@article{park_adler_2007, title={Potential therapy for mucus hypersecretion in COPD.}, volume={3}, DOI={10.1080/17471060601063231}, abstractNote={Mucus hypersecretion is a major pathophysiologic feature of chronic bronchitis. Although mucus functions as a barrier and a facilitator of mucociliary clearance, persistent mucus hypersecretion results in airway obstruction and compromised clearance of inhaled bacteria and particles from the airways of patients with chronic obstructive pulmonary disease. Treatment of mucus hypersecretion is a major therapeutic target; however, mechanisms of mucus hypersecretion remain unknown. Herein, we present evidence that human neutrophil elastase (HNE), a pathophysiologically relevant stimulant of mucus hypersecretion in the airways of patients with chronic bronchitis, provokes release of mucin (the glycoprotein component of mucus) by human airway epithelial cells in vitro. Signaling molecules involved in HNE-induced mucin hypersecretion include protein kinase C, specifically the delta isoform, and the myristoylated alanine-rich C kinase substrate protein. These molecules represent potential therapeutic targets for regulating mucin secretion in patients.}, journal={404nOtfound}, author={Park, J.-A. and Adler, K. B.}, year={2007}, pages={66–71} }
 @article{park_crews_lampe_fang_park_adler_2007, title={Protein kinase C delta regulates airway mucin secretion via phosphorylation of MARCKS protein}, volume={171}, ISSN={["1525-2191"]}, DOI={10.2353/ajpath.2007.070318}, abstractNote={Mucin hypersecretion is a major pathological feature of many respiratory diseases, yet cellular mechanisms regulating secretion of mucin have not been fully elucidated. Previously, we reported that mucin hypersecretion induced by human neutrophil elastase involves activation of protein kinase C (PKC), specifically the δ-isoform (PKCδ). Here, we further investigated the role of PKCδ in mucin hypersecretion using both primary human bronchial epithelial cells and the human bronchial epithelial 1 cell line as in vitro model systems. Phorbol-12-myristate-13-acetate (PMA)-induced mucin hypersecretion was significantly attenuated by rottlerin, a PKCδ-selective inhibitor. Rottlerin also reduced PMA- or human neutrophil elastase-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in these cells. Both secretion and MARCKS phosphorylation were significantly enhanced by the PKCδ activator bryostatin 1. A dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) transfected into human bronchial epithelial 1 cells significantly attenuated both PMA-induced mucin secretion and phosphorylation of MARCKS, whereas transfection of a wild-type construct increased PKCδ and enhanced mucin secretion and MARCKS phosphorylation. Similar transfections of a dominant-negative or wild-type PKCε construct did not affect either mucin secretion or MARCKS phosphorylation. The results suggest that PKCδ plays an important role in mucin secretion by airway epithelium via regulation of MARCKS phosphorylation. Mucin hypersecretion is a major pathological feature of many respiratory diseases, yet cellular mechanisms regulating secretion of mucin have not been fully elucidated. Previously, we reported that mucin hypersecretion induced by human neutrophil elastase involves activation of protein kinase C (PKC), specifically the δ-isoform (PKCδ). Here, we further investigated the role of PKCδ in mucin hypersecretion using both primary human bronchial epithelial cells and the human bronchial epithelial 1 cell line as in vitro model systems. Phorbol-12-myristate-13-acetate (PMA)-induced mucin hypersecretion was significantly attenuated by rottlerin, a PKCδ-selective inhibitor. Rottlerin also reduced PMA- or human neutrophil elastase-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in these cells. Both secretion and MARCKS phosphorylation were significantly enhanced by the PKCδ activator bryostatin 1. A dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) transfected into human bronchial epithelial 1 cells significantly attenuated both PMA-induced mucin secretion and phosphorylation of MARCKS, whereas transfection of a wild-type construct increased PKCδ and enhanced mucin secretion and MARCKS phosphorylation. Similar transfections of a dominant-negative or wild-type PKCε construct did not affect either mucin secretion or MARCKS phosphorylation. The results suggest that PKCδ plays an important role in mucin secretion by airway epithelium via regulation of MARCKS phosphorylation. Mucus produced by epithelium of respiratory, gastrointestinal, and reproductive tracts provides a barrier between the external environment and cellular components of the epithelial layer. Mucins, the glycoprotein component of mucus, constitute a family of large, highly glycosylated macromolecules that impart physical (aggregation, viscosity, viscoelasticity, and lubrication) and biological (protection) properties to mucus (reviewed in Ref. 1Rose MC Mucins: structure, function, and role in pulmonary diseases.Am J Physiol. 1992; 263: L413-L429PubMed Google Scholar). Airway mucus is an integral component of the mucociliary clearance system in the trachea and bronchi and thus serves to protect the lower airways and alveoli from impingement of particulate matter and pathogens. However, mucin secretion is abnormally augmented in disease states, such as chronic bronchitis, asthma, and cystic fibrosis, increasing morbidity and mortality in these patients (reviewed in Refs. 1Rose MC Mucins: structure, function, and role in pulmonary diseases.Am J Physiol. 1992; 263: L413-L429PubMed Google Scholar and 2Rogers DF Barnes PJ Treatment of airway mucus hypersecretion.Ann Med. 2006; 38: 116-125Crossref PubMed Scopus (172) Google Scholar). Mucin hypersecretion is potentiated by many pathophysiological mediators, such as bacterial proteinases and endotoxin, adenine and guanine nucleotides, cytokines, inflammatory mediators, and eicosanoids (reviewed in Ref. 3Adler KB Li Y Airway epithelium and mucus: intracellular signaling pathways for gene expression and secretion.Am J Respir Cell Mol Biol. 2001; 25: 397-400Crossref PubMed Scopus (50) Google Scholar). Intracellular mechanisms and signaling molecules involved in the secretory process have not been fully elucidated.Protein kinase C (PKC) is a serine/threonine kinase involved in various exocytotic events in different cell types, including secretion of mucin,4Abdullah LH Bundy JT Ehre C Davis CW Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA.Am J Physiol Lung Cell Mol Physiol. 2003; 285: L149-L160PubMed Google Scholar, 5Plaisancie P Ducroc R El Homsi M Tsocas A Guilmeau S Zoghbi S Thibaudeau O Bado A Luminal leptin activates mucin-secreting goblet cells in the large bowel.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G805-G812Crossref PubMed Scopus (59) Google Scholar insulin,6Yaney GC Fairbanks JM Deeney JT Korchak HM Tornheim K Corkey BE Potentiation of insulin secretion by phorbol esters is mediated by PKC-alpha and nPKC isoforms.Am J Physiol Endocrinol Metab. 2002; 283: E880-E888Crossref PubMed Scopus (34) Google Scholar neurotransmitters,7Shoji-Kasai Y Itakura M Kataoka M Yamamori S Takahashi M Protein kinase C-mediated translocation of secretory vesicles to plasma membrane and enhancement of neurotransmitter release from PC12 cells.Eur J Neurosci. 