CFTR Negatively RegulatesCyclooxygenase-2-PGE2 PositiveFeedback Loop in InflammationJING CHEN,1 XIAO HUA JIANG,1 HUI CHEN,1 JING HUI GUO,1 LAI LING TSANG,1

MEI KUEN YU,1 WEN MING XU,2 AND HSIAO CHANG CHAN1,2*1Epithelial Cell Biology Research Center, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong,

Hong Kong, China2Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases and Birth Defects of Ministry of

Education West China Second University Hospital, Sichuan University, Chengdu, China

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator(CFTR), a cAMP-dependent anion channel mostly expressed in epithelia. Accumulating evidence suggests that CF airway epithelia areoverwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory conditionobserved in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increasedcyclooxygenase-2 (COX-2) expression and over-production of prostaglandin E2 (PGE2) in human CF bronchial epithelia cell line(CFBE41o�) with elevatedNF-kB activity compared to a wild-type airway epithelial cell line (16HBE14o�). Moreover, we demonstratedthat CFTR knockout mice had inherently higher levels of COX-2 and NF-kB activity, supporting the notion that lack of CFTR results inhyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE2 involving PKA and transcriptionfactor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o� cells,and wild-type CFTR protein expression was significantly increased when 16HBE14o� cells were challenged with LPS as well asPGE2, indicating possible involvement of CFTR in negative regulation of COX-2/PGE2. In conclusion, CFTR is a negative regulator ofPGE2-mediated inflammatory response, defect of whichmay result in excessive activation ofNF-kB, leading to over production of PGE2 asseen in inflammatory CF tissues.J. Cell. Physiol. 227: 2759–2766, 2012. � 2011 Wiley Periodicals, Inc.

Cystic fibrosis (CF) is a genetic autosomal recessive disordercaused by mutations in the gene encoding the cystic fibrosistransmembrane conductance regulator (CFTR), which hasbeen known as a cAMP-dependent anion channel conductingboth chloride and bicarbonate (Riordan et al., 1989; Collins,1992). Though more than 1,800 mutations of this gene havebeen identified, deletion of the phenylalanine at position 508(DF508) is themost frequentmutation occurring in over 70% ofCF patients, which results in a temperature sensitive foldingdefect and endoplasmic reticulum-associated degradation(ERAD) (Sheppard et al., 1994). Progressive lung diseasecharacterized by chronic inflammation and bacterial infection isthe prime cause of morbidity in CF; however, the exactunderlying mechanism remains elusive. While it was long heldthat pro-inflammatory substances produced by various celltypes upon bacterial infection, particularly Pseudomonasaeruginosa, are responsible for the over-inflammatory conditionobserved in CF lung disease (Davis et al., 1996), recent studieshave suggested that inflammation may even occur in theabsence of infection. Elevated levels of inflammatory cytokines,such as interleukin 8 (IL-8), 1 (IL-1), 6 (IL-6), and tumor necrosisfactor-a (TNF-a) which are known to be regulated by NF-kB(Bennett et al., 1977; Weber et al., 2001; Andersson et al.,2008; Vij et al., 2009), have been detected in CF sputum andbronchoalveolar lavage fluid (BALF) of patients with milddisease, as well as those in stable clinical conditions withoutmanifestation of infection (Heeckeren et al., 1997;Tirouvanziam et al., 2000; Verhaeghe et al., 2007). Since theintensity of the inflammatory reaction in CF appears to be, atleast in part, independent of the infectious stimulus, it has beensuggested that early and excessive inflammation may be relatedto constitutive abnormalities associated with a defective CFTR(Muhlebach et al., 1999; Muhlebach and Noah, 2002). Thisnotion is supported by a large body of evidence showing

increased activation of NF-kB and subsequent excessiveinflammatory cytokine expression in a variety of CF cell lineswhere infection is not an issue (Bonfield et al., 1999; Zamanet al., 2004).

In addition to increased release of pro-inflammatorycytokines, several studies have also demonstratedoverproduction of prostanoids such as PGE2, PGF2, PGF1, andthromboxane B2 in the BALF, saliva, and urine of patients withCF (Widdicombe et al., 1989; Strandvik et al., 1996; Corvolet al., 2003), suggesting that they may play a part in thepathogenesis of CF. Further evidence indicating thatprostanoids have an important pro-inflammatory role in CF is

Additional Supporting Information may be found in the onlineversion of this article.

Contract grant sponsor: Focused Investment Scheme.Contract grant sponsor: Li Ka Shing Institute of Health Sciences ofthe Chinese University of Hong Kong.Contract grant sponsor: National Natural Science Foundation ofChina;Contract grant number: 30830106.Contract grant sponsor: National 973 Project;Contract grant number: 2009 CB. 522100.Contract grant sponsor: Morningside Foundation.

