Neutrophil elastase stimulates MUC1 gene expression through increased Sp1 binding to the MUC1 promoter
Ippei Kuwahara,1
Erik P. Lillehoj,1
Akinori Hisatsune,1
Wenju Lu,1
Yoichiro Isohama,2
Takeshi Miyata,2 and
K. Chul Kim1,3
1Department of Pharmaceutical Sciences, School of Pharmacy, and 3Department of Medicine, School of Medicine, University of Maryland, Baltimore, Maryland; and 2Department of Chemico-Pharmacology, Graduate School of Medicine and Pharmacy, Kumamoto University, Kumamoto, Japan
Submitted 20 January 2005
; accepted in final form 21 April 2005
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ABSTRACT
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We previously reported MUC1 was a cell surface receptor for Pseudomonas aeruginosa, and binding of bacteria to cells was significantly reduced by pretreatment with neutrophil elastase (NE) (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181L187, 2001). The current study was conducted to ascertain NE effects on MUC1 gene transcription, and MUC1 protein synthesis and degradation. A549 human lung carcinoma cells treated with NE exhibited significantly higher MUC1 protein levels in detergent lysates compared with cells treated with vehicle alone. Also, MUC1 protein shed into cell-conditioned medium was rapidly and completely degraded by NE. Actinomycin D blocked NE-stimulated increase in MUC1 protein expression, suggesting a mechanism of increased gene transcription that was confirmed by measurement of quantitatively greater MUC1 mRNA levels in NE-treated cells compared with controls. However, NE did not alter MUC1 mRNA stability, implying increased de novo transcription induced by the protease. NE increased promoter activity in A549 cells transfected with MUC1 gene promoter-luciferase reporter plasmid. This effect of NE was completely blocked by mithramycin A, an inhibitor of Sp1, as well as mutation of one of the putative Sp1 binding sites in MUC1 promoter located at 99/90 relative to transcription initiation site. EMSA revealed NE enhanced binding of Sp1 to this 10-bp segment in a time-dependent manner. These results indicate the increase in MUC1 gene transcription by NE is mediated through increase in Sp1 binding to 99/90 segment of MUC1 promoter.
mucin; transcription; protease; epithelial; mithramycin A
NEUTROPHILS ARE MAJOR EFFECTOR CELLS in the clearance of bacteria from sites of infection. Their antimicrobial function is directly mediated through phagocytosis and indirectly by release of bacteriostatic and bactericidal mediators (9). Neutrophil elastase (NE) is a serine protease required for host defense against gram-negative bacteria (4). NE is stored in azurophil granules and released extracellularly during the course of an infection (7). Part of the mechanism by which NE is involved in innate immunity at mucosal surfaces involves upregulation of mucin secretion (56) and degradation of bacterial virulence factors, such as flagella (40).
MUC1 was the first member of the mucin gene family to be cloned (21). Currently, 20 MUC genes have been identified, and their encoded mucin glycoproteins are classified as gel forming, secreted, or cell associated (20, 22, 38). Gel-forming mucins (MUC2, 5AC, 5B, 7, and 8) are produced by mucosal epithelial cells and account for the viscoelastic property of the overlying mucus layer. MUC1, 3A, 3B, 4, 13, and 16 are transmembrane glycoproteins localized on the cell surface. MUC1 is unique among the membrane-bound mucins because its cytosolic region contains amino acid sequence motifs mediating signal transduction cascades (11, 20). However, the roles of individual MUC gene products in mucosal cell physiology and their possible involvement in pathology are unknown.
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen responsible for a wide range of pulmonary infections, one of the most debilitating being chronic lung infection in cystic fibrosis. As a result of P. aeruginosa infection, cystic fibrosis patients mount an inflammatory response characterized by airway mucin hypersecretion, high levels of proinflammatory cytokines in bronchoalveolar lavage fluid, and lung neutrophilia (42). Neutrophils infiltrating P. aeruginosa-infected lungs secrete NE, and elevated levels of the protease were documented in cystic fibrosis lungs compared with controls (15).
Our prior studies demonstrated that airway epithelial cells express MUC1 protein (46, 47), and cells expressing MUC1 protein on their surface exhibited increased adhesion of P. aeruginosa by virtue of a specific interaction between the MUC1 extracellular region and bacterial flagellin, the major structural protein of the flagellum (35, 36). Moreover, bacterial adhesion to the cells was significantly reduced by pretreatment of cells with NE (35), indicating that NE has an ability to cleave MUC1 on the cell surface. These results complemented those of others who showed that secreted mucins purified from the sputum of cystic fibrosis patients were tightly bound to P. aeruginosa (10, 48).
Recently, Voynow et al. (17, 56) reported that NE increased the mRNA levels of both a major secretory mucin, MUC5AC, and a transmembrane mucin, MUC4, in airway epithelial cells by increasing their mRNA stabilities without affecting gene transcription. In light of these studies, we were interested in determining whether or not NE also affected MUC1 gene and protein expression, and, if so, ascertaining the molecular mechanism involved. Our results demonstrated that NE also enhanced the expression of MUC1 at both the mRNA and protein levels. However, unlike the other two mucins, the increase in MUC1 mRNA was not due to an increase in stability but was a result of increased transcription mediated by Sp1.
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MATERIALS AND METHODS
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Materials.
All reagents were from Sigma (St. Louis, MO) unless otherwise indicated. A549, a human lung carcinoma cell line, was from American Type Culture Collection (Manassas, VA). NE was from Elastin Products (Owensville, MO). MUC1 monoclonal antibody GP1.4 against the extracellular (EC) tandem repeats was from Biomeda (Foster City, CA).