2002; 15: 1390-1394Crossref PubMed Scopus (50) Google Scholar and platelet dense granules.8Murugappan S Tuluc F Dorsam RT Shankar H Kunapuli SP Differential role of protein kinase C delta isoform in agonist-induced dense granule secretion in human platelets.J Biol Chem. 2004; 279: 2360-2367Crossref PubMed Scopus (92) Google Scholar Previously, we demonstrated that mucin secretion in airway epithelial cells is regulated by PKC via phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS).9Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (150) Google Scholar, 10Singer M Martin LD Vargaftig BB Park J Gruber AD Li Y Adler KB A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma.Nat Med. 2004; 10: 193-196Crossref PubMed Scopus (147) Google Scholar In addition, we demonstrated that mucin hypersecretion in human airway epithelial cells in vitro in response to human neutrophil elastase (HNE) appears to be mediated by the δ-isoform of PKC (PKCδ).11Park 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 (96) Google Scholar Not surprisingly, PKCδ, a novel PKC isoform, has a strong affinity for MARCKS and can phosphorylate MARCKS both in vitro and in vivo.12Fujise A Mizuno K Ueda Y Osada S Hirai S Takayanagi A Shimizu N Owada MK Nakajima H Ohno S Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate.J Biol Chem. 1994; 269: 31642-31648Abstract Full Text PDF PubMed Google Scholar, 13Herget T Oehrlein SA Pappin DJ Rozengurt E Parker PJ The myristoylated alanine-rich C-kinase substrate (MARCKS) is sequentially phosphorylated by conventional, novel and atypical isotypes of protein kinase C.Eur J Biochem. 1995; 233: 448-457Crossref PubMed Scopus (79) Google Scholar, 14Cabell CH Verghese GM Rankl NB Burns DJ Blackshear PJ MARCKS phosphorylation by individual protein kinase C isozymes in insect Sf9 cells.Proc Assoc Am Physicians. 1996; 108: 37-46PubMed Google Scholar Increasing evidence suggests that PKCδ mediates exocytotic secretion in several different cell types.4Abdullah LH Bundy JT Ehre C Davis CW Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA.Am J Physiol Lung Cell Mol Physiol. 2003; 285: L149-L160PubMed Google Scholar, 8Murugappan S Tuluc F Dorsam RT Shankar H Kunapuli SP Differential role of protein kinase C delta isoform in agonist-induced dense granule secretion in human platelets.J Biol Chem. 2004; 279: 2360-2367Crossref PubMed Scopus (92) Google Scholar, 15Ishikawa T Iwasaki E Kanatani K Sugino F Kaneko Y Obara K Nakayama K Involvement of novel protein kinase C isoforms in carbachol-stimulated insulin secretion from rat pancreatic islets.Life Sci. 2005; 77: 462-469Crossref PubMed Scopus (12) Google Scholar, 16Leitges M Gimborn K Elis W Kalesnikoff J Hughes MR Krystal G Huber M Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation.Mol Cell Biol. 2002; 22: 3970-3980Crossref PubMed Scopus (115) Google Scholar, 17Cho SH Woo CH Yoon SB Kim JH Protein kinase Cdelta functions downstream of Ca2+ mobilization in FcepsilonRI signaling to degranulation in mast cells.J Allergy Clin Immunol. 2004; 114: 1085-1092Abstract Full Text Full Text PDF PubMed Scopus (55) Google ScholarHere, we further elucidate the role of PKCδ in the mucin secretory pathway in human airway epithelial cells in vitro. The mucin secretory response and phosphorylation of MARCKS were assessed after exposure of well differentiated normal human bronchial epithelial (NHBE) cells to phorbol-12-myristate-13-acetate (PMA), a general PKC activator, or bryostatin 1, a PKCδ/ε activator. In addition, we used the papilloma virus-transformed human bronchial epithelial 1 (HBE1) cell line for molecular manipulations. A dominant-negative PKCδ construct (K376R) transfected into HBE1 cells attenuated PMA-stimulated mucin secretion and MARCKS phosphorylation, whereas a similar dominant-negative PKCε construct was without effect. The results indicate that PKCδ is a key isoform regulating airway mucin secretion, and the mechanism of its action appears to involve phosphorylation of MARCKS protein.Materials and MethodsCulture of Bronchial Epithelial CellsPrimary NHBE cells purchased from Cambrex Bioscience (Walkersville, MD) were expanded and maintained in a humidified air/5% CO2 incubator as described previously.9Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (150) Google Scholar, 11Park 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 (96) Google Scholar Cells from at least three separate donors were used in these studies. Passage 31 or 32 of human papilloma virus-transformed human bronchial epithelial cells (HBE1)18Yankaskas JR Haizlip JE Conrad M Koval D Lazarowski E Paradiso AM Rinehart Jr, CA Sarkadi B Schlegel R Boucher RC Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype.Am J Physiol. 1993; 264: C1219-C1230PubMed Google Scholar was seeded and maintained as described previously. Transient transfection of HBE1 cells was performed after 10 days of culture in air-liquid interface.Exposure of Cells to Inhibitors or SecretagoguesWell differentiated NHBE cells were exposed to test agents both apically and basolaterally for 15 minutes (unless otherwise indicated). Transfected HBE1 cells were exposed to PMA (EMD Biosciences, La Jolla, CA) applied apically only. Cells were preincubated with the PKCδ-selective inhibitor, rottlerin (EMD Biosciences) for 20 minutes before PMA exposure. Initial stock solutions of rottlerin or PMA were prepared in dimethyl sulfoxide, kept at −20°C, and diluted in growth medium directly before use. When cells were exposed to PMA in the presence or absence of rottlerin, PMA was “spiked” into each well at the indicated concentration.Measurement of Mucin SecretionMucin was collected both at baseline and after treatments as described previously.9Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (150) Google Scholar Baseline mucin secretion was used to normalize well-to-well variation. After baseline mucin samples were collected, cells were rested overnight and exposed to test reagents the next day for indicated periods of time. After each treatment period, secreted mucin was collected as the baseline sample and quantified by sandwich enzyme-linked immunosorbent assay using the 17Q2 antibody (Covance Research Products, Berkeley, CA), a monoclonal antibody that reacts specifically with a carbohydrate epitope on human airway mucins.19Lin H Carlson DM St George JA Plopper CG Wu R An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates.Am J Respir Cell Mol Biol. 1989; 1: 41-48Crossref PubMed Scopus (63) Google Scholar The 17Q2 antibody was purified using an ImmunoPure(G) IgG purification kit (Pierce Biotechnology, Rockford, IL) following the manufacturer's protocol and then conjugated with alkaline phosphatase (EMD Biosciences). To account for variability between cultures and experiments, levels of mucin secretion were reported as percentage of the nontreated control. Actual values for mucin released by control cell cultures in these experiments ranged from 40 to 70 ng/ml in NHBE cells and 7 to 10 ng/ml in HBE1 cells.Subcellular Localization of PKC IsoformsActivation of PKCδ was assessed by subcellular fractionation following the protocol described by Kajstura et al20Kajstura J Cigola E Malhotra A Li P Cheng W Meggs LG Anversa P Angiotensin II induces apoptosis of adult ventricular myocytes in vitro.J Mol Cell Cardiol. 