*Correspondence to: Hsiao Chang Chan, Epithelial Cell BiologyResearch Center, School of Biomedical Sciences, Faculty ofMedicine, The Chinese University of Hong Kong, Hong Kong,China. E-mail: hsiaocchan@cuhk.edu.hk

Received 10 May 2011; Accepted 6 September 2011

Published online in Wiley Online Library(wileyonlinelibrary.com), 12 September 2011.DOI: 10.1002/jcp.23020

ORIGINAL RESEARCH ARTICLE 2759J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

� 2 0 1 1 W I L E Y P E R I O D I C A L S , I N C .

provided by a clinical study, in which broad spectrum COXinhibitor ibuprofen was shown to delay the progression ofCF lung disease (Konstan et al., 1995; Konstan, 2008).Prostaglandin endoperoxide synthase, commonly calledcyclooxygenase (COX), is the key enzyme required for theconversion of arachidonic acid into prostaglandins (PGs). TwoCOX isoforms have been identified: COX-1 and COX-2.COX-1 is constitutively expressed in most tissues where itmaintains the physiological processes, whereas COX-2 is highlyinducible at inflammatory sites and is considered the maintarget for NF-kB activation (Maier et al., 1990; Smith et al.,2000). Interestingly, consistent with the previous studies, arecent report also observed that COX-2 expression wasupregulated in the nasal polyps of CF patients (Roca-Ferreret al., 2006). Collectively, these observations imply that COX-mediated production of PGsmay play a role in the pathogenesisof inflammatory CF conditions. However, what causes theincreased prostanoid release in CF remains unknown.

Intriguingly, recent studies have suggested a positivefeedback loop from PGs to COX-2 during inflammation. Inparticular, PGE2 upregulates COX-2 expression by virtue of itscAMP-elevating capacity in human blood monocytes andpodocytes (Hinz et al., 2000; Faour et al., 2008). In the presentstudy, we focus on the link between CFTR and COX-2/PGE2,and propose an autocrine positive feedback loop of PGE2-cAMP-PKA-p-CREB-COX-2, which contributes to theexaggerated inflammatory response in CF, but could otherwisebe offset by normal function of CFTR. This finding may explain,at least in part, the amplified PGE2-mediated inflammatorycascade presented in CF.

Materials and MethodsDrugs and reagents

Pseudomonas aeruginosa LPS, PGE2, CFTRinh-172, and Bay 11-7082were obtained from Sigma (St. Louis, MO). Forskolin waspurchased from Tocris (Ellisville, MO). Protein kinase A (PKA)inhibitor H89 was obtained from Cell Signaling Technology(Beverly, MA). CFTR antibody was purchased from Almone Labs(Jerusalem, Israel). COX-2 polyclonal antibody was obtained fromCaymanCompany (Cat 160106) (Ann Arbor, MI). NF-kB p65, p50,Histone H1, b-Tubulin antibodies were obtained from Santa CruzBiotechnology (Santa Cruz, CA). P-CREB and CREB antibodieswere from Cell Signaling Technology.

Cells

Wild-type human bronchial epithelial cell line, 16HBE14o�, andCFtracheo-bronchial cell line which is homozygous for the DeltaF508mutation, CFBE41o�, kindly provided by Professor KoWingHung(The Chinese University of Hong Kong), were grown in MEMmedium supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin, and maintained in an atmosphere of5%CO2–95%O2 at 378C.All cell culturemedia and antibiotics werepurchased from Sigma–Aldrich (Gillingham, UK). Fetal bovineserum was purchased from Invitrogen (Paisley, UK).

Transient transfection

CFBE41o� cells were seeded in six-well plates. When the cellsreached 30–40% confluency, they were transiently transfectedwith 500 ng pEGFPC3 plasmid expressing wild-type CFTR usinglipofectamine 2000 reagent (Invitrogen). The pEGFPC3 plasmidexpressingwild-typeCFTRwas kindly provided by Professor Tzyh-Chang Hwang (University of Missouri-Columbia). Mock controland empty vector control groups were included in the study.Seventy-two hours post-transfection, the transfection efficiencywas checked under fluorescent microscope with over 50% cellsshowing green fluorescence. Cells were harvested for Westernblot analysis.

Real-time quantitative RT-PCR (QRT-PCR)

QRT-PCR was carried out as described (Liu et al., 2010).Total RNA (3mg) was reverse transcribed using M-MLVReverse Transcriptase (USB, GE Healthcare, Little Chalfont,Buckinghamshire, UK) in a 20-ml reaction. COX-2 TaqManprimer and probes were obtained from Applied Biosystems,Life Technologies (Carlsbad, CA). A 96-well plate was used for thePCR reactions. Assays were performed in triplicate on an AppliedBiosystems 7500Fast Real-Time PCR System and average Ct valueswere normalized relative to expression of GAPDH.

