Abstract
The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits C2-ceramide-induced cell death through blockade of the mitochondrial apoptotic pathway in rat cerebellar granule neurones. However, the gene induction processes and transcription factors involved in the anti-apoptotic effect of PACAP remain unknown. Here, we show that PACAP and C2-ceramide activate activator protein-1 (AP-1) DNA binding in a dose- and time-dependent manner, but generate different AP-1 dimers. Thus, PACAP increased the proportion of c-Fos and Jun D while C2-ceramide increased c-Jun and reduced c-Fos in AP-1 complexes. In addition, PACAP strongly activated c-Fos gene expression while C2-ceramide markedly increased c-Jun phosphorylation. The effect of PACAP on c-Fos expression was blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor, U0126, while phosphorylation of c-Jun induced by C2-ceramide was abrogated by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid. Transfection of immature granule cells with c-Fos siRNA, which strongly reduced basal and PACAP-stimulated levels of the protein, totally prevented the stimulatory effect of PACAP on Bcl-2 expression. The present study demonstrates that AP-1 complexes containing c-Fos mediate the effect of PACAP on Bcl-2 gene expression in cerebellar granule neurones. Our data also indicate that different AP-1 dimers are associated with the pro-apoptotic effect of C2-ceramide and the anti-apoptotic effect of PACAP.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Animals, Newborn
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Apoptosis / drug effects
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Apoptosis / physiology
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Cell Survival / drug effects
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Cell Survival / physiology
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Cells, Cultured
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Cerebellar Cortex / drug effects
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Cerebellar Cortex / metabolism
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DNA-Binding Proteins / drug effects
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DNA-Binding Proteins / metabolism
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Dose-Response Relationship, Drug
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Down-Regulation / genetics
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Enzyme Inhibitors / pharmacology
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Gene Expression Regulation / drug effects
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Gene Expression Regulation / genetics
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MAP Kinase Signaling System / drug effects
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MAP Kinase Signaling System / physiology*
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Neurons / drug effects
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Neurons / metabolism
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Phosphoprotein Phosphatases / antagonists & inhibitors
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Phosphoprotein Phosphatases / metabolism
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Phosphorylation / drug effects
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Pituitary Adenylate Cyclase-Activating Polypeptide / metabolism*
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Pituitary Adenylate Cyclase-Activating Polypeptide / pharmacology
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Protein Phosphatase 2
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Proto-Oncogene Proteins c-bcl-2 / drug effects
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Proto-Oncogene Proteins c-bcl-2 / metabolism*
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Proto-Oncogene Proteins c-fos / drug effects
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Proto-Oncogene Proteins c-fos / genetics
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Proto-Oncogene Proteins c-fos / metabolism*
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Proto-Oncogene Proteins c-jun / drug effects
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Proto-Oncogene Proteins c-jun / metabolism
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Rats
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Rats, Wistar
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Sphingosine / analogs & derivatives*
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Sphingosine / metabolism
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Sphingosine / pharmacology
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Transcription Factor AP-1 / metabolism*
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Transcriptional Activation / drug effects
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Transcriptional Activation / physiology
Substances
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DNA-Binding Proteins
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Enzyme Inhibitors
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N-acetylsphingosine
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Pituitary Adenylate Cyclase-Activating Polypeptide
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Proto-Oncogene Proteins c-bcl-2
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Proto-Oncogene Proteins c-fos
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Proto-Oncogene Proteins c-jun
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Transcription Factor AP-1
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Phosphoprotein Phosphatases
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Protein Phosphatase 2
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Sphingosine