1997; 29: 859-870Abstract Full Text PDF PubMed Scopus (375) Google Scholar and subsequent Western blot analysis using a PKCδ-specific antibody (Cell Signaling Technology, Inc., Danvers, MA). Briefly, cells were washed with cold PBS and scraped into lysis buffer [20 mmol/L Tris-Cl (pH 7.5), 1 mmol/L ethylenediamine tetraacetic acid, 100 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, 1% (v/v) protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma, St. Louis, MO)]. The lysate was then sonicated and pelleted at 20,000 × g (Eppendorf 5417 centrifuge) for 40 minutes. The supernatant was collected and kept as the cytosolic fraction at −80°C until used. The remaining pellet was resuspended in lysis buffer containing 1% Triton X-100, sonicated, and centrifuged at 20,000 × g for 40 minutes. The supernatant membrane fraction was stored at −80°C until analyzed by Western blot.Western Blot AnalysisTotal MARCKS, phosphorylated MARCKS, PKCδ, and PKCε protein levels were measured via Western blot. The protein concentrations of cell lysates were quantified by a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Sample lysates were prepared by boiling in 2× SDS sample buffer [125 mmol/L Tris-Cl (pH 6.8), 25% glycerol, 4% SDS, 10% β-mercaptoethanol, and 0.04% bromphenol blue] for 10 minutes. Sample lysates (30 to 60 μg) were loaded on 10 or 12% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane (Schleicher & Schuell BioScience, Inc., Keene, NH) following electrophoresis. Polyvinylidene difluoride membranes were blocked with 5% nonfat milk and then probed with an appropriate dilution of primary antibody followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies. Chemiluminescent detection was performed using ECL detection reagents (GE Health care Life Sciences, Piscataway, NJ) following the manufacturer's protocol. Amounts of specific proteins in bands were quantified using Labworks image acquisition and analysis software 4.0. (Ultra Violet Products, Ltd., Upland, CA).Antibodies against α-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and E-cadherin (BD Biosciences, San Jose, CA) were used as loading controls for cytosolic and membrane fractions, respectively. Phosphorylated MARCKS (at serine 152/156) was detected with a specific antibody (Cell Signaling Technology, Inc.). After detection, the membrane was stripped in 62.5 mmol/L Tris-Cl (pH 6.5), 10% SDS, and 100 mmol/L β-mercaptoethanol for 10 minutes at room temperature and reprobed with a monoclonal antibody against total MARCKS protein (clone no. 2F12; Upstate, Charlottesville, VA) to verify equal loading.Transient Transfection of PKC ConstructsTransient transfection of vectors overexpressing wild-type or dominant-negative PKCδ and PKCε in HBE1 cells was performed using the FuGene 6 transfection reagent (Roche Applied Science, Indianapolis, IN) following the manufacturer's protocol. The pEGFP-N1 vectors containing a wild-type PKCδ cDNA21Mischak H Pierce JH Goodnight J Kazanietz MG Blumberg PM Mushinski JF Phorbol ester-induced myeloid differentiation is mediated by protein kinase C-alpha and -delta and not by protein kinase C-beta II, -epsilon, -zeta, and -eta.J Biol Chem. 1993; 268: 20110-20115Abstract Full Text PDF PubMed Google Scholar and a dominant-negative PKCδ mutant cDNA [lysine (AAG)→arginine (AGG) mutation, position 376]22Li L Lorenzo PS Bogi K Blumberg PM Yuspa SH Protein kinase Cdelta targets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector.Mol Cell Biol. 1999; 19: 8547-8558Crossref PubMed Google Scholar were generously provided by Dr. Arti Shukla (University of Vermont, Burlington, VT) and Dr. Peter Blumberg (National Cancer Institute, Bethesda, MD). The K376→R mutation in the ATP binding site of the catalytic domain has been demonstrated previously to inhibit PKCδ kinase activity.23Li W Yu JC Shin DY Pierce JH Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant: an inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity.J Biol Chem. 1995; 270: 8311-8318Crossref PubMed Scopus (89) Google Scholar Briefly, HBE1 cells grown in air/liquid interface were dissociated in versene solution (Invitrogen, Carlsbad, CA) and re-seeded in 12-well culture plates at a density of 1 × 105 cells/cm2. After overnight incubation, cells were transfected with the pEGFP-N1 vector alone or the pEGFP-N1 vector containing either a wild-type or dominant-negative PKCδ cDNA (K376R). Isotype controls for the dominant-negative PKCδ consisted of transient transfection of both wild-type PKCε as well as a catalytically inactive dominant-negative PKCε (K437R) construct-tagged with hemagglutin24Lee YJ Soh JW Jeoung DI Cho CK Jhon GJ Lee SJ Lee YS PKC epsilon -mediated ERK1/2 activation involved in radiation-induced cell death in NIH3T3 cells.Biochim Biophys Acta. 2003; 1593: 219-229Crossref PubMed Scopus (32) Google Scholar (PKCε constructs were kindly provided by Dr. Jae-Won Soh, University of Inha, Inha, Republic of Korea). Cells were subsequently cultured for 48 hours to allow for detectable protein expression. Transfection of PKCδ constructs was confirmed by fluorescent microscopy and assessment of expression of green fluorescence protein (GFP)-tagged PKCδ assessed by Western blot analysis using monoclonal antibodies against PKCδ or GFP (Cell Signaling Technology, Inc.). Transfection of PKCε was confirmed by Western blot analysis using PKCε and HA monoclonal antibodies (Covance Research Products).Cytotoxicity AssayAll treatments used were tested for cytotoxicity using a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI) according to the manufacturer's instructions. The results were expressed as the ratio of released lactate dehydrogenase to total lactate dehydrogenase. Released lactate dehydrogenase never exceeded 10% of total lactate dehydrogenase with any of the treatments (data not shown).Statistical AnalysisData were expressed as the ratio of treatment to the corresponding vehicle (dimethyl sulfoxide or media) control. Results were evaluated using one-way analysis of variance with Dunnett's test and a Bonferroni posttest correction for multiple comparisons.25Kleinbaum DG Kupper LL Muller KE Applied Regression Analysis and Other Multivariable Methods. PWS-Kent Publishing Co., Boston, MA1988Google Scholar A P value of less than 0.05 was considered significant.ResultsEffect of Rottlerin on PMA-Induced Mucin Secretion and MARCKS Phosphorylation in NHBE CellsTo determine whether PKCδ is an important regulatory molecule in mucin secretion, we investigated the effect of rottlerin, a PKCδ-selective inhibitor, on PMA-induced mucin secretion in well differentiated NHBE cells. As illustrated in Figure 1A, 100 nmol/L PMA provoked translocation of PKCδ from cytosol to membrane in these cells. Inhibition of PKCδ activity by pretreatment of cells with rottlerin (1 to 10 μmol/L) for 20 minutes significantly attenuated PMA-induced mucin secretion in a concentration-dependent manner (Figure 1B). Phosphorylation of MARCKS mediated by PMA in the presence or absence of 15 μmol/L rottlerin was analyzed by Western blot. HNE, previously shown to stimulate mucin secretion in NHBE cells via a rottlerin-inhibitable mechanism,11Park 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 (96) Google Scholar was used as an additional control. As shown in Figure 1C, both PMA- and HNE-induced phosphorylation of MARCKS were decreased by pretreatment with rottlerin.Bryostatin 1 Provokes Mucin Secretion and MARCKS Phosphorylation in NHBE CellsBryostatin 1, a PKCδ/ε activator, was used to investigate further the relationship between PKCδ and mucin secretion in NHBE cells. A naturally occurring marine invertebrate-derived cyclic lactone, Bryostatin 1, causes rapid activation of PKCδ and prolonged protection of PKCδ against ubiquitination and proteolysis.26Szallasi Z Denning MF Smith CB Dlugosz AA Yuspa SH Pettit GR Blumberg PM Bryostatin 1 protects protein kinase C-delta from down-regulation in mouse keratinocytes in parallel with its inhibition of phorbol ester-induced differentiation.Mol Pharmacol. 