The mucin-producing human NCI-H292 airway epithelial cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum (FBS; Hyclone Laboratories Inc., Logan, UT, USA). The cells were grown at 37℃ in 5% CO₂ fully humidified air, and were subcultured twice weekly. The cells were seeded in a 6-well plate at 1×10⁵ cells/well. When the cultured cells were in a confluent state, the cultured cells were incubated in RPMI 1640 medium containing 0.5% FBS for 24 hours. The cultured cells were then rinsed with serum-free RPMI 1640 medium and exposed to the indicated concentrations of PMA (Sigma, St. Louis, MO, USA), MMP-9 (R&D Systems Inc., Minneapolis, MN, USA), and doxycycline (Sigma). In order to investigate the signaling pathway of PMA-induced MUC5B expression, SB2035 (BOMOL Research Laboratories, Plymouth Meeting, PA, USA), as specific inhibitors, was used to pretreat the human NCI-H292 airway epithelial cells for 1 hour before exposure to the indicated concentrations of PMA. In the case of the controls, the cultured cells were incubated with medium alone for the same amount of time.

The total RNAs from PMA treated the cultured cells were prepared using the available reagent (TRIzol; Invitrogen) according to the manufacturer's instructions. Fifteen micrograms of the RNA were reverse-transcribed into cDNA at 37℃ for 70 minutes in 60 µL volume of reaction mixture that contained 150 U of reverse-transcriptase (Superscript-II; Invitrogen), 10 mM each of dATP, dTTP, dCTP, and dGTP, and 5 µL of 50 µM Oligo-dT primer/mL (Amersham International PLC, Little Chalfont, Buckinghamshire, UK). The reactions were stopped by heat inactivation at 85℃ for 10 minutes. Two microliters of each cDNA sample, from the reverse transcription, were amplified by PCR (PTC-200, MJ Research Inc., Watertown, MA, USA) in a volume of 50 µL that contained 0.5 U of Taq DNA polymerase, 2 µL of 50 mM MgCl₂, 1 µL each 0f 10 mM dATP, dTTP, dCTP, and dGTP, and 1 µL of the 10 µM primers. The PCR of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed on each individual sample as a positive internal control. The primer sequences used in the PCR were 5'-CAC ATC CAC CCT TCC AAC-3' (sense) and 5'-GGC TCA TTG TCG TCT CTG-3' (antisense) for MUC5B (245 bp), and 5'-CCT CCA AGG AGT AAG ACC CC-3' (sense) and 5'-AGG GGT CTA CAT GGC AAC TG-3' (antisense) for GAPDH (145 bp). After a hot start, the amplification profile was 30 cycles for 1 minute of denaturation at 95℃, 30 seconds of annealing at 60℃, and 1 minute of extension at 72℃ in the presence of 2.5 mg MgCl₂ for MUC5B and MMP-9. The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and visualized by UV fluorescence. Semiquantitative analysis of the RT-PCR product was performed on the scanned gel images, and the intensity of the PCR product was measured using commercially available imaging software (Scion Software; Scion Co., Frederick, MD, USA). The relative intensity of the individual bands on the gel image was determined as the ratio of the intensities of MUC5B and MMP-9 to the intensity of GAPDH.

MUC5B protein was determined by ELISA. Samples of the supernatant or cell lysates from the cultured cells were prepared in phosphate-buffered saline (PBS) at several dilutions, and each sample was incubated at 40℃ in a 96-well plate until dry. The plates were then washed three times with PBS, blocked with 2% bovine serum albumin for 1 hour at room temperature, washed again three times with PBS, and incubated with primary antibody of MUC5B (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted with PBS containing 0.05% Tween 20 for 1 hour. The wells were then washed three times with PBS and treated with horseradish peroxidase-conjugated secondary antibody of MUC5B (Santa Cruz Biotechnology). After 4 hours, the plates were washed three times with PBS. Color was developed using 3,3',5,5'-tetramethylbenzidine peroxidase solution and stopped with 2N-H₂SO₄. The absorbance was read at 450 nm.

MMP-9 protein activity was measured by gelatin zymography. In brief, the cells culture supernatants containing 10 µg of protein were mixed with 2×sodium-dodecyl sulfate loading buffer before being loaded onto 10% polyacrylamide gels (wt/vol) containing 0.1% (wt/vol) gelatin as a substrate (Biotech, Piscataway, NJ, USA). The protein was subjected to electrophoresis at 80 volt for 120 minutes and the gel was then washed twice with zymogram renaturing solution (Biotech) for 45 minutes at room temperature. The gel was preincubated in zymogram developing solution (Biotech) for 30 minutes at 37℃ and subsequently incubated in zymogram developing solution for 24 hours at 37℃. After incubation, the gel was stained by 0.5% Coomassie Blue R-250 (Bio-Rad Laboratories, Hercules, CA, USA) in 30% methanol and 5% acetic acid solution under continuous shaking for 2 hours, then de-stained in 30% methanol and 5% acetic acid solution for 30 minutes, and then rinsed twice with de-staining solution to visualize the digested bands in the gelatin matrix. The gels were photographed and the averages of the band intensity were measured using commercially available imaging software (Scion Software).

