Signaling intermediates required for NF-kappa B activation and IL-8 expression in CF bronchial epithelial cells

Jing Li, Xa Dwight Johnson, Svetlana Iazvovskaia, Alan Tan, Anning Lin, and Marc B. Hershenson

Department of Pediatrics and Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ligation of the asialoGM1 Pseudomonas aeruginosa pilin receptor has been demonstrated to induce IL-8 expression in airway epithelial cells via an NF-kappa B-dependent pathway. We examined the signaling pathways required for asialoGM1-mediated NF-kappa B activation in IB3 cells, a human bronchial epithelial cell line derived from a cystic fibrosis (CF) patient, and C-38 cells, the rescued cell line that expresses a functional CF transmembrane regulator. Ligation of the asialoGM1 receptor with specific antibody induced greater IL-8 expression in IB3 cells than C-38 cells, consistent with the greater density of asialoGM1 receptors in CF phenotype cells. AsialoGM1-mediated activation of NF-kappa B, Ikappa B kinase (IKK), and ERK was also greater in IB3 cells. With the use of genetic inhibitors, we found that IKK-beta and NF-kappa B-inducing kinase are required for maximal NF-kappa B transactivation and transcription from the IL-8 promoter. Finally, although ERK activation was required for maximal asialoGM1-mediated IL-8 expression, inhibition of ERK signaling had no effect on IKK or NF-kappa B activation, suggesting that ERK regulates IL-8 expression in an NF-kappa B-independent manner.

asialoGM1; extracellular signal-regulated kinase; Ikappa B kinase; mitogen-activated protein kinase; mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; nuclear factor-kappa B-inducing kinase; tumor necrosis factor-alpha ; cystic fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHRONIC AIRWAY INFLAMMATION is an important feature of cystic fibrosis (CF). The concentrations of IL-8, TNF-alpha , and other proinflammatory cytokines have been noted to be increased in the sputum and bronchoalveolar lavage of patients with CF (4, 8, 16, 23, 42). Investigators have compared cytokine expression in airway epithelial cells derived from CF individuals with those derived from normal individuals. DiMango and colleagues (15) found a fourfold increase in IL-8 secretion in response to Pseudomonas aeruginosa induction or ligation of the P. aeruginosa pilin receptor, gangliotetraosylceramide (asialoGM1), in IB3 cells, an adenoassociated virus-transformed human bronchial epithelial cell line derived from a CF patient (Delta F508/W1282X) compared with its CF transmembrane regulator (CFTR)-corrected line C-38 cells. These authors later noted that IL-8 production was increased in CF cells not only following stimulation but also in unstimulated cells (14). Increased TNF-alpha -induced IL-8 expression in IB3 cells was confirmed by Venkatakrishnan and coworkers (46). Similar results were recently found in two additional CF phenotypic cell lines, pCEP-R, human tracheal epithelial cells stably transfected with the regulatory domain of CFTR, and 16HBE-AS cells, human bronchial epithelial cells transfected with antisense CFTR (24). In both instances, CF cells demonstrated enhanced IL-8 protein expression in response to bacterial induction, and, in the case of pCEP-R, basal IL-8 production was also increased.

Tabary and coworkers (45) found that IL-8 expression is also increased in CF primary bronchial gland epithelial cells both in vivo and in vitro. On the other hand, Black and colleagues (3) found that IL-8 production in response to TNF-alpha and respiratory syncytial virus stimulation was not different in normal and CF primary nasal epithelial cells. Massengale and co-workers (32) found that CFT1 cells, a CF airway cell line, secreted less IL-8 in response to IL-1beta than an isogenic corrected line (CFT1-LCFSN cells). Although differences in cell type and the nature and duration of cell stimulation make comparisons difficult, together these studies lend support to the view that mutant CFTR may affect endogenous amounts of proinflammatory cytokine expression.

