@article{white_martin_stern_laxman_marroquin_2010, title={Expression of IL-4/IL-13 receptors in differentiating human airway epithelial cells}, volume={299}, ISSN={["1522-1504"]}, DOI={10.1152/ajplung.00422.2009}, abstractNote={IL-4 and IL-13 elicit several important responses in airway epithelium including chemokine secretion and mucous secretion that may contribute to airway inflammation, cell migration, and differentiation. These cytokines have overlapping but not identical effector profiles likely due to shared subunits in their receptor complexes. These receptors are variably described in epithelial cells, and the relative expression, localization, and function of these receptors in differentiated and repairing epithelial cells are not clear. We examined IL-4/IL-13 receptor expression and localization in primary airway epithelial cells collected from normal human lungs and grown under conditions yielding both undifferentiated and differentiated cells inclusive of basal, goblet, and ciliated cell phenotypes. Gene expression of the IL-4Rα, IL-2Rγc, IL-13Rα1, and IL-13Rα2 receptor subunits increased with differentiation, but different patterns of localization and protein abundance were seen for each subunit based on both differentiation and the cell subtypes present. Increased expression of receptor subunits observed in more differentiated cells was associated with more substantial functional responses to IL-4 stimulation including increased eotaxin-3 expression and accelerated migration after injury. We demonstrate substantial differences in IL-4/IL-13 receptor subunit expression and responsiveness to IL-4 based on the extent of airway epithelial cell differentiation and suggest that these differences may have functional consequences in airway inflammation.}, number={5}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, author={White, Steven R. and Martin, Linda D. and Stern, Randi and Laxman, Bharathi and Marroquin, Bertha A.}, year={2010}, month={Nov}, pages={L681–L693} }
 @article{manzo_slade_richards_mcgee_martin_dye_2010, title={Susceptibility of Inflamed Alveolar and Airway Epithelial Cells to Injury Induced by Diesel Exhaust Particles of Varying Organic Carbon Content}, volume={73}, ISSN={["1087-2620"]}, DOI={10.1080/15287390903566625}, abstractNote={Exposure to traffic-related ambient air pollution, such as diesel exhaust particles (DEP), is associated with adverse health outcomes, especially in individuals with preexisting inflammatory respiratory diseases. Using an analogous novel in vitro system to model both the healthy and inflamed lung, the susceptibility of epithelial cells exposed to DEP of varying organic carbon content was studied. Murine LA-4 alveolar type II-like epithelial cells, as well as primary murine tracheal epithelial cells (MTE), were treated with exogenous cytokines (tumor necrosis factor [TNF] α + interleukin [IL]-1 β + interferon [IFN] γ) to model a mild inflammatory state. Epithelial cells were subsequently exposed to DEP of varying organic carbon content, and the resultant cytotoxic, cytoprotective, or antioxidant cell responses were inferred by changes in lactate dehydrogenase (LDH) release, heme oxygenase-1 (HO-1) expression, or glutathione levels, respectively. Data showed that exposure of healthy LA-4 cells to organic carbon-rich DEP (25 μg/cm2; 24 h) induced adaptive cytoprotective/antioxidant responses with no apparent cell injury. In contrast, exposure of inflamed LA-4 cells resulted in oxidative stress culminating in significant cytotoxicity. Exposure of healthy MTE cells to organic carbon-rich DEP (20 μg/cm2; 24 h) was seemingly without effect, whereas exposure of inflamed MTE cells resulted in increased epithelial solute permeability. Thus, surface lung epithelial cells stressed by a state of inflammation and then exposed to organic carbon-rich DEP appear unable to respond to the additional oxidative stress, resulting in epithelial barrier dysfunction and injury. Adverse health outcomes associated with exposure to traffic-related air pollutants, like DEP, in patients with preexisting inflammatory respiratory diseases may be due, in part, to similar mechanisms.}, number={8}, journal={JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH-PART A-CURRENT ISSUES}, author={Manzo, Nicholas D. and Slade, Ralph and Richards, Judy H. and McGee, John K. and Martin, Linda D. and Dye, Janice A.}, year={2010}, pages={565–580} }
 @article{white_martin_abe_marroquin_stern_fu_2009, title={Insulin receptor substrate-1/2 mediates IL-4-induced migration of human airway epithelial cells}, volume={297}, ISSN={["1040-0605"]}, DOI={10.1152/ajplung.90453.2008}, abstractNote={Migration of airway epithelial cells (AEC) is an integral component of airway mucosal repair after injury. The inflammatory cytokine IL-4, abundant in chronic inflammatory airways diseases such as asthma, stimulates overproduction of mucins and secretion of chemokines from AEC; these actions enhance persistent airway inflammation. The effect of IL-4 on AEC migration and repair after injury, however, is not known. We examined migration in primary human AEC differentiated in air-liquid interface culture for 3 wk. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Concurrent treatment with IL-4 up to 10 ng/ml accelerated migration significantly in fully differentiated AEC. As expected, IL-4 treatment induced phosphorylation of the IL-4 receptor-associated protein STAT (signal transducer and activator of transcription)6, a transcription factor known to mediate several IL-4-induced AEC responses. Expressing a dominant negative STAT6 cDNA delivered by lentivirus infection, however, failed to block IL-4-stimulated migration. In contrast, decreasing expression of either insulin receptor substrate (IRS)-1 or IRS-2 using a silencing hairpin RNA blocked IL-4-stimulated AEC migration completely. These data demonstrate that IL-4 can accelerate migration of differentiated AEC after injury. This reparative response does not require STAT6 activation, but rather requires IRS-1 and/or IRS-2.}, number={1}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, author={White, Steven R. and Martin, Linda D. and Abe, Mark K. and Marroquin, Bertha A. and Stern, Randi and Fu, Xiaoying}, year={2009}, month={Jul}, pages={L164–L173} }
 @article{booth_sandifer_martin_martin_2007, title={IL-13-induced proliferation of airway epithelial cells: mediation by intracellular growth factor mobilization and ADAM17}, volume={8}, journal={Respiratory Research}, author={Booth, B. W. and Sandifer, T. and Martin, E. L. and Martin, L. D.}, year={2007} }
 @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_crews_li_adler_minnicozzi_martin_2006, title={Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro}, volume={7}, journal={Respiratory Research}, author={Chorley, B. N. and Crews, A. L. and Li, Y. H. and Adler, K. B. and Minnicozzi, M. and Martin, L. D.}, year={2006}, pages={35} }
 @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{mexas_hess_hawkins_martin_2006, title={Pulmonary lesions in cats with diabetes mellitus}, volume={20}, ISSN={["1939-1676"]}, DOI={10.1892/0891-6640(2006)20[47:PLICWD]2.0.CO;2}, abstractNote={Diabetes mellitus (DM) is a common endocrinopathy of cats and humans. Although few studies have examined the effects of DM on the pulmonary system, changes in pulmonary function and immunology in humans with type I and II diabetes, and pulmonary lesions in a murine diabetic model have been documented. Our objective was to determine whether pulmonary lesions occurred in cats with DM. Medical records and necropsy evaluations of 42 cats with DM were compared with those of 45 age-matched, nondiabetic cats for the presence of clinical evidence of respiratory disease and pulmonary histopathological findings at the time of necropsy. No statistical difference was noted in the presence of clinical evidence of respiratory disease between cats with diabetes and control cats. Nevertheless, there was a significant association between the presence of abnormal pulmonary histopathology and DM (P= .018, odds ratio = 3 inclusive of all cats; P= .005, odds ratio = 5 when non-DM cats with overt clinical evidence of respiratory disease were excluded). Pulmonary abnormalities detected by histopathological examination in cats with diabetes included congestion and edema, histiocytosis, pneumonia, smooth muscle hypertrophy, fibrosis, mineralization, neoplasia, and type II pneumocyte hyperplasia. The observed association between DM and pulmonary lesions in cats, independent of clinical evidence of respiratory disease, emphasizes the need for careful assessment of the respiratory tract in sick cats with diabetes.}, number={1}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Mexas, AM and Hess, RS and Hawkins, EC and Martin, LD}, year={2006}, pages={47–51} }
