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Impact of CFTR F508 mutation on prostaglandin E₂ production and type IIA phospholipase A₂ expression by pulmonary epithelial cells

Institut Pasteur, Unité de Défense Innée et Inflammation; and Institut National de la Santé et de la Recherche Médicale, E336, Paris, France

Submitted 15 December 2004 ; accepted in final form 14 June 2005

	ABSTRACT

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Cystic fibrosis (CF) is characterized by an exacerbated inflammatory pulmonary response with excessive production of inflammatory mediators. We investigated here the impact of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction on prostaglandin E₂ (PGE₂) production and type IIA secreted phospholipase A₂ (sPLA₂-IIA) expression. We show that both resting and LPS-stimulated human respiratory epithelial cell line bearing F508 mutation on CFTR (CF cells) released more PGE₂ than control cell line. This was accompanied by enhanced expression and activity of cyclooxygenase-2 in CF cells. PGE₂ release was attenuated after experimentally induced retrafficking of the F508-CFTR at the plasma membrane. sPLA₂-IIA expression occurred at higher levels in CF cells than in control cells and was enhanced by LPS and PGE₂. Suppression of PGE₂ synthesis by aspirin led to an inhibition of LPS-induced sPLA₂-IIA expression. Higher activation of NF-B was observed in CF cells compared with control cells and was enhanced by LPS. However, addition of PGE₂ or aspirin had no effect on NF-B activation. LPS-induced sPLA₂-IIA expression was reduced by an NF-B inhibitor. We suggest that the lack of the CFTR in the plasma membrane results in a PGE₂ overproduction and an enhanced sPLA₂-IIA expression. This expression is upregulated by NF-B and amplified by PGE₂ via a unidentified signaling pathway.

cystic fibrosis; inflammation; arachidonic acid metabolism; cystic fibrosis transmembrane conductance regulator

The pulmonary disease in CF is characterized by an excessive inflammation in response to bacterial infection in particular by Pseudomonas aeruginosa (12). Bacterial lipopolysaccharides (LPS) are known to induce the expression of various genes involved in inflammation, including those coding for phospholipase A₂ (PLA₂). The latter belongs to a family of enzymes that catalyze the hydrolysis of phospholipids at the sn-2 position, leading to the generation of lysophospholipids and free fatty acids, such as arachidonic acid (AA) (7). These products are the precursors of lipid mediators (platelet-activating factor and eicosanoids) endowed with various biological activities and involved in the pathophysiological changes observed in a number of inflammatory diseases (3, 6). PLA₂ are classified into two major classes: the low-molecular-weight secreted forms, termed sPLA₂, and the high-molecular-weight cytosolic forms, termed cPLA₂. In mammals, sPLA₂ are classified into several different groups including the type IIA sPLA₂ (sPLA₂-IIA), which seems to play a role in the pathogenesis of various inflammatory and infectious diseases (29). sPLA₂-IIA has been also shown to exhibit antibacterial activity and to play a role in host defense immunity (29). Previous studies have shown the existence of cross talk between cPLA₂ and sPLA₂-IIA leading to the modulation of the expression of the latter via AA metabolite such as prostaglandin E₂ (PGE₂) and leukotriene B₄ (19). Clinical studies reported increased levels of eicosanoids in bronchoalveolar lavage fluid (BALF) from CF patients (18), but whether this increase is a direct or a secondary consequence of CFTR dysfunction has not been investigated.

The present study was undertaken to investigate the impact of the F508 mutation of CFTR on PGE₂ production and sPLA₂-IIA expression in human respiratory epithelial cell lines. The rationale of this study is that the production of PGE₂ by CF pulmonary epithelial cells may upregulate sPLA₂-IIA expression by these cells.