The mechanism by which cytokine expression may be upregulated in CF cells has recently been studied. DiMango and colleagues (14) examined the contribution of NF-kappa B to IL-8 expression in control (1HAEo- and C-38) and CF (CFTEo- and IB3) cell lines. Relative to corrected or control cells, the DNA binding of NF-kappa B was increased in both unstimulated and stimulated CF cell lines. Venkatakrishnan and colleagues (46) attributed increased NF-kappa B activation in IB3 cells to increased basal levels of Ikappa Bbeta . Tabary and coworkers (45) noted increased NF-kappa B activation and Ikappa Balpha phosphorylation in CF airway gland cells. Schwiebert and colleagues (43) demonstrated in airway epithelial cells that CFTR activates the RANTES (for regulated upon activation, normal T cell expressed, and secreted) promoter via an NF-kappa B-mediated pathway. Finally, Weber and colleagues (48) recently demonstrated increased basal NF-kappa B transactivation in pCEP-R and 16HBE-AS CF phenotype cells. In addition, they found that expression of Delta F508 CFTR in Chinese hamster ovary cells increases NF-kappa B transcriptional activity, consistent with the notion that NF-kappa B plays a significant role in the dysregulated inflammatory response in CF.

Few studies have examined the upstream signaling intermediates regulating NF-kappa B activation in response to P. aeruginosa. With the use of a series of chemical inhibitors, Li and colleagues (28) concluded that P. aeruginosa lipopolysaccharide induces NCI-H292 pulmonary epithelial cell MUC2 expression via a Src/Ras/ERK/90-kDa ribosomal S6 kinase (RSK)/NF-kappa B pathway. Denning and coworkers (10) showed that chemical inhibition of two MAP kinase family members, ERK and p38, each attenuated IL-8 expression in P. aeruginosa pyocyanin-treated A549 pulmonary epithelial cells. Similarly, Ratner and colleagues (40), using a series of chemical inhibitors, found that activation of NF-kappa B, ERK, and p38 was each required for maximal IL-8 expression in 1HAEo- human airway epithelial cells incubated with P. aeruginosa or antibody to asialoGM1. As far as we aware, there is no information examining P. aeruginosa or anti-asialoGM1 antibody-induced signaling in CF phenotype cells.

In the present study, we hypothesized that Ikappa B kinase (IKK), NF-kappa B-activating kinase (NIK), MAP kinase/ERK kinase kinase-1 (MEKK1), and ERK, each of which have been implicated in NF-kappa B signaling, would be required for P. aeruginosa-induced NF-kappa B activation and IL-8 expression in IB3 CF phenotype cells. We found that ligation of the asialoGM1 receptor with specific antibody induced greater IL-8 expression in IB3 cells than C-38 cells, consistent with the greater density of asialoGM1 receptors in CF phenotype cells (5, 7, 20). AsialoGM1-mediated activation of NF-kappa B, IKK, and ERK was also greater in IB3 cells. IKK-beta and NIK, but not MEKK1, were required for maximal NF-kappa B transactivation and transcription from the IL-8 promoter. Finally, although ERK was required for maximal IL-8 expression, inhibition of ERK signaling had no effect on IKK-beta or NF-kappa B activation, suggesting that ERK regulates IL-8 expression in an NF-kappa B-independent manner.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
RESULTS
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Materials. Anti-asialoGM1 antibody was purchased from Wako Chemical (Richmond, VA). [gamma -32P]ATP and an enhanced chemiluminescence kit were obtained from DuPont/NEN Research Products (Wilmington, DE). Human TNF-alpha was obtained from R&D Systems (Minneapolis, MN). Antibodies against IKK-gamma and the NF-kappa B family transcription factors p65, p50, c-Rel, and RelB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Myelin basic protein (MBP) was purchased from Sigma Chemical (St. Louis, MO). Activating transcription factor (ATF)-2 was obtained from Cell Signaling Technology (Beverly, MA). An antihemagglutinin (anti-HA) monoclonal antibody (HA.11) was purchased from Babco (Berkeley, CA). RNA was isolated using a RNAqueous-4PCR kit (Ambion, Austin, TX). RT-PCR reagents were obtained from Clontech (Palo Alto, CA). An NF-kappa B reporter plasmid NF-kappa B-TATA-luc was purchased from Stratagene (La Jolla, CA). TransIT-LT was obtained from Mirus (Madison, WI). Optimem transfection medium was obtained from Invitrogen (Gaithersburg, MD). Luciferase assay buffer, U0126, and an oligonucleotide probe encoding the consensus sequences of NF-kappa B were purchased from Promega (Madison, WI). IL-8 ELISA reagents were obtained from Endogen (Woburn, MA).