Effect of NE on MUC1 protein expression and shedding.
A549 cells were seeded at 5 x 104 cells/well in 24-well plates in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Invitrogen, Carlsbad, CA) and incubated at 37°C in a humidified CO2 incubator. Confluent monolayers were washed with serum-free DMEM and treated with 100 nM (2.6 U/ml) NE or vehicle control (PBS) for 24 h. In some experiments, the cells were pretreated for 1 h with 5 µg/ml of actinomycin D or 10 µg/ml of cycloheximide (Chx) before NE treatment. After treatment, cell-conditioned media were collected, and the cells were washed with PBS and lysed with 500 µl/well of PBS containing 1.0% Triton X-100, 1% sodium deoxycholate, 1% protease inhibitor cocktail, and 2 mM PMSF. Lysates and conditioned media were incubated for 20 min at 80°C to inactivate residual NE, insoluble material was removed by centrifugation at 14,000 g for 10 min at 4°C, and protein concentrations were measured by the procedure of Bradford (8) using BSA as standard. MUC1 protein levels in cell lysates and conditioned media were determined by ELISA. Cytotoxicity by mithramycin A was monitored by lactate dehydrogenase (LDH) assay kit (Sigma). Briefly, confluent cultures were treated with 100 or 500 nM mithramycin A for 30 min, and the LDH activities in spent media and cell lysates were measured according to the manufacturer's protocol. The ratios of LDH activities in the spent media to the total LDH activities in the culture were compared between control and treated groups to assess significant differences.
ELISA.
A549 cell lysates or cell-conditioned culture media were centrifuged at 10,000 g for 10 min at 4°C, added in triplicate to 96-well ELISA plates (MaxiSorb; Nalge Nunc, Rochester, NY), and incubated overnight at 4°C. Wells were blocked for 1 h at room temperature (
22°C) with PBS, pH 7.0, containing 10 mg/ml BSA and 50 mg/ml sucrose and washed with PBS containing 0.05% Tween 20 (PBS-T). The samples were reacted for 2 h at room temperature with 200 µg/ml of primary MUC1 antibody (GP1.4), washed with PBS-T, reacted for 2 h at room temperature with 200 µg/ml of secondary antibody (peroxidase-conjugated goat anti-mouse IgG; KPL, Gaithersburg, MD), and washed with PBS-T. Bound antibodies were detected with tetramethylbenzidine substrate (SureBlue, KPL), the substrate reaction was stopped with 1 N HCl, and absorbencies at 450 nm were measured using a microplate spectrophotometer (Dexall, Gaithersburg, MD).
Effect of NE on MUC1 mRNA expression.
A549 cells were seeded in DMEM plus 10% FBS in 24-well plates at 5.0 x 104 cells/well, cultured to confluence for 24 h at 37°C, and treated for 24 h with various concentrations of NE (0, 10, 25, 50, 100, or 150 nM) or vehicle control. In experiments to determine mRNA stability, the cells were treated with 100 nM NE for 24 h and then chased in the presence of 5.0 µg/ml of actinomycin D for 0, 2, 4, 8, or 24 h. At the end of each chase period, the cells were washed with PBS and MUC1 transcripts were quantified by real-time RT-PCR.
Real-time RT-PCR.
Total RNA was isolated using RNeasy (Qiagen, Valencia, CA), and 2 µg were reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA) in a total volume of 20 µl. Real-time PCR was carried out using the Taqman probes and iQ Supermix (Bio-Rad) according to the manufacturer's instructions. Briefly, 2 µl of cDNA or control plasmid were used as template for amplification in the iCycler (Bio-Rad) with 200 nM MUC1 or GAPDH (internal control) primers and probes. The primers and Taqman 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-13'; 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-13'). PCR cycles (n = 40) consisted of a 15-s melt at 95°C, followed by annealing at 60°C for 30 s and extension at 72°C for 30 s. 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. Standard curves for the MUC1 and GAPDH transcripts were generated by serial dilution of a pCMV vector containing the MUC1 cDNA and the pBluescriptSK(-) vector containing the GAPDH cDNA. The levels of MUC1 and GAPDH mRNAs were calculated from the standard curves, and the expression of MUC1 transcripts was normalized to GAPDH transcripts.
Preparation of the MUC1 promoter-luciferase reporter plasmid.
The MUC1 promoter was cloned by PCR using primers designed to amplify between nucleotides 2830 and +33 relative to the transcription initiation site and incorporating KpnI and HindIII restriction sites (30). The PCR product was digested with KpnI and HindIII, isolated by agarose gel electrophoresis, and ligated into the corresponding sites of the pGL2-basic luciferase vector (pGL2b; Promega, Madison, WI) to give the MUC1-pGL2b reporter plasmid. MUC1 promoter deletion-luciferase constructs were prepared as follows: 1) digestion with BsmBI, EagI, PvuII, AvrII, or AgeI, 2) treatment with mung bean nuclease (New England Biolabs, Beverly, MA) or Klenow enzymes (New England Biolabs) to get blunt ends, and 3) re-ligation. Point-mutated MUC1 promoter 97/96(GG
AA)-luciferase construct was synthesized using QuickChange II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The primers for mutagenesis were forward: 5'-GTAGGGGAGGGAACGGGGTTTTGTCACCTG-3' and reverse: 5'-CAGGTGACAAAACCCCGTTCCCTCCCCTAC-3'. PCR cycles (n = 20) consisted of melting at 95°C for 45 s followed by annealing at 55°C for 2 min and extension at 68°C for 10 min. Putative Sp1 binding sites were searched using TFSEARCH software (http://mbs.cbrc.jp/research/db/TFSEARCH.html). Fidelity of the MUC1 promoter-luciferase constructs was confirmed by automated DNA sequencing analysis at the University of Maryland Biopolymer Core Facility.
