Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A 2 Dan Zhang 1,5,8 , Peiman Shooshtarizadeh 1 , Benoıˆt-Joseph Laventie 2 , Didier Andre ´ Colin 2 , Jean-Franc ¸ois Chich 1,3 , Jasmina Vidic 3 , Jean de Barry 4 , Sylvette Chasserot-Golaz 4 , Franc ¸ois Delalande 1 , Alain Van Dorsselaer 6 , Francis Schneider 5 , Karen Helle 7 , Dominique Aunis 1 , Gilles Pre ´ vost 2 , Marie-He ´le ` ne Metz- Boutigue 1 * 1 INSERM U575, Physiopathologie du Syste ` me Nerveux, Strasbourg, France, 2 UPRES-EA 3432, Institut de Bacte ´ riologie de la Faculte ´ de Me ´ decine, Universite ´ Louis Pasteur, Ho ˆ pitaux Universitaires de Strasbourg, Strasbourg, France, 3 INRA, Virologie et Immunologie Mole ´ culaires, Jouy-en-Josas, France, 4 Institut des Neurosciences Cellulaires et Inte ´ gratives, UMR 7168 CNRS-Universite ´ Louis Pasteur, Strasbourg, France, 5 De ´partement de Re ´ animation Me ´dicale, Ho ˆ pital de Hautepierre, Strasbourg, France, 6 Laboratoire de spectrome ´trie de masse BioOrganique, IPHC-DSA, ULP, CNRS, UMR7178, Strasbourg, France, 7 Department of Biomedicine, University of Bergen, Bergen, Norway, 8 First Hospital, Chongqing University of Medical Sciences, Chongqing, China Abstract Background: Antimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides. Methodology/Principal Findings: PMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM- binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity. Conclusions/Significance: For the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems. Citation: Zhang D, Shooshtarizadeh P, Laventie B-J, Colin DA, Chich J-F, et al. (2009) Two Chromogranin A-Derived Peptides Induce Calcium Entry in Human Neutrophils by Calmodulin-Regulated Calcium Independent Phospholipase A 2 . PLoS ONE 4(2): e4501. doi:10.1371/journal.pone.0004501 Editor: Mauricio Rojas, Emory University, United States of America Received June 11, 2008; Accepted January 15, 2009; Published February 19, 2009 Copyright: ß 2009 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Inserm, CNRS, Universite Louis Pasteur Strasbourg, The Delegation a ` la Recherche clinique des Hopitaux universitaires de Strasbourg (Grant 3150), Fondation Transplantation, French ministery de la recherche et de l’enseignement superieur, BRAHMS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: metz@neurochem.u-strasbg.fr Introduction Chromogranin A (CgA) is a well-studied member of the chromogranin/secretogranin family, present in secretory cells of the nervous, endocrine and immune systems [1]. CgA was the first chromogranin to be characterized as an acidic protein co-stored and co-released with the catecholamine hormones from the chromaffin cells of the adrenal medulla. The discovery that Pancreastatin, a CgA-derived peptide (bCGA 248–293 ) was able to inhibit the glucose-evoked insulin secretion from pancreatic beta- cells [2] initiated the concept of a prohormone function for this protein [3]. Numerous endogenous cleavage products of CgA have since been identified in the intragranular matrix of chromaffin cells, resulting from the proteolytic digestion at 13 sites [4] by intragranular enzymes, such as prohormone convertases PC1/3, PC2, neuroendocrine-specific carboxypeptidase E/H, Lys and Arg-aminopeptidases [5]. Among the CGA derived fragments, several induce biological activities [1] an their in vitro actions PLoS ONE | www.plosone.org 1 February 2009 | Volume 4 | Issue 2 | e4501