Cell culture. IB3-1 cells, an adeno-12-SV40-immortalized human bronchial epithelial cell line derived from a CF patient (Delta F508/W1282X), and C38 cells, the rescued cell line that expresses an episomal copy of a truncated but functional CFTR, were obtained from P. Zeitlin (Johns Hopkins University, Baltimore, MD) (14, 51). Cells were grown in LHC-8 (Biofluids, Rockville, MD) supplemented with 5% FBS.

Plasmid vectors. The -162/+44 fragment of the full-length human IL-8 promoter, subcloned into luciferase (-162/+44 hIL-8/Luc) (18), was provided by A. Brasier (University of Texas, Galveston, TX). The reporter activities of this fragment have been shown to be identical to the full-length promoter in response to respiratory syncytial virus infection (18). Construction of cDNAs encoding dominant negative IKK-beta (in which Ser-177 and -181 were replaced by alanines), NIK-KM (KK429/AA430), MEKK1-KM (K432M), and HA-tagged IKK-beta has been described elsewhere (34). A plasmid encoding dominant negative (pCMV-MEK-2A) forms of MEK1, in which serine-218 and -222 phosphorylation sites were modified to alanine, was provided by D. Templeton (Case Western Reserve University) (50). A bacterial expression vector encoding recombinant GST-Ikappa Balpha was provided by M. Karin (University of California, San Diego, CA) (12). A cDNA encoding beta -galactosidase was provided by M. Rosner (University of Chicago).

IL-8 protein abundance. Confluent C38 and IB3 cells were serum starved for 24 h and treated with either TNF-alpha (10 ng/ml) or anti-asialoGM1 antibody (1:50) overnight. Growth media was collected and IL-8 protein was measured by ELISA.

Immunoblotting. Cell lysates were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose by semidry transfer. After incubation with antibody, signals were amplified and visualized by enhanced chemiluminescence.

Reporter assays. Cells were transfected with cDNAs encoding the NF-kappa B reporter or IL-8 promoter using TransIT-LT, a polyamine transfection reagent. Cells were grown to 50-80% confluence, washed in Optimem, and incubated with a solution of plasmid DNA (~1.0 µg total DNA per 35-mm dish), TransIT-LT (2 µl/dish), and Optimem. To normalize for transfection efficiency, cells were cotransfected with a cDNA encoding beta -galactosidase. After 3.5 h, the transfection solution was replaced with 5% FBS/LHC-8. Twenty-four hours after transfection, cells were serum starved for 8 h and treated with either TNF-alpha (10 ng/ml) or anti-asialoGM1 (1:50). Sixteen hours after treatment, cells were harvested and analyzed for luciferase activity using a luminometer, as described (39). Luciferase content was assessed by measuring the light emitted during the initial 30 s of the reaction, and the values were expressed in arbitrary light units. The background activity from cell extracts was typically <0.2 units, compared with basal signals on the order of 102 units. beta -Galactosidase activity was assessed by colorimetric assay using o-nitrophenyl-beta -D-galactoside as a substrate (37).

PT-PCR analysis. Confluent C38 and IB3 cells were stimulated with human TNF-alpha and anti-asialoGM1 antibody. Total RNA was isolated by ethanol precipitation and treated with DNase I before RT-PCR. Each RT reaction was carried out by combining 5-10 µl of total RNA mix with 2 µl 10× RT buffer, 3 µl dNTP mix, 2 µl random primer, 1 µl Moloney murine leukemia virus RT, and 1 µl RNase inhibitor (42°C for 1 h). The PCR reaction was performed by combining 10 µl cDNA, 2 µl primer mix (1 µl of each 5' and 3' primer), 5 µl 10× buffer, 1 µl 50× Taq DNA polymerase, and 5 µl 10× dNTPs. Conditions for PCR consisted of incubation at 94°C for 3 min; 30 cycles each of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, and incubation at 72°C for 7 min. Samples were analyzed by gel electrophoresis on a 1.5% agarose gel. PCR primers for human IL-8 were 5'-TACTCCAAACCTTTCCAACCC-3' and 5'-AACTTCTCCACAACCCTCTG-3'. beta -Actin was used as a normalization marker.