 @misc{martin_adler_macchione_akley_mckane_2005, title={Culture system for mouse tracheal epithelial cells}, volume={6,933,149}, number={2005 Aug. 23}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Martin, L. D. and Adler, K. B. and Macchione, M. and Akley, N. J. and McKane, S. A.}, year={2005} }
 @article{fischer_cuellar_byrd_rice_bonner_martin_voynow_2005, title={ErbB2 activity is required for airway epithelial repair following neutrophil elastase exposure}, volume={19}, number={7}, journal={FASEB Journal}, author={Fischer, B. M. and Cuellar, J. G. and Byrd, A. S. and Rice, A. B. and Bonner, J. C. and Martin, L. D. and Voynow, J. A.}, year={2005} }
 @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 MUC 5 AC and MUC 5 B, but not MUC 2, 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, PKCalpha, delta, zeta, lambda, iota, and epsilon were constitutively expressed in NHBE cells while PKCbeta, eta, and mu were PMA-inducible. PKCdelta was the only isoform to translocate from cytoplasm to membrane in response to HNE. Inhibition of PKCdelta 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 delta isoform.}, 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{deshmukh_case_wesselkamper_borchers_martin_shertzer_nadel_leikauf_2005, title={Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation}, volume={171}, ISSN={["1535-4970"]}, DOI={10.1164/rccm.200408-1003OC}, abstractNote={Chronic obstructive pulmonary disease is marked by alveolar enlargement and excess production of airway mucus. Acrolein, a component of cigarette smoke, increases mucin 5AC (MUC5AC), a prevalent airway mucin in NCI-H292 cells by transcriptional activation, but the signal transduction pathways involved in acrolein-induced MUC5AC expression are unknown. Acrolein depleted cellular glutathione at doses of 10 μM or greater, higher than those sufficient (0.03 μM) to increase MUC5AC mRNA, suggesting that MUC5AC expression was independent of oxidative stress. In contrast, acrolein increased MUC5AC mRNA levels by phosphorylating epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 3/2, or MAPK 3/2(ERK1/2). Pretreating the cells with an EGFR-neutralizing antibody, or a metalloproteinase inhibitor, decreased the acrolein-induced MUC5AC mRNA increase. Small, interfering RNA directed against ADAM17 or MMP9 inhibited the acrolein-induced MUC5AC mRNA increase. Acrolein increased the release and subsequent activation of pro-MMP9. Acrolein increased MMP9 and decreased tissue inhibitor of metalloproteinase 3 (TIMP3), an endogenous inhibitor of ADAM17, transcripts. Together, these data suggest that acrolein induces MUC5AC expression via an initial ligand-dependent activation of EGFR mediated by ADAM17 and MMP9. In addition, a prolonged effect of acrolein may be mediated by altering MMP9 and TIMP3 transcription.}, number={4}, journal={AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE}, author={Deshmukh, HS and Case, LM and Wesselkamper, SC and Borchers, MT and Martin, LD and Shertzer, HG and Nadel, JA and Leikauf, GGD}, year={2005}, month={Feb}, pages={305–314} }
 @article{lankford_macchione_crews_mckane_akley_martin_2005, title={Modeling the airway epithelium in allergic asthma: Interleukin-13-induced effects in differentiated murine tracheal epithelial cells}, volume={41}, number={7}, journal={In Vitro Cellular & Developmental Biology. Animal}, author={Lankford, S. M. and Macchione, M. and Crews, A. L. and McKane, S. A. and Akley, N. J. and Martin, L. D.}, year={2005}, pages={217–224} }
 @article{willens_stoskopf_martin_lewbart_2005, title={Viability of glycerol-preserved and cryopreserved anuran skin}, volume={41}, number={09-Aug}, journal={In Vitro Cellular & Developmental Biology. Animal}, author={Willens, S. and Stoskopf, M. K. and Martin, L. D. and Lewbart, G. A.}, year={2005}, pages={258–263} }
 @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}, 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{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{krunkosky_martin_fischer_voynow_adler_2003, title={Effects of TNF alpha on expression of ICAM-1 in human airway epithelial cells in vitro: Oxidant-mediated pathways and transcription factors}, volume={35}, ISSN={["1873-4596"]}, DOI={10.1016/S0891-5849(03)00498-2}, abstractNote={We demonstrate that two different cell-permeable antioxidants, pyrrolidine dithiocarbamate (PDTC) and dimethylthiourea (DMTU), inhibit TNFalpha-induced ICAM-1 surface and gene expression in primary cultures of differentiated normal human bronchial epithelial (NHBE) cells. In addition, TNFalpha stimulates binding of nuclear proteins to the nuclear factor kappa beta (NFkappaB) and the CAAT/enhancer binding protein (C/EBP) consensus sites in the ICAM-1 promoter in these cells. Because these transcription factors have been suggested to be oxidant-sensitive and important in ICAM-1 expression, the potential involvement of reactive oxygen species (ROS) in the response to TNFalpha was investigated. Interestingly, neither PDTC nor DMTU altered binding of NFkappaB complexes. In contrast, either the proteasome inhibitor carbobenzoxy-L-leucy-L-leucy-L-leucinal (MG 132) or the IkappaBalpha inhibitor BAY 11-7082 ablated TNFalpha-induced ICAM-1 gene expression and MG132 inhibited TNFalpha-induced NFkappaB complexes. Surprisingly, either PDTC or DMTU inhibited the binding of TNFalpha-enhanced C/EBP complexes to the consensus site directly adjacent to the NFkappaB site. These results suggest that although TNFalpha enhances binding of C/EBP and NFkappaB complexes in NHBE cells, C/EBP binding seems to involve an oxidant-dependent mechanism, whereas activation of NFkappaB complexes utilizes the ubiquitin-proteasome pathway, a mechanism that seems to be unaltered by the presence of antioxidants. Because interference with either signaling pathway abrogates TNFalpha-induced ICAM-1 expression, activation of both complexes seems to be involved in this response to TNFalpha, but this activation occurs via different intracellular pathways.}, number={9}, journal={FREE RADICAL BIOLOGY AND MEDICINE}, author={Krunkosky, TM and Martin, LD and Fischer, BM and Voynow, JA and Adler, KB}, year={2003}, month={Nov}, pages={1158–1167} }
 @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{little_dean_young_mckane_martin_jones_blikslager_2003, title={PI3K signaling is required for prostaglandin-induced  mucosal recovery in ischemia-injured porcine ileum}, volume={284}, ISSN={0193-1857 1522-1547}, url={http://dx.doi.org/10.1152/ajpgi.00121.2002}, DOI={10.1152/ajpgi.00121.2002}, abstractNote={We have previously shown that PGE(2) and PGI(2) induce recovery of transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in Cl(-) secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight junction assembly, we postulated that this signaling pathway is involved in PG-induced mucosal recovery. Porcine ileum was subjected to 45 min of ischemia, after which TER was monitored for a 180-min recovery period. Endogenous PG production was inhibited with indomethacin (5 microM). PGE(2) (1 microM) and PGI(2) (1 microM) stimulated recovery of TER, which was inhibited by serosal application of the osmotic agent urea (300 mosmol/kgH(2)O). The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in response to PGs or mucosal urea. Immunofluorescence imaging of recovering epithelium revealed that PGs restored occludin and zonula occludens-1 distribution to interepithelial junctions, and this pattern was disrupted by pretreatment with wortmannin. These experiments suggest that PGs stimulate recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of tight junction protein distribution.}, number={1}, journal={American Journal of Physiology-Gastrointestinal and Liver Physiology}, publisher={American Physiological Society}, author={Little, Dianne and Dean, Rebecca A. and Young, Karen M. and McKane, Shaun A. and Martin, Linda D. and Jones, Samuel L. and Blikslager, Anthony T.}, year={2003}, month={Jan}, pages={G46–G56} }
