Proteomic Analysis Reveals That the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Inhibits Megakaryopoiesis by Preventing Apoptosis
Authors: Di Michele Michela , Peeters Karen , Loyen Serena , Waelkens Etienne , Overbergh Lutberg , Hoylearts Marc , Van Geet Christel , Freson Kathleen 
Published: October 2019

Abstract
Abstract 4791

Introduction:
The pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belonging to the secretin and glucagon family, is implicated in a wide range of biological processes. In previous studies we found PACAP and its Gs-coupled receptor VPAC1 to be negative regulators of megakaryopoiesis and platelet function (Freson K et al, Blood 2008 and J Clin Invest 2004), but further studies were needed to identify the downstream players of this signaling pathway. To better elucidate the molecular mechanisms and players underlying PACAP response in megakaryocytic cells, a proteomic approach was applied, a powerful tool to explore global protein alterations in cells.

Results:
i) In CHRF cells PACAP modulated several proteins that are involved in apoptosis. We applied an innovative and highly reproducible proteomic technology, i.e. difference in gel electrophoresis (2D-DIGE), to detect protein changes in CHRF megakaryocytic cells by PACAP at four different time-points. 159 proteins modulated by PACAP in a statistically significant way (p<0.05, FDR) were identified by mass spectrometry (MALDI TOF-TOF), the majority belonging to the class of “cell cycle and apoptosis” proteins. The up- or down-regulated expression of some proteins in DIGE/MS was confirmed by immunoblot and immunofluorescence analysis. ii) Meta-analysis revealed that PACAP-regulated genes/proteins are implicated in apoptosis, with NFkB playing a major role. Meta-analysis of our work and 12 other published studies was performed to evaluate the main pathways involved in different models of PACAP response. Of 2384 genes/proteins analysed we selected 83 which were modulated by PACAP in at least 3 different studies and performed Ingenuity Pathway Analysis (IPA). The resulting highest scored network was implicated in the apoptosis process, with NFkB as a key-node player. The reduced viability of CHRF cells grown in absence of serum was restored by PACAP at several concentrations, indicating that PACAP protects CHRF cells from apoptosis induced by serum starvation. After PACAP treatment an increased level of NFkB p50 and p65, the two most abundant components of the NFkB complex, was observed by immunoblotting and immunofluorescence in CHRF cells. These findings were further validated in another cell line, CACO-2, an enterocytic cells expressing VPAC1 abundantly. The molecular partners of NFkB p50 and p65 were also characterized by immunoprecipitation and mass spectrometry. iii) Change in expression of apoptotic genes and involvement of NFkB were validated in megakaryocytes from PACAP overexpressing patients by transcriptomic microarray analysis. An apoptosis array was carried out on megakaryocytes of a patient characterized by increased plasmatic PACAP levels and a normal control. Of the 84 key genes of the apoptotic process screened, 15 were increased or decreased by at least 4 fold. The overexpression of one of these genes, Bcl-2, was also confirmed by immunoblotting analysis. Moreover, to elucidate which relevant pathways were represented among the genes identified by the apoptotic array, we applied IPA. Again NFkB revealed to be the central key-node of the resulting highest score network.

Conclusion:
Our proteomic and bioinformatic analysis on CHRF megakaryocytic cells treated with PACAP together with microarray technology performed on PACAP overexpressing patients revealed that PACAP regulates the apoptotic pathway in megakaryocyte cells, thus explaining its inhibitory effect on megakaryopoiesis. PACAP stimulates the NFkB pathway that plays a key role in the regulation of this complex process.

Disclosures:
No relevant conflicts of interest to declare.