	MATERIALS AND METHODS

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Reagents. Dulbecco's modified Eagle's minimum essential medium with Earle's salt and Ham’s F-12 cell culture media, Hanks' balanced salt solution, and trypsin-ethylenediaminetetraacetic acid (EDTA) were from Invitrogen (Cergy-Pontoise, France). Fetal calf serum (FCS) was from Hyclone (Logan, UT). Phenylmethylsulfonyl fluoride (PMSF), benzamidine, dithiothreitol (DTT), EDTA, P. aeruginosa LPS, methyl arachidonyl flurophosphonate (MAFP), MG-132, and phorbol 12-myristate 13-acetate (PMA) were from Sigma (St. Louis, MO). LY-311727 was a gift from Eli Lilly (Indianapolis, IN). The anti-p65 subunit and anti-cyclooxygenase (COX)-2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antiphospho-cAMP response element binding protein (CREB) antibody was purchased by Cell Signaling Technology (Beverly, MA). The anti--actin antibody was purchased from Sigma.

Cell culture. The human tracheal fetal epithelial cell lines CFT-2 and NT-1 were a kind gift of Dr. Annick Paul (INSERM U402, Paris, France). CFT-2 cell line was derived from primary tracheal epithelia homozygous for the common CF mutation F508, and NT-1 cell line was derived from normal primary tracheal epithelial cells (20). Both cell lines were grown in Dulbecco's modified Eagle's medium/Ham’s F-12 (vol/vol) supplemented with 10% FCS at 37°C in 5% CO₂/95% air. The human bronchial epithelial cell line IB3 and C38 cell lines, obtained from ATCC, were grown in minimum essential medium with Earle's salt and L-glutamine supplemented with 10% FCS at 37°C in 5% CO₂/95% air. IB3 express mutant CFTR (F508/W1282X), and C38 is derived from IB3 cells stably transfected with wild-type CFTR (33). The cells were pretreated with various drugs [MAFP (21), LY-311727 (27), aspirin, thapsigargin, and MG-132 (9)] before incubation with LPS or PGE₂ as detailed in the figure legends.

RNA extraction and RT-PCR analysis. Cells were grown on a cell culture plate (Techno Plastic Products, Trasadingen, Switzerland), and total RNA was extracted using an RNeasy kit (Qiagen, Courtaboeuf, France). DNase treatment was performed with 3 µg of extracted RNA, 1 µl of DNase I (Amersham Biosciences, Orsay, France), and 0.5 µl of RNasin (Promega, Madison, WI) in a total volume of 20 µl in the manufacturer's buffer. We obtained cDNA by incubating RNA with 1 mM dNTP (Eurobio, Les Ulis, France), 1.5 µl of hexamers as primers, 20 units of RNasin (Promega), and 300 units of Moloney murine leukemia virus reverse transcriptase RNase H minus (Promega) in a total volume of 40 µl in the manufacturer's buffer for 1 h at 42°C and 10 min at 70°C. PCR was performed using specific primers (Genset, Evry, France) for human sPLA₂-IIA (sense, 5'-AGG GAG GGA GGG TAT GAG AG-3'; antisense, 5'-GAC AGG AAA GGA AGC CGC AC-3'), human COX-1 (sense, 5'-GCA TTG ACA CAA ACT CCC AGA AC-3'; antisense, 5'-TTC TTG CTG TTC CTG CTC CTG-3'), and human COX-2 (sense, 5'-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3'; antisense, 5'-AGA TCA TCT CTG CCT GAG TAT CTT-3'). As an internal control, we used primers for the detection of human -actin (sense, 5'-AAG GAG AAG CTG TGC TAC GTC GC-3'; antisense, 5'-TCT AGA CTA ATT TGA ATT AGG TTG GTG TAG GAT GAC AAA C-3'). Amplifications were performed in a Peltier thermal cycler apparatus (MJ research, Watertown, MA) using Q-BioTaq polymerase (Qbiogene, Ilkirch, France). For the detection of sPLA₂-IIA, the thermocycling protocol was as follows: denaturation at 95°C for 45 s, annealing at 59°C for 45 s for 40 cycles, and extension at 72°C; for human COX-1 the annealing temperature was 55°C for 36 cycles; for human COX-2 annealing temperature was 63°C for 38 cycles; and for -actin the annealing was 64°C for 28 cycles.

