AJP - Lung Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 293: L693-L701, 2007. First published June 15, 2007; doi:10.1152/ajplung.00491.2006
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/L693    most recent
00491.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koga, T.
Right arrow Articles by Kim, K. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koga, T.
Right arrow Articles by Kim, K. C.

TNF-{alpha} induces MUC1 gene transcription in lung epithelial cells: its signaling pathway and biological implication

Takeshi Koga,1,2 Ippei Kuwahara,1,2 Erik P. Lillehoj,3 Wenju Lu,4 Takeshi Miyata,2 Yoichiro Isohama,2 and K. Chul Kim1

1Lovelace Respiratory Research Institute, Albuquerque, New Mexico; 2Kumamoto University, Kumamoto, Japan; 3University of Maryland, Baltimore; and 4Johns Hopkins University, Baltimore, Maryland

Submitted 22 December 2006 ; accepted in final form 6 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The current study was conducted to elucidate the mechanism through which TNF-{alpha} stimulates expression of MUC1, a membrane-tethered mucin. A549 human lung alveolar cells treated with TNF-{alpha} exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Increased MUC1 protein levels were correlated with significantly higher levels of MUC1 mRNA in TNF-{alpha}-treated cells compared with controls. However, TNF-{alpha} did not alter MUC1 transcript stability, implying increased de novo transcription induced by the cytokine. TNF-{alpha} increased MUC1 gene promoter activity in A549 cells transfected with a promoter-luciferase reporter plasmid. Both U0126, an inhibitor of MEK1/2, and dominant negative ERK1 prevented TNF-{alpha}-induced MUC1 promoter activation, and anti-TNFR1 antibody blocked TNF-{alpha}-stimulated ERK1/2 activation. MUC1 promoter activation by TNF-{alpha} also was blocked by mithramycin A, an inhibitor of Sp1, as well as either deletion or mutation of a putative Sp1 binding site in the MUC1 promoter located between nucleotides –99 and –90. TNF-{alpha}-stimulated binding of Sp1 to the MUC1 promoter in intact cells was demonstrated by chromatin immunoprecipitation assay. We conclude that TNF-{alpha} induces MUC1 gene transcription through a TNFR1 -> MEK1/2 -> ERK1 -> Sp1 pathway.

tumor necrosis factor-{alpha}; Sp1; mitogen-activated protein kinase

THE AIRWAY EPITHELIUM is the first layer of cellular interaction with airborne antigens and plays a key role in initiating allergic responses (45, 46). The airway surface liquid, often referred to as mucus, is a thin layer of fluid covering the luminal surface that plays an important defensive role against foreign particles entering the lung. Removal of inspired particles occurs by the combined action of adherence to mucus and cilia beating (mucociliary clearance). The principle protein constituents of airway mucus are the highly hydrated mucin (MUC) glycoproteins (designated MUC in humans, Muc in animals). Currently, 20 MUC genes have been identified and their encoded glycoproteins classified as gel-forming, secreted, or cell-associated mucins (12, 23, 35). Gel-forming mucins (MUC2, 5AC, 5B, 7, and 8) are produced by respiratory epithelial cells and likely account for the viscoelastic property of the mucus layer. MUC1, 4, 13, and 16 are transmembrane glycoproteins localized on the surface of airway epithelial cells.

MUC1 was the first member of the mucin gene family to be cloned (13). MUC1 expression is polarized to the apical surface of most simple epithelia, as well as uniformly on hematopoietic cells. MUC1 is unique among the membrane-bound mucins because its cytoplasmic region possesses a canonical receptor tyrosine kinase-like structure, particularly with respect to the presence of phosphoamino acid sequence motifs mediating signal transduction cascades (5, 12). Phosphorylation of the MUC1 cytoplasmic tail (CT) at serine, threonine, and tyrosine residues has been extensively characterized (22, 23, 43, 44). Although the role of MUC1 in airway cell physiology remains to be determined, a recent study by our group (25) demonstrated that MUC1 exhibits anti-inflammatory properties in the airways, suggesting that it may be involved in resolving ongoing immune responses.

We previously reported that primary hamster tracheal epithelial cells abundantly express Muc1 protein on their surface (30, 31) and that Muc1-expressing cells exhibited increased adhesion of Pseudomonas aeruginosa by virtue of a specific interaction of the Muc1 extracellular region with flagellin, the major structural protein of the bacterial flagellum (20, 21). P. aeruginosa is a gram-negative opportunistic pathogen responsible for a wide range of acute and chronic pulmonary infections, particularly in patients with cystic fibrosis or who are mechanically ventilated, elderly, or immunosuppressed. Patients with P. aeruginosa lung infections mount an inflammatory response characterized by airway mucin hypersecretion, lung neutrophilia, and high levels of proinflammatory cytokines. Prominent among the latter is tumor necrosis factor (TNF)-{alpha}, which is produced by diverse cell types including macrophages, lymphocytes, fibroblasts, and epithelial cells (2, 24). Following binding to the TNF receptor (TNFR), TNF-{alpha} activates multiple intracellular signal transduction cascades culminating in a diverse array of biological responses (9). Noteworthy in this regard, TNF-{alpha} upregulates MUC1 expression in epithelial cells derived from airway, breast, prostate, and uterine tissues (17, 29, 38, 41). In the case of respiratory cells, Shirasaki et al. (38) reported that TNF-{alpha} increased MUC1 mRNA levels in human primary nasal epithelial cells. However, no studies have been reported examining the effects of TNF-{alpha} on MUC1 expression in cells derived from the lower airways.

The purpose of this study was twofold: 1) to determine whether TNF-{alpha} induced MUC1 expression in A549 cells, a human lung carcinoma (19), and if so, 2) to elucidate the mechanism involved. Our results demonstrated that TNF-{alpha} increased the expression of MUC1 in A549 cells at both the mRNA and protein levels, and this effect was mediated through a TNFR1 -> MEK1/2 -> ERK1 -> Sp1 pathway. We speculate that TNF-{alpha} plays a dual role during microbial infection of the airways, serving as a proinflammatory cytokine during the initial stages of the immune response, followed by an anti-inflammatory effect as a consequence of upregulation of MUC1 expression.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. A549 cells were seeded in RPMI 1640 medium containing 10% FBS (Invitrogen, Carlsbad, CA) and 25 mM HEPES in 24-well plates at 5.0 x 104 cells/well and cultured to confluence at 37°C, 5% CO2. Mouse tracheal surface epithelial (MTSE) cells were harvested and cultured on 12-mm Millicell inserts (Millipore, Billerica, MA) at an air-liquid interface as described for rat TSE cells (26).

