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【摘要】
Elevated expression of gel-forming mucin (MUC) genes MUC5AC and MUC5B is a major pathological feature in various airway diseases. In this study, we show that phorbol 12-myristate 13-acetate (PMA) is a potent stimulator for MUC5B gene expression under air-liquid interface conditions in three airway epithelial cell systems: primary cultures of normal human bronchial epithelial cells, the immortalized normal bronchial epithelial cell line HBE1, and the human lung adenocarcinoma cell line A549. Stimulation was time- and dose-dependent, could be demonstrated by promoter-reporter gene transfection, and was sensitive to mithramycin A, suggesting the involvement of a specificity protein 1-based transcriptional mechanism in the stimulation. PMA-induced MUC5B message and promoter-reporter gene activity were specifically sensitive to inhibition of protein kinase C , which was further confirmed by the forced expression of dominant-negative mutant of protein kinase C . Regarding downstream transduction, PMA-induced MUC5B expression was sensitive to inhibitors and dominant-negative expression of signaling molecules involved in Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase1-mediated c-Jun N-terminal kinase and p38 pathways. This contrasted with the inhibition of PMA-induced MUC5AC expression by inhibitors of the Ras/epidermal growth factor receptor/extracellular regulated kinase signaling pathway. These results demonstrate for the first time that PMA-stimulated MUC5AC and MUC5B expressions are regulated through distinctive epidermal growth factor receptor/extracellular regulated kinase-dependent and -independent signaling pathways.
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Mucus secretion is essential for proper mucociliary function and homeostatic control in the airways.1 Mucus lining the airways traps inhaled dust particles, chemicals, and microbes.2 However, airway mucus hypersecretion and accumulation in the airway lumen are pathological symptoms associated with various chronic airway diseases.3,4 Mucus obstruction of the airway is the major cause of morbidity and mortality in patients with chronic airway diseases.5 To date, 20 different mucin (MUC) genes have been identified. Among these MUC genes, at least nine of them (MUC1, -2, -4, -5AC, -5B, -7, -8, -13, and -19) are expressed in human airways. MUC2, MUC5AC, MUC5B, and the newly found MUC19 are gel-forming mucin genes6-8 expressed by the airway epithelium, but only MUC5AC and MUC5B gene products have been convincingly demonstrated in human airway secretions.2,6,9,10
In normal human airways, MUC5AC is mainly expressed by surface goblet epithelial cells, whereas MUC5B is predominantly expressed by mucous cells of submucosal glands.11 Cumulative studies have demonstrated the aberrant elevation and accumulation of MUC5AC and MUC5B in airway secretions from patients with lung diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis.12,13 However, MUC5B gene products in diseased airways are also found in the surface epithelium, rather than just being limited to the submucosal glands. Using an ovalbumin-induced mouse asthma model, our laboratory has shown expression of the glandular MUC5B message in surface airway epithelial cells.14 A similar disease-related gene trans-expression has also been demonstrated in patients with emphysema and diffuse panbronchiolitis.15,16 Thus, a change in cell type-specific MUC5B gene expression is a significant feature associated with the pathogenesis of airway diseases.
Phorbol 12-myristate 13-acetate (PMA) can induce protein kinase C (PKC) activation by acting as an alternative stimulus to diacylglycerol. PMA has been demonstrated as a model inflammatory stimulus that can modulate a variety of cellular events including gene transcription,17 cell growth, and differentiation.18 It has also been used as a tumor-promoting agent.19 PKC activation in airway epithelial cells occurs frequently in airways after cigarette smoking, oxidant exposure, and microorganism infections and during various inflammatory process.17,20 The role of PMA in the induction of mucins has been demonstrated for MUC2 and MUC5AC using NCI-H292 and HM3 colon cell lines.17,21 The results have suggested a PKC-, epidermal growth factor receptor (EGFR)-, Ras/Raf-, extracellular regulated kinase (ERK)-mediated specificity protein 1 (Sp1)-based transcriptional mechanism. Unlike for MUC2 and MUC5AC, there is very little information in regard to the effect of PMA on MUC5B expression. Recent completion of the MUC5B gene cloning and the characterization of its promoter sequence make it feasible to define molecular mechanisms that regulate the transcription of MUC5B.22 In this communication, we show that PMA is a potent mediator for the expression of MUC5B in primary human bronchial epithelial cell cultures and in two cell lines: an immortalized normal bronchial epithelial cell line, HBE1, and a lung adenocarcinoma cell line, A549. In contrast to the signaling cascade of MUC5AC induction, PMA-enhanced MUC5B expression occurs through an EGFR/ERK-independent but PKC-, Ras-, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase (MEKK) 1-mediated, c-Jun N-terminal kinase (JNK)/p38-dependent signaling pathway. These are the first data to identify the molecular signaling mechanism involved in the regulation of MUC5B expression in airway epithelial cells.
【关键词】 distinctive epidermal receptor/extracellular regulated kinase-independent -dependent signaling pathways induction expression -myristate -acetate
Materials and Methods
Cell Culture
Normal human primary tracheobronchial epithelial cells (NHBE) were isolated from human bronchi and trachea obtained from organ donors or autopsy at the University of California, Davis, Medical Center (Sacramento, CA). Tissue procurement and utilization were approved and periodically reviewed by the University of California Davis Human Subject Research Review Committee. Two airway epithelial cell lines were used in this study: HBE1, a papilloma virus-immortalized bronchial epithelial cell line, generated by Dr. James Yankaskas (University of North Carolina, Chapel Hill, NC),23 and A549, a human lung adenocarcinoma cell line obtained from the American Type Culture Collection (Manassas, VA).
Cell isolation and culture methods were performed as described previously,24,25 with some modifications. NHBE cells (1 x 104 cells/cm2) were plated on a Costar Transwell chamber (25 mm2 ) in Ham??s F12/Dulbecco??s modified Eagle??s medium (1:1) supplemented with insulin (5 µg/ml), transferrin (5 µg/ml), epidermal growth factor (EGF) (10 ng/ml), dexamethasone (0.1 µmol/L), cholera toxin (10 ng/ml), bovine hypothalamus extract (15 µg/ml), and bovine serum albumin (0.5 mg/ml).26,27 All-trans-retinoic acid (0.03 µmol/L) was added approximately 2 days after plating when the cells reached confluence. Approximately 1 week after plating, the immersed primary NHBE cells were changed to an air-liquid interface. After 3 weeks in culture (2 weeks under an air-liquid interface), NHBE cells underwent mucociliary differentiation, including formation of cilia and mucus-secreting granules.24 HBE1 cells were cultured under the same air-liquid interface (biphasic) condition as described for primary NHBE cells. In the presence of retinoic acid, HBE1 cells expressed comparable levels of MUC5AC and MUC5B gene products, except they did not undergo ciliogenesis. A549 cells, which constitutively expressed MUC5AC and MUC5B gene products, were also cultured under an air-liquid interface in RPMI (GIBCO, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. Most experiments, except the transient transfection studies, were done 21 days after passage.
