@misc{sheats_yin_fang_park_crews_parikh_dickson_adler_2019, title={MARCKS and Lung Disease}, volume={60}, ISSN={["1535-4989"]}, DOI={10.1165/rcmb.2018-0285TR}, abstractNote={Abstract MARCKS (myristoylated alanine‐rich C kinase substrate) is a prominent PKC substrate expressed in all eukaryotic cells. It is known to bind to and cross‐link actin filaments, to serve as a bridge between Ca2+/calmodulin and PKC signaling, and to sequester the signaling molecule phosphatidylinositol 4,5‐bisphosphate in the plasma membrane. Since the mid‐1980s, this evolutionarily conserved and ubiquitously expressed protein has been associated with regulating cellular events that require dynamic actin reorganization, including cellular adhesion, migration, and exocytosis. More recently, translational studies have implicated MARCKS in the pathophysiology of a number of airway diseases, including chronic obstructive pulmonary disease, asthma, lung cancer, and acute lung injury/acute respiratory distress syndrome. This article summarizes the structure and cellular function of MARCKS (also including MARCKS family proteins and MARCKSL1 [MARCKS‐like protein 1]). Evidence for MARCKS's role in several lung diseases is discussed, as are the technological innovations that took MARCKS‐targeting strategies from theoretical to therapeutic. Descriptions and updates derived from ongoing clinical trials that are investigating inhalation of a MARCKS‐targeting peptide as therapy for patients with chronic bronchitis, lung cancer, and ARDS are provided.}, number={1}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Sheats, Mary K. and Yin, Qi and Fang, Shijing and Park, Joungjoa and Crews, Anne L. and Parikh, Indu and Dickson, Brian and Adler, Kenneth B.}, year={2019}, month={Jan}, pages={16–27} } @article{yin_fang_park_crews_parikh_adler_2016, title={An Inhaled Inhibitor of Myristoylated Alanine-Rich C Kinase Substrate Reverses LPS-Induced Acute Lung Injury in Mice}, volume={55}, ISSN={["1535-4989"]}, DOI={10.1165/rcmb.2016-0236rc}, abstractNote={Intratracheal instillation of bacterial LPS is a well-established model of acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). Because the myristoylated alanine-rich C kinase substrate (MARCKS) protein is involved in neutrophil migration and proinflammatory cytokine production, we examined whether an aerosolized peptide that inhibits MARCKS function could attenuate LPS-induced lung injury in mice. The peptide, BIO-11006, was delivered at 50 μM via inhalation either just before intratracheal instillation of 5 μg of LPS into Balb/C mice, or 4, 12, 24, or 36 hours after LPS instillation. Effects of BIO-11006 were evaluated via analysis of mouse disease-related behavior, lung histology, bronchoalveolar lavage fluid total protein, neutrophil counts and percentages, cytokine (KC [CXCl1, mouse IL-8 equivalent] and TNF-α) expression, and activation of NF-κB in lung tissue. Treatment with aerosolized BIO-11006 at 0, 4, 12, 24, and even 36 hours after LPS instillation reversed the disease process: mouse behavior returned to normal after two treatments 12 hours apart with the inhaled peptide after LPS injury, whereas control LPS-instilled animals treated with PBS only remained moribund. Histological appearance of inflammation, bronchoalveolar lavage fluid protein levels, leukocyte and neutrophil numbers, KC and TNF-α gene and protein expression, and NF-κB activation were all significantly attenuated by inhaled BIO-11006 at all time points. These results implicate MARCKS protein in the pathogenesis of ALI/ARDS and suggest that MARCKS-inhibitory peptide(s), delivered by inhalation, could represent a new and potent therapeutic treatment for ALI/ARDS, even if administered well after the disease process has begun.}, number={5}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Yin, Qi and Fang, Shijing and Park, Joungjoa and Crews, Anne L. and Parikh, Indu and Adler, Kenneth B.}, year={2016}, month={Nov}, pages={617–622} } @article{fang_crews_chen_park_yin_ren_adler_2013, title={MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro}, volume={304}, ISSN={["1522-1504"]}, DOI={10.1152/ajplung.00337.2012}, abstractNote={Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.