Transient transfection and luciferase assay.
A549 cells were seeded in DMEM plus 10% FBS in 24-well plates, incubated for 24 h at 37°C to
7080% confluence, and transfected with the MUC1-pGL2b plasmid or empty vector control using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, the DNA sample was mixed with 2 µl of Lipofectamine 2000 and diluted with OPTI-MEM I (Invitrogen) to 100 µl. After a 20-min incubation at room temperature, DNA-Lipofectamine 2000 mixture in a final volume of 600 µl was added to each well and incubated for 24 h. The DNA sample consisted of 800 ng of the MUC1-pGL2b plasmid or empty vector plus 10 ng of the phRL-TK internal control plasmid (Promega). After transfection, the cells were washed with serum-free DMEM and treated with 100 nM NE or vehicle control for 24 h at 37°C. In some experiments, the transfected cells were pretreated for 30 min with mithramycin A, an Sp1 inhibitor (5, 49), before NE treatment. 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.
EMSA.
Confluent A549 cells were treated with 100 nM NE for 0, 10, 20, 30, 60, or 120 min. After treatment, nuclear extracts were prepared as previously described (31). Briefly, cells were washed three times with ice-cold PBS containing 1 mM PMSF and lysed with buffer A (10 mM HEPES-KOH, pH 7.8, 1.5 mM MgCl2, 10 mM NaCl, 1 mM DTT, 0.25% Igepal CA-630, 1 mM PMSF, and 1% protease inhibitor cocktail) on ice for 10 min. Nuclei were pelleted by centrifugation (1 min at 1,250 g at 4°C) and lysed in buffer B (20 mM HEPES-KOH, pH 7.8, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1% protease inhibitor cocktail) for 30 min on ice. The nuclear lysate was centrifuged at 13,200 g for 5 min at 4°C. The supernatant was collected and dialyzed against buffer C (20 mM HEPES-KOH, pH 7.8, 20% glycerol, 50 mM KCl, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF, and 1% protease inhibitor cocktail) for 2 h at 4°C. The dialyzed nuclear lysate was referred to as the nuclear extract and used for EMSA. Protein concentrations were measured by the procedure of Bradford (8). Twenty micrograms of the nuclear extract were incubated for 30 min on ice with a combination of 1 µg of poly(dI-dC) (Pharmacia, Piscataway, NJ) as a nonspecific competitor, 100-fold excess of the unlabeled oligonucleotide, or 2 µg of rabbit anti-Sp1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA) for supershift analysis, and the mixture was incubated with a [
-32P]-labeled oligonucleotide representing the human MUC1 promoter between nucleotides 104 and 83. The DNA-protein complexes were resolved on 4.5% polyacrylamide gels and analyzed by autoradiography as described (31).
Statistical analysis.
Differences in means ± SE values among groups were assessed using the Student's t-test for unpaired samples and considered significant at P < 0.05.
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RESULTS
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NE increases MUC1 protein expression and degrades shed MUC1.
As shown in Fig. 1A, the amount of MUC1 released into the culture medium during a 24-h period is almost negligible compared with the cellular MUC1 levels that remained almost unchanged even in the presence of 5 µg/ml of actinomycin D or 10 µg/ml of Chx. These results suggest that the rates of MUC1 production (transcription and translation) are very low and cellular MUC1 protein is quite stable in confluent A549 cells. Treatment of A549 cells with NE, however, increased MUC1 levels in cell lysates by 33% (P < 0.05) compared with its 24-h control. The increase in the cellular MUC1 protein content following NE could be due to an increase in transcription, translation, stability of MUC1 mRNA or protein, or a decrease in release. Because NE has been shown to induce MUC1 release from the cell surface (25), we ruled out the possibility of decreased release. To determine whether the higher MUC1 levels were due to increased transcription or translation, cells were pretreated for 1 h with actinomycin D or Chx before 24-h NE treatment to block MUC1 transcription or MUC1 translation, respectively. Figure 1A shows that pretreatment of the cells with actinomycin D or Chx before NE treatment decreased the amounts of cellular MUC1 by >95% with no significant difference between the two treatment groups. Because treatment with either actinomycin D or Chx alone did not significantly affect cellular MUC1 levels, these results suggest that the increase in cellular MUC1 protein levels following treatment with NE was due mainly to an increase in transcription. The fact that the cellular content of MUC1 in the actinomycin D plus NE group was not greater than that in the Chx plus NE group indicates that NE did not increase MUC1 translation during the 24-h treatment period. Likewise, it is highly unlikely that the increase in cellular MUC1 levels was induced by an increase in stability of either MUC1 mRNA or protein based on these same data. Therefore, we decided to focus on increased transcription of the MUC1 gene to explain the NE-induced increase in cellular MUC1 levels.

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Fig. 1. Neutrophil elastase (NE) increases MUC1 protein expression and degrades shed MUC1. A and B: confluent A549 cells were incubated in the presence or absence of 5 µg/ml of actinomycin D (AcD) or 10 µg/ml of cycloheximide (Chx) for 1 h and were either nontreated or treated for 24 h with 100 nM NE. MUC1 protein levels in cell lysates (A) and spent media (B) were measured by ELISA. Each bar represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *Significantly different from 24-h non-NE-treated sample (P < 0.05). Significantly different from 24-h NE-treated samples (P < 0.05). C: aliquots of spent media obtained from confluent A549 cells were nontreated (0 h) or treated for 24 h with 100 nM NE or vehicle control, and MUC1 levels in the spent media were determined by ELISA. Each bar represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *Significantly different from 0 h (P < 0.05).
