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

Florence Borot,Lhousseine Touqui,Maryvonne Baudouin-Legros,Aleksander Edelman,Julien Colas,Mario Ollero,Diane-Lore Vieu,Marc Chanson,Jesus Ayala-Sanmartin,Janine Fritsch,Sandra Moriceau,Grazyna Faure,Franck Brouillard

doi:10.1371/journal.pone.0007116

Abstract

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.

Highlights

Among the multisystemic clinical manifestations of cystic fibrosis (CF), an abnormal inflammatory condition at the airways represents one of the most prominent morbidity factors [1,2,3], resulting in bronchiectasis and respiratory insufficiency
The aim of this work was to examine the hypothesis that CFTR interacts with major protein(s) of the cPLA2a /eicosanoid pathway and that it may function as a modulator of eicosanoid production
The results show that the connection between membrane microdomains and CFTR/cPLA2a/ANXA1/

Summary

Introduction

Among the multisystemic clinical manifestations of cystic fibrosis (CF), an abnormal inflammatory condition at the airways represents one of the most prominent morbidity factors [1,2,3], resulting in bronchiectasis and respiratory insufficiency. Many authors consider it secondary to recurrent infections and airway colonization by opportunistic pathogens [4,5,6]. A growing body of evidence indicates that inflammation and infection in CF can be dissociated, and that a basal inflammatory status preexists to pathogen infections [7,8,9]. Reduced macrophage phagosome acidification related to defective Cl2 conduction has been reported as a potential cause of recurrent infections associated with chronic inflammation in CF [10], this point has recently been contested [11]. It has been suggested that increased inflammation and Pseudomonas aeruginosa colonization in CF could be secondary to intestinal malnutrition and decreased production of the anti-inflammatory cytokine IL-10 [12]. There is evidence that proinflammatory cytokines can regulate CFTR expression [13,14]

Objectives

Methods

Results

Conclusion