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 ΔF508 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 ΔF508-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-κB 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-κB activation. LPS-induced sPLA2-IIA expression was reduced by an NF-κB 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-κB and amplified by PGE2 via a unidentified signaling pathway.
- cystic fibrosis
- arachidonic acid metabolism
- cystic fibrosis transmembrane conductance regulator
cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (25). CF patients express typical phenotype characterized by recurrent excessive inflammation and infection. Pulmonary infection is one of the most frequent processes that ultimately leads to pulmonary failure and death of patients (5, 24). CFTR, a member of the ATP-binding cassette superfamily, contains two similar units, each including membrane-spanning domains and two nucleotide-binding domains linked by a single regulatory domain. It functions as a cyclic AMP (cAMP)-dependent chloride channel that regulates epithelial surface fluid secretion in the respiratory and gastrointestinal tracts (15). The deletion of the phenylalanine at position 508 (ΔF508) in the first nucleotide-binding domain of CFTR is the most common mutation (70% of the mutated alleles) in CF patients. The ΔF508-CFTR is unable to fold correctly and to assume its appropriate tertiary conformation, leading to its retention in the endoplasmic reticulum and to its degradation by the proteasome. However, if the CFTR is permitted to reach the cell membrane by artificial procedures, it becomes able to function as a cAMP-dependent chloride channel (6, 8).
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 ΔF508 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
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-β-actin antibody was purchased from Sigma.
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 ΔF508, 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 (ΔF508/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 β-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 β-actin the annealing was 64°C for 28 cycles.
Nuclear extract and EMSA.
Nuclear proteins were extracted from 2 × 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-κB double-stranded oligonucleotides corresponded to an NF-κB binding site consensus sequence of 5′-GATCATGGGGAATCCCA-3′. The overhanging ends were γ-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 2× 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-κB 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 × 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; Mr range, 7,600–216,000). β-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 1× 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 (ΔOD450).
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.
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.
PCR and Western blot analysis of cell extracts revealed a marked expression of COX-2, the inducible form of COX, whose intensity was more pronounced in CFT-2 compared with NT-1 cells (Fig. 2). In contrast, the expression of the constitutive form COX-1 occurred at similar levels in the two cell lines (Fig. 2A). Similar results were observed in IB3 vs. C38 cell lines (data not shown). COX activity was measured after the addition of AA (10 μM) for 24 h (Fig. 2D). This led to an increased release of PGE2 whose level was higher in CFT-2 compared with NT-1 cells. Surprisingly, LPS had no significant effect either on COX-2 expression or on COX activity.
Consequences of ΔF508-CFTR membrane retrafficking on PGE2 production by epithelial cells.
In the case of the ΔF508 mutation, the CFTR dysfunction is due to the retention of this molecule in the endoplasmic reticulum, leading to its lack of expression at the plasma membrane. It is known that this mutated protein is functional and can be addressed experimentally at the cell membrane. Therefore, we examined whether experimental procedures (thapsigargin and low temperature) known to permit a normal intracellular traffic of ΔF508-CFTR (6, 8) are able to reduce the enhanced PGE2 release observed in CFT-2 cells. Having established that CFTR was readdressed at the plasma membrane (Fig. 3), we looked at the synthesis of PGE2 under those experimental conditions. Interestingly, we observed that both thapsigargin and low temperature reduced the levels of PGE2 released by CFT-2 cells under LPS stimulation. However, there is no significant difference in PGE2 release by unstimulated cells after temperature and thapsigargin treatment (Figs. 4 and 5). These procedures also reduced, although to a lesser extent, the levels of PGE2 release by NT-1 cells.
Consequences of PGE2 release on sPLA2-IIA expression in CF epithelial cells.
As PGE2 is known to modulate sPLA2-IIA expression in different cell types (29), we next examined the consequences of the enhanced PGE2 release on sPLA2-IIA expression in CFT-2 and NT-1 cells. The results showed that CFT-2 cells secreted more sPLA2 than NT-1 cells either at the basal level or upon stimulation with LPS or PGE2 (Fig. 6A). This sPLA2 activity was strongly inhibited after we treated the incubation medium from LPS-stimulated CFT-2 cells with 20 μM of the sPLA2-IIA inhibitor LY-311727 [the levels of sPLA2-IIA activity were 2,610 ± 381 and 437 ± 246 cpm/ml of released oleic acid in untreated and LY-311727-treated media, respectively (n = 3, P < 0.05)]. Similarly, IB3 cells released more sPLA2-IIA-like activity than C38 cells both in stimulated and nonstimulated conditions (Fig. 6B).
These findings were confirmed with the study of sPLA2-IIA mRNA expression, which showed that, under basal conditions, CFT-2 cells expressed sPLA2-IIA mRNA at a higher level than NT-1 cells (Fig. 7). This level was increased by the addition of LPS or PGE2 to both cell types (Fig. 7). Preincubation of the cells for 24 h with 200 μM aspirin, known to inhibit PGE2 synthesis, led to a marked inhibition of LPS-induced sPLA2-IIA mRNA expression (Fig. 7) and reduced the levels of sPLA2-IIA activity released in the medium (data not shown). We verified that aspirin was able to abolish PGE2 release in both cell types under our experimental conditions (data not shown).
It was not possible to investigate the effect of thapsigargin on LPS-induced sPLA2-IIA expression, since the restoration of normal intracellular traffic of CFTR by thapsigargin is a transitory process that cannot be maintained for more than 8 h (8).
Role of NF-κB in ΔF508-CFTR-induced sPLA2-IIA upregulation in CFT-2 cells.
In the next step, we investigated the possible implication of NF-κB 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-κB 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-κB site. Moreover, supershift studies showed that an antibody directed against the NF-κB p65 subunit displaced this band, thus confirming that the complex belongs to the NF-κB family (Fig. 8A). This nuclear translocation was increased after the addition of LPS. However, PGE2 had no effect on NF-κB activation (Fig. 8B). In addition, inhibition of PGE2 synthesis by aspirin failed to interfere with LPS-induced NF-κB activation in these cells (Fig. 8C). Preincubation of the cells with MG-132, a NF-κB inhibitor, before addition of LPS markedly reduced the sPLA2-IIA mRNA levels in LPS-stimulated CFT-2 and NT-1 cells (Fig. 7).
In CF, the inflammatory component is responsible for the pulmonary failure and death of the patients (24). Lipid mediators including eicosanoids play a major role in the induction and/or progression of the inflammatory reaction in various models of inflammatory diseases (16). Previous studies reported an increase of eicosanoid concentrations in BALF, saliva, and urine from CF patients (17, 18, 23), but the molecular mechanisms involved in this increase have not been investigated.
We showed here that CFT-2, a cell line bearing the ΔF508-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 ΔF508-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 ΔF508-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 ΔF508-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 ΔF508-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-κB 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-κB 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-κB 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-κB translocation. However, PGE2-induced sPLA2-IIA expression seems to occur via an NF-κB-independent process in both cell lines. Indeed, PGE2 had no effect on NF-κB activation and inhibition of PGE2 synthesis by aspirin failed to interfere with LPS-induced NF-κB activation in these cells. This led us to suggest that CFTR dysfunction results in an abnormal NF-κB 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-κB-independent pathway. Both NF-κB-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.
This work was supported by the nonprofit associations Vaincre la Mucoviscidose.
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.”
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.
- Copyright © 2005 the American Physiological Society