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Distinctive Epidermal Growth Factor Receptor/Extracellular Regulated Kinase-Independent and -Dependent Signaling Pathways in the Induction of Airway Mucin 5B and Mucin 5AC Expression by Phorbol 12-Myristate 13-Acetate

2007; Elsevier BV; Volume: 170; Issue: 1 Linguagem: Inglês

10.2353/ajpath.2007.060452

ISSN

1525-2191

Autores

Daphne Yuan-chen Wu, Reen Wu, Sekhar P. Reddy, Yong Chan Lee, Mary Mann‐Jong Chang,

Tópico(s)

Infectious Diseases and Mycology

Resumo

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 δ, which was further confirmed by the forced expression of dominant-negative mutant of protein kinase C δ. 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. 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 δ, which was further confirmed by the forced expression of dominant-negative mutant of protein kinase C δ. 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. Mucus secretion is essential for proper mucociliary function and homeostatic control in the airways.1West JB Respiratory Physiology: The Essentials. ed 6. Lippincott Williams & Wilkins, Philadelphia, PA2000Google Scholar Mucus lining the airways traps inhaled dust particles, chemicals, and microbes.2McDonald JA Lung growth and development.in: Lung Biology in Health and Disease. vol 100. M. Dekkar, New York1997Google Scholar However, airway mucus hypersecretion and accumulation in the airway lumen are pathological symptoms associated with various chronic airway diseases.3Vestbo J Prescott E Lange P Association of chronic mucus hypersecretion with FEV1 decline and chronic obstructive pulmonary disease morbidity: Copenhagen City Heart Study Group.Am J Respir Crit Care Med. 1996; 153: 1530-1535Crossref PubMed Scopus (633) Google Scholar, 4Aikawa T Shimura S Sasaki H Ebina M Takishima T Marked goblet cell hyperplasia with mucus accumulation in the airways of patients who died of severe acute asthma attack.Chest. 1992; 101: 916-921Crossref PubMed Scopus (444) Google Scholar Mucus obstruction of the airway is the major cause of morbidity and mortality in patients with chronic airway diseases.5Wang J Lory S Ramphal R Jin S Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients.Mol Microbiol. 1996; 22: 1005-1012Crossref PubMed Scopus (82) Google Scholar 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 genes6Hovenberg HW Davies JR Herrmann A Linden CJ Carlstedt I MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions.Glycoconj J. 1996; 13: 839-847Crossref PubMed Scopus (212) Google Scholar, 7Thornton DJ Howard M Khan N Sheehan JK Identification of two glycoforms of the MUC5B mucin in human respiratory mucus: evidence for a cysteine-rich sequence repeated within the molecule.J Biol Chem. 1997; 272: 9561-9566Crossref PubMed Scopus (160) Google Scholar, 8Chen Y Zhao YH Kalaslavadi TB Hamati E Nehrke K Le AD Ann DK Wu R Genome-wide search and identification of a novel gel-forming mucin MUC19/Muc19 in glandular tissues.Am J Respir Cell Mol Biol. 2004; 30: 155-165Crossref PubMed Scopus (178) Google Scholar expressed by the airway epithelium, but only MUC5AC and MUC5B gene products have been convincingly demonstrated in human airway secretions.2McDonald JA Lung growth and development.in: Lung Biology in Health and Disease. vol 100. M. Dekkar, New York1997Google Scholar, 6Hovenberg HW Davies JR Herrmann A Linden CJ Carlstedt I MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions.Glycoconj J. 1996; 13: 839-847Crossref PubMed Scopus (212) Google Scholar, 9Wickström C Davies JR Eriksen GV Veerman EC Carlstedt I MUC5B is a major gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage.Biochem J. 1998; 334: 685-693Crossref PubMed Scopus (271) Google Scholar, 10Meezaman D Charles P Daskal E Polymeropoulos MH Martin BM Rose MC Cloning and analysis of cDNA encoding a major airway glycoprotein, human tracheobronchial mucin (MUC5).J Biol Chem. 1994; 269: 12932-12939Abstract Full Text PDF PubMed