Enzyme-Linked Immunosorbent Assay for MUC5B Protein
An antibody to MUC5B protein (5B#19-2E) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal antibody recognizes a 19-amino acid sequence (QREELPYSRTGLLVEQSGD) that is unique to the N terminus of human MUC5B protein. The antibody has been used previously to detect MUC5B secretion in primary airway epithelial cell cultures.28 We confirmed the authenticity of the antibody by Western blot analysis; the antibody reacted with salivary secretion and purified MUC5B protein but not MUC5AC protein as provided by Dr. D. Thornton7 (data not included). With this antibody, an enzyme-linked immunosorbent assay (ELISA) method, similar to the work of Rousseau et al29 and Park et al,28 was developed with a slight modification, using the synthetic 19-amino acid peptide as a referenced standard. In brief, day-21 cultures of NHBE, HBE1, and A549 cells were treated with or without 10 nmol/L PMA. Transwell cultures were lysed in 100 µl of radioimmunoprecipitation assay buffer, (1% NP-40, 0.25% deoxycholic acid, 1 mmol/L ethylenediamine tetraacetic acid, 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.4, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 1 mmol/L NaF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstain) at 0, 8, 16, and 24 hours after PMA treatment. Diluted lysates (1:10 in water) and different amounts of referenced 19-amino acid peptide standards were added (100 µl/well) to Maxisorp microtiter plates (Nunc, Rochester, NY) and dried overnight at 37??C. The plates were briefly exposed to UV light for 1 minute (UV Stratalinker 2400; Stratagene, La Jolla, CA) to ensure that the proteins were cross-linked to the substratum and were retained in the well during subsequent washings. The plates were deglycosylated with 200 mmol/L NaIO4 and sodium-acetate buffer (0.33 mol/L NaCl and 0.1 mol/L glacial acetic acid, pH 4.5) and kept overnight at 4??C. On the following day, a sodium-thiosulfate solution (0.133 mol/L Na2S2O3, 0.033 mol/L NaI, and 0.033 mol/L NaHCO3, pH 7.6) was added for 30 minutes at 4??C. The wells were blocked with 1x Blocker BSA (Pierce, Rockford, IL) for 30 minutes, followed by three washes with phosphate-buffered saline (PBS) (100 µl/well for 10 minutes). Next, wells were washed three times with PBS/0.05% Tween 20 and incubated for 1 hour with MUC5B antibody diluted 1:30 in PBS/0.05% Tween 20. After five washes with PBS/0.05% Tween 20, an anti-mouse antibody conjugated with horseradish peroxidase was added to the wells and incubated for 30 minutes at room temperature. After five washes with PBS/0.05% Tween 20, a color reaction was developed with 100 µl of 3,3',5,5'-tetramethylbenzidine substrate (Novagen, San Diego, CA), which was read at 450 nm.
A standard curve was generated with known amounts of serially diluted synthetic 19-amino acid peptide. Cell lysate data were converted according to the standard curve and expressed as moles of MUC5B equivalent per milligram of protein. Experiments were performed in three separate, independent primary NHBE cultures from cells derived from different sources and with two separate passages of HBE1 and A549 cells. Each cell extract was assayed in triplicate wells of ELISA.
MUC5B Promoter-Reporter Transfection Study and Luciferase Assay
A luciferase reporter construct, pGL3-MUC5B, containing 4169 bp of 5'-flanking region of MUC5B previously constructed in our laboratory30 was used for the MUC5B promoter study. For transient transfection studies, NHBE, HBE1, and A549 cells were plated on 24-well plates at 5 x 104 cells/well. The cells were transfected with 0.2 µg of pGL3-MUC5B clone plus 0.2 µg of pSV-ß-galactosidase (ß-gal) plasmid for normalizing transfection efficiency. Transfection was performed by FuGENE 6 (Roche Diagnostics, Indianapolis, IN) according to the manufacturer??s protocol. Cells were transfected in Opti-MEM reduced serum media (Invitrogen). Sixteen hours after transfection, cells were treated with 10 nmol/L PMA in Ham??s F12/Dulbecco??s modified Eagle??s medium (1:1) supplemented with 5 µg/ml insulin, 10 ng/ml EGF, 0.1 µmol/L dexamethasone, 5 µg/ml transferrin, 20 ng/ml cholera toxin, and 15 µg/ml bovine hypothalamus extract. Cells were harvested at 0, 8, 16, 24, and 48 hours after PMA treatment for reporter assays. Luciferase activity was determined using Luclite (PerkinElmer, Boston, MA) and counted in a luminometer. ß-Galactosidase activities were assayed and read at OD405. For each transfection, relative luciferase activity was normalized to ß-galactosidase activity.
For the forced mutant protein expression studies, various dominant-negative (dn) and constitutively active (ca) clones were co-transfected with pGL3-MUC5B. All dn and ca expression clones used were as previously described.31 dn-Ras (Ras-N17) was generated by introducing a point mutation at the 17-position of Ha-Ras. Ca-Ras (Ha-Ras-V12) was cloned in a pBCMG vector.32 dn-MEKK1 (367MEKK1-KR), ca-MEKK1 (367MEKK1), and dn-SEK1/mitogen-activated protein kinase kinase (MKK) 4 (SEK1-AL, serine 220, and threonine 224 mutated to alanine and leucine, respectively) cloned in pEECMV were generously provided by Dr. Dennis Templeton (Case Western Reserve University, Cleveland, OH).33,34 dn-MKK7 (F.MKK7), dn-JNK1 (APF), dn-JNK2, and dn-MKK6 (ala) cloned in pCDNA3 vector; dn-p38 (AGF) cloned in pCMV5 vector; and dn-MKK3 (ala) cloned in pRSV vector were kindly provided by Dr. Roger Davis (University of Vermont, Burlington, VT).35-37 dn-ERK1 (Lys71Arg) and dn-ERK2 (Lys53Arg) mutants, each cloned in a pCEP4 vector, were generously provided by Dr. Melanie Cobb (University of Texas, Austin, TX).38 dn-c-Raf (dn-cRaf-C4) mutant and dn-MKK3 (ala), each cloned in a pRSV vector, were kindly provided by Dr. Stephan Ludwig (Universitat Wurzburg, Wurzburg, Germany).39 dn-PKC and dn-PKCß, each cloned in a SRD vector,40 and dn-PKC cloned in pCMV vector were kindly provided by Drs. Motoi Ohba, Toshio Kuroki, and Shigeo Ohno (Showa University, Tokyo, Japan).41,42
Inhibitor Studies
To conduct inhibitor studies, 21-day-old cultures of NHBE, HBE1, and A549 cells were starved 1 day in serum- and hormone-free medium, except for the supplementation of retinoic acid. After starvation, cells were pretreated with various inhibitors or vehicles 1 hour before PMA treatment. RNA and protein extract were prepared from these cultures 24 hours after PMA treatment for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and luciferase quantification, respectively.
