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

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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Journalistes et chercheurs : l'investigation de terrain, enjeux et stratégies d'accès

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Cystic fibrosis transmembrane conductance regulator (CFTR) regulates the production of osteoprotegerin (OPG) and prostaglandin (PG) E2 in human bone.

International audienceBone loss is an important clinical issue in patients with cystic fibrosis (CF). Whether the cystic fibrosis transmembrane conductance regulator (CFTR) plays a direct role in bone cell function is yet unknown. In this study, we provide evidence that inhibition of CFTR-Cl(-) channel function results in a significant decrease of osteoprotegerin (OPG) secretion accompanied with a concomitant increase of prostaglandin (PG) E(2) secretion of primary human osteoblast cultures (n=5). Our data therefore suggest that in bone cells of CF patients, the loss of CFTR activity may result in an increased inflammation-driven bone resorption (through both the reduced OPG and increased PGE(2) production), and thus might contribute to the early bone loss reported in young children with CF

Effect of TNF-α and DRM destabilization on AA release.

<p>Calu-3 cells were incubated overnight with either ³H-labelled AA, treated with 100 U/mL TNF-α for 10 min, 10 mM mβCD for 1 h, with or without preincubation with 15 µM pyrrolidine for 45 min, or with a combination of the different treatments. For combined treatments, TNF-α was added for the last 10 min of incubation. After incubation, supernatants were collected and radioactivity measured by a scintillation counter. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are expressed as percent increment with respect to control. Asterisks denote p<0.05 with respect to control unless indicated otherwise, n = 3.</p

Hypothetical model linking CFTR/DRM interaction with cytokine and eicosanoid release.

<p>TNF-α exerts two effects that seem to be dissociated: eicosanoid release and IL-8 synthesis with the participation of DRM, in which CFTR, cPLA2α, ANXA1, TNFR1 and c-Src are transiently recruited. Both DRM destabilization (mβCD) and CFTR inhibition (Inh172) lead to increased eicosanoid release. However, they counteract in some conditions the other effect of TNF-α -in the case of Inh172, only at long term (12 h).</p