Western blot analysis

Cells were collected and lysed in buffer A (10mM HEPES, pH 7.9;10mM KCl; 1mM EDTA; 1mM EGTA; 0.2% NP-40; 10% glycerol;1:100 PMSF; and 1:200 PImix). After centrifugation, supernatantswere collected as cytoplasmic extracts. The nuclear pellets wereresuspended in high salt buffer B (20mM HEPES, pH 7.9; 420mMNaCl; 10mM KCl; 1mM EDTA; 1mM EGTA; 20% glycerol; 1:100PMSF; and 1:200 PImix) and collected as nuclear extracts. Equalamounts of cytoplasmic extracts were separated by SDS–PAGEand immunodetected for COX-2 and b-Tubulin; while nuclearextracts were immunoblotted for NF-kB p65, p50, and HistoneH1. Cells were lysed in RIPA buffer (150mM NaCl; 50mM Tris–Cl, pH 8.0; 1% NP-40; 0.5% DOC; 0.1% SDS), and total proteinextracts were immunoblotted for p-CREB, CREB, and b-Tubulin.

Immunohistochemistry

Tissues were fixed in 4% PFA and paraffin-embedded. Forimmunohistochemistry, sections were stained overnight at 48Cwith COX-2 polyclonal rabbit antibody (1:100 diluted, Santa CruzBiotechnology) using the microwave antigen retrieval technique.Afterwashing, sectionswere stainedwith the secondary antibodieswith peroxidase-conjugated anti-rabbit followed by rabbitperoxidase anti-peroxidase (Dako Corporation, Carpinteria, CA)for 1 h at room temperature. After being washed, color wasdeveloped with 3,3-diaminobenzidine and counterstained withhematoxylin. Sections with the primary antibody omitted wereused as a negative control throughout the study.

Prostaglandin E2 EIA assay

Prostaglandin E2 EIA Kit was obtained from Cayman Company andused according to manufacturer’s instructions.

Cytokine array

Human Cytokine Antibody Array was purchased from Panomics(Santa Clara, CA) and used as manufacturer’s instructions.

Animals and procedure

Cftrm1UNC mice were used in the current study (Snouwaert et al.,1992). All procedures were approved by the Animal EthicalCommittee of the Chinese University of Hong Kong. Afterintraperitoneal anesthesia with a mixture of ketamine (75mg/kg)and xylazine (10mg/kg), P. aeruginosa LPS was administered toþ/þand�/�CFTRmice intranasally according to previously describedmethods. Briefly, LPS (10mg in 50ml sterile PBS) or sterile PBS(control) was instilled intranasally into the lungs of the anesthetizedmice steadily. Animals were allowed to recover on a heated plateafter treatment until conscious and were then returned to theircages, with food and water available ad libitum. Six hours afterLPS exposure, the mice were terminated by cervical translocationand lung tissue was collected for subsequent experiments.

Whole-cell patch clamp recording

CFTR channel activity was recorded using the conventionalwhole-cell patch-clamp technique with a patch-clamp amplifier

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(Axopatch-200B, Axon Instruments, Foster City, CA). The controlof commanding voltages was carried out using an IBM-ATcompatible computer equipped with interface (DigiData 1322A,Axon Instruments) and pClamp Version 9 software. The pipetteswere filled with a solution containing (in mM): 140 CsCl, 5 NaCl,2 MgCl2�6H2O, 10 EGTA, and 10 Hepes (pH 7.2). The bathsolution contained (in mM): 70 NaCl, 70 Na-gluconate, 5 KCl, 2CaCl2�2H2O, 2 MgCl2�6H2O, 10 Hepes, and 5 glucose (pH 7.3). Toavoid swelling-activated Cl� currents, the osmolarities of the bathsolution and pipette solutionwere adjusted to 310 and 290mOsm/kg, respectively, with mannitol. All patch-clamp recordings wereperformed at room temperature (228C).

Statistical analysis

Data were expressed as the mean� SEM. Differences in measuredvariables between two groups were assessed by using Student’st-tests. One-way ANOVA was deployed when there were morethan two groups. Results were considered statistically significant atP< 0.05.