1994; 46: 840-850PubMed Google Scholar, 27Heit I Wieser RJ Herget T Faust D Borchert-Stuhltrager M Oesch F Dietrich C Involvement of protein kinase Cdelta in contact-dependent inhibition of growth in human and murine fibroblasts.Oncogene. 2001; 20: 5143-5154Crossref PubMed Scopus (33) Google Scholar Bryostatin 1 interacts with the diacylglycerol binding site on PKC,28Mutter R Wills M Chemistry and clinical biology of the bryostatins.Bioorg Med Chem. 2000; 8: 1841-1860Crossref PubMed Scopus (179) Google Scholar but its complete mode of action has not been fully elucidated, and it can affect PKCs in a cell-type specific manner.29Szallasi Z Smith CB Pettit GR Blumberg PM Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts.J Biol Chem. 1994; 269: 2118-2124Abstract Full Text PDF PubMed Google Scholar As illustrated in Figure 2, exposure of NHBE cells to bryostatin 1 over a range of concentrations (10 to 1000 nmol/L) for 15 minutes resulted in translocation of PKCδ from cytosol to membrane in response to all concentrations tested (Figure 2A). Mucin secretion was also significantly increased by bryostatin 1, with maximal stimulation at 100 nmol/L (Figure 2B). As illustrated in Figure 2C, phosphorylation of MARCKS also was induced in these cells in response to bryostatin 1 (from 10 to 1000 nmol/L) with maximal phosphorylation at 100 nmol/L. None of these treatments induced cytotoxicity as measured by lactate dehydrogenase release assay (data not shown).Figure 2Effect of bryostatin 1, a PKCδ activator, on mucin secretion in well differentiated NHBE cells. NHBE cells were exposed to bryostatin 1 over a range of concentrations from 1 to 1000 nmol/L for 15 minutes. A: PKCδ translocates from cytosol to membrane in response to bryostatin 1. α-Tubulin and E-cadherin were used as controls for the cytosolic and membrane fractions, respectively. Blots are representative of three replicate experiments. B: Bryostatin 1 provokes mucin secretion by NHBE cells in a concentration-dependent manner. Significantly different from vehicle control: *P < 0.05; †P < 0.001; ‡P < 0.005. Data are presented as mean ± SEM (n = 4). C: Phosphorylation of MARCKS in NHBE cells is induced by bryostatin 1 in a concentration-dependent manner. Blots are representative of three replicate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PKC Activation Stimulates Mucin Secretion in HBE1 CellsTo investigate further a role for PKCδ as a regulator of mucin secretion using molecular manipulation of PKCδ activity, the HBE1 cell line was used. As illustrated in Figure 3, exposure of HBE1 cells to 500 nmol/L PMA for 15 minutes significantly increased mucin secretion (by ∼1.7-fold compared with medium vehicle control) and also induced phosphorylation of MARCKS in these cells.Figure 3HBE-1 cells secrete mucin in response to PKC activation. HBE-1 cells maintained in air/liquid interface were exposed to 100 or 500 nmol/L PMA for 15 minutes. Mucin secretion and phosphorylation of MARCKS were assessed by enzyme-linked immunosorbent assay and Western blot analysis, respectively. A: Mucin secretion is significantly enhanced by PMA at 500 nmol/L (but not 100 nmol/L) in HBE1 cells. *Significantly different from vehicle control (P < 0.05). Data are presented as mean ± SEM (n = 4). B: Phosphorylation of MARCKS is increased by exposure of HBE1 cells to 500 nmol/L PMA. Blots are representative of three replicate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PKCδ Appears to Regulate Mucin Secretion in Airway Epithelial CellsTransient transfection of the PKCδ and PKCε constructs into HBE1 cells was confirmed by fluorescent microscopy and Western blot analysis. After 48 hours of transfection, GFP expressed in the transfected cells was detected with a fluorescent microscope [Eclipse TE300 (Nikon, Tokyo, Japan) or Axiovert 35 (Zeiss, Welwyn Garden City, UK)] before PMA exposure (data not shown). After exposure to PMA, cells were lysed to detect expression of PKCδ protein fused with GFP via Western blot analysis using anti-PKCδ and -GFP antibodies (data not shown). Transfection of PKCε was confirmed by Western blot analysis using an antibody against PKCε and an HA tag. Transfection efficiency of all constructs and controls was about 20 to 25% as determined by quantification of GFP and X-gal assay for PKCδ and PKCε, respectively (data not shown).As illustrated in Figure 4A, mucin secretion by transfected HBE1 cells was stimulated by exposure to 500 nmol/L PMA. Transfection of HBE1 cells with the dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) resulted in significant reduction of PMA-induced mucin secretion (∼45%), whereas cells transfected with the wild-type PKCδ construct (pEGFP-N1/PKCδ) showed a significant enhancement of PMA-induced mucin secretion (∼40%) compared with control cells transfected with no DNA or empty vector (pEGFP-N1). In additional control studies, the effects of Bryostatin 1 on both mucin secretion and phosphorylation of MARCKS were markedly attenuated in cells transfected with the dnPKCδ construct (data not shown). PKCε constructs, either wild type or mutated, did not affect the secretory response to PMA. As shown in Figure 4B, phosphorylation of MARCKS in response to PMA was decreased in HBE1 cells transfected with the dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) but increased in cells transfected with the wild-type PKCδ construct (pEGFP-N1/PKCδ). PKCε constructs, either wild type or mutated, had no effect on PMA-induced MARCKS phosphorylation when transfected into HBE1 cells (Figure 4C).Figure 4Transient transfection of HBE1 cells with a dominant-negative PKCδ construct results in reduction of mucin hypersecretion. HBE1 cells were transiently transfected with empty vector (pEGFP-N1), a wild-type PKCδ construct (pEGFP-N1/PKCδ), or a dominant-negative construct (pEGFP-N1/PKCδK376R) using the FuGene 6 transfection reagent as described in Materials and Methods. As an additional control, a wild-type (pHACE/PKCε) and a dominant-negative PKCε construct (pHACE/PKCεK437R) were also transfected. A: After 48 hours transfection, cells were exposed to 500 nmol/L PMA (lanes 2 to 8) or vehicle control (lane 1) for 15 minutes, at which time media were collected and mucin secretion assessed by enzyme-linked immunosorbent assay. Data are significantly different from media control (*P < 0.05; **P < 0.001); significantly different from cells transfected with n}, number={6}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Park, Jin-Ah and Crews, Anne L. and Lampe, William R. and Fang, Shijing and Park, Joungjoa and Adler, Kenneth B.}, year={2007}, month={Dec}, pages={1822–1830} }
 @article{park_crews_adler_2007, title={Protein kinase C delta regulates airway mucin secretion via phosphorylation of MARCKS protein.}, volume={175}, journal={404nOtfound}, author={Park, J. A. and Crews, A. L. and Adler, K. B.}, year={2007}, pages={A752} }