Mithramycin A was purchased from Sigma (St. Louis, MO). Manumycin A, radicicol, JNK inhibitor II (SP600125), SB203580, U0126, AG9, 4--6,7-diaminoquinazoline (BPDQ), and AG1478 were purchased from EMD Biosciences-Calbiochem (San Diego, CA). Calphostin C, Rottlerin, and Gö6979 were purchased from Biomol International (Plymouth Meeting, PA). Inhibitor concentrations were based on the manufacturer??s suggested ranges, and at these concentrations, inhibitor toxicity to the cultured cells was minimal as determined by trypan blue exclusion testing (>95% of cultured cells excluding this dye).
RNA Isolation and Quantitative RT-PCR
RNA was extracted from NHBE, HBE1, and A549 cells after PMA and inhibitor treatments by using RNA Trizol reagent (Invitrogen) according to the manufacturer??s protocol. Extracted RNA (2 µg) was converted to cDNA by adding MMLV-reverse transcriptase (Promega, Madison, WI) and oligo-dT primers for a total volume of 20 µl. The reaction was further diluted to 40 µl with water and used for real-time PCR analysis. The PCR reaction was performed in a 96-well optical PCR plate, and each reaction contained a 50-µl mixture consisting of 25 µl of 2x SYBR Green PCR Master mix (Applied Biosystems, Inc., Foster City, CA), 2 µl of cDNA sample, 1 µl of 5 µmol/L each of forward and reverse primer, and 21 µl of RNase-free water. Results were analyzed by an ABIPRISM 7900HT Sequence Detection system (Applied Biosystems, Inc.). The following PCR primers were used: ß-actin forward, 5'-CTGGAACGGTGAAGGTGACA-3'; ß-actin reverse, 5'-AAGGGACTTCCTGTAACAATGCA-3'; MUC5B forward, 5'-GCTGCTGCTACTCCTGTGAGG-3'; MUC5B reverse, 5'-AGGTGATGTTGACCTCGGTCTC-3'; MUC5AC forward, 5'-GGAACTGTGGGGACAGCTCTT-3'; and MUC5AC reverse, 5'-GTCACATTCCTCAGCGAGGTC-3'. The relative mRNA amount in each sample was calculated based on its Ct value normalized with the Ct value of housekeeping gene (ß-actin). The calculation formula is 2C(Ct of MUC5B or MUC5AC C Ct of ß-actin). Results are the arbitrary unit of PMA-treated samples compared with the arbitrary unit of no treatment controls and presented as induction percentage or fold induction.
Western Blot Analysis
Fifty micrograms of protein lysate prepared in radioimmunoprecipitation assay solution was subjected to electrophoresis on a sodium dodecyl sulfate/12.5% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA). The polyvinylidene difluoride membrane was blocked with 5% milk in Tris-buffered saline/Tween 20 (TBST) and then probed with anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-stress-activated protein kinase/JNK, anti-stress-activated protein kinase/JNK, anti-phospho-p38 mitogen-activated protein kinase (MAPK), and anti-p38 MAPK antibodies (Cell Signaling Technology, Inc., Danvers, MA). The membrane was washed with TBST and then probed for 1 hour with secondary antibodies conjugated with horseradish peroxidase. After four intensive washes with TBST, immunoreactive bands were visualized by chemiluminescence with the use of Western blotting luminal reagent (Santa Cruz Biotechnology).
Results
PMA Stimulates MUC5B Mucin Production
PKC activation has been suggested as one of the major pathways involved in the regulation of MUC2 and MUC5AC gene expression.21,43,44 There are very few studies related to the effect of PMA on MUC5B expression. In this experiment, we examined whether PMA could stimulate MUC5B mucin gene expression under a long-term air-liquid interface condition in NHBE, HBE1, and A549 cells. As shown in Figure 1 , PMA induced MUC5B mRNA in all primary NHBE, HBE1, and A549 cells, but 4-PMA, a negative control for phorbol ester activation of PKC, failed to do any induction. MUC5B mRNA induced by PMA occurred in a dose- and time-dependent manner (Figure 1, A and B , respectively). Maximum stimulation of MUC5B expression occurred 24 hours after the addition of 10 nmol/L PMA. The induction of MUC5B mRNA also resulted in an increase in MUC5B secretion as detected by an ELISA for MUC5B mucin (Figure 2) . The concentration of MUC5B-equivalent mucin protein in NHBE, A549, and HBE1 cells was 37.03, 33.05, and 53.14 pmol/mg cell lysate, respectively, under basal conditions without PMA. However, after incubation with PMA (10 nmol/L) for 24 hours, MUC5B-equivalent protein increased two- to threefold compared with the protein levels in unstimulated cells. These results confirm that PMA, a protein kinase C activator, is a potent stimulator for MUC5B gene and protein expression in cultured airway epithelial cells.
Figure 1. Stimulation of MUC5B mRNA by PMA. A: Dose-response study of PMA effect on MUC5B mRNA expression. HBE1, A549, and NHBE cultures grown in air-liquid interface for 2 weeks supplemented with retinoic acid (day 21) were starved for 16 hours (without growth factors) before being treated with different concentrations of PMA (0 to 10 nmol/L) or 4-PMA (0 to 10 nmol/L). Twenty-four hours after PMA or 4-PMA treatment, total RNA was harvested, and MUC5B mRNA was analyzed using SYBR Green quantitative real-time RT-PCR as described in Materials and Methods. Triplicate dishes were used for each concentration, and experiments were repeated at least three times for different cultures derived from different donors and passages of cell lines. Statistical significance: *P < 0.05 and **P < 0.01, respectively, compared with the unstimulated control. B: Time course study of PMA effects on MUC5B mRNA expression. Day-21 cultures of HBE1, A549, and NHBE cells as described in A were treated with either 10 nmol/L PMA or 10 nmol/L 4-PMA and harvested at different time points (0, 8, 12, 16, 24, and 48 hours). Samples were measured with SYBR Green quantitative PCR. Triplicate dishes were used for each time point, and experiments were repeated at least three times in different cultures derived from different donors and passages of cell lines. Statistical significance: *P < 0.05 and **P < 0.01, respectively, compared with the unstimulated control.
