@misc{lin_park_crews_li_adler_2008, title={Protease-activated receptor-2 (PAR-2) is a weak enhancer of mucin secretion by human bronchial epithelial cells in vitro}, volume={40}, ISSN={["1878-5875"]}, DOI={10.1016/j.biocel.2007.10.031}, abstractNote={PAR-2, a member of a family of G-protein-coupled receptors, can be activated by serine proteases via proteolytic cleavage. PAR-2 expression is known to be upregulated in respiratory epithelium subsequent to inflammation in asthma and chronic obstructive pulmonary disease (COPD). Since these diseases also are characterized by excessive mucus production and secretion, we investigated whether PAR-2 could be linked to mucin hypersecretion by airway epithelium. Normal human bronchial epithelial (NHBE) cells in primary culture or the human bronchial epithelial cell lines, NCI-H292 and HBE-1, were used. NHBE, NCI-H292, and HBE-1 cells expressed prominent levels of PAR-2 protein. Short-term (30min) exposure of cells to the synthetic PAR-2 agonist peptide (SLIGKV-NH2) elicited a small but statistically significant increase in mucin secretion at high concentrations (100microM and 1000microM), compared to a control peptide with reversed amino acid sequence (VKGILS-NH2). Neither human lung tryptase nor bovine pancreatic trypsin, both PAR-2 agonists, affected NHBE cell mucin secretion when added over a range of concentrations. Knockdown of PAR-2 expression by siRNA blocked the stimulatory effect of the AP. The results suggest that, since PAR-2 activation only weakly increases mucin secretion by human airway epithelial cells in vitro, PAR-2 probably is not a significant contributor to mucin hypersecretion in inflamed airways.}, number={6-7}, journal={INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY}, author={Lin, Ko-Wei and Park, Joungjoa and Crews, Anne L. and Li, Yuehua and Adler, Kenneth B.}, year={2008}, pages={1379–1388} }
 @misc{li_martin_adler_2007, title={Method and compositions for altering mucus secretion}, volume={7,265,088}, number={2007 Sept. 4}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Li, Y.-H. and Martin, L. D. and Adler, K. B.}, year={2007} }
 @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} }
 @misc{takashi_parikh_adler_martin_y._2006, title={Methods for regulating inflammatory mediators and peptides useful therein}, volume={7,544,772}, number={2006 Sep. 28}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Takashi, S. and Parikh, I. and Adler, K. B. and Martin, L. D. and Y., Li}, year={2006} }
 @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{singer_martin_vargaftig_park_gruber_li_adler_2004, title={A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma}, volume={10}, ISSN={["1546-170X"]}, DOI={10.1038/nm983}, abstractNote={Mucus hypersecretion is a crucial feature of pulmonary diseases such as asthma, chronic bronchitis and cystic fibrosis. Despite much research, there is still no effective therapy for this condition. Recently, we showed that the myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is required for mucus secretion by human bronchial epithelial cells in culture. Having synthesized a peptide corresponding to the N-terminal domain of MARCKS, we now show that the intratracheal instillation of this peptide blocks mucus hypersecretion in a mouse model of asthma. A missense peptide with the same amino acid composition has no effect. Based on quantitative histochemical analysis of the mouse airways, the peptide seems to act by blocking mucus release from goblet cells, possibly by inhibiting the attachment of MARCKS to membranes of intracellular mucin granules. These results support a pivotal role for MARCKS protein, specifically its N-terminal region, in modulating this secretory process in mammalian airways. Intratracheal administration of this MARCKS-related peptide could therapeutically reduce mucus secretion in the airways of human patients with asthma, chronic bronchitis and cystic fibrosis.}, number={2}, journal={NATURE MEDICINE}, author={Singer, M and Martin, LD and Vargaftig, BB and Park, J and Gruber, AD and Li, YH and Adler, KB}, year={2004}, month={Feb}, pages={193–196} }
 @misc{martin_adler_li_2004, title={Blocking peptide for inflammatory cell secretion}, volume={WO/2003/000027}, number={2004 Sep. 16}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Martin, L. D. and Adler, K. B. and Li, Y}, year={2004} }
 @article{vargaftig_singer_martin_li_adler_2003, title={A myristoylated peptide directed against the N-terminal region of MARCKS protein inhibits mucin secretion in ovalbumin sensitized/challenged mice in vivo.}, volume={167}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Vargaftig, B. and Singer, M. and Martin, L. D. and Li, Y. and Adler, K. B.}, year={2003}, pages={A17} }
 @article{lin_park_li_adler_2003, title={Activation of protease-activated receptors?2 (PAR-2) is not associated with enhanced mucin secretion by well-differentiated normal human bronchial epithelial cells in vitro.}, volume={167}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Lin, K. W. and Park, J. J. and Li, Y. and Adler, K. B.}, year={2003}, pages={A204} }