Electrophoretic mobility shift assays. Nuclear extracts were prepared by the method of Dignam et al. (13) with some modifications. Electrophoretic mobility shift assays were performed using nuclear extracts (4 µg) and binding buffer containing 5 mM Tris · HCl (pH 7.5), 37.5 mM KCl, 0.5 mM EDTA, 2% Ficoll, 50 µg/ml poly (dI-dC), and 30-100,000 cpm of gamma -32P-labeled probe, and incubated on ice for 15 min. Nuclear extracts were added and the mixture was incubated at room temperature for 15 min. In some instances, antibodies against either p65 RelA or p50, the NF-kappa B family of binding proteins, were added (10 min at room temperature). The protein-DNA complexes were analyzed by electrophoresis through a 5% polyacrylamide gel. The gels were dried and exposed to radiographic film.

IKK in vitro phosphorylation assays. Endogenous IKK was immunoprecipitated from cell extracts with an anti-IKK-gamma antibody (34). Activity of the immune complex was assayed in 30 µl of kinase buffer in the presence of 10 µM ATP, 5 µCi [gamma -32P] ATP, and GST-Ikappa Balpha (3 µg/sample) as a substrate (30°C for 15 min). Reactions were terminated with 4× Laemmli sample buffer. Samples were resolved by 10% SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane by semidry transfer. After Ponceau staining, the membrane was exposed to film, and substrate phosphorylation was assessed by optical scanning. Equal levels of immunoprecipitated IKK were confirmed by immunoblotting using an anti-IKK antibody.

To validate the function of the NIK-KM plasmid, we determined the requirement of NIK-KM for IKK-beta activation. Cells were transfected with HA-IKK-beta and NIK-KM or empty vector. HA-IKK-beta was immunoprecipitated from cell extracts with an anti-HA monoclonal antibody. IKK-beta activity was assessed as above. The level of HA-IKK-beta expression was monitored by immunoblotting using the HA antibody.

MAP kinase in vitro phosphorylation assays. Cells were transfected with cDNAs encoding HA-tagged p42 ERK2 or p38 MAP kinase and empty vector. In selected experiments, cells were cotransfected with MEKK1-KM or empty vector. The epitope tag was immunoprecipitated from cell extracts with an anti-HA monoclonal antibody, and an in vitro kinase assay was performed using recombinant MBP or ATF-2 as substrates, as described (36).

Data analysis. Each experiment was performed at least three times. Data were described as means ± SE. The significance of changes in luciferase activity and protein abundance was assessed by analysis of variance. Differences identified by analysis of variance were pinpointed by Student-Newman-Keuls multiple range test. For reporter assays, changes in promoter activity were calculated as arbitrary light units per beta -galactosidase calorimetric units per hour and reported as fold increase vs. baseline.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Treatment of C-38 and IB3 cells with TNF-alpha and anti-asialoGM1 induces IL-8 mRNA and protein expression. C-38 and IB3 cells were treated with either TNF-alpha (10 ng/ml) or anti-asialoGM1 (1:50) overnight, total mRNA was isolated, and RT-PCR was performed. Treatment with TNF-alpha and anti-asialoGM1 each increased IL-8 mRNA expression (Fig. 1A). However, asialoGM1-stimulated IL-8 mRNA was increased in IB3 cells relative to C-38 cells, consistent with the greater density of asialoGM1 receptors in CF phenotype cells (5, 7, 20).


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Fig. 1.   IL-8 expression in cystic fibrosis (CF) (IB3) and corrected (C-38) cells. A: cells were treated with TNF-alpha (10 ng/ml for 16 h) or anti-asialoGM1 (1:50 dilution for 16 h). IL-8 mRNA expression was measured by RT-PCR. These data are representative of 3 experiments. B: IL-8 protein abundance was measured by ELISA (n = 5-9, means ± SE; * different from C-38 cells, P < 0.05, ANOVA).

To determine whether changes in mRNA were reflected in protein expression, cells were incubated with either TNF-alpha or anti-asialoGM1 overnight, and the growth medium was examined for IL-8 protein by ELISA. Treatment with TNF-alpha and anti-asialoGM1 each increased IL-8 protein abundance (Fig. 1B). Again, asialoGM1-stimulated IL-8 protein abundance was increased in IB3 cells relative to C38 cells, reflecting changes in mRNA expression.