Figure 2. Regulation of MUC5B secretion by PMA. Cells were treated with or without 10 nmol/L PMA. Cell lysates were harvested and prepared at 8, 16, and 24 hours after treatment. MUC5B protein was measured by ELISA as described in Materials and Methods. The quantity of MUC5B protein was presented as picomoles of MUC5B-equivalent/milligram of total protein, using the synthetic 19-amino acid peptide immunogen as the reference. Triplicate dishes were used for each protein ELISA, and experiments were repeated at least three times in different cultures derived from different donors and passages of cell lines, with similar results. Significance: *P < 0.05 and **P < 0.01, respectively, compared with unstimulated control at each time point. A: NHBE cells. B: A549 cells. C: HBE1 cells.
PMA Stimulates MUC5B Promoter Activity, and the Promoter Activity Is Sensitive to Mithramycin A
To elucidate the molecular basis of PMA-stimulated MUC5B expression, we used pGL3-MUC5B promoter-luciferase chimeric constructs for transient transfections. Freshly passaged cultures of NHBE, A549, and HBE1 cell systems were used. Despite the low-level expression of MUC5B message in the freshly passaged cultures, reasonably high promoter activity, at least 10 times higher than the promoterless pGL3 construct, was found in these three cell systems (data not included). As shown in Figure 3A , there was a three- to fourfold induction of MUC5B promoter-luciferase activity 24 hours after PMA treatment in all three cell systems. The data are in agreement with the results from the PMA-stimulated mRNA (Figure 1) and protein expression (Figure 2) experiments, suggesting that the stimulation of MUC5B expression by PMA is transcriptionally regulated.
Figure 3. PMA induced MUC5B expression at the promoter level, and the induction was sensitive to mithramycin. A: Effects of PMA on MUC5B promoter-mediated reporter gene expression activity. HBE1, A549, and NHBE cells were transiently transfected with full-length pGL3-MUC5B luciferase chimeric construct (4.17 kb) and co-transfected with pSV-ß-galactosidase to normalize the transfection efficiency. Six to 12 hours after transfection, 10 nmol/L PMA was added to the cells, and cells were then harvested at 0, 8, 16, 24, and 48 hours after PMA treatment for luciferase and ß-galactosidase assays. MUC5B promoter activity was expressed as luciferase/ß-galactosidase activity (%). Triplicate dishes were used for each time point, and experiments were repeated at least three times in different cultures derived from different donors and passages of cell lines. Statistical significance: *P < 0.05 and **P < 0.01, respectively, compared with control (time 0). B: Effects of mithramycin A on PMA-induced MUC5B promoter activity. HBE1 cells were plated on 24-well plates at 5 x 104 cells/well. Cells were transfected with full-length (4.17-kb) pGL3-MUC5B and pSV-ß-galactosidase as described in A. On the 2nd day, the cells were treated with 10 nmol/L PMA for 24 hours before being harvested for a reporter assay. Different concentrations of mithramycin A (0.1, 1, and 10 µmol/L) were added 1 hour before PMA treatment. Statistical significance: *P < 0.05 and **P < 0.01, respectively, compared with PMA-treated, uninhibited controls. #P < 0.05 compared with no PMA-treated, uninhibited control. C: Effects of mithramycin A on PMA-induced MUC5B mRNA. Day-21 cultures of NHBE, A549, and HBE1 cells were prepared as described in Materials and Methods. One hour before PMA (10 nmol/L) treatment, cultures were pretreated with 1 µmol/L mithramycin A. Cultures were harvested 24 hours after PMA treatment for RNA isolation. MUC5B message and ß-actin were quantified by the SYBR Green quantitative PCR analysis. PMA-induced and noninduced MUC5B mRNA was normalized with ß-actin. Results are expressed as fold of induction (PMA-induced MUC5B mRNA/non-PMA-induced MUC5B mRNA), which are the mean ?? SD of triplicates from two separate primary cultures and different passages of cell line cells. Statistical significance: *P < 0.05.
Several studies have shown the involvement of Sp1 in the regulation of mucin genes, including MUC5AC45 and MUC5B.22 In the study of Van Seuningen et al,22 the Sp1 cis-elements were present not only in the promoter region but also in the first intron; the Sp1 sites in both regions appeared to account for the MUC5B transcription activity. Therefore, mithramycin A, a pharmacological agent that can interfere with Sp1-DNA binding, was used to examine its effect on the PMA-induced MUC5B gene expression.46 As shown in Figure 3B , mithramycin A at 1 and 10 µmol/L inhibited the PMA-induced MUC5B gene activity. Inhibition is consistent with the sensitivity of PMA-induced MUC5B mRNA expression toward this drug. As shown in Figure 3C , PMA-induced MUC5B expression in the air liquid interface cultures of NHBE, A549, and HBE1 cells was suppressed in the presence of mithramycin A. These results further confirm that an Sp1-based transcriptional mechanism is involved in the regulation of PMA-induced MUC5B expression.
Requirement of PKC Isotype for PMA-Induced MUC Gene Expression
To reveal the signaling mechanism involved in PMA-induced mucin gene expression, two approaches were adapted. One approach was to use inhibitors on day-21 cultures to attenuate various signaling pathways. This approach was complemented with transient transfection to examine the effects of forced expression of dn and ca mutant proteins of various signaling molecules on gene expression. Because it was quite difficult to carry out the transient transfection on day-21 cultures, we had to rely on the use of freshly passaged cultures.