 @article{booth_newcomb_mckane_crews_adler_bonner_martin_2003, title={Proliferation of the airway epithelium in asthma - Are inflammatory cells required?}, volume={123}, ISSN={["0012-3692"]}, DOI={10.1378/chest.123.3_suppl.384S}, abstractNote={Asthma is associated with a T helper type 2 phenotype in which interleukin (IL)-4, IL-5, and IL-13 predominate. In addition, the long-term presence of these inflammatory mediators is thought to lead to airway structural changes that are collectively referred to as airway remodeling. Data from our laboratory, and those of others, have suggested a role for IL-13 in the development of mucous cell hyperplasia that is associated with such remodeling. Others also have suggested a role for inflammatory cells such as neutrophils in mediating this process. Using normal human bronchial epithelial (NHBE) cells differentiated in vitro, we have shown recently that IL-13 (10 ng/mL for 24 h) induces the proliferation of NHBE cells via a mechanism that is dependent on the IL-13-induced release of transforming growth factor (TGF)-α by the epithelial cells. This epithelium-derived TGF-α then acts in an autocrine/paracrine manner to bind the epidermal growth factor receptor (EGFR) on these NHBE cells, enhancing proliferation. Specifically, soluble TGF-α is released by NHBE cells in response to IL-13 exposure (1 h), and the immunohistochemical analysis of cells exposed to IL-13 (after 1 and 4 h) has revealed a lack of membrane-bound TGF-α when compared to control cells. The IL-13-induced proliferative response can be blocked in a concentration-dependent manner by AG1478 (0.1, 1, and 5 μg/mL), which is a specific inhibitor of EGFR tyrosine kinase activity, and is eliminated by neutralizing TGF-α antibodies, while control antibodies (ie, anti-platelet-derived growth factor, epidermal growth factor [EGF], and heparin-binding EGF) have no effect.}, number={3}, journal={CHEST}, author={Booth, BW and Newcomb, DC and McKane, SA and Crews, AL and Adler, KB and Bonner, JC and Martin, LD}, year={2003}, month={Mar}, pages={384S–385S} }
 @article{booth_adler_martin_2002, title={IL-13 induces intracellular translocation and release of TGF? in human bronchial epithelial cells in vitro.}, volume={165}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Booth, B. W. and Adler, K. B. and Martin, L. D.}, year={2002}, pages={A814} }
 @article{martin_adler_akley_crews_sharova_2002, title={Secretion-competent mouse tracheal epithelial cell culture from the genetically altered mouse - Pathway analysis via gene array}, volume={121}, DOI={10.1016/S0012-3692(15)35478-7}, abstractNote={2002;121;79S-80S Chest V. Romberger John Spurzem, Ji Zhang PhD, Xiang-der Liu, Stephan I. Rennard and Debra * Epithelial Repair Model of In Vitro Gene Expression in an http://chestjournal.chestpubs.org/content/121/3_suppl/79S.2.full.html can be found online on the World Wide Web at: The online version of this article, along with updated information and services ) ISSN:0012-3692 http://chestjournal.chestpubs.org/site/misc/reprints.xhtml ( distributed without the prior written permission of the copyright holder. All rights reserved. No part of this article or PDF may be reproduced or College of Chest Physicians, 3300 Dundee Road, Northbrook, IL 60062. has been published monthly since 1935. Copyright2002by the American is the official journal of the American College of Chest Physicians. It Chest}, number={3}, journal={Chest}, author={Martin, L. D. and Adler, K. B. and Akley, N. J. and Crews, A. and Sharova, L.}, year={2002}, pages={79S} }
 @article{booth_bonner_adler_martin_2001, title={Autocrine production of TGFa mediates interleukin 13-induced proliferation of human airway epithelial cells during development of a mucous phenotype in vitro.}, volume={163}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Booth, B. and Bonner, J. C. and Adler, K. B. and Martin, L. D.}, year={2001}, pages={A738} }
 @article{macchione_akley_adler_martin_2001, title={Differentiation of murine tracheal epithelial cells in vitro.}, volume={163}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Macchione, M. and Akley, N. J. and Adler, K. B. and Martin, L. D.}, year={2001}, pages={A225} }
 @article{marinakos_anderson_ryan_martin_feldheim_2001, title={Encapsulation, permeability, and cellular uptake characteristics of hollow nanometer-sized conductive polymer capsules}, volume={105}, ISSN={["1520-5207"]}, DOI={10.1021/jp010820d}, abstractNote={The use of nanometer-sized gold particles as templates for the synthesis of hollow poly(pyrrole), poly(N-methylpyrrole), and poly(3-methylthiophene) is described in this paper. Diffusion coefficients of small molecules through the capsule shell were found to vary by almost 3 orders of magnitude depending on the polymer, polymer oxidation state, and counteranion incorporated during polymer synthesis. A small molecule (anthraquinone) and an enzyme (horseradish peroxidase) were trapped inside hollow capsules by attaching them to the template particle prior to polymerization and particle etching. A thin poly(pyrrole) shell protected the enzyme 2 times longer in neat toluene compared to unencapsulated enzyme. Finally, the potential for using conductive polymer nanoparticles for intracellular delivery or diagnostics was examined by administering partice suspensions to 3T3 murine fibroblasts. Particles ranging in size from 25 to 100 nm were engulfed by fibroblasts without compromising cell viability.}, number={37}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Marinakos, SM and Anderson, MF and Ryan, JA and Martin, LD and Feldheim, DL}, year={2001}, month={Sep}, pages={8872–8876} }
 @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={Section:ChooseTop of pageAbstract <<Materials and MethodsResultsDiscussionReferencesCITING ARTICLES}, 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} }
 @inbook{martin_macchione_bonner_booth_akley_adler_2001, title={Interleukin-13 induced mucous cell hyperplasia in airway epithelium.}, booktitle={Cilia and mucus: from development to respiratory disease.}, author={Martin, L. D. and Macchione, M. and Bonner, J. C. and Booth, B. W. and Akley, N. J. and Adler, K. B.}, year={2001}, pages={253–263} }