NF-kappa B binding in C-38 and IB3 cells. To determine whether anti-asialoGM1 induces the binding of NF-kappa B to DNA, nuclear extracts from treated cells were incubated with an oligonucleotide encoding the consensus NF-kappa B binding site. Incubation of C-38 and IB3 cells with a 1:50 dilution of anti-asialoGM1 induced substantial NF-kappa B binding (Fig. 2, A and B). AsialoGM1-induced NF-kappa B binding was significantly increased in IB3 cells compared with C38 cells. Coincubation of nuclear extracts with antibodies against p65 RelA and p50 NF-kappa B1, but not c-Rel and RelB induced supershift of the DNA binding complex, demonstrating the presence of these NF-kappa B family transcription factors.


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Fig. 2.   NF-kappa B activation in corrected (C-38) and CF (IB3) cells. Cells were treated with anti-asialoGM1 (1:50 dilution for 10 min). A: NF-kappa B binding was assessed by electrophoretic mobility gel shift. AP, activator protein. B: group mean data for 3 experiments. Data were normalized to basal activity in C-38 cells (means ± SE; * different from control, P < 0.05, ANOVA).

IKK activation in C-38 and IB3 cells. To examine whether IKK is activated in IB3 and C-38 cells, we measured endogenous IKK activity by immunoprecipitating cell lysates with an antibody against IKK-gamma and incubating the precipitates with [gamma -32P]ATP and recombinant Ikappa Balpha as a substrate. IKK-gamma functions as a regulatory subunit for the IKK complex and serves as a docking site for upstream activators (21, 22). AsialoGM1-induced responses were significantly increased in IB3 cells relative to C-38 cells (Fig. 3, A and B).


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Fig. 3.   Ikappa B kinase (IKK) activation in corrected (C-38) and CF (IB3) cells. A: cells were treated with anti-asialoGM1 (1:50 dilution for 10-60 min). IKK activity was assessed by in vitro phosphorylation assay using recombinant Ikappa Balpha as a substrate (top). To normalize for potential differences in IKK immunoprecipitation, precipitates were also probed with an anti-IKK-gamma antibody (bottom). B: for comparison, TNF-alpha -induced IKK activation (10 ng/ml for 10-60 min) was also assessed. C: group mean data for asialoGM1-induced IKK activation (1:50 dilution for 30 and 60 min). Data were normalized to basal activity in C-38 cells (n = 3, means ± SE; * different from control, P < 0.05, ANOVA).

Requirement of IKK-beta for NF-kappa B transactivation and IL-8 promoter activity in IB3 cells. To test whether IKK-beta activation is required for transactivation of NF-kappa B and transcription from the IL-8 promoter, cells were cotransfected with cDNAs encoding either dominant negative IKK-beta (IKK-beta -AA) or empty vector and either NF-kappa B or IL-8 reporter plasmids. Luciferase activity was measured with a luminometer, and results were normalized for transfection efficiency by measurement of beta -galactosidase activity. Expression of IKK-beta -AA attenuated anti-asialoGM1-induced NF-kappa B transactivation (Fig. 4A) and IL-8 promoter activity (Fig. 4B). These results indicate that, in IB3 cells, maximal asialoGM1-mediated NF-kappa B transactivation and transcription from the IL-8 promoter are each dependent on IKK-beta activation.


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Fig. 4.   Role of IKK-beta in IB3 cell NF-kappa B transactivation and IL-8 promoter activity in IB3 cells. Cells were cotransfected with cDNA encoding either NF-kappa B-TATA-luc or -162/+44 hIL-8 luc and either empty vector or IKK-beta -AA (dominant negative IKK-beta ). Cells were treated with anti-asialoGM1 (1:50 dilution for 16 h). The luciferase activity of cell lysates was assessed by luminometer. To normalize for transfection efficiency, cells were also cotransfected with pCMV-beta -galactosidase. beta -Galactosidase activity was assessed by colorimetric assay. Data represent n = 4, means ± SE (* different from control, P < 0.05, ANOVA).

Requirements of NIK and MEKK1 for NF-kappa B transactivation and IL-8 promoter activity in IB3 cells. To determine the upstream signaling intermediates responsible for IKK-beta activation, IB3 cells were cotransfected with kinase-deficient NIK (NIK-KM), kinase-deficient MEKK1 (MEKK1-KM), or empty vector and NF-kappa B and IL-8 reporter plasmids. NIK-KM attenuated both anti-asialoGM1-induced NF-kappa B transactivation and IL-8 promoter activity (Fig. 5, A and B), but MEKK1-KM had no effect (Fig. 5, C and D). Furthermore, NIK-KM attenuated both TNF-alpha and anti-asialoGM1-induced IKK-beta activity (Fig. 5E). Finally, an in vitro kinase assay assessing the effect of MEKK1-KM on p38 MAP kinase activity confirmed the inhibitory effect of this mutant protein on kinase activity (Fig. 5F). Together, these data suggest that NIK but not MEKK1 is an upstream activator of IKK-beta , NF-kappa B, and IL-8 in IB3 CF phenotype cells.