PMA is known as an activator of PKC. To investigate which specific PKC is involved in the PMA-induced mucin gene expression, inhibitors of Pan-PKC and different PKC isotypes were used. As shown in Figure 4, A and B , PMA-induced MUC5AC and MUC5B expression in these three cell systems was inhibited by the pan inhibitor of protein kinase C, Calphostin C, and the specific inhibitor of PKC and - isotypes, Rottlerin. However, MUC5AC and MUC5B expression was not inhibited by Gö6979, a specific inhibitor of the calcium-sensitive PKC isoforms and ß1. Because NHBE cells do not contain PKC,28 the fact that Rotillerin caused inhibition suggested that only PKC is involved in PMA-induced MUC5B activity. These results are consistent with the previous work of PMA-induced MUC5AC expression in the NCI-H292 cell line.17,20
Figure 4. Effects of PKC signaling on PMA-stimulated MUC gene expression. A: Effects of PKC inhibitors on PMA-stimulated MUC5ACexpression. Day-21 cultures of NHBE (unfilled bars), HBE1 (partially filled bars), and A549 (filled bars) cells were prepared as described in Figure 1 and were treated with 3 µmol/L Rottlerin, 70 nmol/L Gö6976, or 0.5 µmol/L Calphostin C 1 hour before PMA (10 nmol/L) treatment. Cultures were harvested 24 hours after PMA treatment for RNA isolation. MUC5AC message, normalized to ß-actin, was quantified by SYBR Green quantitative PCR analysis as described in Materials and Methods. Results are expressed as the mean ?? SD of triplicates from two separate primary cultures and different passages of cell line cells. Statistical significance: *P < 0.05 compared with the vehicle-treated cells. B: Effects of PKC inhibitors on PMA-stimulated MUC5B expression. Day-21 cultures of NHBE (unfilled bars), HBE1 (partially filled bars), and A549 (filled bars) cells were prepared as described in Figure 1 and were treated with 3 µmol/L Rottlerin, 70 nmol/L Gö6976, or 0.5 µmol/L Calphostin C 1 hour before PMA (10 nmol/L) treatment. Cultures were harvested 24 hours after PMA treatment for RNA isolation. MUC5B message, normalized to ß-actin, was quantified by the SYBR Green quantitative PCR analysis as described in Materials and Methods. The results are expressed as the mean ?? SD of triplicates from two separate primary cultures and different passages of cell line cells. Statistical significance: *P < 0.05 compared with the vehicle-treated cells. C: Effects of PKC inhibitors on PMA-stimulated MUC5B promoter activity. HBE1 cells were transfected and treated with 10 nmol/L PMA as described in Figure 3B . Cell lysates were harvested for reporter assay 24 hours after PMA treatment. Inhibitors for a variety of PKC isomers were added 1 hour before PMA treatment at the following concentrations: Calphostin C, a pan inhibitor of PKC, at 0.2 and 0.5 µmol/L; Gö6979, an inhibitor of the calcium-sensitive isoform and ß1, at 7 and 70 nmol/L; and Rottlerin, a specific inhibitor of PKC and -, at 3 and 30 µmol/L. The results were the mean ?? SD of triplicate samples from two separate cultures. Statistical significance: *P < 0.05 compared with PMA-induced promoter activity in control vehicle-treated condition; #P < 0.05 compared with the basal promoter activity in vehicle-treated cultures. D: Effects of forced dn PKC isoforms expression on the regulation of MUC5B promoter activity. HBE1 cells were transfected with pGL3-MUC5B and co-transfected with dn-PKC, dn-PKCß, dn-PKC, pBRL, dn-PKC, SRD, or CMV10. Ten nmol/L PMA was added after transfection, and cell lysates were harvested for luciferase reporter assays after 24 hours of incubation. ß-Galactosidase activities were used to normalize the transfection efficiency. Results are mean ?? SD of quadruplicate samples. Statistical significance: *P < 0.05 compared with PMA-induced promoter activity in cells co-transfected with vector DNA.
Consistent with the data obtained from day-21 cultures, PMA elevated MUC5B promoter two- to threefold in the promoter-reporter gene transfection assay conducted in the HBE1 cell system (Figure 4C) . This stimulation was blocked by Calphostin C and Rottlerin but not by Gö6979. Inhibition was further supported by co-transfection with forced expression of dn-PKC isotypes. As shown in Figure 4D , PMA-induced MUC5B luciferase activity was reversed in cells co-transfected with the dn-PKC isotype, whereas co-transfection with the dn-PKC, -ß, or - isotypes had no effect on basal or PMA-induced promoter activities.
Requirements for PKC Downstream of EGFR, Ras, Raf, and MEKK1 Signaling in PMA-Induced MUC Gene Expression
To elucidate downstream signaling of PKC, various inhibitors, including the ones previously used for PMA-induced MUC2 and MUC5AC expression,47,48 were used. PMA-treated cultures were treated with manumycin A, a specific inhibitor of farnesyltransferase in the Ras signaling pathway (IC50 = 5 µmol/L); radicicol, an inhibitor that disrupts K-Ras-activated signaling pathways by selectively depleting Raf kinase (IC50 = 5 µmol/L); AG1478, an inhibitor for EGFR kinase (IC50 = 3 nmol/L); AG9, a negative control for the inhibition of EGFR; and BPDQ, a potent and specific inhibitor of the tyrosine kinase activity of EGFR. As shown in Figure 5, A and B , PMA-stimulated MUC5AC and MUC5B messages were all sensitive to manumycin A in day-21 cultures. However, inhibitors targeting Raf and EGFR signaling, namely, radicicol, BPDQ, and AG1478, showed quite different results. All of those inhibitors attenuated PMA-induced MUC5AC expression yet failed to affect the PMA-induced MUC5B expression.