Google Scholar In normal human airways, MUC5AC is mainly expressed by surface goblet epithelial cells, whereas MUC5B is predominantly expressed by mucous cells of submucosal glands.11Reid CJ Gould S Harris A Developmental expression of mucin genes in the human respiratory tract.Am J Respir Cell Mol Biol. 1997; 17: 592-598Crossref PubMed Scopus (129) Google Scholar 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.12Groneberg DA Eynott PR Lim S Oates T Wu R Carlstedt I Roberts P McCann B Nicholson AG Harrison BD Chung KF Expression of respiratory mucins in fatal status asthmaticus and mild asthma.Histopathology. 2002; 40: 367-373Crossref PubMed Scopus (140) Google Scholar, 13Groneberg DA Eynott PR Oates T Lim S Wu R Carlstedt I Nicholson AG Chung KF Expression of MUC5AC and MUC5B mucins in normal and cystic fibrosis lung.Respir Med. 2002; 96: 81-86Abstract Full Text PDF PubMed Scopus (147) Google Scholar 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.14Chen Y Zhao YH Wu R In silico cloning of mouse Muc5b gene and upregulation of its expression in mouse asthma model.Am J Respir Crit Care Med. 2001; 164: 1059-1066Crossref PubMed Scopus (60) Google Scholar A similar disease-related gene trans-expression has also been demonstrated in patients with emphysema and diffuse panbronchiolitis.15Chen Y Zhao YH Wu R Differential regulation of airway mucin gene expression and mucin secretion by extracellular nucleotide triphosphates.Am J Respir Cell Mol Biol. 2001; 25: 409-417Crossref PubMed Scopus (73) Google Scholar, 16Kamio K Matsushita I Hijikata M Kobashi Y Tanaka G Nakata K Ishida T Tokunaga K Taguchi Y Homma S Azuma A Kudoh S Keicho N Promoter analysis and aberrant expression of the MUC5B gene in diffuse panbronchiolitis.Am J Respir Crit Care Med. 2005; 171: 949-957Crossref PubMed Scopus (60) Google Scholar 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,17Hewson CA Edbrooke MR Johnston SL PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf MEK, ERK and Sp1-dependent mechanisms.J Mol Biol. 2004; 344: 683-695Crossref PubMed Scopus (162) Google Scholar cell growth, and differentiation.18Park SJ Kang SY Kim NS Kim HM Phosphatidylinositol 3-kinase regulates PMA-induced differentiation and superoxide production in HL-60 cells.Immunopharmacol Immunotoxicol. 2002; 24: 211-226Crossref PubMed Scopus (44) Google Scholar It has also been used as a tumor-promoting agent.19Busuttil V Bottero V Frelin C Imbert V Ricci JE Auberger P Peyron JF Blocking NF-kappaB activation in Jurkat leukemic T cells converts the survival agent and tumor promoter PMA into an apoptotic effector.Oncogene. 2002; 21: 3213-3224Crossref PubMed Scopus (43) Google Scholar PKC activation in airway epithelial cells occurs frequently in airways after cigarette smoking, oxidant exposure, and microorganism infections and during various inflammatory process.17Hewson CA Edbrooke MR Johnston SL PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf MEK, ERK and Sp1-dependent mechanisms.J Mol Biol. 2004; 344: 683-695Crossref PubMed Scopus (162) Google Scholar, 20Shao MX Nadel JA Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-alpha-converting enzyme.J Immunol. 2005; 175: 4009-4016PubMed Google Scholar The role of PMA in the induction of mucins has been demonstrated for MUC2 and MUC5AC using NCI-H292 and HM3 colon cell lines.17Hewson CA Edbrooke MR Johnston SL PMA induces the MUC5AC respiratory mucin in human bronchial epithelial cells, via PKC, EGF/TGF-alpha, Ras/Raf MEK, ERK and Sp1-dependent mechanisms.J Mol Biol. 2004; 344: 683-695Crossref PubMed Scopus (162) Google Scholar, 21Lee HW Ahn DH Crawley SC Li JD Gum Jr, JR Basbaum CB Fan NQ Szymkowski DE Han SY Lee BH Sleisenger MH Kim YS Phorbol 12-myristate 13-acetate up-regulates the transcription of MUC2 intestinal mucin via Ras ERK, and NF-kappa B.J Biol Chem. 2002; 277: 32624-32631Crossref PubMed Scopus (93) Google Scholar The results have suggested a PKCδ-, 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.22Van Seuningen I Perrais M Pigny P Porchet N Aubert JP Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells.Biochem J. 2000; 348: 675-686Crossref PubMed Scopus (81) Google Scholar 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 PKCδ-, 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. 