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Figure 1B shows that despite the very low levels of released MUC1 in all groups compared with cellular MUC1 levels, the spent media of NE-treated groups contained much less MUC1 compared with the control group; 45% for NE alone, 7% for NE plus actinomycin D, and 7% for NE plus Chx. These results suggest degradation of MUC1 by NE. Because the amount of released MUC1 in the NE alone group was higher than NE plus actinomycin D or NE plus Chx, NE probably induced shedding of both the original and newly synthesized MUC1 induced by NE. To verify the ability of NE to degrade MUC1, aliquots of a spent medium sample containing MUC1 molecules were incubated for 24 h in the presence of NE, and the amounts of MUC1 were measured by ELISA. As seen in Fig. 1C, there were no detectable amounts of MUC1, indicating that all the MUC1 molecules released into the medium during the 24-h period were degraded by NE, consistent with our previous finding (25). Collectively, these results indicate that NE stimulates the production of MUC1, MUC1 molecules transported to the cell surface are constantly shed by a NE proteolytic mechanism (25), and MUC1 released into the culture medium is rapidly degraded.
NE increases MUC1 mRNA levels in dose- and time-dependent manners.
To confirm that the increase in cellular MUC1 content following NE treatment was due to an increase in transcription, we measured MUC1 mRNA levels in the presence or absence of NE. A549 cells treated with increasing doses of NE (Fig. 2A) and for increasing time periods (Fig. 2B) exhibited progressively higher MUC1 mRNA levels as determined by real time RT-PCR.

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Fig. 2. NE increases MUC1 mRNA levels in dose- and time-dependent manners. A: confluent A549 cells were treated for 24 h with 0, 10, 25, 50, 100, or 150 nM NE, and MUC1 mRNA levels were measured by real-time RT-PCR. MUC1 mRNA levels were normalized to GAPDH mRNA levels. Each data point represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *Significantly increased MUC1 mRNA levels in NE-treated cells compared with nontreated (0 nM NE) cells (P < 0.05). B: confluent A549 cells were treated for 0, 2, 4, 8, or 24 h with 100 nM NE or vehicle control (PBS), and MUC1 mRNA levels were measured by real-time RT-PCR. MUC1 mRNA levels were normalized to GAPDH mRNA levels. Each data point represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *Significantly increased MUC1 mRNA levels in NE-treated cells compared with control-treated cells (P < 0.05).
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NE treatment does not alter MUC1 mRNA stability.
Increased MUC1 mRNA levels induced by NE could have been due to higher de novo mRNA synthesis and/or greater mRNA stability. It has been previously shown that NE increases mRNA stability of both a secretory mucin, MUC5AC (56), and a transmembrane mucin, MUC4 (17). Therefore, we examined MUC1 mRNA stability following NE treatment. A549 cells were treated with NE for 24 h and chased in the presence of actinomycin D for various time periods. As shown in Fig. 3, the kinetics of MUC1 mRNA levels was identical in NE- and control-treated cells. These results indicate that increased mRNA stability was not responsible for the increased MUC1 mRNA levels induced by NE, unlike the other MUC genes (17, 56).

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Fig. 3. NE treatment does not alter MUC1 mRNA stability. Confluent A549 cells were treated with 100 nM NE for 24 h and then chased in the presence of 5 µg/ml of AcD for 0, 2, 4, 8, or 24 h. MUC1 mRNA levels were measured by real-time RT-PCR and normalized to GAPDH mRNA levels. Each data point represents the mean ± SE (n = 3). Data are representative of 3 separate experiments.
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NE increases MUC1 promoter activity through an Sp1-dependent mechanism.
To confirm transcriptional stimulation of MUC1 by NE, we assessed MUC1 gene promoter activity in the presence or absence of NE treatment by transfecting A549 cells with a MUC1 promoter-luciferase reporter plasmid and subsequently measuring luciferase activity. Cells transfected with the MUC1-pGL2b reporter construct and treated with NE exhibited significantly higher luciferase activity compared with MUC1-pGL2b-expressing cells treated with the vehicle control or cells transfected with the empty pGL2b plasmid and treated with NE (Fig. 4). These results, therefore, confirmed the stimulatory effect of NE on MUC1 gene transcription and also provided a basis for further analysis of MUC1 transcriptional regulation by NE.

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Fig. 4. NE increases MUC1 promoter activity. A549 cells (7080% confluent) were transfected with the pGL2b empty vector or the MUC1-pGL2b plasmid containing the MUC1 promoter, incubated for 24 h, and treated for 24 h with 100 nM NE or vehicle control, and luciferase activity was determined. Each data point represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *Significantly increased luciferase activity in MUC1-pGL2b-transfected cells treated with NE compared with vehicle control (P < 0.05).
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The mechanisms of transcriptional regulation of MUC1 are poorly understood. Using mutational analysis, Kovarik et al. (27) previously demonstrated that Sp1 plays a crucial role in MUC1 transcriptional regulation. Therefore, we determined whether or not NE-induced increase in MUC1 transcription involves Sp1 binding to the MUC1 promoter. In approaching this question, we used the chemical inhibitor mithramycin A, which is known to inhibit the Sp1 binding to DNA (5, 49). Pretreatment of MUC1-pGL2b-transfected cells with 100 nM mithramycin A reduced baseline MUC1 transcription by 75% and completely abolished NE-induced stimulation of MUC1 transcription (Fig. 5). No cytotoxicity was observed with this concentration of mithramycin A judging from both cell morphology and LDH release (data not shown). These results suggest that both basal and NE-induced transcription of MUC1 is under the control of Sp1, supporting the previous report by Kovarik et al. (28).