Figure 5. Effects of PKC downstream signaling pathways on PMA-stimulated MUC gene expression. A: Effects of PKC downstream signaling inhibitors on PMA-stimulated MUC5AC expression. Day-21 cultures of NHBE (unfilled bars), HBE1 (partially filled bars), and A549 (filled bars) cells were prepared as described in Figure 1 and were treated with 1 µmol/L radicicol, 0.2 µmol/L manumycin A, 5 µmol/L AG1478, 120 pmol/L BPDQ, or 10 µmol/L AG9 1 hour before PMA (10 nmol/L) treatment. Cultures were harvested 24 hours after PMA treatment for RNA isolation. MUC5AC message, normalized to ß-actin, was quantified by the SYBR Green quantitative PCR analysis as described in Materials and Methods. Results are expressed as the mean ?? SD of triplicates from two separate primary cultures and different passages of cell line cells. Statistical significance: *P < 0.05 compared with the vehicle-treated cells. B: Effects of PKC downstream signaling inhibitors on PMA-stimulated MUC5B expression. Day-21 cultures of NHBE (unfilled bars), HBE1 (partially filled bars), and A549 (filled bars) cells were prepared as described in Figure 1 and were treated with 1 µmol/L radicicol, 0.2 µmol/L manumycin A, or 5 µmol/L AG1478, 120 pmol/L BPDQ, or 10 µmol/L AG9 1 hour before PMA (10 nmol/L) treatment. Cultures were harvested 24 hours after PMA treatment for RNA isolation. MUC5B message, normalized to ß-actin, was quantified by the SYBR Green quantitative PCR analysis as described in the text. The results are expressed as the mean ?? SD of triplicates from two separate primary cultures and different passages of cell line cells. Statistical significance: *P < 0.05 compared with the vehicle-treated cells. C: Effects of Ras, Raf, and EGFR inhibitors on MUC5B promoter activity. Experiments were performed with HBE1 cells as described in Figure 4C . Inhibitors, as indicated, were added 1 hour before PMA treatment, and cell lysates were harvested for reporter gene assays 24 hours after PMA treatment. The inhibitors used were AG1478, an inhibitor for EGFR tyrosine kinase; manumycin A, an inhibitor of farnesyltransferase in the Ras signaling pathway; and Radicicol, an inhibitor that disrupts K-Ras-activated signaling pathways by selectively depleting Raf kinase. D: Effects of forced expression of dn and ca clones of Ras, MEKK1, and Raf on MUC5B promoter activity. HBE1 cells transfected with pGL3-MUC5B were co-transfected with the following empty vectors: pEECMV (lane 1), pCDNA3 (lane 2), PRSV (lane 6), and PEECMV (lane 8); or co-transfected with the following dominant-negative mutants: dn-MEKK1 (lane 3), dn-Ras (lane 4), and dn-c-Raf-1 (lane 5); or co-transfected with the following constitutively active mutants: ca-Raf (lane 7) and ca-MEKK1 (lane 9). One day after transfection, PMA (10 nmol/L) was added. Cell lysates were harvested 24 hours after PMA treatment for reporter gene assays. Statistical significance: *P < 0.05 compared with PMA-induced promoter activity in empty vector co-transfected controls; #P < 0.05 compared with empty vector co-transfected controls. E: Effects of dn-cRaf-1 and dn-MEKK1 mutants on ca-Ras-enhanced MUC5B promoter activity. HBE1 cells were transfected with pGL3-MUC5B and pSV-ß-galactosidase constructs along with either vector or ca-Ras plus dn-c-Raf-1 or dn-MEKK1 as described in Figure 4C . Forty-eight hours after transfection, cells were harvested for reporter gene assays. Statistical significance: *P < 0.05 compared with empty vector co-transfected controls; #P < 0.05 compared with ca-Ras-enhanced promoter activity.
To confirm further the inhibitor experiments done with day-21 cultures, transient transfection with pGL3-MUC5B construct was performed. As shown in Figure 5C , PMA-induced MUC5B promoter activity was diminished to basal level by 0.2 µmol/L manumycin A, whereas AG1478 and radicicol produced no effect. These results support the significant role of Ras in mediating PMA-induced MUC5B gene expression. The lack of any effect of AG1478 or radicicol on PMA-induced luciferase activity suggests that neither Raf- nor EGFR-mediated signaling is required for MUC5B expression. Because MEKK1 is another Ras effector,31 we conducted co-transfection studies with dn and ca clones of Ras, Raf, and MEKK1 in MUC5B promoter-luciferase transfected cells. As shown in Figure 5D , both dn-Ras and dn-MEKK1, but not dn-c-Raf-1, were able to reverse PMA-induced promoter activity. Furthermore, when non-PMA-treated HBE1 cells were co-transfected with constitutively active ca-Raf and ca-MEKK1, MUC5B basal promoter activity was induced with ca-MEKK1 but not ca-Raf. Therefore, Ras/MEKK1 but not Ras/Raf appeared to be involved in PMA-induced MUC5B promoter activity. To investigate whether MEKK1 is truly the downstream effector of Ras for MUC5B gene induction, we further transfected the HBE1 cells with ca-Ras along with either dn-c-Raf-1 or dn-MEKK1. As shown in Figure 5E , dn-MEKK1 was able to reverse MUC5B promoter activity induced by ca-Ras, whereas dn-c-Raf-1 failed to do so. These results collectively demonstrated the PKC/Ras/MEKK1 signaling cascade involved in PMA-induced MUC5B expression, whereas PMA-induced MUC5AC involved the PKC/EGFR/Ras/Raf signaling pathway.
Requirements for MAPK Signaling Pathways in PMA-Induced Mucin Gene Expression
To identify which MAPK signaling pathway is involved in MUC5B gene expression regulation, we examined whether PMA could induce the phosphorylation of ERK1/2, JNKs, and p38 in HBE1 cells. Protein lysates of HBE1 cells at day 21 were prepared at 0, 0.5, 1, 2, 4, and 8 hours after PMA treatment. As shown in the Western blots of Figure 6 , phosphorylation of ERK, JNK, or p38 could be induced by PMA, although the induction was modest for JNK.
Figure 6. Western blot analysis of PMA-induced phosphorylation of ERK1/2, JNKs, and p38 of MAPK pathways. Day-21 cultures of HBE1 cells were prepared as described in Figure 1 . Cells were treated with PMA (10 nmol/L) and harvested at various times as indicated for protein lysate preparation. Protein lysates (30 mg/lysate) were electrophoresed on a sodium dodecyl sulfate polyacrylamide gel and blotted to a nitrocellulose membrane for Western blot analysis. Blot membranes were treated with anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies (A), anti-phospho-JNKs and anti-total JNK antibodies (B), and anti-phosphorylated p38 and anti-p38 antibodies (C), as indicated. Similar experiments with identical results were repeated in day-21 primary NHBE cultures and the freshly passaged day 3 cultures of HBE1 cells (data not included).
Using inhibitors, we assessed the effect of various MAPK pathways on PMA-induced mucin gene expression. As shown in Figure 7, A and B , respectively, PMA-induced MUC5AC was sensitive to U0126 but not SP600125 or SB203580, whereas PMA-induced MUC5B expression had an opposite response to these inhibitors in day-21 cultures. Transient transfection assays with pGL3-MUC5B promoter-reporter gene assay were consistent with the data obtained from day-21 cultures. As shown in Figure 7C , JNK and p38 inhibitors (SP600125 and SB203580, respectively) inhibited PMA-induced promoter activity in a dose-dependent manner. In contrast, U0126, an inhibitor of the ERK pathway, had no effect regardless of the concentration used.