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Fig. 5.   Roles of NF-kappa B-activating kinase (NIK) and MAP kinase/ERK kinase kinase-1 (MEKK1) in IB3 cell NF-kappa B transactivation and IL-8 promoter activity in IB3 cells. Cells were cotransfected with cDNA encoding either NF-kappa B-TATA-luc (A and C) or -162/+44 human IL-8 luc (B and D) and either empty vector, NIK-KM (A and B) or MEKK1-KM (C and D). Cells were treated with anti-asialoGM1 (1:50 dilution for 16 h). Data represent n = 4-6, means ± SE (* different from control, P < 0.05, ANOVA). E and F: in vitro kinase assays examining the effects of NIK-KM and MEKK1-KM on IKK-beta and p38alpha MAP kinase activities, respectively. IKK-beta and p38alpha activities were assessed by in vitro phosphorylation assay using recombinant Ikappa Balpha and activating transcription factor (ATF)-2 as substrates, respectively. Expression of NIK-KM attenuated both TNF-alpha - and anti-asialoGM1-induced IKK activity. This experiment was repeated twice. Expression of MEKK1-KM attenuated TNF-alpha -induced p38 MAP kinase activity, confirming the inhibitory function of MEKK1-KM. HA, hemagglutinin.

Requirement of ERK signaling for IL-8 expression but not NF-kappa B activation. We examined the contribution of ERK signaling to asialoGM1-induced IL-8 expression. We measured ERK2 activation in IB3 and C-38 cells by in vitro phosphorylation assay using MBP as a substrate. Ligation of asialoGM1 induced ERK activation in both cell types (Fig. 6A). Consistent with previous results, the level of ERK activation appeared greater in IB3 cells. Pretreatment with a chemical inhibitor of MEK, U0126, reduced ERK activation (Fig. 6A). A concentration of 3 µM U0126 was sufficient for this response and was used for subsequent experiments. Pretreatment with 3 µM U0126 also attenuated IL-8 protein abundance (Fig. 6B) and transcription from the IL-8 promoter (Fig. 6C). Inhibition of ERK signaling by expression of a dominant negative form of MEK1 also attenuated asialoGM1-induced transcription in IB3 cells (Fig. 6D).


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Fig. 6.   Requirement of ERK signaling for IL-8 expression. A: ERK2 activation in IB3 and C-38 cells, as assessed by in vitro phosphorylation assay using myelin basic protein (MBP) as a substrate. Pretreatment with a chemical inhibitor of MEK, U0126, reduced asialoGM1-induced ERK activation. This experiment was repeated twice. B: effect of U0126 (3 µM) on IL-8 protein abundance, as assessed by ELISA (n = 4, *P < 0.05, ANOVA). C: effect of U0126 (3 µM) on asialoGM1-induced transcription from the IL-8 promoter (n = 4, *P < 0.05, ANOVA). D: effect of MEK-2A expression on IL-8 transcription (n = 4, *P < 0.05, ANOVA).

We tested whether ERK functions upstream of NF-kappa B in IB3 cells. Pretreatment with U0126 failed to attenuate binding of nuclear proteins to NF-kappa B, as shown by electrophoretic mobility shift assay (Fig. 7A). Inhibition of ERK by U0126 had no effect on NF-kappa B transactivation, as shown by reporter assay (Fig. 7B). Expression of MEK-2A failed to attenuate NF-kappa B transactivation in IB3 cells (Fig. 7C). Pretreatment with U0126 had no effect on endogenous IKK activity (Fig. 7D). Finally, because IKK and MAP kinase kinases share structural elements, including the position of two serine phosphoaccepting sites within the activation loop, it is conceivable that NIK may regulate ERK activation (11). However, expression of NIK-KM had no effect on ERK activity (Fig. 7E). Together, these data suggest that, in IB3 cells, ERK regulates asialoGM1-induced IL-8 expression in an NF-kappa B-independent manner.