MUC1 immunoblotting and immunoprecipitation. A549 cells were washed with PBS, pH 7.4 at 4°C, and extracted with 1.0 ml/100-mm dish of PBS, 1.0% Triton X-100, 1.0% sodium deoxycholate, and 1.0% protease inhibitor cocktail (Sigma, St. Louis, MO). Lysates were incubated for 20 min at 4°C, and insoluble material was removed by centrifugation at 14,000 g for 10 min at 4°C. Protein concentrations were measured using the bicinchoninic acid method with BSA as a standard (Bio-Rad, Hercules, CA). Equal protein aliquots were resuspended in SDS-PAGE Laemmli buffer (0.05 M Tris·HCl, pH 6.8, 2.5% beta-mercaptoethanol, 1.0% SDS, 5% glycerol, and 0.1% bromphenol blue), boiled for 5 min, and resolved on 6% (for the extracellular domain) or 15% (for the CT domain) acrylamide gels. Resolved proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad) for 1 h at 100 V in 0.02 M Tris·HCl, pH 8.3, 0.19 M glycine, and 20% methanol, and the membrane was blocked for 30 min at room temperature with 5% BSA (Sigma)/TBS-T (0.01 M Tris·HCl, pH 7.4, 0.15 M NaCl, and 0.1% Tween 20). Membranes were reacted overnight at 4°C with either the MUC1-reactive monoclonal antibody GP1.4 (20 µg/ml; Biomeda, Foster City, CA) to detect the extracellular subunit or rabbit CT33 antiserum (1: 10,000) to detect the CT subunit. CT33 was prepared against a synthetic peptide corresponding to the COOH-terminal 17 residues of the MUC1 CT subunit and is identical in reactivity to the MUC1 CT1 (32) and CT2 (7) antibodies. Identical dilutions of purified normal mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) were used as negative controls. Following incubation, the membranes were washed three times with TBS-T, reacted 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG; KPL, Gaithersburg, MD), washed three times, and developed with enhanced chemiluminescence reagents (Amersham-Pharmacia, Piscataway, NJ). The bands corresponding to the MUC1 extracellular (>250 kDa) and CT (25 kDa) protein subunits were identified by comigration of prestained protein size markers (Bio-Rad). For MUC1 immunoprecipitation, 0.5 mg of cell lysate was reacted overnight at 4°C with 10 µl of CT33 antiserum. Bound proteins were isolated with 50 µl of protein A-agarose (Invitrogen) and analyzed by immunoblotting. The CT33 antibody heavy chain was detected as a gel loading/transblot efficiency control using anti-rabbit IgG antibody.

Effect of TNF-{alpha} on MUC1 mRNA expression. Confluent A549 cells were treated for 0, 4, 8, or 24 h with 0, 10, 25, 50, or 100 ng/ml of purified human recombinant TNF-{alpha} (ProSpec, Rehovot, Israel) or vehicle control (PBS), and MUC1 mRNA was quantified by real-time RT-PCR. In experiments to determine MUC1 mRNA stability, the cells were treated for 24 h with 50 ng/ml TNF-{alpha}, chased for 0, 4, 8, or 24 h with 5.0 µg/ml actinomycin D (Sigma), and washed with PBS, and MUC1 mRNA levels were quantified.

Real-time RT-PCR. Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA), and 0.2 µg was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad) in a total volume of 20 µl. Real-time PCR was carried out using TaqMan probes and iQ supermix (Bio-Rad) according to the manufacturer's instructions. Briefly, 2.0 µl of cDNA or control plasmid were used as template for amplification in the iCycler (Bio-Rad) with 200 nM of MUC1 or GAPDH (internal control) primers and probes. Primers and probes were designed using Beacon Designer 2.0 software (Biosoft, Palo Alto, CA). The MUC1 forward primer was 5'-TCAGCTTCTACTCTGGTGCACAA-3', and the reverse primer was 5'-ATTGAGAATGGAGTGCTCTTGCT-3'. The MUC1 probe had the fluorescent molecule 6-carboxy-fluorescein (FAM) attached at the 5' end and the black hole quencher-1 (BHQ-1) attached to the 3' end (5'-FAM-TCTGCCAGGGCTACCACAACCC-BHQ-1–3'; IDT, Coralville, IA). The GAPDH forward primer was 5'-AGCCTCAAGATCATCAGCAATG-3', and the reverse primer was 5'-GTTGTCATGGATGACCTTGGC-3'. The GAPDH probe had the fluorescent molecule hexachlorofluorescein (Hex) attached at the 5' end and BHQ-1 attached to the 3' end (5'-Hex-CCTGCACCACCAACTGCTTAGCAC-BHQ-1-3'). PCR was performed with a 15-s melt at 95°C, followed by annealing for 30 s at 60°C and extension for 30 s at 72°C for 40 cycles. All reactions were performed in triplicate. The Ct value was defined as the number of PCR cycles required for the specific fluorescence signal to exceed the detection threshold value set by the software installed in the iCycler. The levels of MUC1 transcripts were normalized to GAPDH transcripts using the 2–{Delta}{Delta}Ct method.

Mitogen-activated protein kinase immunoblot analysis. Confluent A549 cells were serum-starved for 24 h and treated for 0, 5, 10, 20, 30, or 60 min with 50 ng/ml of TNF-{alpha} at 37°C. Equal protein aliquots of cell lysates were subjected to SDS-PAGE on 12% acrylamide gels and analyzed by immunoblotting with anti-JNK or phospho-JNK, anti-p38 or phospho-p38, or anti-ERK1/2 or phospho-ERK1/2 specific antibodies (Cell Signaling, Beverly, MA). Immunoblotting with anti-beta-tubulin antibody was performed as a gel loading control. In some experiments, the cells were pretreated for 30 min with anti-TNFR1 antibody (R&D Systems, Minneapolis, MN) before TNF-{alpha} treatment.