Figure 7. Effects of MAPK pathways on PMA-induced MUC gene expression. A: Effects of MAPK inhibitors on PMA-induced MUC5AC expression. Day-21 cultures of NHBE (unfilled bars), HBE1 (partially filled bars), and A549 (filled bars) cells were prepared as described in Figure 1 and were treated with 10 µmol/L U0126, 1 µmol/L SP600126 (JNKII inhibitor), and 6 µmol/L SB203580 as indicated, 1 hour before PMA (10 nmol/L) treatment. Cultures were harvested 24 hours after PMA treatment for RNA isolation. MUC5AC message, normalized to ß-actin, was quantified by the SYBR Green quantitative PCR analysis as described in Materials and Methods. Results are expressed as the mean ?? SD of triplicates from two separate primary cultures and different passages of cell line cells. Statistical significance: *P < 0.05 compared with the vehicle-treated cells. B: Effects of MAPK inhibitors on PMA-induced MUC5B expression. Day-21 cultures of NHBE (unfilled bars), HBE1 (partially filled bars), and A549 (filled bars) cells were prepared as described in Figure 1 and were treated with 10 µmol/L U0126, 1 µmol/L SP600126 (JNKII inhibitor), and 6 µmol/L SB203580 as indicated, 1 hour before PMA (10 nmol/L) treatment. Cultures were harvested 24 hours after PMA treatment for RNA isolation. MUC5B message, normalized to ß-actin, was quantified by the SYBR Green quantitative PCR analysis as described in Materials and Methods. The results are expressed as the mean ?? SD of triplicates from two separate primary cultures and different passages of cell line cells. Statistical significance: *P < 0.05 compared with the vehicle-treated cells. C: Effects of MAPK inhibitors on MUC5B promoter activity. HBE1 cells were transfected with MUC5B promoter-reporter and ß-gal constructs as described in Figure 4C . One hour before PMA (10 nmol/L) treatment, cells were exposed to various MAPK inhibitors, as indicated. Cells were harvested for the reporter gene assays 24 hours after PMA treatment. Statistical significance: *P < 0.05 compared with PMA-induced promoter activity in vehicle-treated control. D: Effects of forced expression of dominant-negative (dn) clones of MAPK on MUC5B promoter activity. Experiments were performed as described in Figure 4C . Cell lysates were harvested for reporter assay 48 hours after PMA treatment. The results were expressed as the mean ?? SD of triplicates from two independent cultures. Statistical significance: *P < 0.05 compared with PMA-enhanced promoter activity in vector co-transfected cultures. E: Effects of forced expression of dn clones of MAPK on MUC5B expression at the mRNA level. HBE1 cells were transfected with various dn clones, as indicated, and treated with PMA as described in D. After 24 hours of incubation, RNA was harvested for quantitative RT-PCR. ß-Actin message levels were used for normalization of MUC5B mRNA. Statistical significance: *P < 0.05 compared with PMA-induced message expression in the empty vector-transfected controls.
To confirm further the significance of p38 and JNK pathways, dn expression clones of various MAPK signaling molecules were used. As shown in Figure 7D , PMA failed to induce MUC5B luciferase activity in dn-JNK1, dn-JNK2, and dn-p38 co-transfected cells. However, this was not the case for cells co-transfected with dn-ERK1, dn-ERK2, or empty vectors. Consistent with these findings, dn expression clones of MKK3, MKK4, MKK6, and MKK7, all upstream kinases of p38 and JNK, effectively inhibited PMA-induced MUC5B promoter activity (Figure 7D) . These results further confirm the signaling cascade of PKC, Ras, MEKK1, and p38/JNK in the regulation of PMA-induced MUC5B promoter activity.
To show that the inhibitory effect on the promoter activity is reflected in the MUC5B mRNA production, steady-state mRNA level was measured with SYBR quantitative PCR after HBE1 cells were transfected with the dn form of various signaling molecules. As shown in Figure 7E , forced expression of dn-JNK1, dn-JNK2, or dn-p38 but not dn-ERK1 or dn-ERK2 was able to reduce PMA-induced MUC5B message. These results further support the significant roles of p38 and JNK signaling in PMA-induced MUC5B expression.
Discussion
MUC2, MUC5AC, and MUC5B are the three major gel-forming mucin genes found in human airways and are clustered on a single band of chromosome11p15.5.49 Structurally, these three genes show extensive similarity in their 5' and 3' regions. Despite the similarities at the genomic level and the pathological significance of the gene products, most research has been done on the molecular mechanisms of MUC2 and MUC5AC gene expression.6,17,20,21,47 There are very few studies on the regulation of MUC5B gene expression. A major reason for the scarcity of studies may be due to a late completion of MUC5B genomic structure22,30 and the low level of MUC5B expression in the widely used NCI-H292 cell line.47 The NCI-H292 cell line has been the major cell system used to understand the regulation of MUC2/MUC5AC gene expression. However, mucin genes are expressed in a cell- and tissue-specific manner,11,48 and therefore, the information generated for MUC2/MUC5AC expression may not be suitable for MUC5B.
To offset studies based on one cell line, the current study used the following three airway epithelial cell systems: primary NHBE cells, the immortalized normal human bronchial epithelial cell line HBE1, and the human lung epithelial carcinoma cell line A549. The three cell systems were cultured for 3 weeks: 1 week immersed in media followed by 2 weeks in an air-liquid interface culture condition with media supplemented with all-trans-retinoic acid. Under these conditions, reasonable levels of MUC5AC and MUC5B gene expression were observed in the three cell systems. We show that PMA is a potent inducer for MUC5B expression, in addition to being an inducer of MUC5AC. Because promoter-reporter gene expression was elevated in proportion to MUC5B gene products in the transfection studies of all three cell types, PMA induction apparently occurs at the transcriptional level.
Remarkably, under the long-term culture condition, all three cell systems had a similar PMA-inducible response, with the responses to various inhibitors relating to the regulation of MUC5B and MUC5AC expression. Studies done with day-21 cultures of the three cell systems demonstrated that MUC5B and MUC5AC inductions are sensitive to inhibitors of PKC (Figure 4, A and B) , inhibitors of Ras (Figure 5, A and B) , and mithramycin A (Figure 3) . Consistent with the published results in NCI-H292 cell system,47 PMA-induced MUC5AC was attenuated by inhibitors of Raf and EGFR (Figure 5A) and ERK (Figure 6A) but not by inhibitors of JNKs and p38 (Figure 7A) . In contrast, PMA-induced MUC5B expression was sensitive to inhibitors of JNKs and p38 (Figure 7B) but not the inhibitors of Raf and EGFR (Figure 5B) and ERK (Figure 7B) .
Forced expression of various dn and ca mutants of relevant signaling molecules were used to compensate for the nonspecific features of the inhibitors. However, it was nearly impossible to do gene transfer studies in the highly confluent cultures. For this reason, like many other researchers in the field, we chose to do transient transfections with freshly passaged cultures. Despite the lower expression of MUC5B and MUC5AC messages in the freshly passaged cultures, the cultures were still responsive to PMA. Most importantly, the freshly passaged cultures allowed high efficiency of gene transfer for the promoter-reporter gene expression analysis and the co-transfection study that examined the effects of the forced expression of dn and ca mutants in the regulation of gene expression.
