NF-κB is activated during acute inflammatory states as well as in other injury response disease states. Several pathologic states in squamous tissue injury response are characterized by increased squamous proliferation. This study was performed to investigate the hypothesis that Pseudomonas aeruginosa LPS is able to activate a proliferative phenotype in squamous cells via NF-κB induction and that this NF-κB-mediated response may be abrogated with the classic anti-inflammatory agent indomethacin. EMSA, luciferase reporter gene experiments, Western blots, and cellular proliferation assays were performed in normal and transformed human keratinocytes after stimulation with P. aeruginosa LPS. EMSA and luciferase reporter gene assays showed a 3- to 5-fold induction of active NF-κB in human keratinocyte cell lines after stimulation with P. aeruginosa LPS. The stimulation correlated with significantly increased cellular proliferation. As one potential mechanism for this increase in proliferation, an NF-κB-specific activation of cyclin D1 was observed. Both the NF-κB induction and proliferation response were inhibited with indomethacin and in dominant negative stable transfection clones. P. aeruginosa LPS activates proliferation of human keratinocytes, potentially through the induction of NF-κB and cyclin D1. These findings suggest that bacterial components can contribute to proliferative disease states in squamous epithelium through NF-κB activation.

It is becoming clear that bacterial infection and host inflammatory responses are intimately associated with the development of a full range of abnormal proliferative states in host tissues up to and including the development of cancer (1). Prototypically, Helicobacter pylori has been linked to mutagenic free radical formation leading to chronic gastric inflammation and the stimulation of cellular proliferation (2, 3, 4). Mechanisms for H. pylori-induced cellular proliferation and carcinogenesis are under active investigation, but other model systems for complex interactions between bacterial pathogens and host responses, resulting in the development of cellular proliferation and the cancer phenotype are not well described. A variety of pathologic states in squamous tissue injury, including cholesteatoma, chronic burns, and aerodigestive carcinogenesis, are characterized by increased proliferation in response to stimuli that occur during chronic inflammation. One possible reason for this response is NF-κB activation (5).

Many extracellular events can trigger the activation of NF-κB in inflammatory cells. Bacterial LPS was one of the first known activators of B cells and is closely linked to the discovery of NF-κB (6). NF-κB has long been known to be a mediator of the effects of LPS (7). The production of multiple cytokines, such as TNF-α (8), IL-1 (9), IL-6 (10), and GM-CSF (11), is up-regulated in macrophages by NF-κB after LPS stimulation. Reports of bacterial product NF-κB activation in keratinocytes are far less frequent; LPS has been shown to induce the expression of another NF-κB-dependent gene, M-CSF (12). In normal, unstimulated keratinocytes, NF-κB has been shown to inhibit programmed cell death (13). This phenomenon effectively confers protection from premature apoptosis to proliferating benign epidermal cells. To this point, the overexpression of NF-κB’s active subunit, p65, is associated with the production of cytokeratin markers associated with proliferation, such as cytokeratin K6, but mechanisms for proliferative responses of the cells were not investigated (14). Conversely, it has recently been demonstrated that NF-κB inhibition may actually trigger human epidermal invasive cancer, an effect opposite of what might be expected. This result has been observed in other studies (15, 16). Therefore, generalities regarding the effect of NF-κB activation in even a single cell type may be dependent upon the whether the activation is constitutive or induced and may depend on the type of stimulation.

Studies have attempted to elucidate cellular mechanisms and circumstances by which NF-κB is able to mediate changes in cellular proliferation. For example, in a nonepithelial, nonkeratinocyte model system, NF-κB has been shown to induce cyclin D1 gene expression and cellular proliferation by binding to specific sequences in the cyclin D1 promoter (17, 18). Cyclin D1 is an integral protein in cell cycle progression (19). It is synthesized in response to external activators and forms a complex with CDK4 and CDK6 that is then able to phosphorylate the retinoblastoma tumor suppressor protein. Retinoblastoma tumor suppressor protein regulates the transcription of multiple genes that are indispensable for S phase entry. In squamous tissues, cyclin D1 is overexpressed in squamous cell carcinomas of the head and neck (20) and cholesteatoma (21), two epithelial hyperproliferative conditions. Cyclin D1 has been established in vivo as a pure mediator of proliferation in epithelial cells, because its overexpression in the epithelial tissues of transgenic mice results in epidermal hyperproliferation, but has no effect on terminal differentiation (22). The effect of any activators of NFκB on cyclin D1 or cyclin D kinase expression in keratinocytes is unknown.

Cholesteatoma is a squamous disease state characterized by 1) hyperproliferative keratinocyte epithelial in-growth, angiogenesis, and local bony invasion into the middle ear and mastoid cavity; and 2) chronic middle ear infection associated with P. aeruginosa (23). Therefore, it represents one injury response process associated with chronic exposure to bacterial toxins instead. It is also a process that has features in common with aerodigestive carcinogenesis. A large number of NF-κB dependent proinflammatory cytokines, PGs, and growth factors have been identified during squamous tissue inflammatory states, including chronic middle ear infection (24), cholesteatomagenesis (25), and even head and neck cancer (26, 27). Therefore, NF-κB may be an important regulator of events in the squamous inflammatory milieu in a variety of processes. Because P. aeruginosa bacterial products have been linked to NF-κB activation in other epithelial cell experimental models of both chronic and acute inflammatory dysregulation (28, 29), its effects were further studied in this series of experiments.

The purpose of this study was to demonstrate that 1) other models of disorganized epithelial cell proliferation exist during bacterially induced injury exist in addition to H. pylori infection and gastric adenocarcinogenesis; and 2) activation of injury response gene programs by transcription factors such as NF-κB may be responsible for establishing a permissive environment for abnormal cell proliferation during chronic inflammation. Specifically, this study was performed to determine whether P. aeruginosa LPS can induce NF-κB and cell proliferation in a keratinocyte model system as an in vitro model of early response gene activation during squamous inflammation and carcinogenesis and to determine whether this activation results in an NF-κB-dependent, cyclin D1-mediated, induction of proliferation. As a potential therapy for abnormal proliferation, indomethacin was used to examine whether it had any abrogating effect on keratinocyte NF-κB activation and the proliferative response.

Rhek-1A (Rhek) human epidermal keratinocytes were used for most studies (provided by Dr. J. S. Rhim, National Cancer Institute, Frederick Cancer Research and Development Center). The Rhek-1A cell line was immortalized by SV40 and is nontumorigenic in nude mice (30). The Rhek cell line has been used in other studies of keratinocyte proliferation, carcinogenesis, and cytotoxicity (31, 32, 33). Rhek cells were used throughout the project as a prototypic squamous epithelial model system. Human epidermal keratinocytes were used to confirm all findings established in the Rhek cells to demonstrate the suitability of Rhek cells for such experiments. The Rhek cells were maintained in EMEM supplemented with 10% FBS and penicillin/streptomycin (50 μg/ml) at 37°C in 5% CO2 as adherent monolayer cultures. Normal human epidermal keratinocytes were purchased commercially from Cambrex. They were cultured according to the instructions of the manufacturer in keratinocyte serum-free growth medium to maintain a proliferative phenotype. This medium is supplemented with 0.1 μg/ml human recombinant epidermal growth factor, 5.0 mg/ml insulin, 0.5 mg/ml hydrocortisone, 50 mg/ml gentamicin, 50 μg/ml amphotericin B, and 7.5 mg/ml bovine pituitary extract (BioWhittaker) at 37°C in 5% CO2

The cDNA expression plasmid pCMX-IκB-α/M contains a mutation at S36 of the NH2 terminus, and a COOH-terminal PEST sequence mutation and its empty vector pCMX were a kind gift of Dr. Inder Verma (Salk Institute, La Jolla, CA) (34, 35). This stably expressed mutated protein is resistant to extracellular signal-induced phosphorylation and subsequent degradation thus not allowing for activation of NF-κB (36). RHEK cell cultures at 70% confluence were transfected with pCMX-IκB-α/M (2 μg/well) and the pCMV-neo resistant (2 μg/well) or empty vector pCMX (2 μg/well) and pCMV-neo resistant in OptiMEM medium containing 10 μg/ml Lipofectamine (Invitrogen Life Technologies). After 4 h, the medium was removed, and the cells were placed in complete medium for 72 h. Then, cells were placed in selection medium containing G-418 (750 μg/ml). After 14 days, neo-resistant colonies were selected from each well using cloning discs embedded in trypsin and establish as a subcultures.

Extracts from the cell lines in log-growth phase were prepared according to the methods of Dignam et al. (37) and Lee et al. (38) as follows. Cells were grown in 75-cm2 flasks to 60–80% confluence. Cells were stimulated with 0, 10, 50, or 100 μg/ml P. aeruginosa LPS for 60 min. To determine the potential blockade of NF-κB activation, cells were pretreated with 5 or 20 μg/ml indomethacin in DMSO; the groups not pretreated with indomethacin received DMSO only for 90 min. Cell suspensions were prepared by trypsin-EDTA treatment, and the cells were washed twice in cold PBS and pelleted at 500 × g. The cell pellets were then incubated on ice for 5 min with lysis buffer (10 mM Tris-HCl, 60 mM KCl, 1 mM EDTA, 0.5% Nonidet P-40, fresh 0.1 M DTT, and protease inhibitor). The lysates were microfuged for 5 min at 500 × g. The supernatant was collected and saved as the cytoplasmic extract. Next, an equal volume of nuclear extract buffer (20 mM Tris-HCl, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, fresh 0.1 M DTT, and PMSF) was added to the remaining nuclear pellet. This was incubated on ice for 10 min, then centrifuged at 15,000 × g for 10 min. The remaining nuclear extracts were aliquoted and stored at −80°C. The protein concentrations of the extracts were determined in triplicate using the bicinchoninic acid modification of the biuret reaction (Protein Assay Kit; Pierce) scaled for microtiter plate analysis. BSA was used as a standard, and the plates were read at 580 nm on a Biotek 311 microtiter plate reader. Standard curves were generated using computer software. The correlation coefficients for the functions were >0.95 in all experiments.

dsDNA oligonucleotide probes for NF-κB and Oct-1 were synthesized commercially (Promega). The consensus sequences used were 5′-AGTTGAGGGGACTTTCCCAGGC-3′ for NF-κB and 5′-TGTCGAATGCAAATCACTAGAA-3′ for Oct-1. Consensus oligonucleotide probes were labeled with T4 polynucleotide kinase (Promega) and [γ-32P]ATP (6000 Ci/mmol; Amersham Biosciences).