Transient transfection and luciferase assay. A549 cells were seeded in RPMI 1640, 10% FBS, and 25 mM HEPES in 24-well plates and incubated for 24 h at 37°C, 5% CO2, to 70–80% confluence. Medium was replaced with serum-free RPMI 1640 and 25 mM HEPES, and the cells were incubated an additional 24 h. The medium was changed to RPMI 1640, 1% FBS, and 25 mM HEPES, and the cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The DNA sample consisted of 800 ng of a MUC1 promoter-firefly luciferase reporter plasmid (MUC1-pGL2b) (16) or the empty pGL2b vector plus 10 ng of the phRL-TK internal control plasmid encoding Renilla luciferase (Promega). MUC1 promoter deletion-luciferase reporter plasmids were prepared as our group described previously (16). Briefly, sample DNA was mixed with 2.0 µl of Lipofectamine and diluted with OPTI-MEM I (Invitrogen) to a final volume of 100 µl. After 20 min of incubation at room temperature, 600 µl of the DNA-Lipofectamine mixture was added to each well. At 6 h posttransfection, the cells were washed with RPMI 1640, 1% FBS, and 25 mM HEPES and treated for 24 h at 37°C with 50 ng/ml TNF-{alpha} or vehicle control. In some experiments, the transfected cells were pretreated for 1 h before TNF-{alpha} treatment with mithramycin A (Sp1 inhibitor), SB 203580 (p38 inhibitor), JNK inhibitor II, or U0126 (MEK1/2 inhibitor) (Cell Signaling). Luciferase activity was determined using the Dual Luciferase assay system (Promega) according to the manufacturer's instructions and a microplate luminometer (Lmax; Molecular Devices, Sunnyvale, CA). Luciferase activity driven by the MUC1 promoter was normalized to the internal control by calculating the ratio of firefly luciferase activity to Renilla luciferase activity of each sample and expressed as the percentage of MUC1 promoter activity relative to control samples.

Dominant negative ERK1/2 transfection. Dominant negative (DN) expression plasmids for ERK1 and ERK2 (34) were kindly provided by Dr. Jian-Dong Li (Rochester University, Rochester, NY). A549 cells were serum-starved for 24 h and transfected for 6 h as described above with ERK1 DN, ERK2 DN, or pcDNA3.1 vectors before treating with TNF-{alpha} for 12 h. Total RNA was isolated for real-time RT-PCR as described above.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) assay was performed using the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) with slight modification. Briefly, following treatment with TNF-{alpha} or PBS, A549 cells were treated with 1% formaldehyde at 37°C for 10 min to cross-link DNA with its binding proteins. The fixed cells were washed with ice-cold PBS and extracted with 200 µl/100-mm dish of ChIP lysis buffer (50 mM Tris·HCl, pH 8.1, 1% SDS, 10 mM EDTA, and 1.0% protease inhibitor cocktail), and the cell lysates were sonicated to shear DNA to an average size of 500 base pairs. After centrifugation, the supernatant was diluted 10-fold with ChIP dilution buffer (16.7 mM Tris·HCl, pH 8.1, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 150 mM NaCl, and 1.0% protease inhibitor cocktail), and an aliquot of the diluted sample was kept at 4°C after adjusting the NaCl concentration to 200 mM until used as the input sample. The remainder of the diluted sample was mixed with protein A agarose-salmon sperm DNA (1:1) preincubated at 4°C for 1 h to reduce nonspecific binding, the mixture was centrifuged, and the supernatant was immunoprecipitated with Sp1 antibody (Santa Cruz Biotechnology) or control IgG at 4°C overnight. An aliquot of the protein A agarose-salmon sperm DNA slurry was added to the immunoprecipitated sample, followed by constant mixing at 4°C for 1 h. After centrifugation, the resulting agarose pellet was sequentially washed once with low-salt wash buffer (20 mM Tris·HCl, pH 8.1, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 150 mM NaCl), once with high-salt wash buffer (20 mM Tris·HCl, pH 8.1, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 500 mM NaCl), once with LiCl wash buffer (1 mM Tris·HCl, pH 8.1, 1% NP-40, 1% deoxycholate, 1 mM EDTA, and 0.25 M LiCl), and twice with 1x Tris-EDTA buffer (10 mM Tris·HCl, pH 8.0, and 1 mM EDTA). The agarose pellet was incubated at room temperature for 15 min in elution buffer (1% SDS, 0.1 M NaHCO3, and 10 mM DTT), and the protein-DNA cross-linkages were reversed with 200 mM NaCl for 6 h at 65°C. DNA was extracted with phenol-chloroform, precipitated with ethanol, and subjected to PCR using the MUC1 forward primer 5'-TCTTATTTCTCGGCCGCTCTGCTT-3' and the reverse primer 5'-TGGGTAGGGTACAAGGGCTCTAAT-3'. MUC1 amplicons were quantified by densitometry and corrected by background subtraction. Relative intensity values were obtained by normalizing the corrected intensity values to those of the input samples.

Statistical analysis. Differences in mean ± SE values among groups were assessed using the Student's t-test for unpaired samples and considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TNF-{alpha} induces MUC1 protein expression in human lung carcinoma cells. As shown in Fig. 1, TNF-{alpha} significantly increased MUC1 protein expression in A549 cells compared with PBS-treated cells. MUC1 expression by TNF-{alpha} was dose dependent, with a maximum response at 50 ng/ml of the cytokine. As shown by densitometry, ~10-fold increased MUC1 expression was obtained following TNF-{alpha} treatment, compared with constitutive MUC1 expression in cells treated with PBS. This effect of TNF-{alpha} was also observed in primary MTSE cells grown on an air-liquid interface (Fig. 1B). Although A549 cells were originally derived from a carcinoma, their ability to produce various types of mucins, as well as the technical feasibility to transfect various genes into these cells, made it possible to study the regulation of mucin production and mucous cell metaplasia in vitro, which has resulted in a significant contribution to our current understanding of airway mucin cell biology (35). Based on the results in Fig. 1B showing a similar response of MTSE cells to that of A549 cells, we used A549 cells for the remainder of the experiments to identify the signaling pathway involved in TNF-{alpha}-induced MUC1 transcription.


