UNCORRECTED PROOF PACAP and C2-ceramide generate different AP-1 complexes through a MAP-kinase-dependent pathway: involvement of c-Fos in PACAP-induced Bcl-2 expression Nicolas Aubert,* ,  Anthony Falluel-Morel,* David Vaudry,* Xavier Xifro,* , à Jose ´ Rodriguez-Alvarez,à Ce ´cile Fisch, Ste ´phane de Jouffrey, Jean-Franc ¸ois Lebigot,  Alain Fournier,§ Hubert Vaudry* and Bruno J. Gonzalez* *INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research, University of Rouen, Mont-Saint-Aignan, France  CIT, Evreux, France àLaboratory of Biochemistry and Molecular Biology, Neuroscience Institute, University of Barcelona, Spain §INRS-Institut Armand-Frappier, University of Que ´bec, Pointe-Claire, Quebec, Canada Abstract The neuropeptide pituitary adenylate cyclase-activating poly- peptide (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 in- creased 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 phosphoryla- tion. The effect of PACAP on c-Fos expression was blocked by the mitogen-activated protein kinase/extracellular signal- regulated kinase (MEK) inhibitor, U0126, while phosphoryla- tion 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. Keywords: activator protein-1, apoptosis, ceramide, 1 c-Jun, c-Fos, cerebellar granule cells, pituitary adenylate cyclase- activating polypeptide. J. Neurochem. (2006) 10.1111/j.1471-4159.2006.04148.x 2 Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide that belongs to the secretin/ glucagon/vasoactive intestinal polypeptide (VIP) super- family (Miyata et al. 1989). The effects of PACAP are mediated through three types of high affinity G-protein- coupled receptors, i.e. the PACAP-specific receptor, PAC1- R, and the PACAP/VIP mutual receptors, VPAC1-R and J N C 4 1 4 8 B Dispatch: 22.8.06 Journal: JNC CE: Blackwell Journal Name Manuscript No. Author Received: No. of pages: 14 PE: Raymond Received March 13, 2006; revised manuscript received July 20, 2006; accepted July 20, 2006. Address correspondence and reprint requests to Hubert Vaudry, INSERM U413, Laboratory of Cellular and Molecular Neuroendocri- nology, European Institute for Peptide Research (IFRMP 23), University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry@univ-rouen.fr Abbreviations used: AP-1, activator protein-1; CRE, cAMP-response- like element; CREB, CRE binding protein; DMEM, Dulbecco’s modified Eagle’s medium; DTT, dithiothreitol; ERK, extracellular sig- nal-regulated kinase; GFP, green-fluorescent protein; MAP-kinase, mi- togen-activated protein kinase; MEK, MAP-kinase/ERK kinase; PAC1- R, PACAP type I receptor; PACAP, pituitary adenylate cyclase-activa- ting polypeptide; PBS, phosphate-buffered saline; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride; PP1, protein phosphatase 1; PP2A, protein phosphatase 2 A; TRE, 12-O-tetradecanoylphorbol-13- acetate-response element; VIP, vasoactive intestinal polypeptide; VPAC1-R, VIP/PACAP receptor 1; VPAC2-R, VIP/PACAP receptor 2. Journal of Neurochemistry , 2006 doi:10.1111/j.1471-4159.2006.04148.x Ó 2006 The Authors Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 10.1111/j.1471-4159.2006.04148.x 1