title: Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) impairs the regulation of apoptosis in megakaryocytes by activating NF-κB: a proteomic study.
abstract: We previously showed that the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptor VPAC1 are negative regulators of megakaryopoiesis and platelet function, but their downstream signaling pathway that inhibits this process still remained unknown. A combined proteomic, transcriptomic, and bioinformatic approach was here used to elucidate the molecular mechanisms underlying PACAP signaling via VPAC1 in megakaryocytes. Two-dimensional difference gel electrophoresis and tandem MS were applied to detect differentially expressed proteins in megakaryocytic CHRF cells stimulated with PACAP. The majority of the 120 proteins modulated by PACAP belong to the class of "cell cycle and apoptosis" proteins. The up- or down-regulated expression of some proteins was confirmed by immunoblot and immunohistochemical analysis. A meta-analysis of our data and 12 other published studies was performed to evaluate signaling pathways involved in different cellular models of PACAP response. From 2384 differentially expressed genes/proteins, 83 were modulated by PACAP in at least three independent studies and Ingenuity Pathway Analysis further identified apoptosis as the highest scored network with NF-κB as a key-player. PACAP inhibited serum depletion-induced apoptosis of CHRF cells via VPAC1 stimulation. In addition, PACAP switched on NF-κB dependent gene expression since higher nuclear levels of the active NF-κB p50/p65 heterodimer were found in CHRF cells treated with PACAP. Finally, a quantitative real time PCR apoptosis array was used to study RNA from in vitro differentiated megakaryocytes from a PACAP overexpressing patient, leading to the identification of 15 apoptotic genes with a 4-fold change in expression and Ingenuity Pathway Analysis again revealed NF-κB as the central player. In conclusion, our findings suggest that PACAP interferes with the regulation of apoptosis in megakaryocytes, probably via stimulation of the NF-κB pathway.
authors.affiliation: Center for Molecular and Vascular Biology, Leuven, Belgium.
authors.forename: Michela
authors.initials: M
authors.lastname: Di Michele
authors.affiliation: Center for Molecular and Vascular Biology, Leuven, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Peeters
authors.affiliation: Center for Molecular and Vascular Biology, Leuven, Belgium.
authors.forename: Serena
authors.initials: S
authors.lastname: Loyen
authors.affiliation: Center for Molecular and Vascular Biology, Leuven, Belgium.
authors.forename: Chantel
authors.initials: C
authors.lastname: Thys
authors.affiliation: Laboratory of Biochemistry, Leuven, Belgium.
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.affiliation: Laboratory for Experimental Medicine and Endocrinology, Leuven, Belgium.
authors.forename: Lutgart
authors.initials: L
authors.lastname: Overbergh
authors.affiliation: Center for Molecular and Vascular Biology, Leuven, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: Hoylaerts
authors.affiliation: Center for Molecular and Vascular Biology, Leuven, Belgium; Department of Pediatrics, University Hospital Leuven, K.U. Leuven, Leuven, Belgium.
authors.forename: Christel
authors.initials: C
authors.lastname: Van Geet
authors.affiliation: Center for Molecular and Vascular Biology, Leuven, Belgium. Electronic address: kathleen.freson@med.kuleuven.be.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Freson
mesh_terms: D000328:Adult; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008533:Megakaryocytes / Q000187:drug effects* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology*; D040901:Proteomics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D016328:NF-kappa B; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D000242:Cyclic AMP