ResultsIncreased expression of COX-2 and NF-kB in the lungof CF mice

While COX-2 expression has been reported to be upregulatedin the nose polyps of CF patients (Roca-Ferrer et al., 2006), itremains to be answeredwhether or not similar changes inCOXexpression are found in airway epithelium. To answer thisquestion, we examined the expression of COX-2 in the lungtissue derived from CF mice by both QRT-PCR and Westernblot. As shown in Figure 1a,b, significantly higher levels of bothCOX-2 transcript and protein were detected in the lung ofCFTR �/� mice compared to that of littermate mice. Sincelarge amount of data indicate that defect of CFTR in the apicalmembrane of airway epithelial cells results in endogenousactivation of NF-kB (Bonfield et al., 1995; Andersson et al.,2008; Vij et al., 2009), which could transcriptionally activateCOX-2 (Maier et al., 1990; Mitchell et al., 1994), it is reasonablefor us to speculate that the upregulation ofCOX-2 inCFmice isattributed to NF-kB activation. We therefore examined theexpression of NF-kB p65 and p50 in the nuclear fraction fromcontrol and CFTR �/� mice. Consistent with the previousreports, our results exhibited increased NF-kB activation inthe lung of CFTR �/� mice (Fig. 1c). Next, we establisheda LPS-challenged animal model to mimic inflammatoryresponses in vivo and determined COX-2 expression byimmunohistochemistry after 10mg LPS stimulation in controlor CFTR �/� mice. As shown in Figure 1d, COX-2immunostaining was distributed in the cytoplasm ofbronchoalveolar epithelial cells. Strikingly, the expression ofCOX-2 in bronchoalvelolar epithelial cells of CFTR �/� micewas much stronger compared to that of their littermates bothunder basal level (PBS groups) and after LPS challenge.

Defect of CFTR leads to increased COX-2 and PGE2

expression in CF cell line

To further investigate the role of CFTR in the regulation ofCOX-2/PGE2, we took advantage of CFBE41o� cell line, whichwas generated fromCF tracheo-bronchial cells and reported tobe homozygous for the DeltaF508 mutation (Ehrhardt et al.,2006). The CFBE41o� cell line was compared with a wild-typeairway epithelial cell line, 16HBE14o�, which served as amodelfor bronchial epithelial cells in situ. Whole cell lysates derivedfrom 16HBE14o� and CFBE41o� cell lines were analyzed byWestern blot with a CFTR antibody targeting C-terminal of theprotein (Alomone Labs, Jerusalem, Isarel). As expected, twospecific immunoreactive bands (band B 160 kDa and band C180 kDa) were detected in 16HBE14o� cells, whereas only

band B was found in CFBE41o� cells (Fig. 2a). In line withour in vivo data, COX-2 expression is increased in CFBE41o�compared to 16HBE14o� cell lines as detected by both QPCRandWestern blot (Fig. 2b,c).We further tested the direct effectof CFTR inhibition on COX-2 expression by using a specificCFTR inhibitor, CFTRinh-172. Addition of 10mM CFTRinh-172to 16HBE14o� for 24 h caused a significant increase of COX-2expression at both mRNA and protein level (Fig. 2d,e).Accordingly, an increase of PGE2 release was detected in thesupernatant of CFBE41o� compared with that of 16HBE14o�cells, or when 16HBE14o� cells was treated with CFTRinh-172(Fig. 2f). If the upregulation of COX-2 in CFBE41o� cells islargely due to dysfunction of CFTR, the effect should bereversed by normal function of CFTR. As shown in Figure 2g,ectopic expression of wild-type CFTR in CFBE41o� cells

Fig. 1. Increased expression of COX-2 and NF-kB in the lung of CFmice before and after LPS administration in vivo. a: QRT-PCR resultshowing significantly higher level of COX-2 mRNA in the lungs ofCftr�/�mice compared to CftrR/Rmice (MMMP<0.001). Each groupcontains three mice. b: Western blot analysis showing increasedexpression of COX-2 protein in lung homogenates of Cftr R/Rcompared to that in Cftr �/� mice. Representative blots obtainedfrom three pairs ofmice are presented. The corresponding statisticalanalysis is shown in the right part. c: Western blot analysis of nuclearfraction shows increased expression of p65 and p50 protein inlung homogenates of Cftr R/R compared to that in Cftr �/� mice.Representativeblots obtained fromthreepairs ofmicearepresented.The corresponding statistical analyses are shown in the right panel.d: Immunostaining of COX-2 in bronchoalveolar epithelium ofCftr R/R and Cftr �/� mice lungs. Cftr R/R and Cftr �/� mice wereadministered nasally with PBS or LPS, and sacrificed 6h after thetreatment. Negative control represents the control section withprimary antibody omitted.

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significantly suppressed COX-2 expression, further indicatinga regulatory role of CFTR on COX-2 expression. This effectwas further verified by another approach, low temperaturetreatment, which has been demonstrated to be an effective wayof delivering CFTRDF508 to the cell surface to execute itsnormal function. As illustrated in Figure S1, a 48-h low-temperature treatment (278C) markedly decreased COX-2protein expression in CFBE41o� cells, confirming a role ofCFTR in negative regulation of COX-2.

