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.
Highlights
From the ‡Center for Molecular and Vascular Biology, §Laboratory of Biochemistry, ¶Laboratory for Experimental Medicine and Endocrinology, ʈDepartment of Pediatrics, University Hospital Leuven, K.U
Our findings indicate that Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) interferes with apoptosis in CHRF cells via the VPAC1 receptor, as found via a literature-based meta-analysis of other cellular systems after addition of PACAP
PACAP Modulates NF-B Signaling in Megakaryocytes—We previously found that PACAP increases intracellular cAMP levels in CACO-2 cells, platelets, and megakaryocytes via the VPAC1 receptor [3, 5]
Summary
Patient Description—Our trisomy 18p patient with elevated PACAP plasma levels has been previously described [3, 5]. Expressed spots between pairwise comparisons among the different time points following PACAP treatment were identified by mass spectrometry To this aim, after image acquisition, gels were. MS/MS searches were conducted with the following settings: taxonomy set on humans, MSDB (release date: 31.08.2006, locally installed 19.07.2007; number of all entries: 3239079; number of entries for Homo sapiens: 148148) or SwissProt (UniProt_SwissProt, release date: 04.2010; number of all entries: 517000; number of entries for Homo sapiens: 20367) as database, peptide and fragment tolerance equal or lower than 0.4 Da, carbamidomethylation of cysteine as fixed modification, methionine oxidation as variable modification, one missed cleavage allowed in case of incomplete trypsin hydrolysis, precursor charge ϩ1 Using these parameters the probability-based Molecular Weight Search (MOWSE) scores greater than the given cutoff value for MS/MS fragmentation data were taken as significant (p Ͻ 0.05). FACSDiva software was used for immunofluorescence acquisition on the FACSCalibur flow cytometer and data analysis
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