Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

PLOS ONE, Oct 2009

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.

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Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

et al. (2009) Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells. PLoS ONE 4(10): e7116. doi:10.1371/journal.pone.0007116 Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells Florence Borot 0 Diane-Lore Vieu 0 Grazyna Faure 0 Janine Fritsch 0 Julien Colas 0 Sandra Moriceau 0 Maryvonne Baudouin-Legros 0 Franck Brouillard 0 Jesus Ayala-Sanmartin 0 Lhousseine Touqui 0 Marc Chanson 0 Aleksander Edelman 0 Mario Ollero 0 Dominik Hartl, LMU University of Munich, Germany 0 1 INSERM, U845, Universite Paris Descartes, Faculte de Me decine Paris Descartes, Paris, France, 2 Institut Pasteur, Unite d'Immunologie Structurale , CNRS, URA 2185, Paris, France, 3 CNRS, UMR7203 , Groupe N. J. Conte , Laboratoire des BioMole cules, Paris, France, 4 Institut Pasteur, Unite de De fense Inne e et Inflammation, INSERM, U874, Paris, France, 5 Laboratoire d'Investigation Clinique III, Ho pitaux Universitaires et Faculte de Me decine , Gene`ve , Switzerland The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2a) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2a. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-a. This was concomitant with increased IL-8 synthesis and cPLA2a activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-b-cyclodextrin induced further cPLA2a activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-a-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2a and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-a-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis. - Funding: This research was supported by European Commission, FP6-2003-Lifescihealth-I program, grant Development of new methodologies for low abundance proteomics: application to cystic fibrosis Lifesciheath-I (LSHG-CT-2005-512044-NEUPROCF) (http://cordis.europa.eu/fp6/projects.htm). This publication reflects only the authors views. The Commission is not liable for any use that may be made of the information herein. Also supported by European Commission MIRG-CT-2004004153, the French Agence Nationale de la Recherche (Etude de la voie de signalisation des eicosanoid dans la mucoviscidose par les approaches lipidomique et proteomique, EICO-CF, grant No. ANR-07-MRAR-006-01) (http://www.agence-nationale-recherche.fr/), the Swiss National Science Foundation (http://www.snf.ch/F/ Pages/default.aspx), the French foundation for Cystic Fibrosis, Vaincre la Mucoviscidose (http://www.vaincrelamuco.org/), and foundation for Cystic Fibrosis ABCF2 Proteines (http://membres.lycos.fr/abcf/default.htm), and by Legs Poix-University of Paris 5. Florence Borot holds a scholarship from Vaincre la Mucoviscidose. 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. . These authors contributed equally to this work. Among the multisystemic clinical manifestations of cystic fibrosis (CF), an abnormal inflammatory condition at the airways represents one of the most prominent morbidity factors [13], resulting in bronchiectasis and respiratory insufficiency. The origin and nature of this response embodies a controversial subject. On the one side, many authors consider it secondary to recurrent infections and airway colonization by opportunistic pathogens [46]. On the other side, a growing body of evidence indicates that inflammation and infection in CF can be dissociated, and that a basal inflammatory stat (...truncated)


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Florence Borot, Diane-Lore Vieu, Grazyna Faure, Janine Fritsch, Julien Colas, Sandra Moriceau, Maryvonne Baudouin-Legros, Franck Brouillard, Jesus Ayala-Sanmartin, Lhousseine Touqui, Marc Chanson, Aleksander Edelman, Mario Ollero. Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells, PLOS ONE, 2009, Volume 4, Issue 10, DOI: 10.1371/journal.pone.0007116