Nuclear extract and EMSA. Nuclear proteins were extracted from 2 x 10⁶ cells. In brief, cells were washed once and scraped in PBS containing 1 mM PMSF and 2 mM benzamidine before centrifugation for 5 min at 700 g. The cells were suspended in 20 mM HEPES, pH 7, 10 mM KCl, 0.15 mM EDTA, 0.15 mM EGTA, 25% glycerol, 1% Nonidet P-40, and antiproteases; incubated for 5 min at 4°C; and then centrifuged for 5 min at 1,250 g at 4°C. The pellet (nuclear fraction) was resuspended in 10 mM HEPES, pH 8, 400 mM NaCl, 0.1 mM EDTA, 25% glycerol, and antiproteases; incubated for 30 min at 4°C under agitation; and centrifuged for 10 min at 15,000 g at 4°C. Supernatant corresponding to the nuclear extract was quickly frozen at –80°C. The NF-B double-stranded oligonucleotides corresponded to an NF-B binding site consensus sequence of 5'-GATCATGGGGAATCCCA-3'. The overhanging ends were -³²P-labeled with T4 polynucleotide kinase. Protein concentrations are determined by using Nanodrop spectrophotometer (Nyxor Biotech, Paris, France). We performed binding reactions in a total volume of 20 µl for 20 min at room temperature by adding 5 µg of nuclear extract, 10 µl of 2x binding buffer [40 mM HEPES, pH 7, 140 mM KCl, 4 mM DTT, 0.02% Nonidet P-40, 8% Ficoll, 200 µg/ml BSA, 1 µg of poly(dI-dC)], and 1 µl of labeled probe. For specificity control experiments, nuclear extracts were incubated 20 min with 50-fold excess of relevant unlabeled probe or irrelevant oligonucleotides sequence corresponding to 5'-CTAGATGCTGACACAGAACTCACTTTCCGCT-3' before the addition of labeled probe. For supershift assay, 2 µg of a polyclonal anti-p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were added, and the mixtures were incubated for 20 min at room temperature. The specificity control of p65 antibody was performed by using an antiphospho-CREB antibody (Cell Signaling, Beverly, MA). The loading protein control was performed by using KBF1 oligonucleotide, which contains a half-B site recognized by transcription factor CSL, a component of the Notch signaling pathway (13). The reaction mixtures were separated on a 5% polyacrylamide gel in 0.5% Tris-borate-EDTA buffer at 150 V for 2 h. Gels were dried and exposed for 2 to 12 h.

PGE₂ enzyme immunoassay. Epithelial cells were dispensed at 5 x 10⁵ cells/well determined by trypan blue exclusion method. PGE₂ concentrations in culture medium or cell-free BALF were measured with specific enzyme immunoassay kits purchased from Cayman Chemical (Ann Arbor, MI).

COX activity and Western blotting analysis. After treatment with LPS (1 µg/ml) for 6 h, the total proteins were extracted and 30 µg of protein were fractionated on SDS-PAGE gel and transferred as described previously (14). The blot was then incubated with primary monoclonal anti-human COX-2 antibody (1:1,000 in blocking reagent; Santa Cruz Biotechnologies) for 1 h at room temperature. The blot was subsequently washed with TBS-T and incubated with polyclonal anti-mouse immunoglobulin G coupled with horseradish peroxidase (HRP) (1:10,000 in blocking buffer; Perbio, Rockford, IL) for 1 h at room temperature. After washings, HRP activity was developed using the ECL+ reagent kit (Amersham Biosciences, Little Chalfont, UK), and the corresponding luminescence was revealed by exposure of membranes to Kodak X-Omat AR films (Eastman Kodak, Rochester, NY). The position and molecular weight of COX-2 were validated by reference to Kaleidoscope Prestained Standards (Bio-Rad; M_r range, 7,600–216,000). -Actin Western blot analysis using specific antibody AC-74 (Sigma) was performed as internal control. For the measurement of COX activity the cells were incubated with AA (10 µM) for 24 h, in the presence or in the absence of LPS, and then the levels of PGE₂ released were measured as indicated above.