Binding reactions were performed using 5 μg of nuclear extract protein incubated for 25 min at 20°C in buffer containing 20 mM HEPES (pH 7.9), 4.6 mM MgCl2, 63 mM KCl, 11% glycerol, 1 mM DTT, 1 μg of poly(dI-dC) (Amersham Biosciences), and 60,000 cpm 32P-labeled nucleotide probe. For unlabeled competitions, extracts were preincubated with a 100-fold excess of each probe before addition of labeled probe. The binding complexes were resolved from reactions on 5% polyacrylamide gels in 0.25× Tris-borate-EDTA buffer at 20°C and run for 90 min at 200 V. The gels were dried and exposed by autoradiography to intensifying screens (Eastman Kodak) for 12–24 h and imaged with an OptiQuant instant imager (Packard Instruments). All reactions were performed with at least three separate nuclear extract preparations. The procedures for nuclear extract preparation were identical for all nuclear extract preparations. Positive controls for NF-κB p65/p50 heterodimers were included (provided by Dr. K. Brown, National Institutes of Allergy and Infectious Diseases, National Institutes of Health). These proteins were provided as whole cell extracts from p50 and p65 transfected COS cells after PMA stimulation.

Ab supershift analyses of the composition of NF-κB were performed. Anti-p65 and anti-p50 NF-κB subunit Abs were purchased from Rockland Laboratories. Supershift reactions were performed under identical binding conditions for both Abs. Supershift Abs (1 μg) were added 30 min before binding to the reaction mixtures with the labeled probes. All EMSA experiments were repeated three times with similar results.

The pIgκB Luc reporter construct containing two Ig G-κ chain NF-κB binding sites and the luciferase gene have been previously described (39) and were provided by Dr. K. Brown (National Institutes of Allergy and Infectious Diseases, National Institutes of Health). The CD1 reporter plasmids were gifts from Dr. R. Pestell (Albert Einstein College of Medicine). They contain the cyclin D1 promoter upstream form the luciferase gene as previously described (40). The −1745CD1Luc reporter plasmid contains the full sequence cyclin D1 promoter upstream from the luciferase gene. The −66NF-κBmut plasmid has the cyclin D1 promoter with a site-specific mutation at the NF-κB binding site. Cell line cultures at 70% confluence were cotransfected with luciferase reporter plasmid (2 μg/ml) and a pCMV-βGal reporter construct (0.4 μg/ml) in OptiMEM medium containing 5 μg/ml Lipofectamine (Life Technologies). After 4 h, the medium was removed, and the cells were washed and placed in complete medium overnight. The next day the cells were pretreated with indomethacin at 5 or 20 μg/ml in DMSO or with DMSO alone for 90 min. The cells were then stimulated for 12 h with 10, 50, or 100 μg/ml P. aeruginosa LPS. After stimulation, the relative luciferase activity was determined with the Dual Light reporter gene assay (Tropix) and a Monolight 2010 plate luminometer (Analytical Luminescence Laboratories) according to the manufacturers’ instructions. All reporter gene experiments were repeated three times with similar results.

Cell count experiments for Rhek cells were performed by culturing the cells in T-25 flasks at an initial concentration of 35,000 cells/flask. Cells were then pretreated for 90 min with 5 or 20 μg/ml indomethacin in DMSO or in DMSO (0.1%) alone. Cells were stimulated with 10, 50, and 100 μg/ml P. aeruginosa LPS and allowed to grow for 72 h at 37°C in 5% CO2. The cells were then detached from the flask with trypsin-EDTA solution. Cell suspensions were counted using a standard hemocytometer. Each experimental group was repeated in duplicate.

MTT assays (Roche) were performed in 96-well microtiter plates. Rhek cells and normal human epidermal keratinocytes were plated in appropriate medium at a concentration of 5000 cells/well and allowed to grow overnight. The next day, the cells were pretreated with 5 or 20 μg/ml indomethacin in DMSO or in DMSO (0.1%) alone for 90 min before stimulation with 10, 50, or 100 μg/ml P. aeruginosa LPS. Each experimental condition was performed with six replicates in serum-free medium. Cells were then incubated for 3 days at 37°C in 5% CO2. MTT reagent was added to each well, and the plates were reincubated at 37°C in 5% CO2 for 4 h. The resultant cellular precipitant formazan dye was then solubilized with MTT detergent. After overnight incubation at room temperature in the dark, the absorbance was measured using a Biotek microplate reader at 570 nm. All MTT experiments were repeated three times with similar results.

Ten thousand cells per well were seeded in a 96-well plate in appropriate serum-free medium for 24 h. The next day, cells were treated with four serial dilutions of 100 μg/ml P. aeruginosa LPS. Each experimental condition was performed with six replicates in serum-free medium. The cells were then incubated for 24 h at 37°C in 5% CO2. One microcurie of [3H]thymidine in 500 μl was added to each well, and the cells were incubated for 8 h at 37°C in 5% CO2. Medium was the removed, and the cells were washed with 500 μl of ice-cold 1× PBS. Ice-cold 5% TCA (500 μl) was then added to each well, and the plates were left at 4°C for 30 min. The TCA solution was then aspirated, and each well was again washed with 500 μl of ice-cold 1× PBS. At room temperature, 250 μl of 0.5 N NaOH/0.5% SDS was added to each well, solution was pipetted up and down, and each well was counted with a 96-well scintillation counter.

Western blots for cyclin D1 were performed on the cytoplasmic extracts (see above for method of extraction) of the cells exposed to 0, 10, 50, or 100 μg/ml P. aeruginosa LPS for 24 h. After a bicinchoninic acid protein assay, as described above, 20 μg from each sample was incubated with an equal volume of sample buffer for 5 min at 95°C and then loaded onto a 10% acrylamide gel and run by electrophoresis at 20 mAmp for 60–90 min. The protein was transferred onto a nitrocellulose membrane using a semidry gel transfer unit. The membrane was blocked for 1 h, and then primary cyclin D1 Ab (Santa Cruz Biotechnologies) was added overnight. The next morning, the membrane was washed and incubated with secondary Ab for 1–5 min. The horseradish fluoroilluminescence detection protocol was followed. The blots were exposed to Bio-Max film for 10 min and developed.

Similarly, Western blots were performed for IκB-α. Cell lines were grown to 70–80% confluence in T-25 flasks in appropriate medium. The cells were then pretreated with 20 μg/ml indomethacin in DMSO or in DMSO alone for 90 min. Cells were then stimulated for 3 h with 100 μg/ml P. aeruginosa LPS. Cytoplasmic extracts were then prepared as described above. Western blots were performed as described above using primary IκB-α Abs (Santa Cruz Biotechnologies).

The statistical difference between experimental and control groups for all experiments was determined by two-sided Student’s t test. The significance level was set at p < 0.05.

To explore whether NF-κB can be induced by LPS in keratinocytes, the function and composition of NF-κB binding proteins were determined in Rhek cells and normal human epidermal keratinocytes by luciferase reporter assay and EMSA after stimulation with increasing doses of P. aeruginosa LPS. Fig. 1,A demonstrates a statistically significant 2.25- and 3.1-fold functional induction of NF-κB in Rhek cells after 60-min stimulation with 10 and 100 μg/ml LPS, respectively. Additional analysis in this cell line by EMSA demonstrated differences in nuclear NF-κB binding activity consistent with the functional activation shown with the luciferase assay (Fig. 1,B). Competition experiments with unlabeled, wild-type oligonucleotide probe in a 100-fold excess confirm that the binding activity detected in Fig. 1,B was specific for NF-κB. Integrity and equal protein loading amounts of the nuclear extracts were demonstrated by performing Oct-1 DNA binding assays, showing similar Oct-1 binding across experimental groups. To elucidate the specific nature of the EMSA bands, competition experiments were conducted with 1:1, 1:10, 1:25, and 1:50 molar ratios of labeled to unlabeled specific NF-κB oligonucleotides and nonspecific PPAR-γ oligonucleotides (Fig. 1 C). The NF-κB band disappeared with increasing specific cold competition, but did not with increasing nonspecific competition.

FIGURE 1.