 @article{booth_adler_bonner_tournier_martin_2001, title={Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-alpha}, volume={25}, ISSN={["1535-4989"]}, DOI={10.1165/ajrcmb.25.6.4659}, abstractNote={Remodeling of the airways, as occurs in asthmatic patients, is associated with the continual presence of inflammatory mediators and Th2 cytokines, especially interleukin (IL)-13, during cycles of epithelial injury and repair. In this study, we examined the effect of IL-13 on well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture. IL-13 induced proliferation of NHBE cells after 24 h exposure, as reflected by [(3)H]thymidine uptake and cell counts. The effects of IL-13 were mediated through the epidermal growth factor receptor (EGFR), as proliferation was attenuated by AG1478, an EGFR tyrosine kinase inhibitor. Proliferation appeared to be mediated by transforming growth factor (TGF)-alpha, a potent ligand for EGFR, which was released rapidly from NHBE cells in response to IL-13. Neutralizing antibody to TGF-alpha, but not antibodies against other potentially important growth factors (EGF, heparin binding epidermal growth factor-like growth factor [HB-EGF], platelet-derived growth factor [PDGF]), inhibited the mitogenic response to IL-13. This study provides the first experimental evidence that IL-13 can initiate a proliferative response of human airway epithelium in the absence of inflammatory cells or other cell types. The results are consistent with a mechanism whereby IL-13 induces release of TGF-alpha from the epithelial cells, which in turn binds via an autocrine/paracrine-type action to the EGFR, initiating proliferation. IL-13-induced airway remodeling in vivo may involve this epithelium-driven response.}, number={6}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Booth, BW and Adler, KB and Bonner, JC and Tournier, F and Martin, LD}, year={2001}, month={Dec}, pages={739–743} }
 @article{booth_bonner_akley_macchione_adler_martin_2001, title={Interleukin-13, a mediator of subepithelial fibrosis, enhances growth factor production and proliferation in human airway epithelial cells}, volume={120}, ISSN={["0012-3692"]}, DOI={10.1378/chest.120.1_suppl.S15}, abstractNote={Subepithelial fibrosis is a prominent feature of the remodeled asthmatic airway. The cytokine interleukin (IL)-13, implicated as a mediator in the development of asthma, induces a significant degree of subepithelial fibrosis in the lungs of transgenic mice. Since IL-13 has been shown to exert effects on the airway epithelium, including the development of a mucous phenotype, we have begun to determine whether IL-13 provokes production of factors from the epithelium that could elicit the observed subepithelial fibrotic response. In the studies reported herein, injured airways with regions of regenerating/differentiating cells and regions of normal fully differentiated cells have been mimicked by examining the effects of IL-13 on normal human bronchial epithelial cells during mucociliary differentiation in air/liquid interface culture. Exposure of normal human bronchial epithelial cells to IL-13 resulted in increased production of soluble transforming growth factor (TGF)-α, with the growth factor interacting in an autocrine manner with the epidermal growth factor receptor. Production of soluble TGF-α was very rapid, with a threefold increase observed in response to IL-13 (10 ng/mL) by 1 h of exposure. Continuous exposure to IL-13 throughout the course of mucociliary differentiation (a total of 10 days) resulted in a twofold increase in cell number by day 7 when cells are differentiated. Exposure to IL-13 (10 ng/mL; 24 h) provoked a threefold increase in proliferation once the cells were differentiated, an effect that could be duplicated in differ- entiated, but not undifferentiated cells, by the direct addition of TGF-α (5 ng/mL or 25 ng/mL; 24 h). Proliferation of differentiated cells in response to continuous IL-13 treatment was followed 2 days later by a decrease in proliferation compared to control mice. Soluble TGF-α, however, continued to be produced from these nonproliferating cultures. Thus, an increase in soluble TGF-α in response to IL-13 may serve to promote proliferation of injured epithelial cells in an autocrine manner. Once this proliferative effect is no longer necessary, the soluble TGF-α may promote proliferation of other cells. These data suggest that any injury to the airway epithelium resulting in the production of IL-13 from infiltrating inflammatory cells may provoke the release of soluble TGF-α from the airway epithelium. The availability of this growth factor may contribute to the subepithelial fibrosis observed in chronic asthma.}, number={1}, journal={CHEST}, author={Booth, B and Bonner, J and Akley, N and Macchione, M and Adler, K and Martin, LD}, year={2001}, month={Jul}, pages={15S–15S} }
 @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. 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. Mammalian airways are lined by a thin layer of mucus produced and secreted by airway epithelial (goblet) cells and submucosal glands. In diseases such as asthma, chronic bronchitis, and cystic fibrosis, hypersecretion of mucus is a common lesion. Excess mucus can contribute to obstruction, susceptibility to infection, and even to destruction of airway walls and contiguous tissues. The major components of mucus are mucin glycoproteins synthesized by secretory cells and stored within cytoplasmic membrane-bound granules. Upon appropriate stimulation, these granules are released via an exocytotic process in which the granules translocate to the cell periphery where the granule membranes fuse with the plasma membrane, allowing for luminal secretion of the contents. Despite the obvious pathophysiological importance of this process, intracellular signaling mechanisms linking stimulation at the cell surface to mucin granule release have not been elucidated. It is known that a wide variety of agents and inflammatory/humoral mediators can provoke mucin secretion. These include cholinergic agonists, lipid mediators, oxidants, cytokines, neuropeptides, ATP and UTP, bacterial products, neutrophil elastase, and inhaled pollutants (reviewed in Refs. 1Adler K.B. Fischer B.M. Martin L.D. Voynow J.A. Res. Immunol. 1998; 149: 245-248Crossref Scopus (1) Google Scholar and 2Cohn L.A. Adler K.B. Exp. Lung Res. 1992; 18: 299-322Crossref PubMed Scopus (25) Google Scholar). Interestingly, many of these mucin secretagogues are also known to activate several protein kinases, and studies examining the regulation of excess secretion of mucin by airway epithelial cells from various species have consistently implicated involvement of either protein kinase C (PKC) 1PKC, protein kinase C; PKG, cGMP-dependent protein kinase; PKA, protein kinase A; MANS, myristoylated N-terminal sequence; PMA, phorbol 12-myristate 13-acetate; PSD, phosphorylation site domain; 8-Br-GMP, 8-bromo-cyclic GMP; RNS, random N-terminal sequence; MARCKS, myristoylated alanine-rich C kinase substrate; NHBE, normal human bronchial epithelial; PP2A, protein phosphatase 2A; ELISA, enzyme-linked immunosorbent assay. 1PKC, protein kinase C; PKG, cGMP-dependent protein kinase; PKA, protein kinase A; MANS, myristoylated N-terminal sequence; PMA, phorbol 12-myristate 13-acetate; PSD, phosphorylation site domain; 8-Br-GMP, 8-bromo-cyclic GMP; RNS, random N-terminal sequence; MARCKS, myristoylated alanine-rich C kinase substrate; NHBE, normal human bronchial epithelial; PP2A, protein phosphatase 2A; ELISA, enzyme-linked immunosorbent assay.(3Ko K.H. Jo M. McCracken K. Kim K.C. Am. J. Respir. Cell Mol. Biol. 1997; 16: 194-198Crossref PubMed Scopus (22) Google Scholar, 4Abdullah L.H. Conway J.D. Cohn J.A. Davis C.W. Am. J. Physiol. 