Increased expression of COX-2 in CF cells is attributedto NF-kB activation

Our data fromCFmice exhibited an enhancedNF-kB activation(Fig. 1c) in the lung, which was concomitant with COX-2upregulation. To further elucidate whether COX-2upregulation in CF cells is related to NF-kB activation, wecompared the levels of constitutive NF-kB activation between16HBE14o� andCFBE41o� cell lines. As shown, higher level ofconstitutive NF-kB activation was found in CFBE41o� than in

16HBE14o� cells (Fig. 3a). To further evaluate the differentialresponse between normal and CF cell lines upon inflammatorystimuli, we treated both 16HBE14o� and CFBE41o� cellswith LPS (1mg/ml) for 6 h. Of note, LPS treatment significantlyincreased COX-2 expression in CFBE41o� cells at 6 h, whileno significant increase of COX-2 were found in 16HBE14o� atthat time point (Fig. 3b), indicating enhanced inflammatoryresponse upon external stimuli in CF. More importantly,LPS induced upregulation of COX-2 in CFBE41o� cells wascompletely abrogated by a specific NF-kB inhibitor, Bay-11(Fig. 3c), suggesting increased expression of COX-2 in CF cellsis attributed to NF-kB activation.

A positive feedback loop from PGE2 to COX-2 ismediated by PGE2-cAMP-PKA-p-CREB pathway

PGE2 has been shown to create a positive feedback loop thatcan amplify inflammation through elevation of cAMP in othercellular context (Hinz et al., 2000; Faour et al., 2008). In thepresent study, we asked whether PGE2 in bronchial epithelial

Fig. 2. DefectofCFTRleadstoincreasedCOX-2expression inCFcell line.a:Westernblotresultsshowingbothmature, fullyglycosylated formofCFTR (BandC) and immature, core glycosylated CFTR (Band B) in 16HBE14o�, while only Band B in CFBE41o� cells, with beta-tubulin used asloading control. b: Real-time RT-PCR result indicating higher level of COX-2 transcripts in CFBE41o� compared to 16HBE14o� (MMMP<0.001).c:RepresentativeWesternblot result showinghigherCOX-2protein level inCFBE41o�compared to16HBE14o�;Thecorresponding statisticalanalysis is shown in the right part. Datawere from three independent experiments. d:QRT-PCR result showing higher level ofCOX-2 transcriptsexpressed in 16HBE14o� treatedwithCFTR inhibitor, CFTRinh-172 (10mM) for 24 h than in control group; whereas COX-2 expression inCFBEcells is evenhigher (MMP<0.01; MMMP<0.001). e: Lysatesprepared fromCFBE41o�, 16HBE14o�cells treatedwithCFTRinh-172 (10mM)or treatedwith DMSO (0.1%) for 24 h were blotted with COX-2 antibody, representative blot shows increased COX-2 expression with CFTRinh-172treatment in 16HBE14o� and CFBE41o� cells. f: PGE2 EIA assay showing much more PGE2 production in the supernatant of CFBE41o� than16HBE14o�after24h incubation (MP<0.05).PGE2concentration in thesupernatantof16HBE14o� treatedwithCFTRinh-172 is also significantlyhigher than that of the control group. All values were normalized to 1T105 cells (MMP<0.01). g: Representative picture ofWestern blot showingdecreased COX-2 expression in CFBE41o� cells transfected with wild-type CFTR compared to that with vector control.

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cells may act in an autocrinemanner to further enhanceCOX-2expression.We therefore carried out time course experimentsin which cultured 16HBE14o� and CFBE41o� cells wereincubated with 10mM PGE2. As shown in Figure 4a, QRT-PCRanalysis indicated that PGE2 rapidly inducedCOX-2 expression,reaching a significantly higher level after 10min of stimulationin 16HBE14o� cells and the increase subsided afterwards.However, the PGE2-induced expression of COX-2 inCFBE41o� cells increased after 30min and continued toincrease to a significantly higher level compared to thatobserved in 16HBE14o� cells at 2 h (Fig. 4b). In order toexamine the role of cAMP and cAMP-dependent PKA inPGE2-mediated COX-2 induction, we determined COX-2expression using the cAMP-elevating agent, forskolin. As shownin Figure 4c, incubation with 10mM forskolin for 30minsignificantly increased COX-2 expression in 16HBE14o� cellsand subsided at 2 h. Interestingly, in keeping with the inductionof COX-2 by PGE2, the forskolin-mediated activation ofCOX-2was significantly higher in CFBE41o� cells as comparedto 16HBE14o� cells at 2 h (Fig. 4d). Next, we determinedphosphorylation of CREB, a known target of PKA, after eitherPGE2 or forskolin treatment. After administration of PGE2 orforskolin to both cell lines, cell lysates collected at different timepoints (0, 10, 20, and 30min) were analyzed by Western blotwith antibodies raised against p-CREB and CREB. As shown inFigure 4e–h, a rapid increase of CREB phosphorylation wasreadily detected after PGE2 or forskolin challenge in both celllines. To further test the hypothesis that PKA-dependentmechanism underlies PGE2-mediated COX-2 induction, weexamined the inhibitory effect of a specific PKA inhibitor H89onCOX-2 expression. Our results showed that the increase ofCOX-2 transcripts driven by PGE2 could be substantiallyinhibited by H89 in both 16HBE14o� and CFBE41o� cells,suggesting PGE2 upregulates COX-2 expression through itscAMP/PKA-elevating capacity (Fig. 4i,j).