Activation of NF-κB in Rhek cells with P. aeruginosa LPS. A, Luciferase reporter gene activation in Rhek cells after stimulation with P. aeruginosa LPS. The NF-κB luciferase reporter assay was performed as described in Materials and Methods, and the figure shows 2.25- and 3.1-fold activation of NF-κB activity in Rhek cells after 60-min treatment with 10 and 100 μg/ml P. aeruginosa LPS, respectively (∗, p < 0.001). B, EMSA of NF-κB binding activity. Binding in Rhek cells is depicted in lanes 1, 3, 5, and 7 and with unlabeled wild-type (wt) probe in 100-fold excess in lanes 2, 4, 6, and 8. Five micrograms of nuclear extract protein was loaded onto each well. Cells in lanes 3–4, 5–6, and 7–8 had been treated for 60 min with 10, 50, and 100 μg/ml P. aeruginosa LPS, respectively, before nuclear extraction. The results show a 2.99-, 2.12-, and 6.36-fold activation in NF-κB densitometry band intensity with 10, 50, and 100 μg/ml LPS, respectively. EMSA of Oct-1 binding from the same Rhek cell extracts is shown in the bottom panel to demonstrate equal loading and integrity of the extracts. The gel contains labeled Oct-1 probe as noted. C, Competition studies for specificity of EMSA bands. To further elucidate the specific nature of the EMSA bands, competition experiments were conducted with 1:1, 1:10, 1:25, and 1:50 molar ratios of labeled to unlabeled specific NF-κB oligonucleotides (lanes 2–5) and nonspecific PPAR-γ oligonucleotides (lanes 6–9). The NF-κB band disappeared with increasing specific cold competition, but did not with increasing nonspecific competition. All cells had been treated with 100 μg/ml P. aeruginosa LPS before nuclear extraction. D, EMSA supershift analysis of Rhek cell extracts with anti-p50 and anti-p65 Abs. To further characterize the nature of the NF-κ B binding activity, nuclear extracts from Rhek cells stimulated with 100 μg/ml P. aeruginosa LPS (lanes 1–4) were run side-by-side in a gel with 10 μg/ml p50/p65 heterodimer proteins as positive controls (lanes 5–8). The wild-type (wt) probe showed 100-fold competition in lanes 2 and 4 with the p50 and p65 supershift (SS) complexes. Similar results were obtained in two other independent experiments.

FIGURE 1.

Activation of NF-κB in Rhek cells with P. aeruginosa LPS. A, Luciferase reporter gene activation in Rhek cells after stimulation with P. aeruginosa LPS. The NF-κB luciferase reporter assay was performed as described in Materials and Methods, and the figure shows 2.25- and 3.1-fold activation of NF-κB activity in Rhek cells after 60-min treatment with 10 and 100 μg/ml P. aeruginosa LPS, respectively (∗, p < 0.001). B, EMSA of NF-κB binding activity. Binding in Rhek cells is depicted in lanes 1, 3, 5, and 7 and with unlabeled wild-type (wt) probe in 100-fold excess in lanes 2, 4, 6, and 8. Five micrograms of nuclear extract protein was loaded onto each well. Cells in lanes 3–4, 5–6, and 7–8 had been treated for 60 min with 10, 50, and 100 μg/ml P. aeruginosa LPS, respectively, before nuclear extraction. The results show a 2.99-, 2.12-, and 6.36-fold activation in NF-κB densitometry band intensity with 10, 50, and 100 μg/ml LPS, respectively. EMSA of Oct-1 binding from the same Rhek cell extracts is shown in the bottom panel to demonstrate equal loading and integrity of the extracts. The gel contains labeled Oct-1 probe as noted. C, Competition studies for specificity of EMSA bands. To further elucidate the specific nature of the EMSA bands, competition experiments were conducted with 1:1, 1:10, 1:25, and 1:50 molar ratios of labeled to unlabeled specific NF-κB oligonucleotides (lanes 2–5) and nonspecific PPAR-γ oligonucleotides (lanes 6–9). The NF-κB band disappeared with increasing specific cold competition, but did not with increasing nonspecific competition. All cells had been treated with 100 μg/ml P. aeruginosa LPS before nuclear extraction. D, EMSA supershift analysis of Rhek cell extracts with anti-p50 and anti-p65 Abs. To further characterize the nature of the NF-κ B binding activity, nuclear extracts from Rhek cells stimulated with 100 μg/ml P. aeruginosa LPS (lanes 1–4) were run side-by-side in a gel with 10 μg/ml p50/p65 heterodimer proteins as positive controls (lanes 5–8). The wild-type (wt) probe showed 100-fold competition in lanes 2 and 4 with the p50 and p65 supershift (SS) complexes. Similar results were obtained in two other independent experiments.

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The composition of the protein binding to NF-κB was examined by supershift analysis (Fig. 1,D). Nuclear proteins from Rhek cells hybridized with NF-κB consensus oligonucleotides were incubated for 60 min with anti-p65 and anti-p50 Abs and compared by EMSA. Fig. 1 C demonstrates a supershift with both anti-p65 and anti-p50 Abs. Control p50/p65 heterodimer proteins were run side-by-side with the experimental extract, confirming the nature of the EMSA bands.

To determine whether the functional activation of NF-κB seen in the transformed keratinocytes was replicable in normal human keratinocytes, the above experiments were confirmed in normal keratinocytes. Much like in Rhek cells, luciferase reporter assays showed a statistically significant 1.75- and 3.98-fold functional activation of NF-κB in normal keratinocytes with 10 and 100 μg/ml LPS, respectively (Fig. 2,A). Again, the EMSA binding experiments showed similar increases in nuclear NF-κB roughly corresponding to the functional activation seen with the luciferase assays (Fig. 2,B). The integrity of nuclear extracts was also demonstrated in the normal keratinocytes with EMSA for Oct-1 binding (Fig. 2 B). Previously there have been little data linking pathologically important bacterial LPS with NF-κB-associated pathologic states in squamous tissues. We have demonstrated that P. aeruginosa LPS is able to activate NF-κB in two separate keratinocyte systems.

FIGURE 2.

Activation of NF-κB in normal human keratinocytes with P. aeruginosa LPS. A, Luciferase reporter gene activation in normal human keratinocytes after stimulation with P. aeruginosa LPS. The NF-κB luciferase reporter assay was performed as described in Materials and Methods, and the figure shows 1.75- and 3.98-fold activation of NF-κB activity in normal human keratinocytes after 60-min treatment with 10 and 100 μg/ml P. aeruginosa LPS, respectively (∗, p < 0.004; ∗∗, p < 0.0005). B, EMSA of NF-κB binding activity. Binding in Rhek cells is depicted in lanes 1, 3, and 5 and with unlabeled wild-type (wt) probe in a 100-fold excess in lanes 2 and 4. Five micrograms of nuclear extract protein was loaded onto each well. Cells in lanes 3–4 and 5 had been treated for 60 min with 10 and 100 μg/ml P. aeruginosa LPS, respectively, before nuclear extraction. This figure shows 1.79- and 2.59-fold activations of NF-κB densitometry band intensity for 10 and 100 μg/ml LPS stimulation, respectively. EMSA of Oct-1 binding from the same normal human keratinocytes extracts is shown in the bottom panel. The gel contains labeled Oct-1 probe (lanes 1, 3, and 5) and wild-type probe competition in 100-fold excess (lanes 2 and 4).

FIGURE 2.

Activation of NF-κB in normal human keratinocytes with P. aeruginosa LPS. A, Luciferase reporter gene activation in normal human keratinocytes after stimulation with P. aeruginosa LPS. The NF-κB luciferase reporter assay was performed as described in Materials and Methods, and the figure shows 1.75- and 3.98-fold activation of NF-κB activity in normal human keratinocytes after 60-min treatment with 10 and 100 μg/ml P. aeruginosa LPS, respectively (∗, p < 0.004; ∗∗, p < 0.0005). B, EMSA of NF-κB binding activity. Binding in Rhek cells is depicted in lanes 1, 3, and 5 and with unlabeled wild-type (wt) probe in a 100-fold excess in lanes 2 and 4. Five micrograms of nuclear extract protein was loaded onto each well. Cells in lanes 3–4 and 5 had been treated for 60 min with 10 and 100 μg/ml P. aeruginosa LPS, respectively, before nuclear extraction. This figure shows 1.79- and 2.59-fold activations of NF-κB densitometry band intensity for 10 and 100 μg/ml LPS stimulation, respectively. EMSA of Oct-1 binding from the same normal human keratinocytes extracts is shown in the bottom panel. The gel contains labeled Oct-1 probe (lanes 1, 3, and 5) and wild-type probe competition in 100-fold excess (lanes 2 and 4).

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We hypothesized that anti-inflammatory agents may play a therapeutic role in pathologic states of squamous tissues associated with increased proliferation. Therefore, we tested indomethacin, a prototypic, clinically useful, anti-inflammatory drug, as an inhibitor of the observed LPS induction of NF-κB in keratinocytes. Fig. 3 A depicts luciferase reporter assays in Rhek cells, again demonstrating similar 1.68- and 3.06-fold functional activation of NF-κB with 50 and 100 μg/ml LPS. Experimental groups treated for 90 min with 5 and 20 μg/ml indomethacin before stimulation with 100 μg/ml LPS showed significant down-regulation of NF-κB activation. Treatment with 5 μg/ml indomethacin before maximal LPS stimulation reduced NF-κB activation to levels similar to the induction produced by the smaller LPS dose. Pretreatment with 20 μg/ml indomethacin before maximal LPS stimulation decreased the activity of NF-κB to levels below constitutive activity.

FIGURE 3.