1997; 273: L201-L210PubMed Google Scholar, 5Abdullah L.H. Davis S.W. Burch L. Yamauchi M. Randell S.H. Nettesheim P. Davis C.W. Biochem. J. 1996; 316: 943-951Crossref PubMed Scopus (61) Google Scholar, 6Larivee P. Levine S.J. Martinez A. Wu T. Logun C. Shelhamer J.H. Am. J. Respir. Cell Mol. Biol. 1994; 11: 199-205Crossref PubMed Scopus (45) Google Scholar) or cGMP-dependent protein kinase (PKG) (7Fischer B.M. Rochelle L.G. Voynow J.A. Akley N.J. Adler K.B. Am. J. Respir. Cell Mol. Biol. 1999; 20: 413-422Crossref PubMed Scopus (88) Google Scholar) in the secretory process. However, coordinated interactions or “cross-talk” between these two protein kinases in regulation of mucin secretion have not been demonstrated, nor have signaling events downstream of protein kinase activation that ultimately lead to the exocytotic release of mucin granules been elucidated. Previously, we reported (8Krunkosky T.M. Fischer B.M. Martin L.D. Jones N. Akley N.J. Adler K.B. Am. J. Respir. Cell Mol. Biol. 2000; 22: 685-692Crossref PubMed Scopus (133) Google Scholar) development of a procedure to culture normal human bronchial epithelial (NHBE) cells in an air/liquid interface system in which the cells differentiate to a heterogeneous population containing secretory (goblet), ciliated, and basal cells that mimic their in vivo appearance and function. These cell cultures provide an ideal in vitro model system to study mechanisms regulating mucin secretion from human airway epithelium. This report presents direct evidence demonstrating that the myristoylated alanine-rich C kinase substrate (MARCKS), a widely distributed PKC substrate for which a specific biological function has yet to be identified, is a key regulatory molecule mediating mucin granule release by NHBE cells. Secretion of mucin from these cells is maximized by activation of both PKC and PKG, and MARCKS serves as the point of convergence for coordinating the actions of these two protein kinases to control mucin granule release. The mechanism appears to involve PKC-dependent phosphorylation of MARCKS, which releases MARCKS from the plasma membrane into the cytoplasm, where it is in turn dephosphorylated by a protein phosphatase 2A (PP2A) that is activated by PKG. This dephosphorylation would allow MARCKS to regain its membrane-binding capability (9Seykora J.T. Myat M.M. Allen L.A.H. Ravetch J.V. Aderem A. J. Biol. Chem. 1996; 271: 18797-18802Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Swierczynski S.L. Blackshear P.J. J. Biol. Chem. 1996; 271: 23424-23430Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar), enabling its attachment to membranes of cytoplasmic mucin granules. In addition, MARCKS interacts with actin and myosin in the cytoplasm and thus could tether the granules to the cellular contractile apparatus, mediating subsequent granule movement and exocytosis. Expansion, cryopreservation, and culture of NHBE cells in the air/liquid interface were performed as described previously (8Krunkosky T.M. Fischer B.M. Martin L.D. Jones N. Akley N.J. Adler K.B. Am. J. Respir. Cell Mol. Biol. 2000; 22: 685-692Crossref PubMed Scopus (133) Google Scholar). Briefly, NHBE cells (Clonetics, San Diego, CA) were seeded in vented T75 tissue culture flasks (500 cells/cm2) and cultured until cells reached 75–80% confluence. Cells were then dissociated by trypsin/EDTA and frozen as passage-2. Air/liquid interface culture was initiated by seeding passage-2 cells (2 × 104 cells/cm2) in Transwell® clear culture inserts (Costar, Cambridge, MA) that were thinly coated with rat tail collagen, type I (Collaborative Biomedical, Bedford, MA). Cells were cultured submerged in medium in a humidified 95% air, 5% CO2 environment for 5–7 days until nearly confluent. At that time, the air/liquid interface was created by removing the apical medium and feeding cells basalaterally. Medium was renewed daily thereafter. Cells were cultured for an additional 14 days to allow full differentiation. Before collection of “base line” and “test” mucin samples, the accumulated mucus at the apical surface of the cells was removed by washing with phosphate-buffered saline, pH 7.2. To collect the base-line secretion, cells were incubated with medium alone, and secreted mucin in the apical medium was collected and reserved. Cells were rested for 24 h and then exposed to medium containing the selected stimulatory and/or inhibitory reagents (or appropriate controls), after which secreted mucin was collected and reserved as the test sample. Incubation times for the base line and the test were the same but varied depending on the test reagent utilized. Both base line and test secretions were analyzed by ELISA using an antibody capture method described previously (12Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 570-573Google Scholar). The primary antibody for this assay was 17Q2 (Babco, Richmond, CA), a monoclonal antibody that reacts specifically with a carbohydrate epitope on human airway mucins (13Lin H. Carlson D.M. St. George J.A. Plopper C.G. Wu R. Am. J. Respir. Cell Mol. Biol. 1989; 1: 41-48Crossref PubMed Scopus (63) Google Scholar). The ratio of test/base-line mucin, similar to a “secretory index” reported previously (14Wright D.T. Fischer B.M. Li C. Rochelle L.G. Akley N.J. Adler K.B. Am. J. Physiol. 1996; 271: L854-L861PubMed Google Scholar), was used to quantify mucin secretion, allowing each culture dish to serve as its own control and thus minimizing deviation caused by variability among culture wells. Levels of mucin secretion were reported as percentage of the medium control. When labeling with [32P]phosphate, cells were preincubated for 2 h in phosphate-free Dulbecco's modified Eagle's medium containing 0.2% bovine serum albumin and then labeled with 0.1 mCi/ml [32P]orthophosphate (9000 Ci/mmol, PerkinElmer Life Sciences) for 2 h. For labeling with [3H]myristic acid or 3H-amino acids, cells were incubated overnight in medium containing 50 µCi/ml [3H]myristic acid (49 Ci/mmol, PerkinElmer Life Sciences) or 0.2 mCi/ml [3H]leucine (159 Ci/mmol, PerkinElmer Life Sciences) plus 0.4 mCi/ml [3H]proline (100 Ci/mmol, PerkinElmer Life Sciences). Following labeling, cells were exposed to stimulatory reagents for 5 min. When an inhibitor was used, cells were preincubated with the inhibitor for 15 min prior to stimulation. At the end of the treatments, cells were lysed in a buffer containing 50 mmTris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzamidine, 10 µg/ml pepstatin A, and 10 µg/ml leupeptin. The radiolabeling efficiency in each culture was determined by trichloroacetic acid precipitation and scintillation counting. Immunoprecipitation of MARCKS protein was carried out according to the method of Spizz and Blackshear (15Spizz G. Blackshear P.J. J. Biol. Chem. 1996; 271: 553-562Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) using cell lysates containing equal counts/min. Precipitated proteins were resolved by 8% SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. Anti-human MARCKS antibody (2F12) and nonimmune control antibody (6F6) used in this assay were provided by Dr. Perry Blackshear (NIEHS, Research Triangle Park, NC). To assess MARCKS or MARCKS-associated protein complexes in different subcellular fractions, radiolabeled and treated cells were scraped into a homogenization buffer (50 mm Tris-HCl (pH 7.5), 10 mm NaCl, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 1 mm benzamidine, 10 µg/ml pepstatin A, 10 µg/ml leupeptin) and then disrupted by nitrogen cavitation (800 pounds/square inch for 20 min at 4 °C). Cell lysates were centrifuged at 600 × g for 10 min at 4 °C to remove nuclei and unbroken cells. Post-nuclear supernatants were separated into membrane and cytosol fractions by ultracentrifugation at 400,000 × g for 30 min at 4 °C. The membrane pellet was solubilized in the lysis buffer by sonication. Immunoprecipitation was then carried out as described above. Both the myristoylated N-terminal sequence (MANS) and the random N-terminal sequence (RNS) peptides were synthesized at Genemed Synthesis, Inc. (San Francisco, CA), then purified by high pressure liquid chromatography (>95% pure), and confirmed by mass spectroscopy with each showing one single peak with an appropriate molecular mass. The MANS peptide consisted of sequence identical to the first 24 amino acids of MARCKS, i.e. the myristoylated N-terminal region that mediates MARCKS insertion into membranes (9Seykora J.T. Myat M.M. Allen L.A.H. Ravetch J.V. Aderem A. J. Biol. Chem. 1996; 271: 18797-18802Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 10Swierczynski S.L. Blackshear P.J. J. Biol. Chem. 1996; 271: 23424-23430Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 11McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar), MA-GAQFSKTAAKGEAAAERPGEAAVA (where MA = N-terminal myristate chain). The corresponding control peptide (RNS) contained the same amino acid composition as the MANS but arranged in random order, MA-GTAPAAEGAGAEVKRASAEAKQAF. The presence of the hydrophobic myristate moiety in these synthetic peptides enhances their permeability to the plasma membranes, enabling