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 @article{chorley_li_fang_park_adler_2006, title={(R)-Albuterol elicits antiinflammatory effects in human airway epithelial cells via iNOS}, volume={34}, DOI={10.1165/rcmb.2005-03380C}, number={1}, journal={American Journal of Respiratory Cell and Molecular Biology}, author={Chorley, B. N. and Li, Y. H. and Fang, S. J. and Park, J. A. and Adler, K. B.}, year={2006}, pages={119–127} }
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 @article{park_adler_2006, title={Potential therapy for mucus hypersecretion in chronic obstructive pulmonary disease [translated into Chinese].}, volume={86}, journal={404nOtfound}, author={Park, J. A. and Adler, K. B.}, year={2006}, pages={2890–2892} }
 @article{park_chorley_adler_2005, title={Human Neutrophil Elastase provokes mucin secretion by NHBE cells via a Protein Kinase C delta (PKC?)?mediated mechanism.}, volume={2}, journal={404nOtfound}, author={Park, J.-A. and Chorley, B. N. and Adler, K. B.}, year={2005}, pages={A110} }
 @article{park_he_martin_li_chorley_adler_2005, title={Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C delta-mediated mechanism}, volume={167}, ISSN={["1525-2191"]}, DOI={10.1016/S0002-9440(10)62040-8}, abstractNote={The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC5AC and MUC5B, but not MUC2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCα, δ, ζ, λ, ι, and ε were constitutively expressed in NHBE cells while PKCβ, η, and μ were PMA-inducible. PKCδ was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCδ attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the δ isoform. The presence of mucus obstruction and neutrophil-predominant inflammation in several lung disorders, such as cystic fibrosis, suggests a relationship between neutrophils and excess mucus production. Mechanisms of human neutrophil elastase (HNE)-induced mucin secretion by well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air/liquid interface culture were investigated. HNE increased mucin secretion in a concentration-dependent manner, with maximal stimulation (more than twofold) occurring within a short (15 minutes) time period. Mucins MUC5AC and MUC5B, but not MUC2, were released in response to HNE. Stimulation of mucin secretion required partial elastase enzymatic activity and did not appear to involve a soluble product released by the cells. HNE-stimulated secretion involved activation of protein kinase C (PKC), as HNE exposure rapidly provoked PKC enzymatic activity that was attenuated by the general PKC inhibitors calphostin C and bisindoylmaleimide I. Of the different isoforms, PKCα, δ, ζ, λ, ι, and ε were constitutively expressed in NHBE cells while PKCβ, η, and μ were PMA-inducible. PKCδ was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCδ attenuated HNE-mediated mucin secretion. The results suggest HNE stimulation of mucin release by human airway epithelial cells involves intracellular activation of PKC, specifically the δ isoform. Neutrophils are involved in a variety of inflammatory lung disorders including chronic bronchitis, bronchiectasis, cystic fibrosis, and probably asthma. In these diseases, the pathological findings of mucus obstruction and neutrophil-predominant inflammation in airways1Fahy JV Kim KW Liu J Boushey HA Prominent neutrophilic inflammation in sputum from subjects with asthma exacerbation.J Allergy Clin Immunol. 1995; 95: 843-852Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 2Stockley RA Role of inflammation in respiratory tract infections.Am J Med. 1995; 99: 8S-13SAbstract Full Text PDF PubMed Scopus (47) Google Scholar, 3Welsh MD Adair BM Foster JC Effect of BVD virus infection on alveolar macrophage functions.Vet Immunol Immunopathol. 1995; 46: 195-210Crossref PubMed Scopus (54) Google Scholar, 4Mohapatra NK Cheng PW Parker JC Paradiso AM Yankaskas JR Boucher RC Boat TF Alteration of sulfation of glycoconjugates, but not sulfate transport and intracellular inorganic sulfate content in cystic fibrosis airway epithelial cells.Pediatr Res. 1995; 38: 42-48Crossref PubMed Scopus (28) Google Scholar, 5Fahy JV Schuster A Ueki I Boushey HA Nadel JA Mucus hypersecretion in bronchiectasis. The role of neutrophil proteases.Am Rev Respir Dis. 1992; 146: 1430-1433Crossref PubMed Scopus (109) Google Scholar, 6Stockley RA Hill SL Morrison HM Starkie CM Elastolytic activity of sputum and its relation to purulence and to lung function in patients with bronchiectasis.Thorax. 1984; 39: 408-413Crossref PubMed Scopus (93) Google Scholar suggest a relationship between neutrophil recruitment/infiltration and excess mucus production and secretion. Neutrophils store three proteases that have been implicated in airway mucin secretion: elastase,7Breuer R Christensen TG Lucey EC Stone PJ Snider GL An ultrastructural morphometric analysis of elastase-treated hamster bronchi shows discharge followed by progressive accumulation of secretory granules.Am Rev Respir Dis. 1987; 136: 698-703Crossref PubMed Scopus (46) Google Scholar, 8Nadel JA Protease actions on airway secretions. Relevance to cystic fibrosis.Ann NY Acad Sci. 1991; 624: 286-296Crossref PubMed Scopus (25) Google Scholar, 9Kim KC Wasano K Niles RM Schuster JE Stone PJ Brody JS Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells.Proc Natl Acad Sci USA. 1987; 84: 9304-9308Crossref PubMed Scopus (124) Google Scholar cathepsin G,10Sommerhoff CP Nadel JA Basbaum CB Caughey GH Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.J Clin Invest. 1990; 85: 682-689Crossref PubMed Scopus (285) Google Scholar and proteinase-3.11Rao NV Marshall BC Gray BH Hoidal JR Interaction of secretory leukocyte protease inhibitor with proteinase-3.Am J Respir Cell Mol Biol. 1993; 8: 612-616Crossref PubMed Scopus (60) Google Scholar, 12Renesto P Halbwachs-Mecarelli L Nusbaum P Lesavre P Chignard M Proteinase 3. A neutrophil proteinase with activity on platelets.J Immunol. 1994; 152: 4612-4617PubMed Google Scholar Of these, human neutrophil elastase (HNE), a major component of primary or azurophilic granules,13Bainton DF Ullyot JL Farquhar MG The development of neutrophilic polymorphonuclear leukocytes in human bone marrow.J Exp Med. 1971; 134: 907-934Crossref PubMed Scopus (565) Google Scholar is the most widely studied with regard to enhanced mucus secretion. Levels of HNE are elevated in airways of patients with chronic bronchitis and cystic fibrosis,14Fick Jr, RB Naegel GP Squier SU Wood RE Gee JB Reynolds HY Proteins of the cystic fibrosis respiratory tract. Fragmented immunoglobulin G opsonic antibody causing defective opsonophagocytosis.J Clin Invest. 1984; 74: 236-248Crossref PubMed Scopus (153) Google Scholar and levels in patients' sputum may exceed 100 μg/ml (3.3 × 10−6 mol/L).15Doring G Goldstein W Botzenhart K Kharazmi A Schiotz PO Hoiby N Dasgupta M Elastase from polymorphonuclear leucocytes: a regulatory enzyme in immune complex disease.Clin Exp Immunol. 