Activation of NF-κB in Rhek cells and normal human keratinocytes with P. aeruginosa LPS; down-regulation with indomethacin. A, Luciferase reporter gene activation in Rhek cells after stimulation with P. aeruginosa LPS; down-regulation with indomethacin. The NF-κB luciferase reporter assay was performed as described in Materials and Methods, and the figure shows 1.68- and 3.06-fold activation of NF-κB activity in Rhek cells after 60-min treatment with 50 and 100 μg/ml P. aeruginosa LPS, respectively. Note that 90-min pretreatments using 5 and 20 μg/ml indomethacin before maximal stimulation with 100 μg/ml LPS prevented NF-κB activation (L10, I5; L100, I20). Finally, note the blockade of constitutive NF-κB activity with 20 μg/ml indomethacin (all p < 0.01). L, LPS; I, indomethacin. B, EMSA of NF-κB binding activity. Binding in Rhek cells is depicted in lanes 1, 3, 5, and 7 and with unlabeled wild-type (wt) probe in 100-fold excess in lanes 2, 4, 6, and 8. Five micrograms of nuclear extract protein was loaded onto each well. Cells in lanes 3–7 had been treated for 60 min with 100 μg/ml P. aeruginosa LPS before nuclear extraction. Cells in lanes 5–6 and 7–8 had been treated with 5 and 20 μg/ml indomethacin for 90 min, respectively, before LPS stimulation. Note the blockade of NF-κB binding activity with increasing doses of indomethacin. EMSA of Oct-1 binding in Rhek cells is depicted in the bottom panel. The gel contains labeled Oct-1 probe as noted. ns, nonspecific. C, Luciferase reporter gene activation in normal human keratinocytes after stimulation with P. aeruginosa LPS; down-regulation with indomethacin. The NF-κB luciferase reporter assay was performed as described in Materials and Methods, and the figure shows 1.75- and 2.95-fold activation of NF-κB activity in Rhek cells after 60-min treatment with 10 and 100 μg/ml P. aeruginosa LPS, respectively (L10, I0; L100, I0). Inhibition of NF-κB activation with 90-min pretreatments using 5 and 20 μg/ml indomethacin before maximal stimulation with 100 μg/ml LPS (L100, I5; L100, I20; all p < 0.0025). L, LPS; I, indomethacin; ns, nonspecific. D, EMSA of NF-κB binding activity. Binding in Rhek cells is depicted in lanes 1, 3, and 5 and with unlabeled wild-type (wt) probe in 100-fold excess in lanes 2, 4, and 6. Five micrograms of nuclear extract protein was loaded onto each well. Cells in lanes 3–6 had been treated for 60 min with 100 μg/ml P. aeruginosa LPS before nuclear extraction. Cells in lanes 5 and 6 had been treated with 20 μg/ml indomethacin for 90 min, respectively, before LPS stimulation. Note the inhibition of NF-κB binding activity with indomethacin. EMSA of Oct-1 binding in normal human keratinocytes is depicted in the bottom panel and demonstrates equal loading of the extracts. The gel contains labeled Oct-1 probe as noted.

FIGURE 3.

Activation of NF-κB in Rhek cells and normal human keratinocytes with P. aeruginosa LPS; down-regulation with indomethacin. A, Luciferase reporter gene activation in Rhek cells after stimulation with P. aeruginosa LPS; down-regulation with indomethacin. The NF-κB luciferase reporter assay was performed as described in Materials and Methods, and the figure shows 1.68- and 3.06-fold activation of NF-κB activity in Rhek cells after 60-min treatment with 50 and 100 μg/ml P. aeruginosa LPS, respectively. Note that 90-min pretreatments using 5 and 20 μg/ml indomethacin before maximal stimulation with 100 μg/ml LPS prevented NF-κB activation (L10, I5; L100, I20). Finally, note the blockade of constitutive NF-κB activity with 20 μg/ml indomethacin (all p < 0.01). L, LPS; I, indomethacin. B, EMSA of NF-κB binding activity. Binding in Rhek cells is depicted in lanes 1, 3, 5, and 7 and with unlabeled wild-type (wt) probe in 100-fold excess in lanes 2, 4, 6, and 8. Five micrograms of nuclear extract protein was loaded onto each well. Cells in lanes 3–7 had been treated for 60 min with 100 μg/ml P. aeruginosa LPS before nuclear extraction. Cells in lanes 5–6 and 7–8 had been treated with 5 and 20 μg/ml indomethacin for 90 min, respectively, before LPS stimulation. Note the blockade of NF-κB binding activity with increasing doses of indomethacin. EMSA of Oct-1 binding in Rhek cells is depicted in the bottom panel. The gel contains labeled Oct-1 probe as noted. ns, nonspecific. C, Luciferase reporter gene activation in normal human keratinocytes after stimulation with P. aeruginosa LPS; down-regulation with indomethacin. The NF-κB luciferase reporter assay was performed as described in Materials and Methods, and the figure shows 1.75- and 2.95-fold activation of NF-κB activity in Rhek cells after 60-min treatment with 10 and 100 μg/ml P. aeruginosa LPS, respectively (L10, I0; L100, I0). Inhibition of NF-κB activation with 90-min pretreatments using 5 and 20 μg/ml indomethacin before maximal stimulation with 100 μg/ml LPS (L100, I5; L100, I20; all p < 0.0025). L, LPS; I, indomethacin; ns, nonspecific. D, EMSA of NF-κB binding activity. Binding in Rhek cells is depicted in lanes 1, 3, and 5 and with unlabeled wild-type (wt) probe in 100-fold excess in lanes 2, 4, and 6. Five micrograms of nuclear extract protein was loaded onto each well. Cells in lanes 3–6 had been treated for 60 min with 100 μg/ml P. aeruginosa LPS before nuclear extraction. Cells in lanes 5 and 6 had been treated with 20 μg/ml indomethacin for 90 min, respectively, before LPS stimulation. Note the inhibition of NF-κB binding activity with indomethacin. EMSA of Oct-1 binding in normal human keratinocytes is depicted in the bottom panel and demonstrates equal loading of the extracts. The gel contains labeled Oct-1 probe as noted.

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EMSA binding assays confirmed the luciferase findings (Fig. 3 B). Again, increases in nuclear NF-κB DNA binding were seen with LPS stimulation, roughly corresponding to the functional activation demonstrated with the luciferase assays. Pretreatment with indomethacin reduced this nuclear NF-κB DNA binding to below constitutive levels using the maximum indomethacin dose. Oct-1 DNA binding experiments confirmed the integrity and equal loading of the nuclear extracts.

Similar experiments were performed in normal human epidermal keratinocytes. As in the Rhek cells, indomethacin was able to reduce the significant induction of NF-κB after LPS stimulation. Again, pretreatment with 20 μg/ml indomethacin was able to reduce NF-κB activity to below constitutive levels (Fig. 3,C). EMSA binding assays confirmed the luciferase assay findings in the normal keratinocytes by demonstrating an obvious decrease in nuclear NF-κB binding with indomethacin pretreatment (Fig. 3 D). These results indicate that indomethacin has the capacity to block both induced and constitutive NF-κB activities in human keratinocyte cell lines.

Under normal conditions NF-κB is present in the cytoplasm bound to IκB-α, rendering it inactive. Upon extracellular stimulation, IκB-α is phosphorylated, leading to its dissociation from NF-κB subunits (41, 42). IκB-α is then degraded in a proteosome-dependent mechanism (43), and NF-κB becomes activated, translocating into the nucleus. A Western blot was performed to determine whether the observed indomethacin inhibition of NF-κB occurs through prevention of IκB-α degradation. The presence of IκB-α was determined after maximal stimulation (100 μg/ml) with LPS and after pretreatment with 20 μg/ml indomethacin.

IκB-α disappeared in Rhek cells after 3-h stimulation with LPS. Ninety-minute treatment with 20 μg/ml indomethacin before LPS stimulation was able to prevent the disappearance of IκB-α (Fig. 4). These results suggest that indomethacin prevents the release of p50 and p65 from IκB-α, thus blocking the stimulatory effect of LPS.

FIGURE 4.

Western blot: IκB-α ubiquitination with P. aeruginosa LPS, prevention with indomethacin in Rhek cells. A, Western blot depicting the ubiquitination of IκB-α in Rhek cells after 3 h of P. aeruginosa LPS treatment. Rhek cells were incubated in T-75 flasks to confluence. They were then treated for 90 min with 0 or 20 μg/ml indomethacin. P. aeruginosa LPS (100 μg/ml) was added to each flask for 3 h. Control cells were not treated. Whole-cell extracts were performed, and Western blotting was conducted as described in Materials and Methods. Note the expected disappearance of IκB-α protein with P. aeruginosa LPS treatment, indicating that IκB-α is ubiquinated. Also note the prevention of IκB-α protein ubiquitination with indomethacin pretreatment.

FIGURE 4.

Western blot: IκB-α ubiquitination with P. aeruginosa LPS, prevention with indomethacin in Rhek cells. A, Western blot depicting the ubiquitination of IκB-α in Rhek cells after 3 h of P. aeruginosa LPS treatment. Rhek cells were incubated in T-75 flasks to confluence. They were then treated for 90 min with 0 or 20 μg/ml indomethacin. P. aeruginosa LPS (100 μg/ml) was added to each flask for 3 h. Control cells were not treated. Whole-cell extracts were performed, and Western blotting was conducted as described in Materials and Methods. Note the expected disappearance of IκB-α protein with P. aeruginosa LPS treatment, indicating that IκB-α is ubiquinated. Also note the prevention of IκB-α protein ubiquitination with indomethacin pretreatment.

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Experiments were performed to determine whether the functional activation of NF-κB seen in the keratinocyte cell lines correlated with an increased proliferative response. First, MTT assays were performed on Rhek cells. Fig. 5,A demonstrates a significant increase in cell proliferation rates 3 days after stimulation with 50 and 100 μg/ml P. aeruginosa LPS. A similar significant increase in proliferation after 3 days of stimulation with LPS was seen in normal keratinocytes (Fig. 5,B). To determine whether indomethacin is able to block this increase in cell proliferation, the experiments were repeated by pretreating Rhek cells and normal keratinocytes with 5 and 20 μg/ml indomethacin before maximal LPS stimulation (100 μg/ml). Much as indomethacin is able to inhibit the activation of inducible and constitutive NF-κB, it causes a significant decrease in keratinocyte proliferation (Fig. 5, C and D). Other experiments, using p65-overexpressing Rhek cell clones, showed increased proliferation rates similar to those seen with LPS stimulation, as shown in the above experiments (data not shown). This finding coupled with the finding that an NF-κB inhibitor, such as indomethacin, is able to block the LPS-induced increase in keratinocyte proliferation leads us to suspect that the mechanism by which LPS causes the increased keratinocyte cellular proliferative response is by NF-κB activation.