the peptides to be taken up readily by cells. To determine the effects of these peptides on mucin secretion, cells were preincubated with the peptides for 15 min prior to addition of secretagogues, and mucin secretion was then measured by ELISA. MARCKS antisense oligonucleotide and its corresponding control oligonucleotide were synthesized at Biognostik GmbH (Gottingen, Germany). NHBE cells were treated with 5 µm antisense or control oligonucleotide apically for 3 days (in the presence of 2 µg/ml lipofectin for the first 24 h). Cells were then incubated with secretagogues, and mucin secretion was measured by ELISA. Total RNA and protein were isolated from treated cells. MARCKS mRNA was assessed by Northern hybridization according to conventional procedures using human MARCKS cDNA (provided by Dr. Perry Blackshear, NIEHS, Research Triangle Park, NC) as a probe. MARCKS protein level was determined by Western blot using purified anti-MARCKS IgG1 (clone 2F12) as the primary detection antibody. The phosphorylation site domain (PSD) of MARCKS contains the PKC-dependent phosphorylation sites and the actin filament-binding site (16Hartwig J.H. Thelen M. Rosen A. Janmey P.A. Nairn A.C. Aderem A. Nature. 1992; 356: 618-622Crossref PubMed Scopus (618) Google Scholar). To construct a PSD-deleted MARCKS cDNA, two fragments flanking the PSD sequence (coding for 25 amino acids) were generated by polymerase chain reaction and then ligated through the XhoI site that was attached to the 5′-ends of oligonucleotide primers designed for the polymerase chain reaction. The resultant mutant cDNA and the wild-type MARCKS cDNA were each inserted into a mammalian expression vector pcDNA4/TO (Invitrogen, Carlsbad, CA). Isolated recombinant constructs were confirmed by restriction digests and DNA sequencing. HBE1 is a papilloma virus-transformed human bronchial epithelial cell line (17Yankaskas J.R. Haizlip J.E. Conrad M. Koval D. Lazarowski E. Paradiso A.M. Rinehart Jr., C.A. Sarkadi B. Schlegel R. Boucher R.C. Am. J. Physiol. 1993; 264: C1219-C1230Crossref PubMed Google Scholar) capable of mucin secretion when cultured in air/liquid interface. Transfection of HBE1 cells was carried out using the Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions. Briefly, differentiated HBE1 cells grown in air/liquid interface were dissociated by trypsin/EDTA and re-seeded in 12-well culture plates at 1 × 105cells/cm2. After overnight incubation, cells were transfected with the wild-type MARCKS cDNA, the PSD-truncated MARCKS cDNA, or vector DNA. Cells were cultured for 48 h to allow gene expression and then exposed to secretagogues and mucin secretion measured by ELISA. All transfections were carried out in the presence of pcDNA4/TO/lacZ plasmid (Invitrogen) (DNA ratio 6:1, total 1 µg DNA, ratio of DNA to Effectene reagent = 1:25) to monitor variations in transfection efficiency. Results showed no significant difference in β-galactosidase activities in cell lysates isolated from the transfected cells, indicating similar transfection efficiency among different DNA constructs (data not shown). PP1 and PP2A activities were measured using a protein phosphatase assay system (Life Technologies, Inc.) as described (18Huang X.C. Sumners C. Richards E.M. Adv. Exp. Med. Biol. 1996; 396: 209-215Crossref PubMed Scopus (25) Google Scholar) with modification. Briefly, NHBE cells were treated with 8-Br-cGMP or medium alone for 5 min. Cells were then scraped into a lysis buffer (50 mm Tris-HCl (pH 7.4), 0.1% β-mecaptoethanol, 0.1 mm EDTA, 1 mmbenzamidine, 10 µg/ml pepstatin A, 10 µg/ml leupeptin) and disrupted by sonication for 20 s at 4 °C. Cell lysates were centrifuged and the supernatants saved for phosphatase activity assay. The assay was performed using 32P-labeled phosphorylase A as a substrate. Released 32Pi was counted by scintillation. The protein concentration of each sample was determined by the Bradford assay. PP2A activity was expressed as the sample total phosphatase activity minus the activity remaining in the presence of 1 nm okadaic acid. PP1 activity was expressed as the difference between the activities remaining in the presence of 1 nm and 1 µm okadaic acid, respectively. Protein phosphatase activities were reported as nmol of Pireleased per min/mg total protein. All reagents used in treating NHBE cells were examined for cytotoxicity by measuring the total release of lactate dehydrogenase from the cells. The assay was carried out using the Promega Cytotox 96 Kit according to the manufacturer's instructions. All experiments were performed with reagents at non-cytotoxic concentrations. Data were analyzed for significance using one-way analysis of variance with Bonferroni post-test corrections. Differences between treatments were considered significant at p < 0.05. To determine the potential role of PKC and/or PKG in the mucin secretory process, NHBE cells were exposed to the following two specific protein kinase activators: the phorbol ester, phorbol 12-myristate 13-acetate (PMA), for activation of PKC, and the nonhydrolyzable cGMP analogue, 8-Br-cGMP, for activation of PKG. Preliminary studies examining mucin secretion in response to PMA stimulation at various concentrations for different times (up to 1 µm for 2 h) indicated that activation of PKC alone did not induce significant mucin secretion from NHBE cells, although a moderate secretory response was repeatedly observed at PMA concentrations higher than 100 nm (0.05 <p < 0.1). Also, the cells did not respond to the cGMP analogues at concentrations as high as 500 µm for up to 2 h of exposure. However, a combination of PMA + 8-Br-cGMP, affecting dual activation of PKC and PKG, provoked a rapid increase in secretion, approximately doubling it within 15 min of exposure (Fig.1A). This secretory response induced by PMA + 8-Br-cGMP was concentration-dependent, with maximal stimulation at 100 nm PMA + 1 µm8-Br-cGMP (Fig. 1B). UTP is a well defined pathophysiologically relevant mucin secretagogue (19Lethem M.I. Dowell M.L. Van Scott M. Yankaskas J.R. Egan T. Boucher R.C. Davis C.W. Am. J. Respir. Cell Mol. Biol. 1993; 9: 315-322Crossref PubMed Scopus (108) Google Scholar). Our preliminary studies showed that UTP (100 µm) could induce a significant increase in mucin secretion from NHBE cells after a 2-h exposure. To determine whether PKC and PKG were involved in regulation of mucin secretion in response to a pathophysiological stimulus, effects of PKC/PKG inhibitors on UTP-induced mucin secretion were investigated. NHBE cells were preincubated with various inhibitors for 15 min and then exposed to UTP (100 µm) plus the inhibitor for 2 h, and secreted mucin was measured by ELISA. The results indicated that mucin secretion provoked by UTP similarly required both PKC and PKG activities, as the secretory response was attenuated independently by the PKC inhibitor calphostin C (500 nm), the PKG inhibitorRp-8-Br-PET-cGMP (10 µm), or the soluble guanylyl cyclase (GC-S) inhibitor LY83583 (50 µm) but not by the protein kinase A (PKA) inhibitor KT5720 (500 nm) (Fig. 1C). Apparently, mucin secretion in NHBE cells is regulated by a signaling mechanism involving both PKC and PKG. To address the signaling mechanism downstream of protein kinase activation, we turned our attention to a specific cellular substrate of PKC, MARCKS protein, that might play a role in linking kinase activation to granule release. We first confirmed the presence of MARCKS in NHBE cells by [3H]myristic acid-labeled immunoprecipitation assay. As illustrated in Fig.2A, MARCKS was expressed in NHBE cells, and the majority of this protein was membrane-associated under unstimulated conditions. Then a role for MARCKS as a key regulatory component of the mucin secretory pathway was demonstrated in three different ways. NHBE cells were preincubated with either the MANS or the RNS peptide (1–100 µm) for 15 min, and then PMA (100 nm) + 8-Br-cGMP (1 µm) or UTP (100 µm) was added, and cells were incubated for