1986; 64: 597-605PubMed Google Scholar, 16Goldstein W Doring G Lysosomal enzymes from polymorphonuclear leukocytes and proteinase inhibitors in patients with cystic fibrosis.Am Rev Respir Dis. 1986; 134: 49-56PubMed Google Scholar, 17Suter S Schaad UB Tegner H Ohlsson K Desgrandchamps D Waldvogel FA Levels of free granulocyte elastase in bronchial secretions from patients with cystic fibrosis: effect of antimicrobial treatment against Pseudomonas aeruginosa.J Infect Dis. 1986; 153: 902-909Crossref PubMed Scopus (104) Google Scholar Purified HNE has been shown to provoke secretion of mucin by isolated airway epithelial cells and glands from several species.7Breuer R Christensen TG Lucey EC Stone PJ Snider GL An ultrastructural morphometric analysis of elastase-treated hamster bronchi shows discharge followed by progressive accumulation of secretory granules.Am Rev Respir Dis. 1987; 136: 698-703Crossref PubMed Scopus (46) Google Scholar, 8Nadel JA Protease actions on airway secretions. Relevance to cystic fibrosis.Ann NY Acad Sci. 1991; 624: 286-296Crossref PubMed Scopus (25) Google Scholar, 10Sommerhoff CP Nadel JA Basbaum CB Caughey GH Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.J Clin Invest. 1990; 85: 682-689Crossref PubMed Scopus (285) Google Scholar, 18Kim KC Nassiri J Brody JS Mechanisms of airway goblet cell mucin release: studies with cultured tracheal surface epithelial cells.Am J Respir Cell Mol Biol. 1989; 1: 137-143Crossref PubMed Scopus (46) Google Scholar Although there have been suggestions that interactions between HNE and epithelial cell surfaces may be involved in the response,9Kim KC Wasano K Niles RM Schuster JE Stone PJ Brody JS Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells.Proc Natl Acad Sci USA. 1987; 84: 9304-9308Crossref PubMed Scopus (124) Google Scholar, 19Takeyama K Agusti C Ueki I Lausier J Cardell LO Nadel JA Neutrophil-dependent goblet cell degranulation: role of membrane-bound elastase and adhesion molecules.Am J Physiol. 1998; 275: L294-L302PubMed Google Scholar intracellular mechanisms and signaling pathways associated with HNE-induced mucin hypersecretion have not been elucidated. In this study, well-differentiated primary normal human tracheobronchial epithelial (NHBE) cells maintained in vitro in air/liquid interface were exposed to HNE, and the secretory response assessed. Elastase proved to be a potent mucin secretagogue for NHBE cells, eliciting a robust (greater than twofold) increase in mucin secretion within 15 minutes. The mucin gene products released included those of MUC5AC and MUC5B, but not of MUC2. The mechanism appeared to involve activation of protein kinase C (PKC), as HNE exposure rapidly provoked phosphorylation of MARCKS (myristoylated alanine-rich C kinase substrate) protein, a cellular substrate of PKC, and the mucin secretory response to HNE was attenuated by two different PKC inhibitors. Additional studies provided compelling evidence that PKCδ is the specific PKC isoform involved in the secretory pathway. All chemicals were of analytical grade or higher. NHBE cells, bronchial epithelial basal medium, and supplements for air/liquid interface cell cultures were purchased from Cambrex (San Diego, CA). Endotoxin-free HNE purified from human sputum was purchased from Elastin Products Company (EPC, Owensville, MO). Cytotoxicity was evaluated with CytoTox 96 nonradioactive cytotoxicity assay kits obtained from Promega Corp. (Madison, WI). A specific HNE substrate, MeO-SUC-AL-AL-PRO-VAL-PNA, and an HNE inhibitor, chloromethyl ketone-modified tetrapeptide (CMK), also were purchased from EPC and the HNE inhibitor elastatinal was obtained from Calbiochem (La Jolla, CA). 17Q2 pan mucin antibody was purchased from Babco (Richmond, CA) and anti-MUC5AC (45M1) was purchased from Neomarkers (Fremont, CA). A monoclonal antibody (11C1) against human MUC5B was generously provided by Dr. Reen Wu, University of California at Davis, Davis, CA. The epitope for this antibody, which was generated from the secreted mucin of well-differentiated airway epithelial cells, is not known, but by immunohistochemical staining and Western blot analysis, it appears to recognize the MUC5B peptide. A monoclonal antibody that cross reacts with human MUC2, raised against the guinea pig 522-bp gene sequence analogous to the human D4 domain located in the carboxy-terminal region of the Muc2 gene sequence established previously in our laboratory, was used to detect MUC2 mucins.20Li Y Martin LD Minnicozzi M Greenfeder S Fine J Pettersen CA Chorley B Adler KB Enhanced expression of mucin genes in a guinea pig model of allergic asthma.Am J Respir Cell Mol Biol. 2001; 25: 644-651Crossref PubMed Scopus (32) Google Scholar An ImmunoPure (G) IgG purification kit used for purification of antibodies for enzyme-linked immunosorbent assay (ELISA) was from Pierce (Rockford, IL). For Western blot analysis of PKC isoforms expressed in NHBE cells, a PKC sampler kit and E-cadherin antibody were obtained from BD Biosciences (San Jose, CA). Goat anti-PKCζ and mouse anti-α-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphorylated (ser) PKC substrate and phosphorylated MARCKS were from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated goat anti-mouse IgG and donkey anti-goat IgG also were purchased from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-rabbit IgG was purchased from Upstate Biotechnology (Lake Placid, NY). Enhanced chemiluminescence development kits and Hyperfilm were from Amersham Pharmacia Biotech (Piscataway, NJ). All PKC-related inhibitors (ie, calphostin C, bisindoylmaleimide, PKC epsilon and zeta inhibitor peptides, rottlerin) were purchased from Calbiochem. A PepTag assay for nonradioactive detection of PKC activity was purchased from Promega. Other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO). Transwell-Clear culture inserts and high-binding 96-well assay plates were purchased from Corning Inc. (Corning, NY). Primary cultures of NHBE cells were established using an air/liquid interface cell culture system described previously.21Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (155) Google Scholar Briefly, NHBE cells were expanded once and cells collected and frozen in liquid nitrogen (referred to as passage-2 cells). Air/liquid interface cultures of NHBE cells were established on Transwell-Clear culture inserts thin-coated with rat-tail type I collagen. The basic medium used for NHBE cells was a 1:1 mixture of bronchial epithelial basal medium and high glucose (4.5 g/L) Dulbecco's modified Eagle's medium. The complete medium was composed of basic medium containing a final concentration of 0.5 ng/ml human recombinant epidermal growth factor, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10 μg/ml transferrin, 0.5 μg/ml epinephrine, 6.5 ng/ml triiodothyronine, 50 μg/ml gentamicin, and 50 ng/ml amphotericin-B. In addition, the media contained 0.13 mg/ml bovine pituitary extract made according to the protocol of Bertolero and colleagues,22Bertolero F Kaighn ME Gonda MA Saffiotti U Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium.Exp Cell Res. 1984; 155: 64-80Crossref PubMed Scopus (61) Google Scholar 5 × 10−8 mol/L all-trans retinoic acid, 1.5 μg/ml bovine serum albumin, and 20 U/ml nystatin. Frozen NHBE cells were recovered and seeded at a density of ∼2 × 104 cells/cm2 onto the apical surface of the inserts. Media were changed the next day, then every other day until the cells reached ∼90% confluence. At this point, the air/liquid interface was established by removing the apical media, whereas basolateral media were changed daily for up to 21 days. A mucin phenotype was observed at ∼14 days in culture (∼7 days in air-liquid interface culture) and cilia were apparent by 18 days in culture. Mucin secretion reached maximal levels at ∼18 days in culture, so cells cultured for ∼18 to 21 days were used for the experiments described below. HNE stock was made as 10 mg/ml (339 μmol/L) in a 1:1 mixture of glycerol and 