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. TNF-{alpha} induces MUC1 protein expression. Confluent A549 human cells (A) or primary mouse tracheal surface epithelial cells (MTSE; B) were serum-starved for 24 h and either nontreated or treated for 24 h with the indicated concentrations of TNF-{alpha}. Equal protein aliquots of cell lysates were subjected to immunoprecipitation with antibody against the MUC1 cytoplasmic tail (CT33) and immunoblot analysis with antibody against the MUC1 extracellular region (GP1.4). Both CT33 antibody heavy chain (A) and beta-tubulin (B) were used to verify equal gel loading and transblot efficiencies. Data are representative of 3 experiments.

 
TNF-{alpha} increases MUC1 mRNA levels in dose- and time-dependent manners. The increase in cellular MUC1 protein content following TNF-{alpha} treatment could have been due to increased gene transcription, translation, or stability of MUC1 mRNA or protein. To determine the effect of TNF-{alpha} on MUC1 transcription, A549 cells were treated with various concentrations of TNF-{alpha} and MUC1 transcript levels were determined using real-time RT-PCR. TNF-{alpha} significantly increased MUC1 mRNA levels in a dose-dependent manner compared with PBS-treated cells (Fig. 2A). At 50 ng/ml, TNF-{alpha} also significantly increased MUC1 transcripts at all time points examined (Fig. 2B). This concentration of TNF-{alpha} is within the physiological range measured in vivo based on a study with human asthmatics (4).


Figure 2
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 2. TNF-{alpha} induces MUC1 mRNA expression in A549 cells. Confluent A549 cells were serum-starved for 24 h and treated for 6 h with PBS or the indicated concentrations of TNF-{alpha} (A) or treated with 50 ng/ml TNF-{alpha} for the indicated time periods (B). Total RNA was analyzed for the levels of MUC1 mRNA by using real-time RT-PCR, and data were normalized to GAPDH mRNA levels. Each data point represents the mean ± SE (n = 3). Data are representative of 3 experiments. *P < 0.05, significantly increased MUC1 mRNA levels in TNF-{alpha}-treated cells compared with vehicle control (0 ng/ml).

 
TNF-{alpha} does not affect MUC1 mRNA stability. To determine whether TNF-{alpha} treatment altered MUC1 mRNA stability, A549 cells were treated for 24 h with 50 ng/ml of TNF-{alpha} and chased in the presence of PBS or actinomycin D to block RNA polymerase activity. As shown in Fig. 3, MUC1 mRNA levels in cells treated with actinomycin D for 4, 8, or 24 h were identical to those in PBS-treated cells. To verify that the effect of TNF-{alpha} was to induce MUC1 gene transcription, A549 cells were transfected with a MUC1 promoter-firefly luciferase reporter plasmid (MUC1-pGL2b) and treated with PBS or TNF-{alpha}, and luciferase activity was determined relative to that of cotransfected Renilla luciferase. TNF-{alpha}-treated MUC1-pGL2b-transfected cells exhibited ~170% increased luciferase activity compared with PBS-treated cells (Fig. 4).


Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 3. TNF-{alpha} does not alter MUC1 mRNA stability. Confluent A549 cells were treated for 24 h with PBS or 50 ng/ml TNF-{alpha} and chased in the presence of 5.0 µg/ml actinomycin D for various time periods. MUC1 mRNA levels were measured using real-time RT-PCR, and data were normalized to GAPDH mRNA levels. Each data point represents the mean ± SE (n = 3). Data are representative of 3 experiments.

 

Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 4. TNF-{alpha} activates MUC1 gene promoter activity. A549 cells (70–80% confluent) were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid containing the MUC1 promoter, incubated for 6 h, and treated for 24 h with PBS or 50 ng/ml TNF-{alpha}, and then luciferase activity was determined. Each data point represents the mean ± SE (n = 3). Data are representative of 3 experiments. *P < 0.05, significantly increased luciferase activity in MUC1-pGL2b-transfected cells treated with TNF-{alpha} compared with PBS-treated cells.

 
TNF-{alpha} induces MUC1 transcription through ERK1. TNF-{alpha} is a potent activator of mitogen-activated protein (MAP) kinases (2, 9, 24). As shown in Fig. 5, the levels of phosphorylated p38, JNK, and ERK1/2 were increased in A549 cells treated with TNF-{alpha} compared with PBS treatment. Phosphorylation of all three MAP kinases was maximal at 10 min after cytokine treatment and decreased to basal levels by 60 min. Next, to determine which MAP kinase(s) was responsible for TNF-{alpha}-induced MUC1 expression, MUC1-pGL2b-transfected A549 cells were pretreated with selective kinase inhibitors before TNF-{alpha} treatment, and relative luciferase activity was determined. Only the MEK1/2-specific inhibitor U0126 completely blocked the ability of TNF-{alpha} to induce MUC1 promoter activity compared with PBS (Fig. 6). To confirm the involvement of ERK1/2 in this pathway, A549 cells were transfected with ERK1 DN, ERK2 DN, or their control plasmid (pcDNA3.1) before TNF-{alpha} treatment. As shown in Fig. 7, TNF-{alpha}-induced MUC1 transcription was completely suppressed with ERK1 DN but not with ERK2 DN. To confirm that the effect of the cytokine was not due to a potential impurity in the TNF-{alpha} preparation, A549 cells were pretreated with anti-TNFR1 antibody before TNF-{alpha} treatment, and the levels of phospho-ERK1/2 were determined. As shown in Fig. 8, anti-TNFR1 antibody completely blocked the ability of TNF-{alpha} to stimulate ERK1/2 phosphorylation compared with anti-TNFR1 alone. The anti-TNFR1 antibody used in this experiment exhibits a partial TNF-{alpha} agonist activity in A549 cells according to the manufacturer's description of the antibody. From the collective results presented in Figs. 5–8, we concluded that TNF-{alpha} stimulated MUC1 expression through TNFR1, MEK1/2, and ERK1.