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Fig. 7.   ERK regulates IL-8 expression in an NF-kappa B-independent manner. A: pretreatment of IB3 cells with U0126 failed to attenuate asialoGM1-mediated binding of nuclear proteins to NF-kappa B (arrow), as shown by electrophoretic mobility shift assay. B: effect of U0126 on NF-kappa B transactivation, as shown by reporter assay (n = 4). C: effect of MEK-2A expression of MEK-2A on asialoGM1-mediated NF-kappa B transactivation in IB3 cells (n = 4). D: effect of U0126 on endogenous IKK activity in IB3 cells. IKK activation was assessed by immunoprecipitation of IKK-gamma followed by in vitro kinase assay using Ikappa B as a substrate. E: effect of NIK-KM on IKK-beta activity in IB3 cells. Activation of ectopically expressed IKK-beta was assessed by immunoprecipitation of the epitope tag followed by in vitro kinase assay using Ikappa B as a substrate.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Investigators have compared cytokine expression in airway epithelial cells derived from CF individuals with those derived from normal individuals. In at least three cell line pairs, IL-8 expression has been found to be increased in CF phenotype cells relative to corrected cells (15, 24). Further studies examining the mechanism by which cytokine expression is upregulated in these cells have demonstrated a potential role for NF-kappa B signaling (14, 46, 48). However, the upstream intermediates responsible for P. aeruginosa-induced NF-kappa B responses in CF cells have not been studied. To accomplish this, we examined the responses to ligation of the P. aeruginosa pilin receptor, gangliotetraosylceramide (asialoGM1) in IB3 cells, an adeno-associated virus-transformed human bronchial epithelial cell line derived from a CF patient (Delta F508/W1282X), compared with its CFTR-corrected line, C-38 cells. In the present study, we found that ligation of the asialoGM1 receptor with specific antibody induced greater IL-8 expression in IB3 cells than C-38 cells, consistent with the greater density of asialoGM1 receptors in CF phenotype cells (5, 7, 20). As in previous studies (14, 48), asialoGM1-mediated activation of NF-kappa B was also greater in IB3 cells.

The basic NF-kappa B complex is a dimer of two members of the Rel family of proteins, p50 (NF-kappa B1) and p65 (RelA). In unstimulated cells, NF-kappa B is sequestered in the cytoplasm by Ikappa B proteins. Phosphorylation and degradation of Ikappa B allow translocation of NF-kappa B to the nucleus, where it regulates gene transcription by binding to specific sequences of DNA. Although Ikappa Balpha is the best characterized family member, Ikappa Bbeta and Ikappa Bepsilon may also regulate NF-kappa B (21). The "classical" signaling pathway to NF-kappa B activation includes the successive phosphorylation and activation of NIK and IKK (21, 22). IKK consists of two catalytic subunits (IKK-alpha and IKK-beta ) and a regulatory subunit (IKK-gamma ). Although IKK-alpha and IKK-beta contain similar kinase domains with essentially identical activation loops (33), they are functionally distinct. Recent studies suggest that IKK-beta serves as the target for proinflammatory signals, whereas IKK-alpha plays a critical role in development (6, 9, 19). On the other hand, it has recently been shown that UV-C irradiation (2, 29), hepatitis B protein X (38), and p21-activated kinase (17) may each activate NF-kappa B via as yet unidentified IKK-independent mechanisms. In the present study, we found that ligation of the asialoGM1 receptor with specific antibody induced greater IKK activation in IB3 than C-38 cells, consistent with the notion that IKK is a downstream target of the asialoGM1 receptor in CF phenotype cells. Furthermore, expression of a dominant negative form of IKK-beta (IKK-beta -AA) significantly attenuated asialoGM1-mediated NF-kappa B transactivation and IL-8 promoter activity, strongly suggesting that IKK-beta is required for maximal asialoGM1-mediated Ikappa B phosphorylation.

The most potent IKK activator yet identified is the serine-threonine kinase NIK. NIK was originally identified as a protein that interacts with TNF receptor-associated factor-2 (TRAF2). Constitutive activation of NIK causes Ikappa Balpha degradation and NF-kappa B activation (31), suggesting that NIK is involved in NF-kappa B signaling. NIK is required for activation of NF-kappa B by non-typeable Hemophilus influenzae (44) and micrococci (47). On the other hand, NIK strongly and preferentially interacts with and activates IKK-alpha rather than IKK-beta (30, 49). In addition, replacement of the COOH-terminal TRAF domain of TRAF2 or TRAF6 with a heterologous oligodimerization domain results in chimeric proteins that retain the ability to activate IKK, although they no longer interact with NIK (1).