Data from the co-transfection studies further demonstrate a signal pathway for MUC5B that is distinctively different from the documented signaling mechanisms for MUC2 and MUC5AC17,20,21,48 (Figure 8) . Our study establishes an EGFR/ERK-independent but PKC/Ras/MEKK1-mediated JNK and p38 signaling-dependent regulation through an Sp1-based transcriptional mechanism for PMA-stimulated MUC5B expression. This signaling mechanism is in contrast to the prevailing PKC/EGFR/Ras/Raf/ERK/Sp1 signaling pathway for induced MUC5AC expression, as studied by others.17,20,21,48
Figure 8. A schematic summary of signal transduction pathways exerted by PMA involved in the regulation of MUC5B and MUC5AC expression in airway epithelial cells. Shaded boxes indicate the molecules involved in PMA-induced MUC5B expression.
PMA, an analog of diacylglycerol, is known to activate PKC, which in turn initiates signaling cascades that lead to a variety of cell events, including gene expression, cell differentiation, cell proliferation, and respiratory secretions. Adler and colleagues50 have identified a PKC-dependent phosphorylation of myristoylated alanine-rich C-kinase substrate as being the key element in the exocytosis of mucin granules. A similar conclusion has also been reported by Abdullah et al.51 Studies of PMA-induced MUC5AC and MUC2 expressions have also demonstrated the important role of PKC in mucin gene expression.17,20 Using various PKC isoform-specific inhibitors, we have shown that Rottlerin, a specific inhibitor of PKC and -, diminished PMA-enhanced MUC5B promoter activity. Because NHBE cells do not contain the isoform of PKC,28 the rotillerin effect should be on PKC only. This conclusion is supported by the use of dn mutants of various PKC isotypes in a MUC5B promoter activity assay. Both studies demonstrated the involvement of PKC in the regulation of MUC5B expression. Because PKC activation is a prevalent event that is frequently found in inflammatory airways associated with the exposure to smoke, particulates, oxidant air pollutants, and microbial organism infection, our findings further strengthen the role of PKC, especially the PKC isoform, in disease-associated pathogenesis involving mucin overproduction and accumulation as is found in various airway diseases.
Using a combination approach of inhibitor treatment and co-transfections with dn and ca expression constructs, the Ras, MEKK1, and p38/JNK signaling cascade downstream of PKC was characterized. The involvement of Ras/MEKK1 rather than Ras/Raf was clearly demonstrated using dn and ca clones. For the MAPK pathway, we have shown that PMA treatment can induce the phosphorylation of ERKs, JNKs, and p38 in cells. However, the inhibitor U0126, which is specific to ERK signaling, had no effect on PMA-enhanced MUC5B expression. These results were further supported by the transfection studies with dn-ERK1 and dn-ERK2 clones, which also failed to abrogate the enhanced MUC5B expression. On the other hand, dn clones of JNK and p38 were able to abrogate PMA-enhanced MUC5B promoter activity as well as mRNA production, suggesting that the MAPK signaling pathways involved in PMA-enhanced MUC5B expression are JNK and p38, which are quite different from the known ERK-dependent MUC5AC expression.
It is noteworthy that both JNK and p38 pathways, instead of just one single pathway, are required for the transcriptional induction of MUC5B. One possible explanation is that the two pathways are controlling two different events for one function, that is, to enhance MUC5B expression, especially related to the Sp1-mediated transcriptional mechanism. It is possible that one of those two molecules, JNK or p38, is involved in the activation of Sp1, whereas the other one is responsible for an activation of accessory protein(s). Our study here, along with other documented reports,17,47 demonstrates the involvement of the SP1 sites in MUC5AC and MUC5B gene regulation. We speculate that the difference in the SP1-mediated transcription of those two genes resides on the fact that MUC5B transcriptional induction requires an accessory protein for SP1 activation, and the accessory protein involves a second MAPK pathway, either JNK or P38. MUC5AC may not require a secondary protein, and therefore, only the ERK pathway is involved.
The EGFR signaling pathway is considered to be a common pathway by which many stimuli induce MUC5AC production.17,20,52-57 The same outcome has also been demonstrated for tumor necrosis factor--induced48 and adenosine-induced58 MUC2 gene expression. AG1478, the pharmacological agent that was used frequently in studies related to the inhibition of EGFR tyrosine kinase activity previously,17,20,52-57 has been shown to be toxic in certain system. For that reason, another EGFR inhibitor, BPDQ, was also used to inhibit the PMA-induced MUC5AC or MUC5B. Both inhibitors attenuated MUC5AC gene expression but failed to abolish PMA-mediated MUC5B expression in all three cell types (Figure 5) , indicating that PMA-induced MUC5B gene expression is EGFR independent. This is inconsistent with the study of MUC5B gene expression by Perrais et al59 on colon cancer cell lines. Therefore, our study supports the notion that MUC5B expression is independent from EGFR- and ERK-activated pathways, a key step that has been proposed in PMA and various mediator-induced MUC5AC expression.17
In summary, we have identified distinctive signaling pathways involved in the stimulation of MUC5AC and MUC5B expression by PMA in three airway epithelial cell systems. For MUC5B expression, this is the first study to examine the molecular signaling pathway involved in its regulation. We have demonstrated a PKC-, Ras-, MEKK1-, and JNK/p38-dependent but EGF receptor/ERK-independent signaling pathway in the regulation of PMA-enhanced MUC5B expression. This signaling pathway is different from the known EGF receptor/ERK-dependent pathway for MUC2 and MUC5AC gene expression. Because JNK and p38 pathways are generally activated under stress-related conditions, as in various airway diseases, we propose that such activation may be responsible for the trans-differentiation of epithelial cells from non-MUC5B-producing cells to MUC5B-expressing cells in disease states. Mucins are expressed in a cell- and tissue-specific manner. Therefore, an altered expression of mucin gene products, especially the gel-forming ones, could impair the mucociliary function such that the pathogenesis of various airway diseases is enhanced.
Acknowledgements
We thank Dr. Suzette Smiley-Jewell for her critical reading and editing of this manuscript.
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作者单位：From the Center for Comparative Respiratory Biology and Medicine,* University of California, Davis, California; and the Department of Environmental Health Sciences, School of Public of Health, The Johns Hopkins University, Baltimore, Maryland