FIGURE 5.

Stimulation of Rhek and normal human keratinocyte proliferation with P. aeruginosa LPS. Inhibition with indomethacin pretreatment. A and B, Proliferation of Rhek cells after P. aeruginosa LPS stimulation. Cells were plated in 96-well plates at a density of 5,000 cells/well and were incubated overnight as described in Materials and Methods. The following day, they were treated with P. aeruginosa LPS at the indicated doses. After 3 days, MTT assays were performed. There was a significant increase in Rhek cell (A) and normal human keratinocyte (B) proliferation over controls with increasing doses of P. aeruginosa LPS (∗, all p < 0.02). C and D, The same MTT proliferation assay experiments were repeated as described above. In addition, some wells were treated with indomethacin at the indicated amounts in micrograms per milliliter. Note the significant decrease in stimulated proliferation rates with indomethacin in both Rhek cells (C) and normal human keratinocytes (D; ∗, all p < 0.003). L, P. aeruginosa LPS; I, indomethacin. E, Cell count experiment. Rhek cells were plated in T-75 flasks at a density of 35,000 cells/flask. They were then incubated for 3 days in 5% CO2. Cell counts were performed from each flask. The absolute cell number in each flask was determined (cells per flask).

FIGURE 5.

Stimulation of Rhek and normal human keratinocyte proliferation with P. aeruginosa LPS. Inhibition with indomethacin pretreatment. A and B, Proliferation of Rhek cells after P. aeruginosa LPS stimulation. Cells were plated in 96-well plates at a density of 5,000 cells/well and were incubated overnight as described in Materials and Methods. The following day, they were treated with P. aeruginosa LPS at the indicated doses. After 3 days, MTT assays were performed. There was a significant increase in Rhek cell (A) and normal human keratinocyte (B) proliferation over controls with increasing doses of P. aeruginosa LPS (∗, all p < 0.02). C and D, The same MTT proliferation assay experiments were repeated as described above. In addition, some wells were treated with indomethacin at the indicated amounts in micrograms per milliliter. Note the significant decrease in stimulated proliferation rates with indomethacin in both Rhek cells (C) and normal human keratinocytes (D; ∗, all p < 0.003). L, P. aeruginosa LPS; I, indomethacin. E, Cell count experiment. Rhek cells were plated in T-75 flasks at a density of 35,000 cells/flask. They were then incubated for 3 days in 5% CO2. Cell counts were performed from each flask. The absolute cell number in each flask was determined (cells per flask).

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Finally, the MTT assay results were confirmed by cell count experiments (Fig. 5 E). Rhek cells were plated in T-25 flasks at an initial concentration of 35,000 cells/flask, then stimulated with increasing doses of LPS with or without indomethacin pretreatment in serum-free medium. The cells were allowed to grow for 3 days in culture. Again, there was a marked increase in the flasks in which the cells had been treated with LPS. Indomethacin was able to block this increase.

Because the actual mechanism for inhibition of NF-κB by indomethacin is not known, more direct inhibition of NF-κB with a dominant negative IκB construct was performed. Rhek cells (Fig. 6,A), Rhek cell clones stably transfected with pCMX empty vector (Rhek-pCMX; Fig. 6B), and Rhek cell clones stably transfected with pCMX-IκB-α/M plasmids (Rhek-pCMX-IκB-α/M) were stimulated with 0, 5, 10, and 25 μg/ml LPS for 60 min before nuclear extraction. EMSA experiments were then performed as described above. These gel results are depicted in the top panels of Fig. 6. Note how Rhek (Fig. 6,A, top panel) and Rhek-CMX (Fig. 6,B, top panel) cells demonstrated activation of nuclear NF-κB with increasing doses of LPS as expected. Rhek-pCMX-IκB-α/M (Fig. 6 C, top panel) cells, in contrast, showed little constitutive or stimulated NF-κB activity.

FIGURE 6.

Activation of NF-κB and cellular proliferation in Rhek cells and Rhek-CMX cells with P. aeruginosa LPS. Prevention of NF-κB activation and cellular proliferation in Rhek-pCMX-IκB-α/M dominant negative clones with P. aeruginosa LPS stimulation. A, Activation of NF-κB and cellular proliferation in Rhek cells with P. aeruginosa LPS. The top panel depicts an EMSA experiment after stimulation with 0, 5, 10, and 25 μg/ml (lanes 1–4) P. aeruginosa LPS. Note the increasing NF-κB signal with increasing dose of LPS, as expected. The bottom panel depicts an MTT experiment with the same doses of LPS stimulation for 3 days. The experimental conditions were exactly as in the above MTT assay results. There was a statistically significant increase in cell proliferation with increasing doses of LPS (∗, all p < 0.01). B, Activation of NF-κB and cellular proliferation in Rhek-MT2T cell clones with P. aeruginosa LPS. The experiments shown in A were repeated under the same conditions in the empty vector Rhek cell clones. The top panel depicts similar increases in nuclear NF-κB activity with increasing doses of LPS (lanes 1–4). Also, the MTT assay results shown in the bottom panel demonstrate statistically significant increases in cell proliferation with increasing doses of LPS (∗, all p < 0.01). C, Prevention of NF-κB activation and cellular proliferation in Rhek-pCMX-IκB-α/M dominant negative clones with P. aeruginosa LPS stimulation. Again, the same experiments as those shown in A and B were performed in the IκB dominant negative cell clones. The top panel demonstrates minimal constitutive nuclear NF-κB activity (lane 1). Stimulation with 5, 10, and 25 μg/ml LPS was not able to activate NF-κB as it did in the nontransfected and empty vector clones. Similarly, the bottom panel demonstrates that the dominant negative clones did not exhibit increases in cell proliferation with increasing doses of LPS.

FIGURE 6.

Activation of NF-κB and cellular proliferation in Rhek cells and Rhek-CMX cells with P. aeruginosa LPS. Prevention of NF-κB activation and cellular proliferation in Rhek-pCMX-IκB-α/M dominant negative clones with P. aeruginosa LPS stimulation. A, Activation of NF-κB and cellular proliferation in Rhek cells with P. aeruginosa LPS. The top panel depicts an EMSA experiment after stimulation with 0, 5, 10, and 25 μg/ml (lanes 1–4) P. aeruginosa LPS. Note the increasing NF-κB signal with increasing dose of LPS, as expected. The bottom panel depicts an MTT experiment with the same doses of LPS stimulation for 3 days. The experimental conditions were exactly as in the above MTT assay results. There was a statistically significant increase in cell proliferation with increasing doses of LPS (∗, all p < 0.01). B, Activation of NF-κB and cellular proliferation in Rhek-MT2T cell clones with P. aeruginosa LPS. The experiments shown in A were repeated under the same conditions in the empty vector Rhek cell clones. The top panel depicts similar increases in nuclear NF-κB activity with increasing doses of LPS (lanes 1–4). Also, the MTT assay results shown in the bottom panel demonstrate statistically significant increases in cell proliferation with increasing doses of LPS (∗, all p < 0.01). C, Prevention of NF-κB activation and cellular proliferation in Rhek-pCMX-IκB-α/M dominant negative clones with P. aeruginosa LPS stimulation. Again, the same experiments as those shown in A and B were performed in the IκB dominant negative cell clones. The top panel demonstrates minimal constitutive nuclear NF-κB activity (lane 1). Stimulation with 5, 10, and 25 μg/ml LPS was not able to activate NF-κB as it did in the nontransfected and empty vector clones. Similarly, the bottom panel demonstrates that the dominant negative clones did not exhibit increases in cell proliferation with increasing doses of LPS.

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MTT assays for cellular proliferation after stimulation with 0, 5, 10, and 25 μg/ml P. aeruginosa LPS for 72 h were also performed in these cells as described above. These proliferation results are depicted in the bottom panels of Fig. 6. Rhek (Fig. 6,A, bottom panel) and Rhek-CMX (Fig. 6,B, bottom panel) showed statistically increased proliferation as measured by MTT assay compared with the dominant negative Rhek-pCMX-IκB-α/M (Fig. 6 C, bottom panel) clones, which did not.

To confirm the results found with the MTT assays, thymidine incorporation assays for cellular proliferation were performed on Rhek cells and pCMX-IκB-α/M dominant negative clones. Fig. 7,A demonstrates a statistically significant increase in irradiated thymidine incorporation after simulation with P. aeruginosa LPS in Rhek cells. In the dominant negative cell clones, however (Fig. 7 B), no such increase in irradiated thymidine incorporation was noted. These results demonstrate that with direct, specific NF-κB inhibition, the increases in cellular proliferation due to LPS stimulation do not occur.

FIGURE 7.

