further experiments indicated that ceramide-induced apoptosis in PC12 cells requires new protein synthesis, and that nerve growth factor and zVAD-FMK can prevent apoptosis after JNK activity has been detected. These results indicate that ceramide-induced JNK activation is an early event and may be required for the expression of essential components of the apoptotic machinery. It is anticipated that neurotrophic factors inhibit ceramide-induced apoptosis by affecting signaling events downstream of JNK activation.

Abbreviations used: bFGF, basic fibroblast growth factor; CPT-cAMP, 4-(8-chlorophenylthio)cyclic AMP; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; NGF, nerve growth factor; PACAP, pituitary adenylyl cyclase-activating peptide 1–38; PAGE, polyacrylamide gel electrophoresis; PI, phosphatidylinositol; SDS, sodium dodecyl sulfate; zVAD-FMK, benzyloxycarbonyl-Val-Ala-dl-Asp fluoromethyl ketone.