PGE2 increase the expression of CFTR protein in16HBE14o� but not in CFBE41o� cells

It has been shown that basal CFTR gene transcription iscontrolled by intracellular cAMP (Davis et al., 1996; Baudouin-Legros et al., 2008). If PGE2 could induce COX-2 expressionthrough cAMP-PKA-p-CREB pathway, CFTR transcriptionshould also be increased by PGE2 in cell lines with normal CFTRfunction. Thus, we proceeded to test whether expressionof wild-type CFTR protein could be increased by PGE2,which would negatively regulate NF-kB activity to suppressthe positive feedback loop of PGE2. As expected, afteradministration of PGE2, significant increase of mature form of

CFTR (band C) was detected in 16HBE14o�, but not inCFBE41o� cells (Fig. 5a,b). Taken together, these resultssuggest that only wild-type CFTR could be upregulated by PGE2to in turn switch off the PGE2-mediated positive feedback loop,defect of which would lead to an exaggerated inflammatoryresponse as seen in CF.

Discussion

It has long been recognized that the abnormal inflammatoryresponse exhibited by bronchial epithelial cells in CFpredisposes to the development of bronchial damage andinflammation. The mechanism is still under debate, butincreased inflammatory cytokine secretion, altered function ofthe mucociliary escalator and antibacterial defence molecules,such as defensins, are all thought to play a part (Clayton, 1996;Smith et al., 1996). What is less clear is whether inflammationitself can have feed back to further compromise theabnormalities leading to an amplified noxious cycle of lungdestruction. In the present study, an intrinsic correlationbetween CFTR and inflammation mediator PGE2 has beenidentified in bronchial epithelia cells. Moreover, our studyreveals a positive feedback loop from PGE2 to COX-2, whichmay ensure sufficient production of PGE2 in airway immuneresponse. We have further demonstrated that this positivefeedback loop could be abrogated by the normal function ofCFTR to dampen the inflammatory response to avoidunwanted damage due to over inflammation (Fig. 6). Thesefindings provide novel insight into the inflammatory cascadesleading to the exacerbated inflammation in CF airway from theperspective of PGE2, and emphasize the role of CFTR as anegative regulator of excessive inflammation throughsuppression of NF-kB activation.

PGE2 plays an essential role in both the induction andprogression of the inflammatory reaction in a variety ofinflammatory diseases (Kemp, 2003). Overproduction of PGE2in CF airway and their pathophysiological consequences in CFhave been demonstrated in a number of studies (Widdicombeet al., 1989; Dakin et al., 2002; Caristi et al., 2005; Chen et al.,2006). In this study, we first investigated the expression of a keyenzyme required for the production of PGE2, COX-2, in airwayepithelium of CFTR knockout mice. The striking feature of ourfindingswas thatCOX-2protein expressionwasmuch higher inCF than in non-CF mice (Fig. 1). Our data for the first timedemonstrate that CFTR knockout mice have inherently higherlevels of COX-2, supporting the notion that lack of CFTRresults in hyper-inflammatory signaling by compromising theregulatory mechanisms of innate immunity. Subsequentexperiments using both normal and CF bronchial epithelial cell

Fig. 3. Increased expression of COX-2 in CF cells is attributed to NF-kB activation. a: Nuclear extracts of 16HBE14o� and CFBE41o� wereblottedwith the indicated antibodies. IncreasedNF-kBactivationwas detected inCFBE41o� compared to 16HBE14o� cells; nuclear histoneH1wasused as control. b: Representative picture ofWesternblot showing increasedCOX-2 expression inCFBE41o� cells 6 h after LPS stimulation,while no significant change of COX-2 was observed in 16HBE14o� after the same treatment. c: CFBE41o� cells were pretreated with a NF-kBinhibitor, Bay-11 (2mM) or DMSO as control for 12 h. Then, the cells were treated with LPS (1mg/ml) for another 6 h. Cell lysates were appliedfor Western blot analysis with an antibody targeting COX-2. Six hours after LPS administration, significant increase of COX-2 was observed,which could be reversed to basal level by NF-kB inhibitor pretreatment.