CFTR cell ELISA. Cells were grown to confluence on 24-well plates. After three washes with PBS, nonspecific binding was blocked with 1x TBS and 5% milk for 1 h at 4°C. All incubations were followed by three washes with PBS. CFTR was detected by incubation with specific CFTR antibody CF-3 raised against first extracellular loops of human CFTR (1:500 in blocking buffer; Abcam, Cambridge, UK) at 4°C. Cells were then fixed with 3% paraformaldehyde for 15 min at 4°C. CFTR antibody was detected by incubation with an anti-mouse Ig-HRP (1:10,000 in blocking buffer, Perbio) for 1 h at 4°C. 3,3',5,5'-Tetramethylbenzidine substrate buffer was added at room temperature in the dark. The reaction was stopped with 2 N H₂SO₄, and color change of substrate was read at 450 nm [optical density at 450 nm (OD₄₅₀)] in a plate reader. For the low-temperature CFTR retrafficking studies, the cells were incubated at 28°C for 36 h. For the thapsigargin studies, the cells were incubated with 1 µM thapsigargin for 90 min, and the detection was performed 3 h later. The results are expressed as subtraction OD₄₅₀ values obtained with specific CFTR by OD₄₅₀ values obtained with isotypic antibody (OD₄₅₀).

sPLA₂ activity assay. The sPLA₂ activity was assayed using [³H]oleate-labeled membranes of Escherichia coli, following a modification of the method of Franson et al. (11). E. coli strain CECT 101 was seeded in medium containing 1% tryptone, 0.5% (wt/vol) NaCl, and 0.6% (wt/vol) sodium dihydrogen orthophosphate, pH 0.5, and grown for 6–8 h at 37°C in the presence of 5 µCi/ml [³H]oleic acid (specific activity 10 Ci/mmol) until growth approached the end of the logarithmic phase. After centrifugation at 2,500 g for 10 min, the membranes were washed in buffer (0.7 M Tris·HCl, 10 mM CaCl₂, 0.1% BSA, pH 8.0), resuspended in saline, and autoclaved for 30–45 min. After washing and centrifugation, the membranes were frozen at –80°C. The phospholipid fraction incorporated at least 95% of the radioactivity. Cell culture media (30 µl) were incubated with 20 µl of autoclaved oleate-labeled membranes in a final volume of 250 µl of 100 mM Tris·HCl, 1 mM CaCl₂ buffer, pH 7.5. Incubation proceeded for 30 min at 37°C with gentle shaking to prevent sedimentation of membranes. The reaction was stopped by adding 100 µl of ice-cold solution of 0.25% BSA in saline to a final concentration of 0.07% (wt/vol). After centrifugation at 2,500 g for 10 min at 4°C, the radioactivity in the supernatants was determined by liquid scintillation counting. For the inhibition experiments, samples were preincubated with 20 µM LY-311727 for 15 min at 37°C before addition of labeled membranes.

Calculation and statistical analyses. Data are expressed as means ± SE of separate experiments, and statistical analyses were performed by unpaired Student's t-test.

	RESULTS

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Consequences of CFTR dysfunction on PGE₂ release and COX expression in epithelial cells. To determine the impact of CFTR on AA metabolism, we measured PGE₂ release by two pulmonary epithelial CF cell lines, CFT-2 and IB3, versus two control cell lines, NT-1 and C38, respectively. Basal PGE₂ release occurred at higher levels in both CF cell lines compared with control cell lines (Fig. 1, A and B). Upon P. aeruginosa LPS stimulation, PGE₂ release was increased and remained higher in CF cells compared with control cells. This enhanced PGE₂ release was not due to a higher responsiveness of CF cells to LPS since similar results were observed when cells were stimulated with PMA instead of LPS. Indeed, PMA (50 ng/ml) induced the production of 519 ± 3.4 pg/ml and 622 ± 8.2 pg/ml (n = 3, P < 0.05) of PGE₂ from NT-1 and CFT-2 cells, respectively. The subsequent studies were performed with the NT-1 and CFT-2 cells because they produced much more PGE₂ than C38 and IB3 cells.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Production of PGE2 by epithelial cell lines. A: NT-1 and CFT-2 cells were stimulated for 24 h by Pseudomonas aeruginosa LPS (1 µg/ml). Supernatants were collected, and PGE2 concentrations were determined by an enzyme immunoassay. The results are expressed as means ± SE of 3 distinct experiments performed in duplicate. *CFT-2 vs. NT-1 (P < 0.05); #LPS vs. control (P < 0.05). B: C38 and IB3 cells were stimulated for 24 h by P. aeruginosa LPS (10 µg/ml). The results are expressed as means ± SE of 3 distinct experiments performed in duplicate. *IB3 vs. C38 (P < 0.05); #LPS vs. control (P < 0.05).