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),23Verma M Murthy VV Mathew S Banerji D Kurl RN Olnes MJ Yankaskas JR Blass C Davidson EA Promoter of the canine tracheobronchial mucin gene.Glycoconj J. 1996; 13: 797-807Crossref PubMed Scopus (3) Google Scholar 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,24Wu R Zhao YH Chang MM Growth and differentiation of conducting airway epithelial cells in culture.Eur Respir J. 1997; 10: 2398-2403Crossref PubMed Scopus (93) Google Scholar, 25Wu R Martin WR Robinson CB St George JA Plopper CG Kurland G Last JA Cross CE McDonald RJ Boucher R Expression of mucin synthesis and secretion in human tracheobronchial epithelial cells grown in culture.Am J Respir Cell Mol Biol. 1990; 3: 467-478Crossref PubMed Scopus (95) Google Scholar with some modifications. NHBE cells (1 × 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).26Chang MM Juarez M Hyde DM Wu R Mechanism of dexamethasone-mediated interleukin-8 gene suppression in cultured airway epithelial cells.Am J Physiol. 2001; 280: L107-L115Google Scholar, 27Wu R Smith D Continuous multiplication of rabbit tracheal epithelial cells in a defined, hormone-supplemented medium.In Vitro. 1982; 18: 800-812Crossref PubMed Scopus (133) Google Scholar 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.24Wu R Zhao YH Chang MM Growth and differentiation of conducting airway epithelial cells in culture.Eur Respir J. 1997; 10: 2398-2403Crossref PubMed Scopus (93) Google Scholar 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. 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.28Park JA He F Martin LD Li Y Chorley BN Adler KB Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C{delta}-mediated mechanism.Am J Pathol. 2005; 167: 651-661Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar 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. Thornton7Thornton DJ Howard M Khan N Sheehan JK Identification of two glycoforms of the MUC5B mucin in human respiratory mucus: evidence for a cysteine-rich sequence repeated within the molecule.J Biol Chem. 1997; 272: 9561-9566Crossref PubMed Scopus (160) Google Scholar (data not included). With this antibody, an enzyme-linked immunosorbent assay (ELISA) method, similar to the work of Rousseau et al29Rousseau K Wickstrom C Whitehouse DB Carlstedt I Swallow DM New monoclonal antibodies to non-glycosylated domains of the secreted mucins MUC5B and MUC7.Hybrid Hybridomics. 2003; 22: 293-299Crossref PubMed Scopus (48) Google Scholar and Park et al,28Park JA He F Martin LD Li Y Chorley BN Adler KB Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C{delta}-mediated mechanism.Am J Pathol. 2005; 167: 651-661Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar 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 1× 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. A luciferase reporter construct, pGL3-MUC5B, containing 4169 bp of 5′-flanking region of MUC5B previously constructed in our laboratory30Chen Y Zhao YH Di YP Wu R Characterization of human mucin 5B gene expression in airway epithelium and the genomic clone of the amino-terminal and 5′-flanking region.Am J Respir Cell Mol Biol. 2001; 25: 542-553Crossref PubMed Scopus (105) Google Scholar was used for the MUC5B promoter study. For transient transfection studies, NHBE, HBE1, and A549 cells were plated on 24-well plates at 5 × 104 cells/well. The cells were transfected with 0.2 μg of pGL3-MUC5B clone plus 0.2 μg of pSV-β-galactosidase (β-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. β-Galactosidase activities were assayed and read at OD405. For each transfection, relative luciferase activity was normalized to