an additional 15 min or 2 h, respectively. Mucin secretion was measured by ELISA. As shown in Fig. 2B, incubation of NHBE cells with the MANS peptide resulted in a concentration-dependent suppression of mucin secretion in response to PKC/PKG activation or UTP stimulation, whereas the control peptide (RNS) did not affect secretion at these same concentrations. Effects of the MANS peptide were not related to cytotoxicity or general repression of cellular metabolic activity, as neither the MANS nor the RNS peptide affected lactate dehydrogenase release or [3H]deoxyglucose uptake by the cells (data not shown). To demonstrate further MARCKS as a key signaling component of the mucin secretory pathway, the effect of an antisense oligonucleotide directed against MARCKS on mucin secretion was examined. As illustrated in Fig.3, this antisense oligonucleotide down-regulated both mRNA and protein levels of MARCKS in NHBE cells and significantly attenuated mucin secretion induced by PMA + 8-Br-cGMP, whereas a control oligonucleotide had no effect. Transfection of HBE1 cells with the PSD-truncated MARCKS cDNA resulted in significant inhibition of mucin secretion in response to PMA + 8-Br-cGMP stimulation, whereas transfection with the wild-type MARCKS cDNA or the vector DNA had no effect (Fig. 4). To reveal molecular events by which MARCKS links kinase activation to mucin secretion, phosphorylation of MARCKS in response to PKC/PKG activation was investigated in depth. As illustrated in Fig. 5A, PMA (100 nm) induced a significant increase (3–4-fold) in MARCKS phosphorylation in NHBE cells, and this phosphorylation was attenuated by the PKC inhibitor calphostin C (500 nm). Once phosphorylated, MARCKS was translocated from the plasma membrane to the cytoplasm (Fig. 5B). Activation of PKG by 8-Br-cGMP (1 µm), another kinase activation event necessary for provoking mucin secretion, did not lead to MARCKS phosphorylation, but, in fact, the opposite effect was observed: MARCKS phosphorylation induced by PMA was reversed by 8-Br-cGMP (Fig.6A). This effect of 8-Br-cGMP was not due to suppression of PKC activity, as the PMA-induced phosphorylation could be reversed by subsequent addition of 8-Br-cGMP to the cells (Fig. 6B). Therefore, PKG activation clearly resulted in dephosphorylation of MARCKS.Figure 6PKG induces dephosphorylation of MARCKS by activating PP2A. NHBE cells were labeled with [32P]orthophosphate and then exposed to the indicated reagents. MARCKS phosphorylation in response to the treatments was evaluated by immunoprecipitation assay. A, 8-Br-cGMP reversed MARCKS phosphorylation induced by PMA, and this effect of 8-Br-cGMP could be blocked by Rp-8-Br-PET-cGMP (PKG inhibitor) or okadaic acid (PP1/2A inhibitor). B,PMA-induced phosphorylation of MARCKS was reversed by subsequent exposure of cells to 8-Br-cGMP. Lane 1, medium alone for 8 min; lane 2, 100 nm PMA for 3 min; lane 3, 100 nm PMA for 3 min and then with 1 µm 8-Br-cGMP for 5 min; lane 4, 100 nm PMA for 8 min; lane 5, medium alone for 3 min and then 100 nm PMA + 1 µm 8-Br-cGMP for 5 min. C, 8-Br-cGMP-induced MARCKS dephosphorylation was attenuated by fostriecin in a concentration-dependent manner. ◒ from left to right: 500, 100, 10, and 1 nm fostriecin; the last lane: 500 nm fostriecin alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Further investigation showed that PKG-induced MARCKS dephosphorylation was blocked by 500 nm okadaic acid, a protein phosphatase (type 1 and/or 2A (PP1/2A)) inhibitor (Fig. 6A, lane 6). Thus, it appeared that the dephosphorylation was mediated by PP1 and/or PP2A. To define the subtype of protein phosphatase involved, a novel and more specific inhibitor of PP2A, fostriecin (IC50 = 3.2 nm) (20Walsh A.H. Cheng A. Honkanen R.E. FEBS Lett. 1997; 416: 230-234Crossref PubMed Scopus (198) Google Scholar), was utilized in additional phosphorylation studies. As illustrated in Fig.6C, fostriecin inhibited PKG-induced MARCKS dephosphorylation in a concentration-dependent manner (1–500 nm), suggesting that PKG induced the dephosphorylation via activation of PP2A. To confirm further activation of PP2A by PKG in NHBE cells, cytosolic PP1 and PP2A activities were determined after exposure of the cells to 8-Br-cGMP. PP2A activity was increased approximately 3-fold (from 0.1 to 0.3 nmol/min/mg proteins,p < 0.01) at concentrations of 8-Br-cGMP as low as 0.1 µm, whereas PP1 activity remained unchanged. These data clearly indicate that PP2A is activated by PKG and is responsible for the dephosphorylation of MARCKS. Accordingly, this PP2A activity appeared critical for mucin secretion to occur; when PKG-induced MARCKS dephosphorylation was blocked by okadaic acid or fostriecin, the secretory response to PKC/PKG activation or UTP stimulation was ameliorated (Fig. 7). Radiolabeled immunoprecipitation assay revealed that MARCKS associated with two other proteins (∼200 and ∼40 kDa) in the cytoplasm (Fig. 8). Matrix-assisted laser desorption ionization/time of flight mass spectrometry/internal sequence analysis indicated that these two MARCKS-associated proteins were myosin (heavy chain, non-muscle type A) and actin, respectively. Transformed cell lines of airway epithelium tend to contain altered signaling pathways, and cell lines or nondifferentiated cells may not respond to exogenous stimuli in a manner similar to differentiated cells in vivo. The NHBE cells utilized in the present study were cultured at the air/liquid interface, resulting in fully differentiated primary cell cultures that maintained a well documented structure and function similar to the in vivo one (8Krunkosky T.M. Fischer B.M. Martin L.D. Jones N. Akley N.J. Adler K.B. Am. J. Respir. Cell Mol. Biol. 2000; 22: 685-692Crossref PubMed Scopus (133) Google Scholar, 21Adler K.B. Cheng P.W. Kim K.C. Am. J. Respir. Cell Mol. Biol. 1990; 2: 145-154Crossref PubMed}, 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} }
 @inbook{li_martin_adler_2001, title={MARCKS protein: a potential modulator of airway mucin secretion.}, booktitle={Cilia and mucus: from development to respiratory disease.}, author={Li, Y. and Martin, L. D. and Adler, K. B.}, year={2001}, pages={179–193} }
 @article{zhang_rice_adler_sannes_martin_gladwell_koo_gray_bonner_2001, title={Vanadium stimulates human bronchial epithelial cells to produce heparin-binding epidermal growth factor-like growth factor - A mitogen for lung fibroblasts}, volume={24}, ISSN={["1044-1549"]}, DOI={10.1165/ajrcmb.24.2.4096}, abstractNote={Section:ChooseTop of pageAbstract <<Materials and MethodsResultsDiscussionReferencesCITING ARTICLES}, number={2}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Zhang, LM and Rice, AB and Adler, K and Sannes, P and Martin, L and Gladwell, W and Koo, JS and Gray, TE and Bonner, JC}, year={2001}, month={Feb}, pages={123–131} }
 @inbook{he_adler_akley_fischer_jiang_krunkosky_martin_2000, title={Air-liquid interface culture systems for exposure of differentiated cells to oxidant stress.}, booktitle={Models and methods in cell signaling and gene expression applications to oxidative stress research.}, author={He, F. and Adler, K. B. and Akley, N. J. and Fischer, B. M. and Jiang, N. and Krunkosky, T. M. and Martin, L. D.}, year={2000}, pages={15–30} }
 @article{martin_krunkosky_adler_2000, title={Differential generation of intracellular oxidants by TNF? and exogenous superoxide affords distinct expression of IL-6 in human airway epithelium in vitro.}, volume={161}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Martin, L. D. and Krunkosky, T. M. and Adler, K. B.}, year={2000}, pages={A152} }
 @article{krunkosky_fischer_martin_jones_akley_adler_2000, title={Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro - Signaling pathways controlling surface and gene expression}, volume={22}, ISSN={["1535-4989"]}, DOI={10.1165/ajrcmb.22.6.3925}, abstractNote={Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.}, number={6}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Krunkosky, TM and Fischer, BM and Martin, LD and Jones, N and Akley, NJ and Adler, KB}, year={2000}, month={Jun}, pages={685–692} }