Figure 5
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 5. TNF-{alpha} activates MAP kinases in A549 cells. Confluent A549 cells were treated for the indicated times with PBS or 50 ng/ml TNF-{alpha}, and equal protein aliquots of cell lysates were examined by immunoblotting with antibodies against phospho-JNK, phospho-p38, phospho-ERK, total MAPKs, or beta-tubulin. Data are representative of 3 experiments.

 

Figure 6
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 6. TNF-{alpha} stimulates MUC1 promoter activity through MEK1/2. A549 cells (70–80% confluent) were serum-starved for 24 h, transfected with MUC1-pGL2b plasmid, and incubated for 6 h in the presence of 1% serum. The cells were pretreated for 1 h with the indicated concentrations of JNK inhibitor II (A), SB 203580 (B), or U0126 (C) and treated for 24 h with PBS or 50 ng/ml TNF-{alpha}, and relative luciferase activity was determined. Each data point represents the mean ± SE (n = 3). Data are representative of 3 experiments. *P < 0.05, significantly increased luciferase activity in MUC1-pGL2b-transfected cells treated with TNF-{alpha} compared with PBS-treated cells. {dagger}P < 0.05, significantly suppressed luciferase activity compared with MUC1 promoter activity induced by TNF-{alpha}.

 

Figure 7
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 7. ERK1 is crucial for MUC1 mRNA induction by TNF-{alpha}. A549 cells (70–80% confluent) were serum-starved for 24 h, transfected with pcDNA3.1 control plasmid or with dominant negative ERK1 (ERK 1 DN) or ERK2 DN in pcDNA3.1 and incubated for 6 h in the presence of 1% serum. Cells were treated with 50 ng/ml TNF-{alpha} or vehicle control for 12 h, and total RNA was isolated for real-time RT-PCR. Each data point represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *P < 0.05, significantly increased MUC1 mRNA expression in the cells treated with TNF-{alpha} compared with PBS-treated cells. {dagger}P < 0.05, significantly suppressed MUC1 mRNA expression compared with MUC1 mRNA induced by TNF-{alpha}.

 

Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 8. Anti-TNF receptor 1 (TNFR1) antibody blocks TNF-{alpha}-stimulated ERK1/2 activation. Confluent A549 cells were serum-starved for 24 h, pretreated for 1 h with PBS or 10 µg/ml anti-TNFR1 antibody, and treated for 30 min with 50 ng/ml TNF-{alpha}, and equal protein aliquots of cell lysates were examined by immunoblotting with antibodies against phospho-ERK1/2 or beta-tubulin. Data are representative of 3 experiments.

 
TNF-{alpha} induces MUC1 transcription through Sp1. Although the mechanism of transcriptional regulation of the MUC1 gene remains to be completely determined, it is well-known that the Sp1 transcription factor plays a major role downstream of ERK1/2 (14, 16). Consequently, to determine whether TNF-{alpha}-induced MUC1 transcription involves Sp1, MUC1-pGL2b-transfected A549 cells were pretreated with mithramycin A, a specific Sp1 inhibitor (3), before TNF-{alpha} treatment, and relative luciferase activity was determined. Mithramycin A (500 nM) completely prevented MUC1 promoter induction by TNF-{alpha} (Fig. 9A). No cytotoxicity was observed with this concentration of mithramycin A judging from both cell morphology and cellular lactate dehydrogenase release (data not shown).


Figure 9
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 9. TNF-{alpha} increases MUC1 promoter activity through Sp1. A549 cells (70–80% confluent) were serum-starved for 24 h and transfected with MUC1-pGL2b plasmid alone (A), 2 different MUC1 promoter deletion mutants (DM), and a mutant MUC1-pGL2b plasmid containing nucleotides changes at positions –97 and –96 (GG->AA) (B and C) for 6 h in the presence of 1% serum. The cells were treated with PBS or 50 ng/ml TNF-{alpha} for 24 h, and relative luciferase activity was determined. Each data point represents the mean ± SE (n = 3). Data are representative of 3 experiments. *P < 0.05, significantly increased luciferase activity in MUC1-pGL2b-transfected cells treated with TNF-{alpha} compared with PBS-treated cells. {dagger}P < 0.05, significantly suppressed luciferase activity compared with MUC1 promoter activity induced by TNF-{alpha}.

 
The Sp1 binding site at –99/–90 in the MUC1 promoter is crucial for TNF-{alpha}-induced MUC1 transcription. Ten putative Sp1 binding sites are present in the MUC1 promoter, and our group (16) previously reported that only the site between nucleotides –99 and –90 (relative to the transcription initiation site) was required for neutrophil elastase (NE)-induced MUC1 transcription. Based on the report by Sivko et al. (39), we made several deletion mutants and point mutations at positions –97 and –96 (GG to AA) (Fig. 9B) to block the binding of Sp1 to this region and determined the effect on TNF-{alpha}-stimulated MUC1 promoter activation. MUC1 promoter activity induced by TNF-{alpha} was completely abolished in the cells transfected with a deletion mutant that does not contain the putative Sp1 binding site (Fig. 9, B and C) or a point-mutated MUC1 promoter plasmid (Fig. 9C). These data suggest that the –99/–90 Sp1 binding site is the only one that is crucial for MUC1 promoter activity induced by TNF-{alpha}.

TNF-{alpha} induces Sp1 binding to the MUC1 promoter region in vivo. To determine whether Sp1 binding to the MUC1 promoter is induced by TNF-{alpha}, we performed a ChIP assay on the MUC1 promoter with Sp1 antibody or control IgG. Sp1-responsive elements in the MUC1 promoter after immunoprecipitation were analyzed by semiquantitative PCR. TNF-{alpha} significantly induced the binding of Sp1 to the MUC1 promoter region (Fig. 10A) by 2.3-fold compared with vehicle control (Fig. 10B).