 @article{chorley_martin_crews_li_adler_2003, title={Differential effects of albuterol isomers on normal human bronchial epithelial cells in vitro.}, volume={167}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Chorley, B. N. and Martin, L. D. and Crews, A. C. and Li, Y. and Adler, K. B.}, year={2003}, pages={A205} }
 @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} }
 @article{adler_li_2001, title={Airway epithelium and mucus - Intracellular signaling pathways for gene expression and secretion}, volume={25}, ISSN={["1044-1549"]}, DOI={10.1165/ajrcmb.25.4.f214}, abstractNote={It is the rare scientific paper dealing with any aspect of airway mucus that does not open with a statement about the contribution of excess mucus to the pathogenesis of airway obstruction, susceptibility to infection, or compromised defense in a myriad of inflammatory airway diseases, such as chronic bronchitis, asthma, bronchiectasis, or cystic fibrosis. Excess mucus in the airways can result from any of three different lesions, and in most cases various combinations of these: ( 1 ) enhanced production through overexpression of mucin (MUC) genes; ( 2 ) excess production secondary to mucus cell hyperplasia, hypertrophy, or even metaplasia; or ( 3 ) hypersecretion of formed and stored mucin by goblet cells or glands in the airways. In context of a perspective, it may be instructive to trace the historical pathways that have led to our present understanding of the mechanisms associated with mucus-related phenomena. Clearly, the importance of studying production and secretion of mucus (or its glycoprotein component, mucin) was not lost on early researchers. In the 1960s and early 1970s, several groups looked at mucus production and secretion in the airways. However, lack of appropriate in vitro or in vivo model systems made these early studies mostly descriptive and limited, for the most part, to characterization of alcian blue/PAS-stained cells in different regions of the airways in health and disease (1-4). In the mid-1970s, with the introduction of organ culture techniques to study isolated rings or explants of bronchi or trachea from several species, it became possible to investigate mechanisms related to production and secretion of mucin (5, 6). Unfortunately, there were serious problems with explant cultures, not the least of which was quantification of produced or released mucin. The “state-of-the-art” at that time was either to measure carbohydrate components of secreted or retained mucin in the explants (such as sialic acid; fucose, or glucosamine [7]) or to incubate the explants with a radiolabeled sugar (such as tritiated glucosamine) for a time period allowing for incorporation of the label into the mucin glycoproteins, and then measure the released radiolabeled activity as a reflection of secreted mucin, or radioactivity within the tissue as a measure of mucin synthesis (8). For greater specificity, the homogenate or spent medium was either precipitated with trichloroacetic acid, sometimes with the addition of phosphotungstic acid, prior to counting of radioactivity. More accurate quantification was achieved by treatment of the homogenate or spent medium with enzymes to digest other contaminating sugar-containing proteins, such as hyaluronic acid or chondroitin sulfate, with hyaluronidase or chondroitinase ABC, respectively. Separation via column chromatography also improved detection, as the high molecular weight mucins would appear in the void volume (9). A second problem related to organ cultures was the large number of cell types present in the explants, confounding interpretation of effects of added agents on epithelium and making it difficult to attribute responses to any particular cell type. There were some major advancements in the field of mucin research during the 1980s, both in the development of better cell culture techniques and in detection of intraand extracellular (secreted) mucin. The first was a great improvement in our ability to culture cells from airway epithelium from several species. Prior to this time, it was difficult to culture airway epithelial cells so as to maintain differentiated characteristics in vitro , but the development of defined, serum-free medium, as well as improvements in the types of substrata beneath the cultured cells, gave researchers the ability to culture airway epithelial cells that looked and acted somewhat like their in vivo counterparts. Maintaining cells in a defined medium atop a collagen gel provided improved model systems, and in the latter part of the 1980s, the concept of air/liquid interface culture was first introduced. Starting with guinea pig tracheal epithelial cells (10, 11), it was discovered that cells grown on a collagen substrate, atop a permeant filter, with all medium placed beneath the cells and only a humidified air environment above, would result in well-differentiated