View larger version (39K): [in this window] [in a new window]   Fig. 2. Expression of human cyclooxygenase (hCOX)-1 and hCOX-2 in NT-1 and CFT-2 cells. NT-1 and CFT-2 cells were stimulated or not by LPS (1 µg/ml) for 6 h. A: total RNA were analyzed by RT-PCR with specific probes for COX-1, COX-2, and -actin. B: quantitation of PCR signal intensity normalized against -actin. C: total protein extracts were analyzed by Western blot with specific COX-2 and -actin antibodies. D: NT-1 and CFT-2 cells were incubated for 24 h with arachidonic acid (10 µM) and stimulated or not by LPS (1 µg/ml). PGE2 concentrations were determined by enzyme immunoassay. The results are expressed as means ± SE and are representative of 3 distinct experiments performed in duplicate. *CFT-2 vs. NT-1 (P < 0.05).

Consequences of F508-CFTR membrane retrafficking on PGE₂ production by epithelial cells.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Effect of low temperature and thapsigargin on F508-CFTR and CFTR membrane retrafficking. CFTR and F508-CFTR were detected by specific CFTR antibody (CF3). NT-1 and CFT-2 cells were incubated at low temperature (28°C) for 36 h (A) or pretreated with thapsigargin (1 µM) for 90 min and incubated in fresh medium for 3 h (B), and cell ELISA was performed as detailed in MATERIALS AND METHODS. The results expressed were obtained after subtraction of optical density at 450 nm (OD450) values obtained with isotypic antibody (OD450) ± SE. The figure is representative of 3 independent experiments performed in triplicate. *NT-1 vs. CFT-2 (P < 0.05), #treated vs. control cells (P < 0.05).

View larger version (10K): [in this window] [in a new window]   Fig. 4. Effect of F508-CFTR membrane retrafficking induced by low-temperature culture on PGE2 production by NT-1 and CFT-2 cells. NT-1 and CFT-2 cells were cultured at 28°C for 48 h and then incubated with LPS (1 µg/ml) for 24 h. Supernatants were collected, and PGE2 concentrations were determined by an enzyme immunoassay. The results are expressed as means ± SE of 3 distinct experiments performed in duplicate. *NT-1 vs. CFT-2 (P < 0.05); 28°C vs. 37°C (P < 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effect of F508-CFTR membrane retrafficking induced by thapsigargin on PGE2 production by NT-1 and CFT-2 cells. NT-1 and CFT-2 cells were preincubated with thapsigargin (1 µM) for 90 min and incubated with LPS (1 µg/ml) for 10 h. Supernatants were collected and PGE2 concentrations were determined by an enzyme immunoassay. The results are means ± SE of 3 distinct experiments performed in duplicate. *CFT-2 vs. NT-1 (P < 0.05); #LPS vs. control (P < 0.05); LPS + thapsigargin vs. LPS alone (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Measurement of type IIA secreted phospholipase A2 (sPLA2-IIA) secretion by NT-1 and CFT-2 cells. Supernatants were collected after 24-h incubation of the cells with LPS (1 µg/ml) or PGE2 (10 µM). sPLA2-IIA activity was measured as detailed in MATERIALS AND METHODS. The results show the release of sPLA2-IIA activity by CFT-2 vs. NT-1 cells (A) and IB3 vs. C38 cells (B). They are expressed as percentages of sPLA2-IIA released by nonstimulated NT-1 or C38 cells and are representative of 3 independent experiments performed in duplicate. *CFT-2 vs. NT-1 or IB3 vs. C38 (P < 0.05); #stimulated vs. nonstimulated corresponding cell line (P < 0.05).