0.02 mol/L NaOAc, pH 5.0. The stock was diluted into the culture medium to the final concentration indicated. In all studies, the above solvent appropriately diluted was used as a negative control. NHBE cells were exposed to HNE from both apical and basolateral sides for 15 minutes (unless otherwise indicated). At the end of each treatment, apical medium containing the secreted mucin was collected and quantified. Briefly, 0.25 ml of media containing secreted mucin was collected, 0.5 ml of 1 mmol/L dithiothreitol in phosphate-buffered saline (PBS) was added into each well, and the plates were gently agitated and allowed to stand for 3 minutes before the dithiothreitol/PBS plus mucin was collected in the same tube. Finally, 0.5 ml of 10 μmol/L CMK in PBS was added and collected the same way. Approximately 1.25 ml of the collected mucin mixture with dithiothreitol and CMK was centrifuged at 8000 rpm for 5 minutes to remove cell debris, and then collected in a fresh tube. Phenylmethyl sulfonyl fluoride was added to a final concentration of 1 mmol/L. Baseline and treatment mucin secretions were collected from each culture plate. Baseline mucin secretion was collected to normalize variations from well to well, and to control for possible release of mucin in response to the stress of media change or washing. After the baseline mucin secretion sample was collected, the cells were rested overnight and exposed to test agents the next day for indicated periods of time. Mucin samples were quantified using specific ELISA methods. Firstly, total mucin was quantified by a double-sandwich ELISA using a pan-mucin antibody, 17Q2, that cross reacts with a carbohydrate epitope on human mucins, as described previously.21Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (155) Google Scholar Additional studies were performed using ELISAs for secreted protein products of the mucin genes MUC5AC, MUC5B, and MUC2 to determine which mucin gene products were being released on exposure to HNE. MUC5AC was measured via ELISA as described by Takeyama and colleagues23Takeyama K Dabbagh K Lee HM Agusti C Lausier JA Ueki IF Grattan KM Nadel JA Epidermal growth factor system regulates mucin production in airways.Proc Natl Acad Sci USA. 1999; 96: 3081-3086Crossref PubMed Scopus (522) Google Scholar using the 45M1 antibody. MUC5B protein was assayed via a standard double-sandwich ELISA method using the 11C1 monoclonal antibody against MUC5B provided by Dr. Reen Wu, University of California, Davis, Davis, CA, as described previously.24Groneberg DA Eynott PR Oates T Lim S Wu R Carlstedt L 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 (154) Google Scholar, 25Crowther JR ELISA. Theory and practice.Methods Mol Biol. 1995; 42: 1-218PubMed Google Scholar The MUC2 gene product was quantified by modification of an ELISA as described previously.20Li Y Martin LD Minnicozzi M Greenfeder S Fine J Pettersen CA Chorley B Adler KB Enhanced expression of mucin genes in a guinea pig model of allergic asthma.Am J Respir Cell Mol Biol. 2001; 25: 644-651Crossref PubMed Scopus (32) Google Scholar HNE activity assays were performed following the manufacturer's protocol (EPC). HNE substrate was prepared in substrate buffer (Tris-NaCl buffer: 0.1 mol/L Tris, pH 7.5, containing 0.5 mol/L NaCl and 0.01% Na3N). Briefly, 3 ml of substrate solution at 25°C was added to test tubes, 1.0 μg of HNE then was added, and the developed color was read immediately and continuously thereafter at 1 minute intervals. Elastase activity was reflected by the rate increase in absorbance in time units (minutes). Color development was read at 410 nm on a spectrophotometer UV160U (Shimadzu, Kyoto, Japan). The specific activity of HNE was expressed as U/mg, and results expressed as percentage of activity of native HNE for each treatment. Effects of enzymatic inhibition of HNE were investigated using three different elastase inhibitors: 1) elastatinal, a natural HNE inhibitor produced by Actinomycetes;26Umezawa H Structures and activities of protease inhibitors of microbial origin.Methods Enzymol. 1976; 45: 678-695Crossref PubMed Scopus (303) Google Scholar 2) CMK, a synthetic tetrapeptide;27Rees DD Brain JD Wohl ME Humes JL Mumford RA Inhibition of neutrophil elastase in CF sputum by L-658,758.J Pharmacol Exp Ther. 1997; 283: 1201-1206PubMed Google Scholar and 3) α1-antitrypsin (α1-AT), a physiological HNE inhibitor.28Gadek JE Fells GA Zimmerman RL Rennard SI Crystal RG Antielastases of the human alveolar structures. Implications for the protease-antiprotease theory of emphysema.J Clin Invest. 1981; 68: 889-898Crossref PubMed Scopus (311) Google Scholar The inhibitors were added directly to HNE, incubated for 15 minutes at 37°C, and then added directly to the cells for another 15 minutes. At the end of this exposure, secreted mucin was collected and quantified as described above. To determine whether HNE enzymatic activity was directly required for stimulated mucin secretion, or if a secondary product(s) released by NHBE cells after exposure to HNE could be involved in the secretory response, NHBE cells were exposed to HNE (or vehicle) for 5 minutes. After exposure, the conditioned medium was collected and treated with 5 μmol/L of the HNE enzymatic inhibitor, α1-AT, for 15 minutes, at which time this α1-AT-treated medium was added to a new set of NHBE cells and effects on mucin secretion quantified as described above. The PKC inhibitors, bisindolylmaleimide I (10, 100, 1000 nmol/L)29Martiny-Baron G Kazanietz MG Mischak H Blumberg PM Kochs G Hug H Marme D Schachtele C Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976.J Biol Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar or calphostin C (5, 50, 500 nmol/L)30Takahashi I Saitoh Y Yoshida M Sano H Nakano H Morimoto M Tamaoki T UCN-01 and UCN-02, new selective inhibitors of protein kinase C. II. Purification, physico-chemical properties, structural determination and biological activities.J Antibiot (Tokyo). 1989; 42: 571-576Crossref PubMed Scopus (168) Google Scholar were used to determine PKC involvement in HNE-induced mucin secretion. NHBE cells were preincubated with these agents (or vehicle control) for 15 minutes, then HNE was added for another 15 minutes before mucin secretion was quantified as described above. PKC activity in NHBE cells after exposure to HNE was assessed using a PepTag assay for nonradioactive detection of PKC (following the manufacturer's protocol). Briefly, 10 μg of protein extracted from each treatment of NHBE cells was added into the PKC reaction buffer (20 mmol/L HEPES, pH 7.4, 1.3 mmol/L CaCl2, 1 mmol/L dithiothreitol, 10 mmol/L MgCl2, 1 mmol/L ATP) containing 1 mg/ml phosphatidylserine and PepTag C1 PKC substrate peptide (P-L-S-R-T-L-S-V-A-A-K) conjugated with fluorescent dye, and incubated for 30 minutes at 30°C. The reaction was stopped by boiling at 100°C for 10 minutes. Reaction mixtures were separated on 0.8% agarose gels and proteins quantified by Labworks image acquisition and analysis software (UVP Bioimaging System, Upland, CA). Phosphorylation of MARCKS was detected by Western blot using an antibody against phophospecific-MARCKS. After treatments, NHBE cells were washed with ice-cold PBS twice and then scraped into lysis buffer (50 mmol/L Tris, pH 7.5, 1 mmol/L ethylenediamine tetraacetic acid, 100 mmol/L NaCl, 1 mmol/L phenylmethyl sulfonyl fluoride) using a rubber policemen. The collected cells