Figure 10
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 10. TNF-{alpha} induces Sp1 recruitment to the MUC1 promoter in viable cells. A549 cells (100% confluent) were serum-starved for 24 h and treated with vehicle control or 50 ng/ml TNF-{alpha} for 1 h at 37°C. Cell lysates were sonicated, and the resulting disrupted DNA was chromatin immunoprecipitated (ChIP) with Sp1 antibody or control IgG as described in MATERIALS AND METHODS. After reversal of cross-linking, target DNA was amplified using MUC1 primers with 40 cycles of PCR. Each data point represents the mean ± SE (n = 3). Data are representative of 3 experiments. *P < 0.05, significantly different from vehicle control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we demonstrated that MUC1 mRNA and protein synthesis in A549 cells were enhanced by TNF-{alpha} treatment. Increased MUC1 transcript levels following TNF-{alpha} treatment were due to increased de novo transcription, not elevated mRNA stability. With the use of a combination of biochemical, pharmacological, and molecular biological approaches, a TNFR1 -> MEK1/2 -> ERK1 -> Sp1 pathway was demonstrated to mediate TNF-{alpha}-induced MUC1 promoter activity. Finally, a specific cis regulatory element located between nucleotides –99 and –90 in the MUC1 promoter that possessed a consensus Sp1 binding sequence motif was required for TNF-{alpha}-stimulated MUC1 promoter activation. It is noteworthy that the mechanism described in this study was obtained using a lung adenocarcinoma that may have a different signaling pathway for TNF-{alpha}-induced MUC1 transcription compared with normal airway epithelial cells.

The identification of a MUC1 transcriptional mechanism induced by TNF-{alpha} corroborates and extends a previous study by our group (18) and others (1, 11) that revealed positive regulatory elements in the MUC1 gene promoter, which contains five putative NF-{kappa}B and 10 potential Sp1 binding sites. As a mechanism for TNF-{alpha}-induced MUC1 transcription in the presence of IFN-{gamma}, it was demonstrated that binding of NF-{kappa}B to the MUC1 promoter occurred at nucleotides –589/–581 (17). On the other hand, binding of Sp1 at the –99/–90 site was crucial for the regulation of MUC1 transcription (14). In particular, we previously demonstrated that NE induced MUC1 transcription via Sp1 binding at this same site (16). On the basis of the present results, increased Sp1 binding to the –99/–90 sequence also appears responsible for TNF-{alpha}-induced MUC1 transcription. Five phosphorylation sites on Sp1, either serine or threonine, have been confirmed (6). Mutation studies revealed a role for Sp1 phosphorylation in DNA binding and promoter activation in mammalian cells (6). Activated ERK has been shown to be involved in serine or threonine phosphorylation of Sp1, resulting in transcriptional activation of either the urokinase plasminogen activator receptor gene (42) or the vascular endothelial growth factor gene (27). Therefore, it is possible that increased Sp1 binding to the –99/–90 segment of the MUC1 promoter is mediated by Sp1 phosphorylation by ERK. Experiments are underway in our laboratory to delineate the site(s) of Sp1 phosphorylation in the context of ERK-induced MUC1 transcription.

Although we cannot formally rule out the possible involvement of NF-{kappa}B in TNF-{alpha}-stimulated MUC1 expression, this mechanism appears unlikely by analogy to the effect of NE, where protease-induced MUC1 expression was retained in cells expressing a MUC1 promoter deletion mutant lacking all of the NF-{kappa}B sites (16). Thus the detailed mechanism through which TNF-{alpha} induces increased Sp1 binding to the –99/–90 promoter segment remains to be determined. TNF-{alpha}-induced Sp1 phosphorylation may be involved, because it has been shown that Sp1 binding to DNA is enhanced by serine/threonine phosphorylation of the transcription factor (6, 27). Further investigation of the signaling pathway responsible for MUC1 transcription induced by TNF-{alpha} is presently in progress in our laboratory.

The current paradigm of airway inflammation dictates that following inhalation and colonization of the lungs by pathogenic microorganisms, respiratory epithelial cells and resident leukocytes become activated to express many different genes, including the proinflammatory cytokines IL-1beta, IL-6, IL-8, and TNF-{alpha} (15, 33), which attract neutrophils from the circulation into the airways. In particular, IL-1beta and TNF-{alpha} induce the transcription of genes necessary to perpetuate the innate immune response that ultimately results in activation and extravasation of neutrophils to the lungs. Accumulated pathogens are killed mainly by neutrophils through phagocytosis, as well as by the release of various antimicrobial products such as NE and reactive oxygen species (46). Although a plethora of information is available with regard to the pathology of airway inflammation, the manner in which inflammation is controlled is less clearly understood. It is known, however, that IL-10 plays an important role in resolving inflammation and is considered the major anti-inflammatory cytokine in the lung (36). The relationship among IL-10, TNF-{alpha}, and MUC1 in controlling respiratory inflammatory responses, however, remains to be clarified. In 1999, Skerrett et al. (40) reported that TNFR1 null mice treated intranasally with P. aeruginosa showed significantly increased inflammation compared with wild-type mice, as measured by enhanced bacterial clearance, increased numbers of neutrophils in bronchoalveolar lavage fluid (BALF), and higher levels of TNF-{alpha} in BALF. Interestingly, these phenotypes were identical to those described in our prior report (25) following experimental P. aeruginosa lung infection of Muc1 knockout mice. In that study, we demonstrated that MUC1/Muc1 expression by human/mouse airway epithelial cells was associated with an anti-inflammatory response, mediated through cross talk with Toll-like receptor 5 (TLR5).

Based on the result of the current study demonstrating that exogenous TNF-{alpha} stimulated MUC1 production, one possible explanation for the fact that TNFR1 knockout mice display phenotypes similar to those of Muc1 null mice following P. aeruginosa infection is that TNFR1 knockout mice failed to upregulate MUC1 levels following P. aeruginosa infection. Because NE activates TNF-{alpha} expression by cultured lung epithelial cells (37), we theorize that release of the protease from infiltrating neutrophils is an initial event that is responsible for an early proinflammatory response following P. aeruginosa infection mediated, in part, by TNF-{alpha}. Subsequently, TNF-{alpha} induces MUC1 expression to downregulate inflammation, possibly through MUC1's ability to block TLR signaling (25) and/or to stimulate IL-10 production by airway dendritic cells (8, 28). Moreover, IL-10 inhibits TNF-{alpha} production (10), suggesting a feedback mechanism may operate to control the excessive and prolonged inflammatory effect by TNF-{alpha}. Thus the interactions among TNF-{alpha}, MUC1, and IL-10 may play a crucial role controlling the balance between proinflammatory and anti-inflammatory responses during P. aeruginosa lung infection. Further studies in our laboratory are directed at elucidating the functional relationships between these three key airway effector molecules.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants R01 HL-47125 (to K. C. Kim) and R21 ES-013483 (to E. P. Lillhoj) and the Cystic Fibrosis Foundation (to E. P. Lillehoj).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Chul Kim, Lovelace Respiratory Research Institute, 2425 Ridgecrest Dr, S.E., Albuquerque, NM 87108 (e-mail: kckim{at}lrri.org)