View larger version (29K): [in this window] [in a new window]   Fig. 7. sPLA2-IIA mRNA expression by NT-1 and CFT-2 cells. Total RNA were extracted 24 h after cell treatment with LPS (1 µg/ml) or PGE2 (1 µM), and RT-PCR were performed with specific sPLA2-IIA and -actin primers. Cells were preincubated with aspirin (200 µM) for 24 h or with MG-132 (1 µM) for 2 h before addition of LPS. A: electrophoretic migration of actin and sPLA2-IIA PCR products in ethidium bromide gel. B: densitometric analysis was carried out using the NIH Image version 1.62 software. The results are means ± SE of 3 independent experiments. *CFT-2 vs. NT-1 (P < 0.05); #LPS + inhibitor vs. LPS alone (P < 0.05).

Role of NF-B in F508-CFTR-induced sPLA₂-IIA upregulation in CFT-2 cells. In the next step, we investigated the possible implication of NF-B in the enhanced expression of sPLA₂-IIA in CFT-2 cells as a consequence of CFTR dysfunction. EMSA analysis showed that, in the absence of any added stimuli, a higher nuclear translocation of NF-B was observed in CFT-2 compared with NT-1 cells (Fig. 8). The specificity of the complex was verified by inhibition with an excess of unlabeled relevant oligonucleotides and irrelevant unlabeled oligonucleotides containing a mutated NF-B site. Moreover, supershift studies showed that an antibody directed against the NF-B p65 subunit displaced this band, thus confirming that the complex belongs to the NF-B family (Fig. 8A). This nuclear translocation was increased after the addition of LPS. However, PGE₂ had no effect on NF-B activation (Fig. 8B). In addition, inhibition of PGE₂ synthesis by aspirin failed to interfere with LPS-induced NF-B activation in these cells (Fig. 8C). Preincubation of the cells with MG-132, a NF-B inhibitor, before addition of LPS markedly reduced the sPLA₂-IIA mRNA levels in LPS-stimulated CFT-2 and NT-1 cells (Fig. 7).

View larger version (66K): [in this window] [in a new window]   Fig. 8. NF-B nuclear translocation in NT-1 and CFT-2 cells. Nuclear extracts were obtained and gel shift analysis was performed as described in MATERIALS AND METHODS. A: shift analyses were performed by using 1) 50-fold excess of unlabeled oligonucleotide KBF1, 2) 50-fold excess of labeled irrelevant oligonucleotide, 3) a specific antibody direct against the p65 subunit (supershift analyses), 4) relevant labeled oligonucleotide. B: control of anti-p65 antibody specificity was performed using an anti-phospho-CREB antibody with nuclear protein extract from CFT-2 cells. C: NT-1 and CFT-2 cells were incubated with PGE2 (1 µM) or LPS (1 µg/ml) for 1 h, and nuclear proteins were extracted. CSL was used as internal control. D: NT-1 and CFT-2 cells were incubated with aspirin (200 µM) for 24 h or its vehicle and then incubated with LPS (1 µg/ml) for 1 h before extraction of nuclear proteins. The results are representative of 3 separate experiments.

	DISCUSSION

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We showed here that CFT-2, a cell line bearing the F508-CFTR mutation, produced more PGE₂ than the control cell line NT-1. This finding is not simply due to a genetic background difference between these cells since enhanced PGE₂ release was also observed in IB3 cells bearing the F508-CFTR mutation, compared with C38 cells, which are corrected IB3 cells complemented with wild-type CFTR. In all these cell lines, the production of PGE₂ was enhanced after LPS stimulation and remained higher in CF cells compared with corresponding control cells. Together, these findings suggest that CFTR dysfunction leads to enhanced PGE₂ release and that the latter is amplified by LPS. These findings are in agreement with the report of Fink et al. (10), which showed that stimulation by isolates of Burkholderia cepacia, an important lung pathogen in CF patients, induces a release of PGE₂ at higher levels in IB3 cells than in C38 cells. However, the bacterial component(s) and the mechanisms involved in the induction of PGE₂ release by B. cepacia have not been analyzed in this study.