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Fig. 5. NE increases MUC1 promoter activity through a Sp1-dependent mechanism. A549 cells (7080% confluent) were transfected with the pGL2b empty vector or the pGL2b plasmid containing the MUC1 promoter, incubated for 24 h, and treated for 30 min with 0, 50, 100, or 500 nM mithramycin A. After being treated, the cells were incubated for 24 h with 100 nM NE or vehicle control, and luciferase activity was determined. Each data point represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *Significantly increased luciferase activity in MUC1-pGL2b-transfected cells treated with NE compared with vehicle control (P < 0.05).
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The Sp1 binding site at 99/90 in the MUC1 promoter is crucial for NE-induced MUC1 transcription.
Because the pharmacological approach suggested involvement of Sp1 in NE-induced MUC1 transcription, we examined binding of Sp1 to the MUC1 promoter. Ten putative Sp1 binding sites are present on the MUC1 promoter. Detailed promoter analysis using several deletion mutants (Fig. 6A) revealed that deletion of promoter sequences from 167 to 66 completely abolished NE-induced MUC1 transcription (Fig. 6B). Because there is only one putative Sp1 binding site (99/90) in this 102-bp region, we determined whether or not Sp1 binding to this segment is responsible for NE-induced MUC1 transcription. Based on a recent report by Sivko et al. (53), we made point mutations at positions 97 and 96 (GG to AA). As seen in Fig. 7, NE-induced MUC1 transcription was completely abolished in cells transfected with this mutated construct, indicating that this Sp1 binding site is crucial for NE-induced MUC1 transcription. Second, EMSA using a radiolabeled nucleotide probe corresponding to nucleotides 103 to 84 revealed that, in the absence of NE, Sp1 specifically bound to this segment (Fig. 8A), and in the presence of NE, the binding to this segment increased dramatically in a time-dependent manner reaching maximum binding at 60 min (Fig. 8B). Together, these results indicate that NE induces MUC1 transcription through an increase in Sp1 binding to its specific cis element located at 99/90 of the MUC1 promoter.

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Fig. 6. MUC1 promoter segment at 167/66 is important for NE-induced MUC1 transcription. A: constructs of MUC1 promoter deletion mutants (DM). White boxes are putative Sp1 binding sites. B: A549 cells (7080% confluent) were transfected with the pGL2b empty vector or pGL2b containing MUC1 promoter deletion mutants. At 24 h following transfection, cells were treated for 24 h with 100 nM NE or vehicle control, and luciferase activity was determined. Each data point represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *Significantly increased luciferase (Luc) activity in MUC1-pGL2b-transfected cells treated with NE compared with vehicle control (P < 0.05).
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Fig. 7. Putative Sp1 binding site at 99/90 in the MUC1 promoter is crucial for NE-induced MUC1 transcription. A549 cells (7080% confluent) were transfected with the pGL2b empty vector or pGL2b containing a MUC1 promoter with point mutations at nucleotides 97 and 96 (GG AA). At 24 h following transfection, cells were treated for 24 h with 100 nM NE or vehicle control, and luciferase activity was determined. Each data point represents the mean ± SE (n = 3). Data are representative of 3 separate experiments. *Significantly increased luciferase activity in MUC1-pGL2b-transfected cells treated with NE compared with vehicle control (P < 0.05).
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Fig. 8. NE enhances the binding of Sp1 to the 99/90 segment in the MUC1 promoter. Confluent A549 cells were treated with vehicle control (A) or 100 nM NE (B) for 0, 10, 20, 30, 60, or 120 min. Cells were lysed, and nuclear extracts were incubated with or without 100-fold molar excess of unlabeled probe, poly(dI-dC) as a nonspecific competitor, or Sp1 antibody for 30 min on ice, and then incubated with a [ -32P]-labeled oligonucleotide corresponding to the human MUC1 promoter between nucleotides 104 and 83. DNA-protein complexes were resolved on 4.5% polyacrylamide gels and analyzed by autoradiography.
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DISCUSSION
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In this study, we demonstrated that MUC1 protein synthesis in confluent A549 cells was almost negligible, but greatly enhanced, following treatment with NE, and this effect was blocked by pretreatment with actinomycin D as well as Chx. By real-time RT-PCR, we observed that MUC1 mRNA levels, but not mRNA stability, were increased by NE. We also confirmed our earlier observation that MUC1 released into the spent medium was rapidly degraded by NE. These results indicate that NE stimulates both synthesis and release of MUC1 and degrades MUC1 released in the culture medium. Using a promoter-luciferase reporter assay, we showed that NE increased MUC1 promoter activity, and this effect was completely blocked by an inhibitor of the Sp1 transcription factor. Based on promoter analysis, we identified a potential Sp1 binding site as being responsible for NE-induced MUC1 transcription. Finally, using EMSA, we demonstrated that NE increased Sp1 binding to this segment in a time-dependent fashion. Collectively, these observations indicated that increased cellular MUC1 levels induced by NE were due to increased MUC1 protein synthesis as a consequence of elevated transcription mediated by Sp1 binding to a specific cis element located at 99/90 of the MUC1 promoter.