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lines verified the negative correlation of CFTR with COX-2expression, namely defect of CFTR results in upregulation ofCOX-2 (Fig. 2b,c). Moreover, we also detected increase ofPGE2 production in CF cell lines compared with normal celllines, which is consistent with the previous report (Fig. 2f).These results suggest that themarked upregulation ofCOX-2 islikely to be responsible for the increased prostanoid levelsfound by others in CF. Importantly, the negative associationbetween normal CFTR function and COX-2 was ascertainedby the fact that CFTR inhibitor significantly increased bothCOX-2 expression and PGE2 production in 16HBE14o� cells(Fig. 2d–f), whereas ectopic introduction of wild-type CFTR inCFBE41o� cells led to decrease of COX-2 expression (Fig. 2g).Interestingly, in support of our findings, one recent studycorrelated the heterozygosity status of identifiedpolymorphisms of COX with better lung function in 94 CFpatients, suggesting the critical role of COX as a modifier of

pulmonary disease in CF patients (Czerska et al., 2010).Nevertheless, we cannot exclude the possibility that othereicosanoid pathways also contribute to the exaggeratedinflammatory response observed in CF. In fact, it has beenreported that upon intrabronchial LPS treatment, arachidonicacid (AA) release is enhanced in BALF of CFTR �/� versusCFTRþ/þmice (Wu et al., 2010). Moreover, Leukotriene B4,which is the product of lipoxygenase, has also been shown tobe elevated by CFTR dysfunction (Konstan et al., 1993). Thus,the involvement of other eicosanoid signaling pathways willneed to be investigated more in details in future studies.

There is currently no evidence that alterations in CFTRcan directly regulate COX-2 whereas COX-2 upregulationand PG overproduction are commonly seen in inflammatorymicroenvironments such as the CF lung. Since bacteriallipopolysaccaride can induce COX-2 expression in a variouscellular context (Breder and Saper, 1996; Korhonen et al.,

Fig. 4. Apositive feedback loop fromPGE2 toCOX-2 ismediatedbyPGE2-cAMP-PKA-p-CREBpathway (a,b).TheeffectsofPGE2 treatmentonlevelsofCOX-2mRNAwereevaluatedbymultipleQ-PCRanalyses in thesamereplicatesamples (nU 9).Dataarenormalizedwith18Svaluesandare expressed in arbitrary units relative to theCt value ofHBEgroup,which is set as 1.QRT-PCR results showing elevationofCOX-2mRNA levelafterPGE2 (10mM)administration inboth16HBE14o�andCFBE41o� (MP<0.05; MMP<0.01; MMMP<0.001).One-wayANOVAandTurkeyposthoctest were used to analyze the data. c,d: QRT-PCR results showing elevation of COX-2mRNA level after forskolin (10mM) administration in both16HBE14o� andCFBE41o� (MP<0.05; MMP<0.01; MMMP<0.001), One-wayANOVA andTurkey post hoc test were used to analyze the data (e–h).Westernblot showing the increase of p-CREBafterPGE2 (e,f) or forskolin (g,h) treatment in 16HBE14o� (e,g), orCFBE41o� cells (f,h). i,j:QPCRanalysis shows that The PKA inhibitor, H89 (20mM), readily and significantly reduces the increase of COX-2mRNA in both cell lines (MMP<0.01;MMMP<0.001).

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2004), one explanation is that bacterial products and/or pro-inflammatory cytokines in the inflamed CF lung are responsiblefor inducing COX-2 through NF-kB. In the current study, weclearly demonstrated that CFTR defect leads to both NF-kBactivation and COX-2 induction in CF cell line and CFTR �/�mice.More importantly, LPS-induced upregulation of COX-2 inCFBE41o� cells was completely abrogated by specific NF-kBinhibitor (Fig. 3C), indicating increased expression of COX-2 inCF cells is attributed to NF-kB activation. Increasing evidenceindicates that defect of CFTR in the apical membrane of airwayepithelial cells results in endogenous activation ofNF-kB, whichin turn results in excessive pro-inflammatory cytokines, such asIL-1b, IL-6, and IL-8 (DiMango et al., 1998; Weber et al., 2001;Hunter et al., 2010). These cytokines could further activateNF-kB to form a self-perpetuating inflammatory cycle. Thus,functional CFTR is required for controlling the NF-kB-mediated inflammatory signaling (Schwiebert et al., 1999; Estellet al., 2003; Vij et al., 2009). It should be noted that this linkbetween CFTR and NF-kB is even stronger during bacterialinfection or LPS challenge (Machen, 2006; Saadane et al., 2006;Dechecchi et al., 2007). In consistency with the previousreports, we have demonstrated that inflammatory cytokines,such as IL-8, and IL-4, were increased in CFBE41o� cells asdetected by our cytokine antibody array (Fig. S3).Moreover,werevealed that apart from inflammatory cytokines, activation ofNF-kB was responsible for the upregulation of COX-2 in CFdefect bronchial epithelial cells.