epithelial cells essentially identical in structure and function to airway epithelium in situ . In quick succession, techniques for air/liquid interface culture of airway epithelium from rat (12), bovine (13), canine (14), primate (15), and eventually human (16-18) cells were developed. At present, culturing human airway epithelial cells in air/liquid interface provides a model system in which the epithelial cells are similar if not identical to human airway epithelium in vivo , and such cells now can be purchased commercially. With regard to detection of intracellular or secreted ( Received in original form September 10, 2001 )}, number={4}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Adler, KB and Li, YH}, year={2001}, month={Oct}, pages={397–400} }
 @article{li_martin_minnicozzi_greenfeder_fine_pettersen_chorley_adler_2001, title={Enhanced expression of mucin genes in a guinea pig model of allergic asthma}, volume={25}, ISSN={["1535-4989"]}, DOI={10.1165/ajrcmb.25.5.4485}, abstractNote={The ovalbumin (OVA)-sensitized guinea pig is often used as an animal model of asthma and airway hyperreactivity. A characteristic lesion of asthma is excessive production of mucin in the airways. Mechanistic studies of this lesion in guinea pigs have been limited due to lack of mucin gene probes for this species. The aim of the present study was to clone the cDNAs encoding two major airway mucins (Muc2 and Muc5ac) from the guinea pig, and investigate mucin gene expression in lungs of sensitized animals in response to antigen challenge. We isolated and sequenced two cDNA fragments coding for the sequences located within the carboxyl-terminal cysteine-rich region of guinea pig Muc2 and Muc5ac mucins. Comparison of cloned cDNAs with those from other species revealed high degrees of sequence identity and conservation of all cysteine residues in deduced primary sequences. Based on the resultant sequence information, we also designed oligonucleotide primers for specific detection of guinea-pig Muc2 and Muc5ac steady-state mRNA levels via reverse transcriptase/ polymerase chain reaction (RT-PCR). Levels of both Muc2 and Muc5ac mRNA in lungs of OVA-sensitized guinea pigs increased significantly by 30 min after an acute exposure to 0.3% OVA. In addition, levels of eotaxin mRNA also increased in these tissues, but the increases were not significant until 2 h after challenge. Correspondingly, the number of eosinophils in bronchoalveolar lavage fluid did not increase until 4 h postchallenge. Results of these studies suggest that the OVA-sensitized guinea pig responds to allergic challenge with enhanced expression of genes (e.g., eotaxin, Muc2, and Muc5ac) that likely play a role in increased airway inflammation and mucin overproduction, and enhanced mucin gene expression appears to occur before eosinophil infiltration.}, number={5}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Li, YH and Martin, LD and Minnicozzi, M and Greenfeder, S and Fine, J and Pettersen, CA and Chorley, B and Adler, KB}, year={2001}, month={Nov}, pages={644–651} }
 @article{li_pettersen_martin_adler_2001, title={MARCKS protein interaction with the cellular contractile machinery may regulate mucin secretion by human airway epithelium.}, volume={163}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Li, Y. and Pettersen, C. A. and Martin, L. D. and Adler, K. B.}, year={2001}, pages={A225} }
 @article{li_martin_spizz_adler_2001, title={MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro}, volume={276}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.M105614200}, abstractNote={Hypersecretion of airway mucin characterizes numerous respiratory diseases. Although diverse pathological stimuli can provoke exocytotic release of mucin from secretory cells of the airway epithelium, mechanisms involved remain obscure. This report describes a new paradigm for the intracellular signaling mechanism regulating airway mucin secretion. Direct evidence is provided that the myristoylated alanine-rich C kinase substrate (MARCKS) is a central regulatory molecule linking secretagogue stimulation at the cell surface to mucin granule release by differentiated normal human bronchial epithelial cells in vitro. Down-regulation of MARCKS expression or disruption of MARCKS function in these cells inhibits the secretory response to subsequent stimulation. The intracellular mechanism controlling this secretory process involves cooperative action of two separate protein kinases, protein kinase C and cGMP-dependent protein kinase. Upon stimulation, activated protein kinase C phosphorylates MARCKS, causing translocation of MARCKS from the plasma membrane to the cytoplasm, where it is then dephosphorylated by a protein phosphatase 2A that is activated by cGMP-dependent protein kinase, and associates with both actin and myosin. Dephosphorylated cytoplasmic MARCKS would also be free to interact with mucin granule membranes and thus could link granules to the contractile cytoskeleton, mediating their movement to the cell periphery and subsequent exocytosis. These findings suggest several novel intracellular targets for pharmacological intervention in disorders involving aberrant secretion of respiratory mucin and may relate to other lesions involving exocytosis of membrane-bound granules in various cells and tissues.}, number={44}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Li, YH and Martin, LD and Spizz, G and Adler, KB}, year={2001}, month={Nov}, pages={40982–40990} }