Irradiated thymidine incorporation assays. Activation of cellular proliferation with P. aeruginosa LPS in Rhek cells; prevention with NK-κB dominant negative inhibition. A, Increased irradiated thymidine incorporation as a measure of cellular proliferation in Rhek cells after LPS stimulation. Ten thousand cells per well were seeded in a 96-well plate in appropriate serum-free medium for 24 h. The next day, cells were treated with four serial dilutions of 100 μg/ml P. aeruginosa LPS. After 8-h incorporation of 1 μCi of [3H]thymidine, followed by treatment with 5% TCA, then DNA extraction with NaOH/0.5% SDS, radiation counts were measured using a 96-well scintillation counter. There were significant increases in cellular proliferation at all doses of LPS stimulation (all p < 0.01). B, Prevention of increased irradiated thymidine incorporation with NF-κB dominant negative inhibition. The same experiments as those in A were performed on Rhek-pCMX-IκB-α/M dominant negative clones. In contrast to the increased proliferation seen in the Rhek cells, no significant increases were noted in the stably transfected NF-κB dominant negative cell clones.

FIGURE 7.

Irradiated thymidine incorporation assays. Activation of cellular proliferation with P. aeruginosa LPS in Rhek cells; prevention with NK-κB dominant negative inhibition. A, Increased irradiated thymidine incorporation as a measure of cellular proliferation in Rhek cells after LPS stimulation. Ten thousand cells per well were seeded in a 96-well plate in appropriate serum-free medium for 24 h. The next day, cells were treated with four serial dilutions of 100 μg/ml P. aeruginosa LPS. After 8-h incorporation of 1 μCi of [3H]thymidine, followed by treatment with 5% TCA, then DNA extraction with NaOH/0.5% SDS, radiation counts were measured using a 96-well scintillation counter. There were significant increases in cellular proliferation at all doses of LPS stimulation (all p < 0.01). B, Prevention of increased irradiated thymidine incorporation with NF-κB dominant negative inhibition. The same experiments as those in A were performed on Rhek-pCMX-IκB-α/M dominant negative clones. In contrast to the increased proliferation seen in the Rhek cells, no significant increases were noted in the stably transfected NF-κB dominant negative cell clones.

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We hypothesized that the LPS-induced increase in cell proliferation was mediated through an NF-κB-dependent pathway. NF-κB is known to have binding sequences in the cyclin D1 promoter and is a mediator of cyclin D1 effects in mesodermally derived transformed cell lines (17, 44). To investigate this hypothesis, we transiently transfected Rhek cells with a cyclin D1 promoter luciferase plasmid and stimulated the transfected cells with 100 μg/ml P. aeruginosa LPS for 60 min. Fig. 8,A shows a significant increase in cyclin D1 promoter activity in Rhek cells after LPS stimulation. LPS is unable to significantly cause this activation of the cyclin D1 promoter in the presence of indomethacin. Because we have shown that indomethacin blocks the LPS-induced activation of NF-κB, it is likely that this increase in cyclin D1 promoter activity is a reflection of NF-κB activation and binding. To further confirm this suspicion, we repeated the experiment, this time transiently transfecting Rhek cells with a cyclin D1 promoter luciferase plasmid with a mutation at the NF-κB binding site (−66CD1Luc). Fig. 8 B shows that in these cells, LPS was unable to induce activation of the cyclin D1 promoter. Thus, when NF-κB is prevented from binding the cyclin D1 promoter, P. aeruginosa LPS is unable to activate the cyclin D1 promoter. This confirms our suspicion that LPS is activating cyclin D1 in Rhek cells via its up-regulation of NF-κB functional activity. This finding probably explains the increase in cellular proliferation that we found in keratinocytes after P. aeruginosa stimulation.

FIGURE 8.

NF-κB mediated activation of cyclin D1 in Rhek cells with P. aeruginosa LPS stimulation in Rhek cells. A, Cyclin D1 luciferase reporter gene activation with P. aeruginosa LPS stimulation in Rhek cells. Rhek cells were transiently transfected with the −1745CD1 cyclin D1-luciferase reporter plasmid constructs. Luciferase reporter assays were then performed as described in Materials and Methods. The figure shows a significant 2-fold increase in functional cyclin D1 promoter activation after overnight stimulation with P. aeruginosa LPS (p < 0.04). No difference in cyclin D1 activity was found in the same transfected cells when indomethacin treatment was performed for 90 min before LPS stimulation (∗, all p < 0.02). L, LPS; I, indomethacin. B, Cyclin D1 luciferase reporter gene activation with P. aeruginosa LPS stimulation in Rhek cells. Rhek cells were transiently transfected with −66CD1 cyclin D1-luciferase reporter plasmids containing site-specific mutations at the NF-κB binding site in the cyclin D1 promoter. Luciferase reporter assays were performed as described in Materials and Methods. Note the absence of activation of cyclin D1 after overnight stimulation with P. aeruginosa LPS (∗, all p < 0.01). C, Western blot: cyclin D1 activation with P. aeruginosa LPS in Rhek cells; inhibition with indomethacin. Rhek cells were incubated in T-75 flasks to confluence. They were then treated for 90 min with 0 or 20 μg/ml indomethacin. P. aeruginosa LPS (100 μg/ml) was added to each flask overnight. Whole-cell extracts were performed, and Western blotting was conducted as described in Materials and Methods. Note the appearance of cyclin D1 protein in cells treated with P. aeruginosa LPS in lane 2 and its absence when cells were pretreated with indomethacin in lane 3.

FIGURE 8.

NF-κB mediated activation of cyclin D1 in Rhek cells with P. aeruginosa LPS stimulation in Rhek cells. A, Cyclin D1 luciferase reporter gene activation with P. aeruginosa LPS stimulation in Rhek cells. Rhek cells were transiently transfected with the −1745CD1 cyclin D1-luciferase reporter plasmid constructs. Luciferase reporter assays were then performed as described in Materials and Methods. The figure shows a significant 2-fold increase in functional cyclin D1 promoter activation after overnight stimulation with P. aeruginosa LPS (p < 0.04). No difference in cyclin D1 activity was found in the same transfected cells when indomethacin treatment was performed for 90 min before LPS stimulation (∗, all p < 0.02). L, LPS; I, indomethacin. B, Cyclin D1 luciferase reporter gene activation with P. aeruginosa LPS stimulation in Rhek cells. Rhek cells were transiently transfected with −66CD1 cyclin D1-luciferase reporter plasmids containing site-specific mutations at the NF-κB binding site in the cyclin D1 promoter. Luciferase reporter assays were performed as described in Materials and Methods. Note the absence of activation of cyclin D1 after overnight stimulation with P. aeruginosa LPS (∗, all p < 0.01). C, Western blot: cyclin D1 activation with P. aeruginosa LPS in Rhek cells; inhibition with indomethacin. Rhek cells were incubated in T-75 flasks to confluence. They were then treated for 90 min with 0 or 20 μg/ml indomethacin. P. aeruginosa LPS (100 μg/ml) was added to each flask overnight. Whole-cell extracts were performed, and Western blotting was conducted as described in Materials and Methods. Note the appearance of cyclin D1 protein in cells treated with P. aeruginosa LPS in lane 2 and its absence when cells were pretreated with indomethacin in lane 3.

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Finally, we performed a Western blot, which confirmed the up-regulation of cyclin D1 in Rhek cells 24 h after treatment with LPS (Fig. 8 C). In the presence of indomethacin (and NF-κB inhibition), cyclin D1 was not up-regulated.

It has been established that bacterial products have the capacity to stimulate NF-κB in inflammatory cells and that these activations are important in host humoral immunity. There are few data linking pathologically important bacterial products with injury response and abnormal proliferation in squamous tissues. In this study we demonstrate that NF-κB is activated in both normal and transformed keratinocytes after exposure to a pathologically important bacterial product, specifically P. aeruginosa LPS. This activation correlates with a significant increase in NF-κB-dependent cell proliferation, because inhibition of NF-κB reverses the proliferative response. LPS activates the cyclin D1 promoter in keratinocytes via the induction of NF-κB binding to the cyclin D1 promoter, a mechanism probably responsible for the observed proliferative response. Finally, a prototypic anti-inflammatory drug, indomethacin, has the capacity to inhibit both the proliferative process as well as the molecular events (NF-κB activation of cyclin D1 promoter) underlying it. NF-κB-mediated activation of cyclin D1 has not been described in epithelially derived cells.

Bacterial toxins have been implicated as mediators of nonepithelial cell proliferation in other systems (45, 46). It is not well established that bacterial products can induce a proliferative phenotype in squamous epithelia. In fact, outside of the field of H. pylori-induced proliferation of gastric mucosa, findings of bacterial products directly contributing to pathologic proliferative states are rare. Our findings have implications for the pathogenesis of a multitude of inflammatory associated squamous disease processes. The activation of NF-κB with bacterial products (such as P. aeruginosa LPS) may trigger a proinflammatory and hyperproliferative cellular phenotype that, when deranged, can lead to neoplasia (such as cholesteatoma) or even contribute downstream gene products to the chronic inflammatory milieus that leads to malignancy (47). H. pylori infection induces cell proliferation (48) and overexpression of IL-8 and cyclooxygenase-2 in gastric epithelial cells (49), both events that may play a causative role in gastric carcinoma. Similarly, NF-κB-mediated proinflammatory dysregulation has been associated with both the development of pancreatic cancer (50) and the progression to metastases (51).

The production of cytokines, angiogenic factors, proliferation markers, cell cycle activators, and apoptosis resistance are all NF-κB-mediated cellular events that can contribute to abnormal cellular proliferation and even the malignant phenotype (52, 53). Constitutive NF-κB activation has been shown to be associated with squamous proliferation and tumor growth (27) and resistance to apoptosis (53). Drugs that inhibit NF-κB may serve as potential therapeutic agents in these abnormal proliferative processes (52, 53, 54, 55). Antioxidants (56), phytochemicals (57), and anti-inflammatory mediators (58) may exert some of their antiproliferative functions through suppression of NF-κB. Our finding of NF-κB inhibition with indomethacin suggests that it may also have a potential therapeutic role in abnormal proliferative states of squamous tissues, including cancer progression. In this matter, indomethacin has been touted as a potential novel chemotherapeutic agent for oral cancer by other investigators (58).