 @article{bonner_zhang_sannes_martin_gladwell_koo_gray_adler_2000, title={Induction of heparin-binding epidermal growth factor (HB-EGF) mRNA in normal human bronchial epithelial cells by metal-induced oxidative stress.}, volume={161}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Bonner, J. C. and Zhang, L. and Sannes, P. and Martin, L. and Gladwell, W. and Koo, J.-S. and Gray, T. and Adler, K.}, year={2000}, pages={A149} }
 @article{martin_bonner_macchione_booth_akley_adler_2000, title={Interaction of TGF? and EGF receptor mediates IL-13 induced mucous cell hyperplasia in human airway epithelium in vitro.}, volume={161}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Martin, L. D. and Bonner, J. C. and Macchione, M. and Booth, B. and Akley, N. and Adler, K. B.}, year={2000}, pages={A779} }
 @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{he_martin_adler_2000, title={Mechanisms of human neutrophil elastase-induced mucin hypersecretion by human airway epithelial cells in vitro.}, volume={161}, journal={American Journal of Respiratory and Critical Care Medicine}, author={He, F. and Martin, L. D. and Adler, K. B.}, year={2000}, pages={A155} }
 @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{martin_norford_voynow_adler_2000, title={Response of human airway epithelium in vitro to inflammatory mediators - Dependence on the state of cellular differentiation}, volume={117}, ISSN={["0012-3692"]}, DOI={10.1378/chest.117.5_suppl_1.267s}, number={5}, journal={CHEST}, author={Martin, LD and Norford, D and Voynow, J and Adler, KB}, year={2000}, month={May}, pages={267S–267S} }
 @article{li_greenfeder_martin_minnicozzi_voynow_adler_1999, title={Cloning and expression of guinea pig MUC2 and MUC5AC genes.}, volume={159}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Li, Y. and Greenfeder, S. and Martin, L. D. and Minnicozzi, M. and Voynow, J. A. and Adler, K. B.}, year={1999}, pages={A852} }
 @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{adler_martin_voynow_krunkosky_1999, title={TNF? effects on human airway epithelial cells in vitro: molecular mechanisms of enhanced mucin production/secretion and ICAM-I expression.}, volume={8}, journal={404nOtfound}, author={Adler, K. B. and Martin, L. D. and Voynow, J. A. and Krunkosky, T. M.}, year={1999}, pages={S55} }
 @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{martin_krunkosky_adler_1999, title={Transcription factor activation during oxidant-regulated interleukin-6 expression in normal human bronchial epithelium in vitro.}, volume={159}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Martin, L. D. and Krunkosky, T. M. and Adler, K. B.}, year={1999}, pages={A447} }
 @article{adler_fischer_martin_voynow_1998, title={Effects of inflammatory mediators and drugs on mucus secretion and mucociliary function.}, volume={149}, journal={404nOtfound}, author={Adler, K. B. and Fischer, B. M. and Martin, L. D. and Voynow, J. A.}, year={1998}, pages={245–248} }
 @article{martin_krunkosky_voynow_adler_1998, title={Intracellular reactive oxygen and nitrogen species as signaling molecules in airway epithelium.}, volume={106}, journal={Environmental Health Perspectives}, author={Martin, L. D. and Krunkosky, T. M. and Voynow, J. A. and Adler, K. B.}, year={1998}, pages={1197–1203} }
 @inbook{martin_krunkosky_voynow_adler_1998, title={Oxidant-regulated gene expression in inflammatory lung disease.}, DOI={10.1007/978-1-4419-8634-4_23}, booktitle={Acute respiratory distress syndrome: cellular and molecular mechanisms and clinical management.}, author={Martin, L. D. and Krunkosky, T. M. and Voynow, J. A. and Adler, K. B.}, year={1998}, pages={187–195} }
 @article{martin_krunkosky_voynow_adler_1998, title={The role of reactive oxygen and nitrogen species in airway epithelial gene expression}, volume={106}, ISSN={["0091-6765"]}, DOI={10.2307/3433986}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Martin, LD and Krunkosky, TM and Voynow, JA and Adler, KB}, year={1998}, month={Oct}, pages={1197–1203} }
 @misc{martin_rochelle_fischer_krunkosky_adler_1997, title={Airway epithelium as an effector of inflammation: molecular regulation of secondary mediators}, volume={10}, ISSN={["0903-1936"]}, DOI={10.1183/09031936.97.10092139}, abstractNote={Deleterious environmental stimuli cause the airway epithelium to respond with increased secretions of mucus, reaction of oxygen/nitrogen species, changes in ciliary beating, and the influx of inflammatory cells. The epithelium is a target for factors released by infiltrating inflammatory cells, and has recently been shown to serve as an effector of such inflammation. Molecular mechanisms regulating production of secondary inflammatory mediators (cytokines, lipid mediators, and reactive oxygen/nitrogen species) have yet to be fully described. This report reviews the production of secondary mediators by epithelial cells and by airway epithelium. Lipid mediators are enzymatically produced by the airway epithelium in response to primary mediators. Molecular mechanisms regulating the production of cyclo-oxygenase, lipoxygenase and prostaglandin synthase are discussed, along with the potential of lipid mediators to produce inflammation. The molecular regulation of nitric oxide production is also described in the context of its role as a signalling molecule in pathways regulating secretion of mucus, ciliary motion, and intercellular adhesion molecule-1 (ICAM-1) expression. The production of cytokines by the airway epithelium is shown to play a role in causing inflammation associated with respiratory diseases. Particular attention is paid to molecular mechanisms governing the expression of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-8 (IL-8).}, number={9}, journal={EUROPEAN RESPIRATORY JOURNAL}, author={Martin, LD and Rochelle, LG and Fischer, BM and Krunkosky, TM and Adler, KB}, year={1997}, month={Sep}, pages={2139–2146} }
 @article{krunkosky_martin_fischer_adler_1997, title={Mechanisms of TNF?-induced ICAM-1 expression in airway epithelium.}, volume={155}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Krunkosky, T. M. and Martin, L. D. and Fischer, B. M. and Adler, K. B.}, year={1997}, pages={A207} }
 @article{martin_krunkosky_rochelle_adler_1997, title={TNF? and oxidant-induced expression of Interleukin-6 in normal human bronchial epithelium in vitro.}, volume={155}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Martin, L. D. and Krunkosky, T. M. and Rochelle, L. G. and Adler, K. B.}, year={1997}, pages={A206} }
 @article{martin_krunkosky_dye_fischer_jiang_rochelle_akley_dreher_adler_1997, title={The role of reactive oxygen and nitrogen species in the response of airway epithelium to particulates}, volume={105}, ISSN={["1552-9924"]}, DOI={10.2307/3433551}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Martin, LD and Krunkosky, TM and Dye, JA and Fischer, BM and Jiang, NF and Rochelle, LG and Akley, NJ and Dreher, KL and Adler, KB}, year={1997}, month={Sep}, pages={1301–1307} }
 @article{adler_krunkosky_fischer_rochelle_martin_dreher_jiang_dye_1996, title={Role or reactive oxygen and nitrogen species in the response of airway epithelium to particulates.}, volume={6}, journal={Proceedings of the 6th International Meeting of the Toxicology of Natural and Man-Made Fibrous and Non-Fibrous Particles.}, author={Adler, K. B. and Krunkosky, T. M. and Fischer, B. M. and Rochelle, L. G. and Martin, L. D. and Dreher, K. L. and Jiang, N. and Dye, J.}, year={1996}, pages={139} }
 @article{krunkosky_fischer_martin_adler_1996, title={Tumor necrosis factor alpha (TNF?) provokes expression of intercellular adhesion molecule-1 (ICAM-1) on human bronchial epithelial cells in primary culture.}, volume={153}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Krunkosky, T. M. and Fischer, B. M. and Martin, L. D. and Adler, K. B.}, year={1996}, pages={A27} }