IKK-beta and the MAP kinase kinases (MKKs) share structural elements, including the position of two serine phosphoaccepting sites within the activation loop. Accordingly, it was later found that MKK kinases, including those of the MAP kinase/MEKK family, may phosphorylate and activate IKK (34). MEKK1 is activated by TNF-alpha and IL-1, and overexpression of a dominant negative MEKK1 inhibits IKK-beta and NF-kappa B activation (26, 34). Other putative IKK kinases include protein kinase C-zeta (25) and Akt/protein kinase B (35, 41). To determine the requirement of NIK and MEKK1 for NF-kappa B activation and IL-8 expression in CF cells, we transiently transfected cells with cDNAs encoding kinase-inactive mutants of NIK and MEKK1. We found that inhibition of NIK but not MEKK1 attenuated TNF-alpha and asialoGM1-mediated NF-kappa B and IL-8 responses, suggesting that NIK is required for maximal IKK-beta activation. Expression of kinase-inactive NIK also inhibited basal and stimulated IKK-beta activity, consistent with the notion that NIK is a major upstream activator of IKK-beta and NF-kappa B in CF airway cells.

With the use of a series of chemical inhibitors, Li and colleagues (28) concluded that P. aeruginosa lipopolysaccharide induces NCI-H292 pulmonary epithelial cell MUC2 expression via a Src/Ras/ERK/RSK/NF-kappa B pathway. Similarly, Ratner and colleagues (40), using a series of chemical inhibitors, found that activation of NF-kappa B, ERK, and p38 was each required for maximal IL-8 expression in 1HAEo- human airway epithelial cells incubated with P. aeruginosa or antibody to asialoGM1. However, the precise relationship between ERK activation and NF-kappa B activation in CF epithelial cells has not been studied. We previously found that in 16HBE14o- human bronchial epithelial cells, ERK regulated IL-8 expression in an AP-1-dependent, NF-kappa B-independent manner (27). In the present study, we found that although ERK was required for maximal IL-8 expression, inhibition of ERK signaling had no effect on IKK-beta or NF-kappa B activation, confirming that in human bronchial epithelial cells ERK regulates IL-8 expression in an NF-kappa B-independent manner.

Finally, as noted previously (14, 46), we found that basal levels of IL-8 expression and NF-kappa B activation were modestly increased in IB3 cells relative to corrected C-38 cells. We did not detect basal increases in endogenous IKK activity in IB3 cells, suggesting that IKK is not responsible for the observed differences in NF-kappa B activation. However, it should be noted that basal levels of IL-8 expression and NF-kappa B activation were relatively small compared with asialoGM1-mediated responses, and therefore we did not choose to investigate the underlying mechanism for these differences.

In summary, we showed that asialoGM1-mediated activation of IKK, NIK, and ERK is each increased in a phenotypic CF bronchial epithelial cell line, relative to the corrected line, consistent with the greater density of asialoGM1 receptors in CF phenotype cells (5, 7, 20). Inhibition of these kinases, each of which was required for maximal IL-8 expression, may attenuate the inflammatory response in CF respiratory epithelia.


    ACKNOWLEDGEMENTS

The authors thank Dr. P. Zeitlin (Johns Hopkins University) for the contribution of IB3-1 and C-38 cells and Dr. A. Brasier (University of Texas, Galveston, TX), Dr. M. Karin (University of California, San Diego, CA), and Dr. M. Rosner (University of Chicago) for the gifts of plasmid vectors.


    FOOTNOTES

These studies were supported by National Institutes of Health Grants CA-73740 (A. Lin) and HL-56399 (A. Lin and M. B. Hershenson), American Cancer Society Grant CCG-98471 (A. Lin), and the Cystic Fibrosis Foundation (M. B. Hershenson).

Address for reprint requests and other correspondence: M. B. Hershenson, Univ. of Michigan, 1150 W. Medical Center Dr., 3570 MSRBII/Box 0688, Ann Arbor, MI 48109-0688 (E-mail: mhershen{at}umich.edu).

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.

First published September 27, 2002;10.1152/ajplung.00086.2002

Received 22 March 2002; accepted in final form 20 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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