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


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abe M, Kufe D. Characterization of cis-acting elements regulating transcription of the human DF3 breast carcinoma-associated antigen (MUC1) gene. Proc Natl Acad Sci USA 90: 282–286, 1993.[Abstract/Free Full Text]
  2. Baud V, Karin M. Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol 11: 372–377, 2001.[CrossRef][ISI][Medline]
  3. Blume SW, Snyder RC, Ray R, Thomas S, Koller CA, Miller DM. Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J Clin Invest 88: 1613–1621, 1991.[ISI][Medline]
  4. Broide DH, Paine MM, Firestein GS. Eosinophils express interleukin 5 and granulocyte macrophage-colony-stimulating factor mRNA at sites of allergic inflammation in asthmatics. J Clin Invest 90: 1414–1424, 1992.[ISI][Medline]
  5. Carraway KL, Ramsauer VP, Haq B, Carothers Carraway CA. Cell signaling through membrane mucins. Bioessays 25: 66–71, 2003.[CrossRef][ISI][Medline]
  6. Chu S, Ferro TJ. Sp1: regulation of gene expression by phosphorylation. Gene 348: 1–11, 2005.[CrossRef][ISI][Medline]
  7. Croce MV, Isla-Larrain M, Remes-Lenicov F, Colussi AG, Lacunza E, Kim KC, Gendler SJ, Segal-Eiras A. MUC1 cytoplasmic tail detection using CT33 polyclonal and CT2 monoclonal antibodies in breast and colorectal tissue. Histol Histopathol 21: 849–855, 2006.[ISI][Medline]
  8. Daftarian PM, Kumar A, Kryworuchko M, Diaz-Mitoma F. IL-10 production is enhanced in human T cells by IL-12 and IL-6 and in monocytes by tumor necrosis factor-{alpha}. J Immunol 157: 12–20, 1996.[Abstract]
  9. Darnay BG, Aggarwal BB. Early events in TNF signaling: a story of associations and dissociations. J Leukoc Biol 61: 559–566, 1997.[Abstract]
  10. Fiorentino DF, Zlotnik A, Mosmann TR, Howard M, O'Garra A. IL-10 inhibits cytokine production by activated macrophages. J Immunol 147: 3815–3822, 1991.[Abstract]
  11. Gaemers IC, Vos HL, Volders HH, van der Valk SW, Hilkens J. A STAT-responsive element in the promoter of the episialin/MUC1 gene is involved in its overexpression in carcinoma cells. J Biol Chem 276: 6191–6199, 2001.[Abstract/Free Full Text]
  12. Gendler SJ. MUC1, the renaissance molecule. J Mammary Gland Biol Neoplasia 6: 339–353, 2001.[CrossRef][ISI][Medline]
  13. Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, Lalani EN, Wilson D. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 265: 15286–15293, 1990.[Abstract/Free Full Text]
  14. Kovarik A, Lu PJ, Peat N, Morris J, Taylor-Papadimitriou J. Two GC boxes (Sp1 sites) are involved in regulation of the activity of the epithelium-specific MUC1 promoter. J Biol Chem 271: 18140–18147, 1996.[Abstract/Free Full Text]
  15. Kube D, Sontich U, Fletcher D, Davis PB. Proinflammatory cytokine responses to P. aeruginosa infection in human airway epithelial cell lines. Am J Physiol Lung Cell Mol Physiol 280: L493–L502, 2001.[Abstract/Free Full Text]
  16. Kuwahara I, Lillehoj EP, Hisatsune A, Lu W, Isohama Y, Miyata T, Kim KC. Neutrophil elastase stimulates MUC1 gene expression through increased Sp1 binding to the MUC1 promoter. Am J Physiol Lung Cell Mol Physiol 289: L355–L362, 2005.[Abstract/Free Full Text]
  17. Lagow EL, Carson DD. Synergistic stimulation of MUC1 expression in normal breast epithelia and breast cancer cells by interferon-gamma and tumor necrosis factor-alpha. J Cell Biochem 86: 759–772, 2002.[CrossRef][ISI][Medline]
  18. Lee IJ, Hyun SW, Nandi A, Kim KC. Transcriptional regulation of the hamster Muc1 gene: identification of a putative negative regulatory element. Am J Physiol Lung Cell Mol Physiol 284: L160–L168, 2003.[Abstract/Free Full Text]
  19. Lieber M, Smith B, Szakal A, Nelson-Rees W, Todaro G. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer 17: 62–70, 1976.[ISI][Medline]
  20. Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, Kim KC. Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol 280: L181–L187, 2001.[Abstract/Free Full Text]
  21. Lillehoj EP, Kim BT, Kim KC. Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin. Am J Physiol Lung Cell Mol Physiol 282: L751–L756, 2002.[Abstract/Free Full Text]
  22. Lillehoj EP, Kim H, Chun EY, Kim KC. Pseudomonas aeruginosa stimulates phosphorylation of the airway epithelial membrane glycoprotein Muc1 and activates MAP kinase. Am J Physiol Lung Cell Mol Physiol 287: L809–L815, 2004.[Abstract/Free Full Text]
  23. Lillehoj EP, Kim KC. Airway mucus: its components and function. Arch Pharm Res 25: 770–780, 2002.[ISI][Medline]
  24. Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104: 487–501, 2001.[CrossRef][ISI][Medline]
  25. Lu W, Hisatsune A, Koga T, Kato K, Kuwahara I, Lillehoj EP, Chen W, Cross AS, Gendler SJ, Gewirtz AT, Kim KC. Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice. J Immunol 176: 3890–3894, 2006.[Abstract/Free Full Text]
  26. Lu W, Lillehoj EP, Kim KC. Effects of dexamethasone on Muc5ac mucin production by primary airway goblet cells. Am J Physiol Lung Cell Mol Physiol 288: L52–L60, 2005.[Abstract/Free Full Text]