Therefore, it was of great interest to investigate the mechanism responsible for the observed increase of PGE₂ release by CF cells. We examined the expression levels of COX-1 and COX-2, two enzyme isoforms involved in the conversion of AA to PGE₂. The results revealed that CFT-2 and IB3 cells constitutively expressed COX-2 at higher levels than NT-1 and C38 cells. In contrast, no significant difference was observed in the expression level of COX-1. However, although LPS clearly induced an increase in PGE₂ release it had no effect on the expression of COX-2 in all the cell lines studied. This is in agreement with studies of Rodgers et al. (26), who reported that bradykinin fails to induce COX-2 expression, although it was able to stimulate PGE₂ release by A549 cell line. Therefore, the observed increase in PGE₂ release by LPS-stimulated cells might be due to the activation by LPS of the release of AA via a PLA₂-dependent process. Indeed, we observed that LPS induces a marked release of AA by NT-1 and CFT-2 cells. Using MAFP and LY-311727, cPLA₂ and sPLA₂-IIA inhibitors, respectively, we showed that cPLA₂ plays a major role in LPS-induced AA release both in NT-1 and CFT-2 cells. However, sPLA₂-IIA had no significant role in this release (Medjane S, Raymond B, and Touqui L, unpublished observations).

The previous study of Berguerand et al. (2) showed that under stimulation with bradykinin, CFT-2 cells release more AA than NT-1 cells. This study suggested that CFTR inhibits cPLA₂ activity and that the retention of CFTR in the endoplasmic reticulum, which occurs in cells bearing F508-CFTR mutation, would lead to the removal of this inhibition and consequently to enhanced AA release. Indeed, procedures that allow CFTR to reach the plasma membrane in CF cells reduced this release (2). In agreement, the present study shows that retrafficking of CFTR leads to a decrease of PGE₂ release in CFT-2 cells. Along the same lines, C38 cells, in which F508-CFTR mutation is corrected by gene transfection, released less PGE₂ than IB3 cells. It is of note that PGE₂ release was also partially reduced after retrafficking of CFTR in NT-1 cells. This might be due to the fact that even in normal cells, part of CFTR is retained in the endoplasmic reticulum. Indeed, only 30% of the total wild-type CFTR is detected in the plasma membrane of normal epithelial cells (31). In agreement, the present study shows that low-temperature or thapsigargin treatment also induced CFTR retrafficking in NT-1 cells.

Together, these findings suggest that the enhanced PGE₂ release observed in CF cells is due to both increased expression of COX-2 and accumulation of free AA, as a consequence of CFTR dysfunction. However, as CF patients are known to overproduce other eicosanoids (e.g., thromboxanes, leukotrienes), which are not products of epithelial cells, it is likely that other mechanisms besides direct effect of CFTR are involved in the exacerbation of AA metabolism in CF.

We next examined the consequences of CFTR dysfunction on sPLA₂-IIA expression in epithelial cells and the possible implication of PGE₂ in this process. Indeed, previous studies have shown that PGE₂ regulates sPLA₂-IIA expression, either negatively or positively, depending on the cell type (29). Our study clearly shows that F508-CFTR mutation leads to an increased synthesis and secretion of sPLA₂-IIA by CFT-2 cells. In agreement, IB3 cells released more sPLA₂-IIA than C38 corrected cells. Our study also clearly demonstrates that PGE₂ is involved, at least in part, in the observed increase of sPLA₂-IIA expression in CFT-2 cells. Indeed, 1) these cells release more PGE₂ than NT-1 cells, 2) added PGE₂ enhances sPLA₂-IIA expression in CFT-2 and NT-1 cells, and 3) aspirin, which inhibits PGE₂ synthesis, abrogates LPS-induced sPLA₂-IIA expression in both cell lines. Thus, based on our results and findings from other groups (2, 22), we suggest that the CFTR dysfunction in epithelial cells leads to an increase of free AA production and to a subsequent enhanced PGE₂ release. In turn, the latter induces an upregulation of sPLA₂-IIA expression. This led us to investigate the signaling pathways that may link CFTR dysfunction to sPLA₂-IIA upregulation.