Our identification of a transcriptional mechanism for NE-stimulated MUC1 expression corroborates and extends our previous study that revealed a positive regulatory element in the MUC1 gene promoter (31). In addition to our report, the presence of several positive regulatory regions in the MUC1 promoter was previously reported (1, 19, 52). In particular, the MUC1 promoter contains 5 putative NF-
B and 10 potential Sp1 binding sites. As a mechanism for TNF-
-induced stimulation of MUC1 transcription in the presence of IFN-
, Lagow and Carson (29) demonstrated binding of NF-
B to the MUC1 promoter at 589/581. On the other hand, Kovarik et al. (28) reported that binding of Sp1 at 99/90 is crucial for the regulation of MUC1 transcription. On the basis of our present results, an increase in Sp1 binding to the MUC1 promoter seems to be responsible for NE-induced stimulation of MUC1 transcription.
How NE induces increased Sp1 binding specifically to the 99/90 promoter segment remains to be answered. NE-induced Sp1 phosphorylation is a plausible mechanism since it has been shown that Sp1 binding to DNA is enhanced by serine/threonine phosphorylation (14). In addition, Fischer and Voynow (18) showed that NE enhances the stability of MUC5AC mRNA via a pathway involving reactive oxygen species. It would be interesting to determine whether oxidative stress is also responsible for an increase in MUC1 transcription mediated by NE. Further investigation of the signaling pathway responsible for NE-induced MUC1 transcription is currently in progress in our laboratory.
Identification of the NE receptor remains to be determined. Uehara et al. (55) showed that NE induced IL-8 production by fibroblasts through the protease-activated receptor-2 (PAR-2) pathway. In contrast, Dulon et al. (16) reported that NE inactivated PAR-2 in both A549 and 16HBE cells and prevented subsequent activation by trypsin, an agonist for PAR-2. Our recent preliminary data showed that trypsin also induced MUC1 transcription, although its activity was <10% of that of NE (data not shown), which suggests involvement of PAR-2, at least in part, in NE-induced increase in MUC1 transcription.
What could be the physiological importance of NE-induced upregulated MUC1 expression? With the use of a cloned hamster Muc1 cDNA, our previous studies indicated that Muc1 is a pattern recognition receptor on the surface of airway epithelial cells with binding specificity for bacterial flagellin (35, 36). Treatment of Muc1-expressing cells with P. aeruginosa or its purified flagellin induced phosphorylation of the Muc1 cytoplasmic tail (CT), and phosphorylation was stimulated both by P. aeruginosa laboratory strains as well as cystic fibrosis clinical isolates (37). In parallel with the hamster Muc1 studies, a CD8/MUC1 fusion protein containing the EC region of CD8 and the MUC1 CT was used to confirm stimulated phosphorylation of the CT and to identify four sites of tyrosine phosphorylation (58). Further analysis of both the hamster Muc1 and CD8/MUC1 cell culture systems indicated that phosphorylation of Muc1/MUC1 leads to activation of the ERK MAP kinase, indicating a role for this membrane mucin in initiating a signal transduction pathway in response to bacterial infection (37, 59). Combined with the results of the current investigation, these results suggest a model whereby NE released from neutrophils at sites of bacterial infection upregulates MUC1 expression resulting in enhanced innate immunity and pathogen clearance. Current studies in our laboratory are directed at testing this hypothesis.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-47125 and a research grant from the Cystic Fibrosis Foundation.
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FOOTNOTES
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Address for reprint requests and other correspondence: K. C. Kim, Dept. of Pharmaceutical Sciences, School of Pharmacy, Univ. of Maryland, 20 Penn St., Baltimore, MD 21201 (e-mail: kkim{at}umaryland.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
|
---|
- Abe M and 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: 282286, 1993.[Abstract/Free Full Text]
- Amitani R, Wilson R, Rutman A, Read R, Ward C, Burnett D, Stockley RA, and Cole PJ. Effects of human neutrophil elastase and Pseudomonas aeruginosa proteinases on human respiratory epithelium. Am J Respir Cell Mol Biol 4: 2632, 1991.[ISI][Medline]
- Belaaouaj A, Kim KS, and Shapiro SD. Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science 289: 11851188, 2000.[Abstract/Free Full Text]
- Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, and Shapiro SD. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med 4: 615618, 1998.[CrossRef][ISI][Medline]
- Blume SW, Snyder RC, Ray R, Thomas S, Koller CA, and 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: 16131621, 1991.[ISI][Medline]
- Bode W, Meyer E Jr, and Powers JC. Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors. Biochemistry 28: 19511963, 1989.[CrossRef][ISI][Medline]
- Boutten A, Dehoux MS, Seta N, Ostinelli J, Venembre P, Crestani B, Dombret MC, Durand G, and Aubier M. Compartmentalized IL-8 and elastase release within the human lung in unilateral pneumonia. Am J Respir Crit Care Med 153: 336342, 1996.[Abstract]
- Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254, 1976.[CrossRef][ISI][Medline]
- Burg ND and Pillinger MH. The neutrophil: function and regulation in innate and humoral immunity. Clin Immunol 99: 717, 2001.[CrossRef][ISI][Medline]
- Carnoy C, Scharfman A, Van Brussel E, Lamblin G, Ramphal R, and Roussel P. Pseudomonas aeruginosa outer membrane adhesins for human respiratory mucus glycoproteins. Infect Immun 62: 18961900, 1994.[Abstract]
- Carraway KL, Ramsauer VP, Haq B, and Carothers Carraway CA. Cell signaling through membrane mucins. Bioessays 25: 6671, 2003.[CrossRef][ISI][Medline]