  27. Milanini-Mongiat J, Pouyssegur J, Pages G. Identification of two Sp1 phosphorylation sites for p42/p44 mitogen-activated protein kinases: their implication in vascular endothelial growth factor gene transcription. J Biol Chem 277: 20631–20639, 2002.[Abstract/Free Full Text]
  28. Monti P, Leone BE, Zerbi A, Balzano G, Cainarca S, Sordi V, Pontillo M, Mercalli A, Di C, V, Allavena P, Piemonti L. Tumor-derived MUC1 mucins interact with differentiating monocytes and induce IL-10highIL-12low regulatory dendritic cell. J Immunol 172: 7341–7349, 2004.[Abstract/Free Full Text]
  29. O'Connor JC, Julian J, Lim SD, Carson DD. MUC1 expression in human prostate cancer cell lines and primary tumors. Prostate Cancer Prostatic Dis 8: 36–44, 2005.[CrossRef][ISI][Medline]
  30. Park H, Hyun SW, Kim KC. Expression of MUC1 mucin gene by hamster tracheal surface epithelial cells in primary culture. Am J Respir Cell Mol Biol 15: 237–244, 1996.[Abstract]
  31. Paul E, Lee DI, Hyun SW, Gendler S, Kim KC. Identification and characterization of high molecular-mass mucin-like glycoproteins in the plasma membrane of airway epithelial cells. Am J Respir Cell Mol Biol 19: 681–690, 1998.[Abstract/Free Full Text]
  32. Pemberton L, Taylor-Papadimitriou J, Gendler SJ. Antibodies to the cytoplasmic domain of the MUC1 mucin show conservation throughout mammals. Biochem Biophys Res Commun 185: 167–175, 1992.[CrossRef][ISI][Medline]
  33. Perez A, Davis PB. Gene profile changes after Pseudomonas aeruginosa exposure in immortalized airway epithelial cells. J Struct Funct Genomics 5: 179–194, 2004.[CrossRef][Medline]
  34. Robbins DJ, Zhen E, Owaki H, Vanderbilt CA, Ebert D, Geppert TD, Cobb MH. Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro. J Biol Chem 268: 5097–5106, 1993.[Abstract/Free Full Text]
  35. Rose MC, Voynow JA. Respiratory tract mucin genes and mucin glycoproteins in health and disease. Physiol Rev 86: 245–278, 2006.[Abstract/Free Full Text]
  36. Shanley TP, Vasi N, Denenberg A. Regulation of chemokine expression by IL-10 in lung inflammation. Cytokine 12: 1054–1064, 2000.[CrossRef][ISI][Medline]
  37. Shao MX, Nadel JA. Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-{alpha}-converting enzyme. J Immunol 175: 4009–4016, 2005.[Abstract/Free Full Text]
  38. Shirasaki H, Kanaizumi E, Watanabe K, Konno N, Sato J, Narita S, Himi T. Tumor necrosis factor increases MUC1 mRNA in cultured human nasal epithelial cells. Acta Otolaryngol 123: 524–531, 2003.[CrossRef][Medline]
  39. Sivko GS, DeWille JW. CCAAT/enhancer binding protein {delta} (C/EBP{delta}) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the C/EBP{delta} growth inhibitory pathway in breast cancer cell lines. J Cell Biochem 93: 830–843, 2004.[CrossRef][ISI][Medline]
  40. Skerrett SJ, Martin TR, Chi EY, Peschon JJ, Mohler KM, Wilson CB. Role of the type 1 TNF receptor in lung inflammation after inhalation of endotoxin or Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol 276: L715–L727, 1999.[Abstract/Free Full Text]
  41. Thathiah A, Brayman M, Dharmaraj N, Julian JJ, Lagow EL, Carson DD. Tumor necrosis factor {alpha} stimulates MUC1 synthesis and ectodomain release in a human uterine epithelial cell line. Endocrinology 145: 4192–4203, 2004.[Abstract/Free Full Text]
  42. Trisciuoglio D, Iervolino A, Candiloro A, Fibbi G, Fanciulli M, Zangemeister-Wittke U, Zupi G, Del Bufalo D. bcl-2 induction of urokinase plasminogen activator receptor expression in human cancer cells through Sp1 activation: involvement of ERK1/ERK2 activity. J Biol Chem 279: 6737–6745, 2004.[Abstract/Free Full Text]
  43. Wang H, Lillehoj EP, Kim KC. Identification of four sites of stimulated tyrosine phosphorylation in the MUC1 cytoplasmic tail. Biochem Biophys Res Commun 310: 341–346, 2003.[CrossRef][ISI][Medline]
  44. Wang H, Lillehoj EP, Kim KC. MUC1 tyrosine phosphorylation activates the extracellular signal-regulated kinase. Biochem Biophys Res Commun 321: 448–454, 2004.[CrossRef][ISI][Medline]
  45. Zaas AK, Schwartz DA. Innate immunity and the lung: defense at the interface between host and environment. Trends Cardiovasc Med 15: 195–202, 2005.[CrossRef][ISI][Medline]
  46. Zhang P, Summer WR, Bagby GJ, Nelson S. Innate immunity and pulmonary host defense. Immunol Rev 173: 39–51, 2000.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
K. C. Kim and E. P. Lillehoj
MUC1 Mucin: A Peacemaker in the Lung
Am. J. Respir. Cell Mol. Biol., December 1, 2008; 39(6): 644 - 647.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
K. Ueno, T. Koga, K. Kato, D. T. Golenbock, S. J. Gendler, H. Kai, and K. C. Kim
MUC1 Mucin Is a Negative Regulator of Toll-Like Receptor Signaling
Am. J. Respir. Cell Mol. Biol., March 1, 2008; 38(3): 263 - 268.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/L693    most recent
00491.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koga, T.
Right arrow Articles by Kim, K. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koga, T.
Right arrow Articles by Kim, K. C.