How does an increase in COX-2 expression and PGE2production contribute to CF pathophysiology? It has beenproposed that PGE2 triggers the release of a number ofcytokines including IL-8 during inflammation condition(Tavakoli et al., 2001; Rodgers et al., 2002; Caristi et al., 2005).In addition, PGE2 has also been reported to possessimmunosuppressive functions (Prescott, 2000; Aronoff et al.,2004), indicating that PGE2 overproduction may contributeto impaired host defense against infection in CF airway.Interestingly, recent studies have demonstrated that PGE2could execute positive feedback loop to activate COX-2through its cAMP-elevating capacity in monocyte andpodocytes, thus amplifying the inflammatory responses (Hinzet al., 2000; Faour et al., 2008). In the present study, we haveidentified a positive feedback loop from PGE2 to COX-2activation in bronchial epithelial cells, which is mediated bycAMP-PKA-p-CREB pathway. We have demonstrated thatboth PGE2 and cAMP activator induce COX-2 transcription,with the concomitant increase of CREB phosphorylation(Fig. 4a–h). SinceCOX-2 gene contains aCREBbinding site in itspromoter, it is very likely that PGE2-dependent induction ofCOX-2 is mediated through PKA/p-CREB pathway. Thishypothesis is proved by the fact that PKA inhibitor significantlydecreases the PGE2-mediated COX-2 induction (Fig. 4i,j).Given the fact that PGE2-mediated COX-2 induction exists inboth normal and CF bronchial epithelial cell lines, which is alsoobserved in other cellular context, this positive feedback loopfrom PGE2 to COX-2 seems to be necessary for the persistentand sufficient production of PGE2 in normal innate immuneresponse. However, the present study has demonstrated thatthis positive feedback loop is normally safe guarded by anegative regulation by CFTR, which is defective in CF. This issupported by the present observation that wild-type CFTRprotein expression was significantly increased when16HBE14o� cells was challenged with LPS as well as PGE2(Fig. 5). In addition, it has beenwell established that cAMP couldinvoke CFTR channel function which subsequently activatesNF-kB-mediated inflammatory responses. In support of ourfindings, increased CFTR expression was also observed inintestinal epithelia infected with Escherichia coli (EIEC 029: NM)or Salmonella dublin (Resta-Lenert and Barrett, 2002; Zamanet al., 2004). Collectively, these observations suggest that PGE2release during inflammation condition activates an autocrineloop involving cAMP-PKA-p-CREB-mediated COX-2induction, which could be dampened/offset by the normalfunction of CFTR, defect of which would lead to noxiousinflammatory cycle as seen in CF. This notion is supported bythe recent study showing that in 16HBE14o� cells,

Fig. 5. PGE2 increases the mature form of CFTR protein in 16HBE14o� but not in CFBE41o� cells. a: Lysates of 16HBE14o� andCFBE41o� treated with PGE2 for different time periods were subjected to immunoblot analysis using antibody against CFTR after SDS–PAGE.b: The corresponding statistical analysis from three independent experiments was shown.

Fig. 6. Schematicdiagramof thepathway.Schematicdiagramof thepathway showing a positive feedback loop of COX-2-PGE2-cAMP-PKA-CREB-COX-2, which is negatively regulated by CFTR.

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pharmacological elevation of cAMP could suppress NF-kBactivity by about 25% below baseline through activating CFTR(Hunter et al., 2010).

Taken together, we propose that CFTR is a negativeregulator of PGE2-mediated inflammatory response and servesas protective mechanism to avoid over inflammation-induceddamage in normal tissues. CFTR dysfunction may result indefective negative regulation of the PGE2-mediated positivefeedback loop through excessive activation ofNF-kB, leading toover production of PGE2 as seen in inflammatory CF tissues.

Acknowledgments

The work was supported in parts by the Focused InvestmentScheme and Li Ka Shing Institute of Health Sciences of theChinese University of Hong Kong, National Natural ScienceFoundation of China (no. 30830106), National 973 Project(2009 CB. 522100), and Morningside Foundation.

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