The mechanisms by which bacterial LPS is able to mediate intracellular proinflammatory responses, such as NF-κB nuclear translocation, have been partially delineated. Genetic studies and gene transfer experiments indicate that LPS intracellular effects are mediated through TLR signaling (59, 60). TLRs are a family of transmembrane proteins with a leucine-rich extracellular domain and an IL-1R-like cytoplasmic homologue domain (61). They appear to be the main intracellular signaling molecules of CD14, an extracellular glycosylphophatidylinositol membrane-anchored protein known to bind to LPS (60, 61). Together, TLR and CD14 form the LPS-receptor complex. The most important TLR family member involved in Gram-negative LPS-receptor complexes appears to be TLR4 (62). Another TLR family member, TLR2, has been reported to signal Gram-positive bacterial lipoprotein-mediated intracellular events (63). Although how TLRs mediate innate immune responses in leukocytes has been studied extensively (64), investigations dealing with their presence and function in keratinocytes are few. Recently, it has been shown that human cornea keratinocytes express functionally active TLR4 (65). Additional studies have shown that cultured skin keratinocytes also express active TLR4 that, when bound to LPS, induces NF-κB nuclear translocation and proinflammatory cytokine secretion (62). Immunohistochemical profiling studies have shown cell membrane TLR4 expression in the midepidermal layers of normal human skin (66). To this end, we performed initial studies in which Rhek cells were grown to 80% confluence directly on slides and subsequently fixed with 4% paraformaldehyde. Immunohistochemical analyses failed to demonstrate the presence of TLR4 in these cells (data not shown). It is possible that immunohistochemistry may not be sensitive enough to detect TLR4 in cultured cells. Future studies using flow cytometry techniques should more sensitively determine whether TLR4 are involved in NFκB signaling processes in cultured keratinocyte systems.

Cholesteatomagenesis is a prototypic state of disorganized squamous proliferation and invasion, although cholesteatomas do not metastasize nor are they genetically unstable. Cholesteatomas almost universally arise in the setting of inflammation and infection, and their formation is related to internal molecular dysregulation and external stimuli from Pseudomonas and other bacterial toxins, proinflammatory cytokines, and growth factors. Inflammatory granulation tissue always appears with invading epithelium in active human cholesteatomas and experimental animal cholesteatomas (25). Many of the proinflammatory mediators and cytokines seen in otitis media and cholesteatoma (24) are under transcriptional control of NF-κB. TNF-α, IL-1, and IL-6 are among these proinflammatory molecules. The most common pathogen in cholesteatoma is P. aeruginosa (23). Our studies demonstrate that P. aeruginosa LPS induces NF-κB activation in human keratinocytes, causing a significant increase in proliferation, a phenomenon likely to contribute to cholesteatoma pathogenesis and behavior.

In conclusion, we have shown that P. aeruginosa LPS up-regulates NF-κB and cellular proliferation in human keratinocytes and RHEK cells. The increase in proliferation is probably mediated by cyclin D1 in an NF-κB-dependent fashion. Indomethacin, a prototypic, clinically useful, anti-inflammatory drug abrogates these events. Clearly, future studies will focus on the degree to which the squamous phenotype is altered by these bacterial toxin-mediated events on the continuum of normal to malignant keratinocyte growth. It is unlikely that LPS alone would result in the conversion of squamous cells to fully malignant cells. However, other products generated in the milieu, in addition to underlying cellular genetic abnormalities, might easily constitute a permissive environment for very disordered growth of cells in a variety of pathologic states, including the development of malignancy. The development of anti-inflammatory strategies in such processes is of obvious benefit.

The authors have no financial conflict of interest.

We acknowledge Drs. Simon Wright and Richard Pestell for technical advice and support.

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

1

This work was supported by the Lion’s 5M Research Foundation.