β-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.31Vuong H Patterson T Shapiro P Kalvakolanu DV Wu R Ma WY Dong Z Kleeberger SR Reddy SP Phorbol ester-induced expression of airway squamous cell differentiation marker, SPRR1B, is regulated by protein kinase Cdelta/Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway.J Biol Chem. 2000; 275: 32250-32259Crossref PubMed Scopus (44) Google Scholar 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.32Kumar CC Prorock-Rogers C Kelly J Dong Z Lin JJ Armstrong L Kung HF Weber MJ Afonso A SCH 51344 inhibits ras transformation by a novel mechanism.Cancer Res. 1995; 55: 5106-5117PubMed Google Scholar dn-MEKK1 (Δ367MEKK1-KR), ca-MEKK1 (Δ367MEKK1), 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).33Yan M Templeton DJ Identification of 2 serine residues of MEK-1 that are differentially phosphorylated during activation by raf and MEK kinase.J Biol Chem. 1994; 269: 19067-19073Abstract Full Text PDF PubMed Google Scholar, 34Yan M Dai T Deak JC Kyriakis JM Zon LI Woodgett JR Templeton DJ Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1.Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar dn-MKK7 (F.MKK7), dn-JNK1 (APF), dn-JNK2, and dn-MKK6 (ala) cloned in pCDNA3 vector; dn-p38α (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).35Dérijard B Raingeaud J Barrett T Wu IH Han J Ulevitch RJ Davis RJ Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms.Science. 1995; 267: 682-685Crossref PubMed Scopus (1409) Google Scholar, 36Dérijard B Hibi M Wu IH Barrett T Su B Deng T Karin M Davis RJ JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2954) Google Scholar, 37Raingeaud J Whitmarsh AJ Barrett T Dérijard B Davis RJ MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway.Mol Cell Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1143) Google Scholar dn-ERK1 (Lys71→Arg) and dn-ERK2 (Lys53→Arg) mutants, each cloned in a pCEP4 vector, were generously provided by Dr. Melanie Cobb (University of Texas, Austin, TX).38Robbins DJ Zhen E Owaki H Vanderbilt CA Ebert D Geppert TD Cobb MH Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro.J Biol Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar 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).39Ludwig S Engel K Hoffmeyer A Sithanandam G Neufeld B Palm D Gaestel M Rapp UR 3pK, a novel mitogen-activated protein (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways.Mol Cell Biol. 1996; 16: 6687-6697Crossref PubMed Scopus (153) Google Scholar dn-PKCα and dn-PKCβ, each cloned in a SRD vector,40Hirai S Izumi Y Higa K Kaibuchi K Mizuno K Osada S Suzuki K Ohno S Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta.EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar and dn-PKCδ cloned in pCMV vector were kindly provided by Drs. Motoi Ohba, Toshio Kuroki, and Shigeo Ohno (Showa University, Tokyo, Japan).41Ohba M Ishino K Kashiwagi M Kawabe S Chida K Huh NH Kuroki T Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C.Mol Cell Biol. 1998; 18: 5199-5207Crossref PubMed Google Scholar, 42Akita Y Ohno S Yajima Y Konno Y Saido TC Mizuno K Chida K Osada S Kuroki T Kawashima S Overproduction of a Ca(2+)-independent protein kinase C isozyme, nPKC epsilon, increases the secretion of prolactin from thyrotropin-releasing hormone-stimulated rat pituitary GH4C1 cells.J Biol Chem. 1994; 269: 4653-4660Abstract Full Text PDF PubMed Google Scholar 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-[(3-bromophenyl)amino]-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 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 2× 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: β-actin forward, 5′-CTGGAACGGTGAAGGTGACA-3′; β-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 (β-actin). The calculation formula is 2−(Ct of MUC5B or MUC5AC − Ct of β-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. 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% mi

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