Our findings revealed an enhanced nuclear translocation of NF-B in CFT-2 compared with NT-1 cells. This is in agreement with the previous studies of Venkatakrishnan et al. (30) reporting an exaggerated NF-B activation in IB3 CF cells compared with corresponding corrected C38 cells. We demonstrated that this abnormal activation is involved, at least in part, in the LPS-induced sPLA₂-IIA upregulation in CFT-2 cells. Indeed, NF-B translocation was increased by LPS, and LPS-induced sPLA₂-IIA expression was abolished by pretreatment of the cells with MG-132, an inhibitor of NF-B translocation. However, PGE₂-induced sPLA₂-IIA expression seems to occur via an NF-B-independent process in both cell lines. Indeed, PGE₂ had no effect on NF-B activation and inhibition of PGE₂ synthesis by aspirin failed to interfere with LPS-induced NF-B activation in these cells. This led us to suggest that CFTR dysfunction results in an abnormal NF-B activation, leading to an upregulation of sPLA₂-IIA expression. In parallel, CFTR dysfunction leads to an increased PGE₂ release that induces sPLA₂-IIA expression via an NF-B-independent pathway. Both NF-B-dependent and -independent sPLA₂-IIA expressions are enhanced by LPS. The signaling pathway(s) and the transcription factors involved in PGE₂-induced sPLA₂-IIA expression in this cell system remains to be investigated. In particular, it would be of interest to examine the possible implication of cAMP/PKA and CEB/P or CREB in PGE₂-induced sPLA₂-IIA expression in CF cells. It is of interest to note that pulmonary tissue from CF mice have a different expression pattern of a number of genes involved in inflammation compared with wild-type mice (32), suggesting that genes other than that encoding for sPLA₂-IIA are also modulated by normal CFTR.

In conclusion, our study suggests that CFTR dysfunction in epithelial cells induces an enhanced AA metabolism leading to PGE₂ overproduction. This overproduction might have some pathophysiological consequences in CF lungs since PGE₂ has been shown to induce the expression of a number of cytokines including IL-6 and IL-8 by epithelial cells and T-lymphocytes (4, 26, 28), which may contribute to the exacerbation of inflammation. However, PGE₂ has also immunosuppressive functions since it has been shown to inhibit microbial phagocytosis and killing by alveolar macrophages (1). Thus PGE₂ overproduction could contribute not only to enhance the inflammatory state of the lung but also to impair its defense against infection. Our studies clearly demonstrated that CF epithelial cells exhibit exaggerated synthesis and secretion of sPLA₂-IIA through an autocrine/paracrine process involving PGE₂. The fact that LPS enhances sPLA₂-IIA expression in CF cells suggests that the sPLA₂-IIA gene is probably upregulated in CF patients during the episodes of infection by gram-negative bacteria, particularly by P. aeruginosa. The importance of this finding is linked to the fact that sPLA₂-IIA is known to exhibit proinflammatory and bactericidal properties and thus may play a role in lung host defense (29). The pathophysiological relevance of the induction of sPLA₂-IIA expression by bronchial epithelial cells in the context of CF remains to be investigated.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by the nonprofit associations Vaincre la Mucoviscidose.

	ACKNOWLEDGMENTS

 
The authors thank Dr. Annick Paul for her generous gift of NT-1 and CFT-2 cell lines and Dr. Miguel Paya, Dr. Sylvie Memet, and Monique Singer for helpful advice. We warmly thank Dr. Michel Chignard for comments and criticisms on the manuscript.

Benoît Raymond was supported by the Fondation pour la Recherche Médicale (Bourse Marianne Josso). Yongzheng Wu was supported by the "Société de Secours des Amis des Sciences."

	FOOTNOTES

 

Address for reprint requests and other correspondence: L. Touqui, Unité de Défense Innée et Inflammation, INSERM E336, Institut Pasteur, 25, rue du Dr. Roux, 75015, Paris, France (e-mail: touqui{at}pasteur.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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