 @article{li_martin_adler_2000, title={MARCKS protein: a key intracellular molecule controlling mucin secretion by human airway goblet cells.}, volume={161}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Li, Y. and Martin, L. D. and Adler, K. B.}, year={2000}, pages={A259} }
 @article{adler_li_martin_2000, title={Myristoylated alanine-rich C-kinase substrate protein: A major intracellular regulatory molecule controlling secretion of mucin by human airway goblet cells.}, volume={117}, number={5 Supplement 1}, journal={Chest}, author={Adler, K. B. and Li, Y. and Martin, L. D.}, year={2000}, pages={266S–267S} }
 @article{jiang_dreher_dye_li_richards_martin_adler_2000, title={Residual oil fly ash induces cytotoxicity and mucin secretion by guinea pig tracheal epithelial cells via an oxidant-mediated mechanism}, volume={163}, ISSN={["0041-008X"]}, DOI={10.1006/taap.1999.8886}, abstractNote={Inhalation of ambient air particulate matter (PM) is associated with pulmonary injury and inflammation. Using primary cultures of guinea pig tracheal epithelial (GPTE) cells as an in vitro model of airway epithelium, we examined effects of exposure to suspensions of six different emission and ambient air PM samples: residual oil fly ash (ROFA) from an electrical power plant; fly ash from a domestic oil burning furnace (DOFA); ambient air dust from St. Louis (STL), Ottawa (OT), and Washington, DC (WDC); and volcanic ash from the eruption of Mount Saint Helens (MSH) in 1980. Effects of these particulates on cell viability (assessed via LDH assay), secretion of mucin (measured by a monoclonal antibody-based ELISA), and steady-state mRNA levels of the mucin gene MUC2 were determined. ROFA was the most toxic of the dusts tested, as it significantly increased LDH release following a 24-h incubation with 50 microg/cm(2) ROFA. ROFA also enhanced MUC2 mRNA after 4-h exposure, and mucin secretion after 8 h. ROFA-induced mucin secretion and cytotoxicity were attenuated by the oxidant scavenger, dimethylthiourea (DMTU). ROFA exposure also depleted cells of glutathione (GSH). Relatedly, depletion of intracellular GSH by treatment of the cells with buthionine sulfoxamine (BSO) also provoked mucin secretion, as well as enhancing the secretory effect of ROFA when the two agents were added together. L-NMA, the nitric oxide synthase (NOS) inhibitor, did not affect ROFA-induced mucin secretion. Of the soluble transition metals in ROFA (nickel, iron, vanadium), only vanadium individually, or combinations of the metals containing vanadium, provoked secretion. The results suggest ROFA enhances mucin secretion and generates toxicity in vitro to airway epithelium via a mechanism(s) involving generation of oxidant stress, perhaps related to depletion of cellular antioxidant capacity. Deleterious effects of inhalation of ROFA in the respiratory tract in vivo may relate to these cellular responses. Vanadium, a component of ROFA, may be important in generating these reactions.}, number={3}, journal={TOXICOLOGY AND APPLIED PHARMACOLOGY}, author={Jiang, NF and Dreher, KL and Dye, JA and Li, YH and Richards, JH and Martin, LD and Adler, KB}, year={2000}, month={Mar}, pages={221–230} }
 @article{li_he_martin_krunkosky_lincoln_cornwell_adler_1999, title={Myristoylated alanine-rich C kinase substrate (MARCKS) is produced by human airway epithelial cells and is phosphorylated by PKC and PKG.}, volume={159}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Li, Y. and He, F. and Martin, L. D. and Krunkosky, T. M. and Lincoln, T. M. and Cornwell, T. L. and Adler, K. B.}, year={1999}, pages={A723} }
 @article{jiang_dreher_li_martin_adler_1999, title={Residual oil fly ash (ROFA) increases mucin secretion and mucin gene expression in guinea pig airway epithelial cells in vitro.}, volume={159}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Jiang, N.-F. and Dreher, K. L. and Li, Y. and Martin, L. D. and Adler, K. B.}, year={1999}, pages={A888} }
 @article{krunkosky_martin_li_adler_1999, title={TNF?-induced ICAM-1 expression in airway epithelium: involvement of IkB?.}, volume={159}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Krunkosky, T. M. and Martin, L. D. and Li, Y. and Adler, K. B.}, year={1999}, pages={A184} }
 @article{li_martin_minnicozzi_adler_1998, title={Cloning of guinea pig Muc2 cDNA and MUC2 gene expression in guinea pig airway epithelium in vitro.}, volume={157}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Li, Y. and Martin, L. D. and Minnicozzi, M. and Adler, K. B.}, year={1998}, pages={A728} }