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Title: Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Impairs the Regulation of Apoptosis in Megakaryocytes by Activating NF-kappaB: a Proteomic Study
Authors: Di Michele, Michela ×
Peeters, Karen
Loyen, Serena
Thys, Chantel
Waelkens, Etienne
Overbergh, Lut
Hoylaerts, Marc
Van Geet, Chris
Freson, Kathleen #
Issue Date: Jan-2012
Publisher: American Society for Biochemistry and Molecular Biology
Series Title: Molecular & Cellular Proteomics vol:11 issue:1 pages:M111.007625
Article number: M111.007625
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.
URI: 
ISSN: 1535-9476
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Molecular and Vascular Biology
Cardiology
Clinical and Experimental Endocrinology
Laboratory of Phosphoproteomics (-)
Laboratory of Protein Phosphorylation and Proteomics
× corresponding author
# (joint) last author

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