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

Identification and functional validation of SRC and RAPGEF1 as new direct targets of miR-203, involved in regulation of epidermal homeostasis

Abstract The epidermis is mostly composed of keratinocytes and forms a protecting barrier against external aggressions and dehydration. Epidermal homeostasis is maintained by a fine-tuned balance between keratinocyte proliferation and differentiation. In the regulation of this process, the keratinocyte-specific miR-203 microRNA is of the outmost importance as it promotes differentiation, notably by directly targeting and down-regulating mRNA expression of genes involved in keratinocyte proliferation, such as ΔNp63, Skp2 and Msi2. We aimed at identifying new miR-203 targets involved in the regulation of keratinocyte proliferation/differentiation balance. To this end, a transcriptome analysis of human primary keratinocytes overexpressing miR-203 was performed and revealed that miR-203 overexpression inhibited functions like proliferation, mitosis and cell cycling, and activated differentiation, apoptosis and cell death. Among the down-regulated genes, 24 putative target mRNAs were identified and 8 of them were related to proliferation. We demonstrated that SRC and RAPGEF1 were direct targets of miR-203. Moreover, both were down-regulated during epidermal morphogenesis in a 3D reconstructed skin model, while miR-203 was up-regulated. Finally silencing experiments showed that SRC or RAPGEF1 contributed to keratinocyte proliferation and regulated their differentiation. Preliminary results suggest their involvement in skin carcinoma hyperproliferation. Altogether this data indicates that RAPGEF1 and SRC could be new mediators of miR-203 in epidermal homeostasis regulation

Effect of TNF-α and DRM destabilization on eicosanoid production.

<p>Calu-3 cells were treated with either 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 the supernatant was collected, either immediately or after 3 h of incubation in fresh medium, and subjected to ELISA for LTB4 and PGE2 determination. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are expressed as percent of control values. Asterisks denote p<0.05 with respect to control, n≥3.</p

The direct interaction between NBD1 and p11 may connect CFTR to the cPLA2α/ANXA1 complex.

<p>A: SPR association curves of NBD1 and p11 (analyte) at serial concentrations of the latter. B: SPR association of NBD1 with p11 and cPLA2α/ANXA1 complex. p11 (400 µg/ml) and a preincubated cPLA2α/ANXA1 (100 µg/ml each) complex were sequentially co-injected as analytes (cPLA2α/ANXA1 was injected before dissociation of p11). C: Negative SPR association of NBD1 with a preincubated cPLA2α/ANXA1 complex (injected as analyte at 100 µg/ml each). D: Negative SPR association of NBD1 with ANXA1 (injected as analyte at 100 µg/ml).</p

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

Effect of CFTR inhibition on eicosanoid, AA and IL-8 release.

<p>A: Iodide efflux (CFTR activity) measurements on Calu-3 cells. Effect of 10 min incubation with 10 µM Inh172 in Calu-3 cells (left). Effect of forskolin activation (right). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are representative of three experiments. B: Calu-3 cells were treated with either 20 µM Inh172 or Gly-101 for 20 min. In a separate experiment, HeLa cells were treated with 20 µM Inh172 for 20 min. The supernatant was collected right after treatment and subjected to ELISA for LTB4 and PGE2 determination. C: Calu-3 cells were incubated with or without 20 µM Inh172 for 20 min. After incubation, supernatants were removed and fresh DMEM medium containing fetal calf serum was added. After 3 h supernatants were harvested for IL-8 determination. All results are expressed as percent of control values.</p

Effect of CFTR inhibition on DRM localization of ANXA1, cPLA2α and CFTR.

<p>A: Calu-3 cells were treated with 100 U/mL TNF-α for 10 min alone or after preincubation with 20 µM Inh172 for 20 min. After treatment, cells were incubated in 1% Triton X-100 and subjected to OptiPrep gradient separation. DRM and non-DRM fractions were pooled separately and subjected to western blotting analysis of CFTR, ANXA1, and cPLA2α. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are representative of at least 3 experiments. B: Same as in A, except preincubation time with 20 µM Inh172 for 12 h. Western blot for CFTR is not shown. C: Densitometric quantification of DRM localization of CFTR, ANXA1 and cPLA2α. Western blot bands corresponding to DRM and non-DRM were quantified. Data are expressed as the percent of each protein present in DRM (means±SEM, n≥3). D: Calu-3 cells were incubated with 20 µM Inh172 for either 1 h, 6 h or 24 h. After incubation the supernatant was collected and subjected to ELISA for LTB4 determination.</p

Impact of TNF-α and DRM destabilization on IL-8 release.

<p>IL-8 production by Calu-3 cells after proinflammatory stimulation, functional inhibition of CFTR and DRM disruption. Calu-3 cells were incubated with either 100 U/mL TNF-α for 10 min, 10 mM mβCD for 1 h, or with a combination of both treatments. After incubation, supernatants were removed and fresh DMEM medium containing fetal calf serum was added. After 3 h of incubation, supernatants were harvested for IL-8 determination. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007116#s3" target="_blank">Results</a> are expressed as percent of control values. Asterisks denote p<0.05 with respect to control, n≥3.</p