were lysed by sonication. For separation of cytosolic and membrane fractions, the lysates were spun at 400,000 × g in a Sorvall Discovery 100S ultracentrifuge (Sorvall, Inc. Newtown, CT) for 1 hour. The supernatant was reserved as the cytosolic sample. The pellet was resuspended in the same lysis buffer containing 0.05% Triton-100, dissolved by sonication, and incubated on ice for 30 minutes. After incubation, the same ultracentrifugation as described above was performed on the pellet mixture, and the supernatant separated from the pellet mixture was reserved as the membrane fraction. For preparation of whole cell crude lysates, the disrupted cellular mixture was centrifuged at 15,000 rpm in an Eppendorf 5417R centrifuge (Eppendorf Corp., Hamburg, Germany) for 1 hour at 4°C. The supernatant was collected as the whole crude NHBE cell lysate. The protein concentration of cell lysate samples was quantified by a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Each sample was boiled in 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for 10 minutes, loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and transferred to a polyvinylidene difluoride membrane (Micron Separation Inc., Westborough, MA). After blocking with 5% skim milk, the antigen was captured by the specific PKC antibody and further amplified by binding to horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies. Anti-α-tubulin and E-cadherin antibodies were used for cytosolic and membrane controls, respectively, for each sample. Final development was accomplished by the enhanced chemiluminescence method. The amount of each PKC isoform was analyzed by Labworks image acquisition and analysis software. Because the studies above indicated that PKCδ was the only isoform to translocate to membranes in response to HNE, additional studies were performed with rottlerin, an inhibitor of PKCδ and θ.31Gschwendt M Muller HJ Kielbassa K Zang R Kittstein W Rincke G Marks F Rottlerin, a novel protein kinase inhibitor.Biochem Biophys Res Commun. 1994; 199: 93-98Crossref PubMed Scopus (767) Google Scholar (Because PKCθ was not expressed in NHBE cells under basal or stimulated conditions, rottlerin is referred to below as a specific inhibitor of PKCδ). Rottlerin has the following potency against PKC isoforms: PKC δ (IC50 = 3 to 6 μmol/L); PKCθ (IC50 = 50 μmol/L); PKCα, PKCβ, and PKC γ (IC50 = 30 to 42 μmol/L); PKCε, PKCη, and PKCζ (IC50 = 80 to 100 μmol/L). It also can inhibit CaM kinase III (IC50 = 5.3 μmol/L).31Gschwendt M Muller HJ Kielbassa K Zang R Kittstein W Rincke G Marks F Rottlerin, a novel protein kinase inhibitor.Biochem Biophys Res Commun. 1994; 199: 93-98Crossref PubMed Scopus (767) Google Scholar, 32Villalba M Kasibhatla S Genestier L Mahboubi A Green DR Altman A Protein kinase C cooperates with calcineurin to induce fas ligand expression during activation-induced T cell death.J Immunol. 1999; 163: 5813-5819PubMed Google Scholar Cells were preincubated with rottlerin (1.5 μmol/L; IC50 = 3 to 6 μmol/L) for 20 minutes before exposure to HNE, and effects on PKC activity [using detection of phosphorylated (ser) PKC substrate] and on HNE-induced mucin secretion were assessed. As additional controls, the potential role of other PKC isoforms present in these cells was assessed: cells were exposed to the following specific inhibitors for 15 minutes before exposure to HNE and assay for mucin secretion: The PKCα/β inhibitor, Gö 6976 (10 nmol/L; IC50 = 2 ∼ 6 nmol/L);29Martiny-Baron G Kazanietz MG Mischak H Blumberg PM Kochs G Hug H Marme D Schachtele C Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976.J Biol Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar a PKCζ peptide inhibitor (50 μmol/L; Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu; IC50 = 10 μmol/L);33Bandyopadhyay G Standaert ML Galloway L Moscat J Farese RV Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes.Endocrinology. 1997; 138: 4721-4731Crossref PubMed Scopus (210) Google Scholar or a PKCε peptide inhibitor (3 ∼ 300 μmol/L; Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr; IC50 = 80.3 μmol/L).34Johnson JA Gray MO Chen CH Mochly-Rosen D A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function.J Biol Chem. 1996; 271: 24962-24966Crossref PubMed Scopus (343) Google Scholar, 35Mendez CF Leibiger IB Leibiger B Hoy M Gromada J Berggren PO Bertorello AM Rapid association of protein kinase C-epsilon with insulin granules is essential for insulin exocytosis.J Biol Chem. 2003; 278: 44753-44757Crossref PubMed Scopus (59) Google Scholar Data were expressed as the ratio of treatment to the corresponding vehicle control. Results were evaluated using one-way analysis of variance with Bonferroni posttest correction for multiple comparisons.36Kleinbaum DG Kupper LL Muller KE Applied Regression Analysis and Other Multivariable Methods. PWS-Kent Pub. Co., Boston1988: 341-386Google Scholar A P value of <0.05 was considered significant. All reagents used were tested for cytotoxicity using a Promega Cytotox 96 nonradioactive cytotoxicity assay kit according to the manufacturer's instructions. The data were expressed as the ratio of released lactate dehydrogenase to total lactate dehydrogenase. Released lactate dehydrogenase never exceeded 10% of total lactate dehydrogenase (data not shown) in any of the experiments below. As illustrated in Figure 1, HNE stimulated mucin secretion by NHBE cells. Maximal mucin secretion was elicited after 15 minutes exposure to HNE (Figure 1A) so this time point was chosen for additional experiments. HNE increased mucin secretion in a concentration-dependent manner, with 0.01 to 1.0 μmol/L HNE increasing secretion significantly over vehicle control (Figure 1B). Secretion of major gel-forming mucins, including MUC2, MUC5AC, and MUC5B, was investigated after exposure to HNE. As illustrated in Figure 2, HNE enhanced release of both MUC5AC and MUC5B mucins from NHBE cells in a concentration-dependent manner. Secretion of MUC2 mucin was significantly decreased by HNE. Elastatinal appeared to be the weakest of the three HNE inhibitors used in this study because the highest concentration used, 100 μmol/L, blocked only 50% of HNE enzymatic activity and did not affect HNE-stimulated mucin secretion (Figure 3A). CMK proved to be a more potent HNE enzymatic inhibitor because 50 μmol/L CMK completely blocked the enzymatic activity of 1 μmol/L HNE, whereas lower concentrations partially inhibited HNE activity in a concentration-dependent manner. CMK also showed an inhibitory effect on HNE-stimulated mucin secretion in a concentration-dependent manner w}, number={3}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Park, JA and He, F and Martin, LD and Li, YH and Chorley, BN and Adler, KB}, year={2005}, month={Sep}, pages={651–661} }
 @article{park_fang_gruber_adler_2004, title={MARCKS protein interaction with the ?secretory module? regulates airway mucin secretion.}, volume={169}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Park, J. and Fang, S. and Gruber, A. D. and Adler, K. B.}, year={2004}, pages={A535} }
 @article{park_he_li_martin_adler_2003, title={Human neutrophil elastase provokes release of MUC5B mucin from normal bronchial epithelial cells in vitro via a PKC-dependent mechanism.}, volume={167}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Park, J. A. and He, F. and Li, Y. and Martin, L. D. and Adler, K. B.}, year={2003}, pages={A203} }