- Chen HC, Lin HC, Liu CY, Wang CH, Hwang T, Huang TT, Lin CH, and Kuo HP. Neutrophil elastase induces IL-8 synthesis by lung epithelial cells via the mitogen-activated protein kinase pathway. J Biomed Sci 11: 4958, 2004.[CrossRef][ISI][Medline]
- Chokki M, Yamamura S, Eguchi H, Masegi T, Horiuchi H, Tanabe H, Kamimura T, and Yasuoka S. Human airway trypsin-like protease increases mucin gene expression in airway epithelial cells. Am J Respir Cell Mol Biol 30: 470478, 2004.[Abstract/Free Full Text]
- Chu S and Ferro TJ. Sp1: regulation of gene expression by phosphorylation. Gene 348: 111, 2005.[CrossRef][ISI][Medline]
- Dosanjh AK, Elashoff D, and Robbins RC. The bronchoalveolar lavage fluid of cystic fibrosis lung transplant recipients demonstrates increased interleukin-8 and elastase and decreased IL-10. J Interferon Cytokine Res 18: 851854, 1998.[ISI][Medline]
- Dulon S, Cande C, Bunnett NW, Hollenberg MD, Chignard M, and Pidard D. Proteinase-activated receptor-2 and human lung epithelial cells: disarming by neutrophil serine proteinases. Am J Respir Cell Mol Biol 28: 339346, 2003.[Abstract/Free Full Text]
- Fischer BM, Cuellar JG, Diehl ML, deFreytas AM, Zhang J, Carraway KL, and Voynow JA. Neutrophil elastase increases MUC4 expression in normal human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 284: L671L679, 2003.[Abstract/Free Full Text]
- Fischer BM and Voynow JA. Neutrophil elastase induces MUC5AC gene expression in airway epithelium via a pathway involving reactive oxygen species. Am J Respir Cell Mol Biol 26: 447452, 2002.[Abstract/Free Full Text]
- Gaemers IC, Vos HL, Volders HH, van der Valk SW, and 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: 61916199, 2001.[Abstract/Free Full Text]
- Gendler SJ. MUC1, the renaissance molecule. J Mammary Gland Biol Neoplasia 6: 339353, 2001.[CrossRef][ISI][Medline]
- Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, Lalani EN, and Wilson D. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem 265: 1528615293, 1990.[Abstract/Free Full Text]
- Gendler SJ and Spicer AP. Epithelial mucin genes. Annu Rev Physiol 57: 607634, 1995.[CrossRef][ISI][Medline]
- Jang BC, Lim KJ, Paik JH, Kwon YK, Shin SW, Kim SC, Jung TY, Kwon TK, Cho JW, Baek WK, Kim SP, Suh MH, and Suh SI. Up-regulation of human
-defensin 2 by interleukin-1
in A549 cells: involvement of PI3K, PKC, p38 MAPK, JNK, and NF-
B. Biochem Biophys Res Commun 320: 10261033, 2004.[CrossRef][ISI][Medline]
- Kawabata A, Morimoto N, Nishikawa H, Kuroda R, Oda Y, and Kakehi K. Activation of protease-activated receptor-2 (PAR-2) triggers mucin secretion in the rat sublingual gland. Biochem Biophys Res Commun 270: 298302, 2000.[CrossRef][ISI][Medline]
- Kim KC, Wasano K, Niles RM, Schuster JE, Stone PJ, and Brody JS. Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells. Proc Natl Acad Sci USA 84: 93049308, 1987.[Abstract/Free Full Text]
- Kohri K, Ueki IF, and Nadel JA. Neutrophil elastase induces mucin production by ligand-dependent epidermal growth factor receptor activation. Am J Physiol Lung Cell Mol Physiol 283: L531L540, 2002.[Abstract/Free Full Text]
- Kovarik A, Lu PJ, Peat N, Morris J, and 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: 1814018147, 1996.[Abstract/Free Full Text]
- Kovarik A, Peat N, Wilson D, Gendler SJ, and Taylor-Papadimitriou J. Analysis of the tissue-specific promoter of the MUC1 gene. J Biol Chem 268: 99179926, 1993.[Abstract/Free Full Text]
- Lagow EL and Carson DD. Synergistic stimulation of MUC1 expression in normal breast epithelia and breast cancer cells by interferon-
and tumor necrosis factor-
. J Cell Biochem 86: 759772, 2002.[CrossRef][ISI][Medline]
- Lee IJ, Han F, Baek J, Hisatsune A, and Kim KC. Inhibition of MUC1 expression by indole-3-carbinol. Int J Cancer 109: 810816, 2004.[CrossRef][ISI][Medline]
- Lee IJ, Hyun SW, Nandi A, and Kim KC. Transcriptional regulation of the hamster Muc1 gene: identification of a putative negative regulatory element. Am J Physiol Lung Cell Mol Physiol 284: L160L168, 2003.[Abstract/Free Full Text]
- Lee PW, Wu S, and Lee YM. Differential expression of µ-opioid receptor gene in CXBK and B6 mice by Sp1. Mol Pharmacol 66: 15801584, 2004.[Abstract/Free Full Text]
- Lee YW, Kuhn H, Hennig B, Neish AS, and Toborek M. IL-4-induced oxidative stress upregulates VCAM-1 gene expression in human endothelial cells. J Mol Cell Cardiol 33: 8394, 2001.[CrossRef][ISI][Medline]
- Lillehoj EP, Han F, and Kim KC. Mutagenesis of a Gly-Ser cleavage site in MUC1 inhibits ectodomain shedding. Biochem Biophys Res Commun 307: 743749, 2003.[CrossRef][ISI][Medline]
- Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol 280: L181L187, 2001.[Abstract/Free Full Text]
- Lillehoj EP, Kim BT, and Kim KC. Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin. Am J Physiol Lung Cell Mol Physiol 282: L751L756, 2002.[Abstract/Free Full Text]
- Lillehoj EP, Kim H, Chun EY, and 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: L809L815, 2004.[Abstract/Free Full Text]
- Lillehoj ER and Kim KC. Airway mucus: its components and function. Arch Pharm Res 25: 770780, 2002.[ISI][Medline]
- Lin YZ, Yao SY, Veach RA, Torgerson TR, and Hawiger J. Inhibition of nuclear translocation of transcription factor NF-
B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J Biol Chem 270: 1425514258, 1995.[Abstract/Free Full Text]
- Lopez-Boado YS, Espinola M, Bahr S, and Belaaouaj A. Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity. J Immunol 172: 509515, 2004.[Abstract/Free Full Text]
- Lundgren JD, Rieves RD, Mullol J, Logun C, and Shelhamer JH. The effect of neutrophil protenase enzymes on the release of mucus from feline and human airway cultures. Respir Med 88: 511518, 1994.[ISI][Medline]
- Lyczak JB, Cannon CL, and Pier GB. Lung infections associated with cystic fibrosis. Clin Microbiol Rev 15: 194222, 2002.[Abstract/Free Full Text]
- Morris JR and Taylor-Papadimitriou J. The Sp1 transcription factor regulates cell type-specific transcription of MUC1 DNA. Cell Biol 20: 133139, 2001.