The cultured cells were seeded in a 6-well plate and treated with PMA and/or MMP-9, and doxycycline for the indicated times. The cells were then washed with cold PBS, exposed to trypsin, and formed into pellets at 700 g at 4℃. The pellets were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail). The preparation was then clarified by centrifugation, and the supernatant was saved as a whole-cell lysate. The proteins (50 µg) were separated using 10% reducing SDS-polyacrylamide gel electrophoresis and electroblotted in 20% methanol, 25 mM Tris, and 192 mM glycine onto a nitrocellulose membrane. The membrane was then blocked with 5% nonfat dry milk in 25 mM Tris-HCl, 150 mM NaCl, and 0.2% Tween 20, and then incubated with the indicated primary antibody of ERK1/2 (Santa Cruz Biotechnology) or p38 (Santa Cruz Biotechnology) for 4 hours. Subsequently, the membrane was washed and incubated for 1 hour with secondary antibody of ERK1/2 (Santa Cruz Biotechnology) or p38 (Santa Cruz Biotechnology) conjugated to HRP, rewashed, and developed using an enhanced chemiluminescence reagent kit (PerkinElmer Life Sciences, Boston, MA, USA) and exposed to X-ray film for 10 seconds.

Predesigned siRNA targeting p38 MAPK and negative control siRNA for p38 MAPK were purchased from Invitrogen (Carlsbad, CA, USA). The sequences of p38 MAPK siRNA were as follows; sense: AUG AAU GAU GGA CUG AAA UGG UCU G and antisense: CAG ACC AUU UCA GUC CAU CAU UCA U. The transfection rate of p38 MAPK siRNA was verified to be over 90% in the human NCI-H292 airway epithelial cells. Transfection was performed according to the manufacture's protocol (Invitrogen). In brief, the human NCI-H292 airway epithelial cells were seeded in a 6-well plate at 1×10⁵ cells/well and incubated overnight in RPMI 1640 medium without antibiotics. When the cells were 80-90% confluent, the following day, and the cells were washed with PBS, and then OPTI-MEN I Reduced Serum Medium (Invitrogen) was added to the cells. Then, p38 MAPK siRNA and a nucleic acid transferring agent, Lipofectamine 2000 (Invitrogen) was incubated together in OPTI-MEM I Reduced Serum Medium for 20 minutes at room temperature to form a p38 MAPK siRNA-Lipofectamine complex. The p38 MAPK siRNA-Lipofectamine complex-containing medium was added to each well containing the cells to a final p38 MAPK siRNA concentration of 20 nM; the cells were then incubated for 24 hours at 37℃ in a CO₂ incubator. The p38 MAPK siRNA-Lipofectamine complex-containing medium was replaced with RPMI 1640 medium after 4 hours without loss of transfection activity. After 24 hours of transfection with p38 MAPK siRNA, the cells were exposed to the indicated concentrations of PMA and then harvested for RT-PCR analysis of MUC5B mRNA expression. The same procedure was performed with a negative control siRNA (Invitrogen).

Statistical analysis was performed using SPSS ver. 10.0 (SPSS Inc., Chicago, IL, USA). The mean for each of the obtained quantitative values was calculated. Comparisons were made using the Student's t-test. For all tests, a P-value of less than 0.05 was considered statistically significant.

The effects of PMA on MUC5B and MMP-9 mRNA expression in a dose-dependent manner were investigated. The MUC5B mRNA expression was significantly increased at all doses of PMA and peaked to 10 nM of PMA for 8 hours, and the MMP-9 mRNA expression was significantly increased about 17 fold at 5, 10, 25, and 50 nM for 8 hours (P<0.05) (Fig. 1).

The effect of MMP-9 on MUC5B mRNA expression in a dose-dependent manner was investigated. MUC5B mRNA expression was significantly increased in a dose-dependent manner (P<0.05) (Fig. 2).

SB203580 as a p38 MAPK inhibitor inhibited the PMA-induced MUC5B expression (P<0.05) (Fig. 3A). In addition, the knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked PMA-induced MUC5B mRNA expression (P<0.05) (Fig. 3B).

Doxycycline significantly inhibited PMA-induced MUC5B expression in a dose-independent manner (P<0.05) (Fig. 4). Doxycycline significantly inhibited PMA-induced MMP-9 mRNA expression (P<0.05) (Fig. 5A). In addition, doxycycline significantly inhibited PMA-induced MMP-9 protein activity (P<0.05) (Fig. 5B).

Doxycycline inhibited PMA induced phosphorylation of p38 in a dose dependent manner; however, it did not change the phosphorylation of ERK1/2 significantly (P<0.05) (Fig. 6A). In addition, doxycyline significantly inhibited MMP-9 induced phosphorylation of p38 (P<0.05) (Fig. 6B).