1
Parsonnet, J..
1995
. Bacterial infection as a cause of cancer.
Environ. Health Perspect.
103
:
263
.
2
Guarner, J., A. Moher, J. Parsonet, D. C. Halperin.
1993
. The association of Helicobacter pylori with gastric cancer and other pre-neoplastic cancer lesions in Chiapas, Mexico.
Cancer
71
:
297
.
3
Parsonnet, J., G. D. Friedman, D. P. Vandersteen, Y. Chang, J. H. Vogelman, N. Orentreich, R. K. Sibley.
1991
. Helicobacter pylori infection and the risk of gastric carcinoma.
N. Engl. J. Med.
325
:
1127
.
4
Cahill, R. J., S. Sant, H. Hamilton.
1993
. Helicobacter pylori and increased cell proliferation: a risk factor for cancer.
Gastroenterology
104
:
1032a
. (Abstr.).
5
Komine, M., L. Rao, T. Kaneko, M. Tomic-Canic, K. Tamaki, I. M. Freedberg, M. Blumenberg.
2000
. Inflammatory versus proliferative processes in epidermis.
J. Biol. Chem.
275
:
32077
.
6
Sen, R., D. Baltimore.
1986
. Multiple nuclear factors interact with the immunoglobulin enhancer sequence.
Cell
46
:
705
.
7
Muller, J. M., H. W. Ziegler-Heitbrock, P. A. Bauerle.
1993
. Nuclear factor κB, a mediator of lipopolysaccharide effects.
Immunobiology
187
:
233
.
8
Yao, J., N. Mackman, T. S. Edgington, S. T. Fan.
1997
. LPS induction of tumor necrosis factor promoter in human monocytic cells: regulation by Erg-1, c-jun and NF-κB.
J. Biol. Chem.
272
:
17795
.
9
Cavallion, J. M., M. Haeffner-Cavallion.
1990
. Signals involved in interleukin-1 synthesis and release by lipopolysaccharide-stimulated monocytes/macrophages.
Cytokine
2
:
313
.
10
Liberman, T. A., D. Baltimore.
1990
. Activation of interleukin-6 gene expression through the NF-κB transcription factor.
Mol. Cell. Biol.
10
:
561
.
11
Schreck, R., P. A. Bauerle.
1990
. NF-κB as inducible transcriptional activator of the granulocyte colony-stimulating factor.
Mol. Cell. Biol.
10
:
1281
.
12
Chodakewitz, J. A., J. Lacy, S. E. Edwards, N. Birchall, D. L. Coleman.
1990
. Macrophage colony stimulating factor production by murine and human keratinocytes.
J. Immunol.
147
:
520
.
13
Seitz, C. S., R. A. Freiberg, K. Hinata, P. A. Khavari.
2000
. NF-κB determines localization and features of cell death in epidermis.
J. Clin. Invest.
105
:
253
.
14
Ma, S., L. Rao, I. M. Freedburg, M. Blumenberg.
1997
. Transcriptional control of K5, K6, K14, and K17 keratin genes by AP-1 and NFK-B family members.
Gene Expression
6
:
361
.
15
Dajee, M., M. Lazarov, J. Y. Zhang, T. Cai, C. L. Green, A. J. Russell, M. P. Marinkovich, S. Tao, Q. Lin, Y. Kubo, et al
2003
. NF-κB blockade and oncogenic Ras trigger invasive human epidermal neoplasia.
Nature
421
:
639
.
16
Seitz, C. S., Q. Lin, H. Deng, P. A. Khavari.
1998
. Alterations in NF-κB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-κB.
Proc. Natl. Acad. Sci. USA
95
:
23072
.
17
Guttridge, D. C., C. Albanese, J. Y. Reuther, R. G. Pestell.
1999
. NF-κB controls cell growth and differentiation through transcriptional regulation of cyclin D1.
Mol. Cell Biol.
19
:
5785
.
18
Hinz, M., D. Krappman, A. Eichten.
1999
. NF-κB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition.
Mol. Cell. Biol.
19
:
2690
.
19
Sherr, C. J..
1996
. Cancer cell cycles.
Science
274
:
1672
.
20
Hall, M., G. Peters.
1996
. Genetic alterations of cyclins, cyclin-dependent kinases and CDKs inhibitors in human cancers.
Adv. Cancer Res.
68
:
67
.
21
Tanaka, Y., H. Kojima, H. Miyazaki, T. Koga, H. Moriyama.
1999
. Roles of cytokines and cell cycle regulating substances in proliferation of cholesteatoma epithelium.
Laryngoscope
109
:
1102
.
22
Robles, A. I., F. Larcher, R. Whalin, R. Murillas, E. Richie, I. B. Gimenez-Conti, J. L. Jorcano, C. J. Conti.
1996
. Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperplasia and severe thymic hyperplasia.
Proc. Natl. Acad. Sci. USA
93
:
7634
.
23
Brook, I..
1981
. Aerobic and anaerobic bacteriology of cholesteatoma.
Laryngoscope
91
:
250
.
24
Ondrey, F. G., S. K. Juhn, G. L. Adams.
1998
. Early-response cytokine expression in adult middle ear effusions.
Otolaryngol. Head Neck Surg.
119
:
342
.
25
Albino, A. P., C. P. Kimmelman, S. C. Parisher.
1998
. Cholesteatomas: a molecular and cellular puzzle.
Am. J. Otol.
19
:
7
.
26
Ondrey, F. G., G. Dong, J. Sunwoo, Z. Chen, J. S. Wolf, C. V. Crowl-Bancroft, N. Mukaida, C. Van Waes.
1999
. Constitutive activation of transcription factors NF-κB, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines.
Mol. Carcinog.
26
:
119
.
27
Duffey, D. C., Z. Chen, G. Dong, F. G. Ondrey, J. S. Wolf, K. Brown, U. Siebenlist, C. Van Waes.
1999
. Expression of a dominant-negative mutant inhibitor-κBα of NF-κB in human head and neck squamous cell carcinoma inhibits survival, pro-inflammatory cytokine expression, and tumor growth in vivo.
Cancer Res.
59
:
3468
.
28
DiMango, E., A. J. Ratner, R. Bryan, S. Tabibi, A. Prince.
1998
. Activation of NF-κB by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells.
J. Clin. Invest.
101
:
2598
.
29
Schroeder, T. H., M. M. Lee, P. W. Yacono, C. L. Cannon, A. A. Gerceker, D. E. Golan, G. B. Pier.
2002
. CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-κB translocation.
Proc. Natl. Acad. Sci. USA
99
:
6907
.
30
Rhim, J. S., G. Jay, P. Arnstein, F. M. Price, K. K. Sanford, S. A. Aaronson.
1985
. Neoplastic transformation of human epidermal keratinocytes by Ad12-SV40 and Kirsten sarcoma viruses.
Science
227
:
1250
.
31
Zheng, X., B. Christensson, B. Drettner.
1993
. Studies on etiological factors of nasopharyngeal carcinoma.
Acta Otolaryngol.
113
:
455
.
32
Babich, H., H. L. Zucerbraun, B. J. Wurzburger, Y. L. Rubin, E. Borenfreund, L. Blau.
1996
. Benzoyl peroxide cytotoxicity evaluated in vitro with the human keratinocyte cell line, RHEK-1.
Toxicology
106
:
187
.
33
Chakraborty, A., J. Pawleek.
1993
. MSH receptors in immortalized human epidermal keratinocytes: a potential mechanism for coordinate regulation of the epidermal-melanin unit.
J. Cell Physiol.
157
:
344
.
34
Van Antwerp, D J., S. J. Martin, T. Kafri, D. R. Green, I. M. Verrma.
1996
. Suppression of TNF-α-induced apoptosis by NF-κB.
Science
274
:
7887
.
35
Lin, R., P. Beauparlant, C. Makris, S. Meloche, J. Hiscott.
1996
. Phosphorylation of IκBα in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability.
Mol. Cell. Biol.
16
:
1401
.
36
Gilmore, T. D., M. Koedood, K. A. Piffat, D. W. White.
1996
. Rel/NF-κBIκ proteins and cancer.
Oncogene
13
:
1367
.
37
Dignam, J. D., R. M. Lebovitz, R. G. Roeder.
1983
. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11
:
1475
.
38
Lee, K. A., A. Bindereif, M. R. Green.
1988
. A small scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA slicing.
Gene Anal. Tech.
5
:
22
.
39
Albanese, C., J. Johnson, G. Watanabe, N. Eklund, D. Vu, A. Arnold, R. G. Pestell.
1995
. Transforming p21 mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions.
J. Biol. Chem.
270
:
23589
.
40
Schreck, R., P. Rieber, P. A. Bauerle.
1991
. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-κB transcription factor and HIV-1.
EMBO J.
10
:
2247
.
41
Beg, A., T. S. Finco, P. V. Nantermet, A. Baldwin.
1993
. Tumor necrosis factor and Interleukin-1 lead to phosphorylation and loss of I-κ-Bα: a mechanism for NF-κB activation.
Mol. Cell. Biol.
13
:
3301
.
42
Roff, M., J. Thompson, M. S. Rodriguez.
1996
. Role of IκBα ubiquitination in signal-induced activation of NF-κB in vivo.
J. Biol. Chem.
271
:
7844
.
43
Joyce, D., B. Bouzahzah, M. Fu, C. Albanese, M. D’Amico, J. Steer, J. U. Klein, R. J. Lee, J. E. Segall, J. K. Westwick, et al
1999
. Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-κB-dependent pathway.
J. Biol. Chem.
274
:
25245
.
44
Goodier, M., M. Londei.
2000
. Lipopolysaccharide stimulates the proliferation of human CD56+CD3 NK cells: a regulatory role of monocytes and IL-10.
J. Immunol.
165
:
139
.
45
Olaya, J., V. Neopikhanov, A. Uribe.
1999
. Lipopolysaccharide of Escherichia coli, polyamines, and acetic acid stimulate cell proliferation in intestinal epithelial cells.
In Vitro Cell. Dev. Biol. Anim.
35
:
43
.
46
Smith, C. W., Z. Chen, G. Dong, E. Loukinova, M. Y. Pegram, L. Nicholas-Figueroa, C. Van Waes.
1998
. The host environment promotes the development of primary and metastatic squamous cell carcinomas that constitutively express proinflammatory cytokines IL-1α, IL-6, GM-CSF, and KC.
Clin. Exp. Metastasis
16
:
655
.
47
Lim, J. W., H. Kim, K. H. Kim.
2001
. Nuclear factor-κB regulates cyclooxygenase-2 expression and cell proliferation in human gastric cancer cells.
Lab. Invest.
81
:
349
.
48
Kim, H., J. W. Lim, K. H. Kim.
2001
. Helicobacter pylori-induced expression of interleukin-8 and cyclooxygenase-2 in AGS gastric epithelial cells: mediation by nuclear factor-κB.
Scand. J. Gastroenterol.
36
:
706
.
49
Farrow, B., B. M. Evers.
2002
. Inflammation and the development of pancreatic cancer.
Surg. Oncol.
10
:
153
.
50
Fujioka, S., G. M. Sclabas, C. Schmidt, W. A. Frederick, Q. G. Dong, J. L. Abbruzzese, D. B. Evans, C. Baker, P. J. Chiao.
2003
. Function of NF-κB in pancreatic cancer metastasis.
Clin. Cancer Res.
9
:
346
.
51
Bharti, A. C., B. B. Aggarwal.
2002
. Nuclear factor-κB and cancer: its role in prevention and therapy.
Biochem. Pharmacol.
64
:
883
.
52
Nakshatri, H., P. Bhat-Nakshatri, D. A. Martin, R. J. Goulet, G. W. Slegde.
1997
. Constitutive activation of NF-κB during progression of breast cancer to hormone independent growth.
Mol. Cell. Biol.
17
:
3629
.
53
Wang, C. Y., J. C. Cusack, R. Liu, A. S. Baldwin.
1999
. Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-κB.
Nat. Med.
5
:
412
.
54
Yamamoto, Y., R. B. Gaynor.
2001
. Therapeutic potential of inhibition of the NF-κB pathway in the treatment of inflammation and cancer.
J. Clin. Invest.
107
:
135
.
55
Lee, J. S., T. Y. Oh, B. O. Ahn, H. Cho, W. B. Kim, Y. B. Kim, Y. J. Surh, H. J. Kim, K. B. Hahm.
2001
. Involvement of oxidative stress in experimentally induced reflux esophagitis and Barrett’s esophagus: clue for the chemoprevention of esophageal carcinoma by antioxidants.
Mutat. Res.
480
:
189
.
56
Surh, Y. J., K. S. Chun, H. S. Cha, S. S. Han, Y. S. Keum, K. K. Park, S. S. Lee.
2001
. Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-κB activation.
Mutat. Res.
480
:
243
.
57
El Attar, T. M., A. S. Virji.
1997
. Effects of indomethacin, cisplatin and Δ12-prostaglandin J2 on growth of oral squamous carcinoma cells.
Anticancer Res.
17
:
4399
.
58
Yetiser, S., B. Satar, N. Aydin.
2002
. Expression of epidermal growth factor, tumor necrosis factor-α, and interleukin-1α in chronic otitis media with or without cholesteatoma.
Otol. Neurotol.
23
:
647
.
59
Abreu, M. T., P. Vora, E. Faure, L. S. Thomas, E. T. Arnold, M. Arditi.
2001
. Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide.
J. Immunol.
167
:
1609
.
60
Wang, P. L., Y. Azuma, M. Shinohara, K. Ohura.
2000
. Toll-like receptor 4-mediated signal pathway induced by Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts.
Biochem. Biophys. Res. Commun.
273
:
1161
.
61
Rock, F. L., G. Hardiman, J. C. Timans, R. A. Kastelein, J. F. Bazan.
1998
. A family of human receptors structurally related to Drosophila Toll.
Proc. Natl. Acad. Sci. USA
95
:
588
.
62
Akashi, S., R. Shimazu, H. Ogata, Y. Nagai, K. Takeda, M. Kimoto, K. Miyake.
2000
. Cutting edge: cell surface expression and lipopolysaccharide signaling via the Toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages.
J. Immunol.
164
:
3471
.
63
Beutler, B..
2000
. Tlr4: central component of the sole mammalian LPS sensor.
Curr. Opin. Immunol.
12
:
20
.
64
Yoshimura, A., E. Lien, R. R. Ingalls, E. Tuomanen, R. Dziarski, D. Golenbock.
1999
. Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2.
J. Immunol.
163
:
1
.
65
Dobrovolskaia, M. A., A. E. Medvedev, K. E. Thomas, N. Cuesta, V. Toshchakov, T. Ren, M. J. Cody, S. M. Michalek, N. R. Rice, S. N. Vogel.
2003
. Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR “homotolerance” versus “heterotolerance” on NF-κB signaling pathway components.
J. Immunol.
170
:
508
.
66
Song, P. I., T. A. Abraham, Y. Park, A. S. Zivony, B. Harten, H. F. Edelhauser, S. L. Ward, C. A. Armstrong, J. C. Angel.
2001
. The expression of functional LPS receptor proteins CD14 and Toll-like receptor 4 in human corneal cells.
Invest. Ophthalmol. Vis. Sci.
42
:
2867
.