- Nakamura H, Yoshimura K, McElvaney NG, and Crystal RG. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J Clin Invest 89: 14781484, 1992.[ISI][Medline]
- O'Riordan TG, Otero R, Mao Y, Lauredo I, and Abraham WM. Elastase contributes to antigen-induced mucociliary dysfunction in ovine airways. Am J Respir Crit Care Med 155: 15221528, 1997.[Abstract]
- Park H, Hyun SW, and Kim KC. Expression of MUC1 mucin gene by hamster tracheal surface epithelial cells in primary culture. Am J Respir Cell Mol Biol 15: 237244, 1996.[Abstract]
- Paul E, Lee DI, Hyun SW, Gendler S, and 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: 681690, 1998.[Abstract/Free Full Text]
- Ramphal R, Guay C, and Pier GB. Pseudomonas aeruginosa adhesins for tracheobronchial mucin. Infect Immun 55: 600603, 1987.[ISI][Medline]
- Ray R, Snyder RC, Thomas S, Koller CA, and Miller DM. Mithramycin blocks protein binding and function of the SV40 early promoter. J Clin Invest 83: 20032007, 1989.[ISI][Medline]
- Rose MC. Epithelial mucous glycoproteins and cystic fibrosis. Horm Metab Res 20: 601608, 1988.[ISI][Medline]
- Shapiro SD. Neutrophil elastase: path clearer, pathogen killer, or just pathologic? Am J Respir Cell Mol Biol 26: 266268, 2002.[Free Full Text]
- Shirotani K, Taylor-Papadimitriou J, Gendler SJ, and Irimura T. Transcriptional regulation of the MUC1 mucin gene in colon carcinoma cells by a soluble factor. Identification of a regulatory element. J Biol Chem 269: 1503015035, 1994.[Abstract/Free Full Text]
- Sivko GS, Sanford DC, Dearth LD, Tang D, and DeWille JW. CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: II. Analysis of activating signal transduction pathways, transcriptional, post-transcriptional, and post-translational control. J Cell Biochem 93: 844856, 2004.[CrossRef][ISI][Medline]
- Tkalcevic J, Novelli M, Phylactides M, Iredale JP, Segal AW, and Roes J. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12: 201210, 2000.[CrossRef][ISI][Medline]
- Uehara A, Muramoto K, Takada H, and Sugawara S. Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2. J Immunol 170: 56905696, 2003.[Abstract/Free Full Text]
- Voynow JA, Young LR, Wang Y, Horger T, Rose MC, and Fischer BM. Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells. Am J Physiol Lung Cell Mol Physiol 276: L835L843, 1999.[Abstract/Free Full Text]
- Walsh DE, Greene CM, Carroll TP, Taggart CC, Gallagher PM, O'Neill SJ, and McElvaney NG. Interleukin-8 up-regulation by neutrophil elastase is mediated by MyD88/IRAK/TRAF-6 in human bronchial epithelium. J Biol Chem 276: 3549435499, 2001.[Abstract/Free Full Text]
- Wang H, Lillehoj EP, and Kim KC. Identification of four sites of stimulated tyrosine phosphorylation in the MUC1 cytoplasmic tail. Biochem Biophys Res Commun 310: 341346, 2003.[CrossRef][ISI][Medline]
- Wang H, Lillehoj EP, and Kim KC. MUC1 tyrosine phosphorylation activates the extracellular signal-regulated kinase. Biochem Biophys Res Commun 321: 448454, 2004.[CrossRef][ISI][Medline]
- Wasano K, Kim KC, Niles RM, and Brody JS. Membrane differentiation markers of airway epithelial secretory cells. J Histochem Cytochem 36: 167178, 1988.[Abstract]
- Weinrauch Y, Drujan D, Shapiro SD, Weiss J, and Zychlinsky A. Neutrophil elastase targets virulence factors of enterobacteria. Nature 417: 9194, 2002.[CrossRef][ISI][Medline]
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