}, number={8}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, author={Fang, Shijing and Crews, Anne L. and Chen, Wei and Park, Joungjoa and Yin, Qi and Ren, Xiu-Rong and Adler, Kenneth B.}, year={2013}, month={Apr}, pages={L511–L518} } @article{lampe_park_fang_crews_adler_2012, title={Calpain and MARCKS protein regulation of airway mucin secretion}, volume={25}, ISSN={["1094-5539"]}, DOI={10.1016/j.pupt.2012.06.003}, abstractNote={Hypersecretion of mucin plays an important role in the pathophysiology of many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Myristoylated alanine-rich C-kinase substrate (MARCKS) protein has been shown to play an important role in regulation of airway mucin secretion, as peptides analogous to the amino (N)-terminus of MARCKS attenuate mucin secretion by airway epithelium in vitro and in vivo. Here, we investigated a potential role for the protease Calpain, a calcium-dependent cysteine protease that can cleave MARCKS, in the MARCKS-related secretory mechanism. We theorized that Calpain might cleave MARCKS near the N-terminus, thereby attenuating the ability of MARCKS to bind to membranes and/or creating a small N-terminal peptide that could act as a competitive intracellular inhibitor to remaining endogenous full-length MARCKS molecules. Primary normal human bronchial epithelial (NHBE) cells and the virally-transformed human bronchial epithelial HBE1 cell line were exposed to phorbol-12-myristate-13-acetate (PMA) to stimulate the Protein Kinase C (PKC) pathway, leading to enhanced mucin secretion, and Calpain activity within the cells was measured with a fluorescent cleavage assay. Calpain activity was increased by PMA, and pretreatment of the cells with Calpain inhibitors reduced both Calpain activity and mucin secretion in a concentration-dependent manner. Thus, as opposed to the original hypothesis, inactivating Calpain caused a decrease rather than an increase in secretion. HBE1 cells transfected with DNA constructs encoding a MARCKS-YFP fusion protein showed cleavage at a putative site near the N-terminus in response to PMA. Cleavage of MARCKS by Calpain may have an important role in regulation of the PKC/MARCKS pathway regulating airway mucin secretion.}, number={6}, journal={PULMONARY PHARMACOLOGY & THERAPEUTICS}, author={Lampe, W. Randall and Park, Joungjoa and Fang, Shijing and Crews, Anne L. and Adler, Kenneth B.}, year={2012}, month={Dec}, pages={427–431} } @article{green_park_yin_fang_crews_jones_adler_2012, title={Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein}, volume={92}, ISSN={0741-5400}, url={http://dx.doi.org/10.1189/jlb.1211604}, DOI={10.1189/jlb.1211604}, abstractNote={Abstract}, number={3}, journal={Journal of Leukocyte Biology}, publisher={Wiley}, author={Green, T. D. and Park, J. and Yin, Q. and Fang, S. and Crews, A. L. and Jones, S. L. and Adler, K. B.}, year={2012}, month={May}, pages={633–639} } @article{seibold_wise_speer_steele_brown_loyd_fingerlin_zhang_gudmundsson_groshong_et al._2011, title={A Common MUC5B Promoter Polymorphism and Pulmonary Fibrosis}, volume={364}, ISSN={["1533-4406"]}, DOI={10.1056/nejmoa1013660}, abstractNote={BACKGROUND The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk. METHODS Using a genomewide linkage scan, we detected linkage between idiopathic interstitial pneumonia and a 3.4-Mb region of chromosome 11p15 in 82 families. We then evaluated genetic variation in this region in gel-forming mucin genes expressed in the lung among 83 subjects with familial interstitial pneumonia, 492 subjects with idiopathic pulmonary fibrosis, and 322 controls. MUC5B expression was assessed in lung tissue. RESULTS Linkage and fine mapping were used to identify a region of interest on the p-terminus of chromosome 11 that included gel-forming mucin genes. The minor-allele of the single-nucleotide polymorphism (SNP) rs35705950, located 3 kb upstream of the MUC5B transcription start site, was present at a frequency of 34% among subjects with familial interstitial pneumonia, 38% among subjects with idiopathic pulmonary fibrosis, and 9% among controls (allelic association with familial interstitial pneumonia, P=1.2×10(-15); allelic association with idiopathic pulmonary fibrosis, P=2.5×10(-37)). The odds ratios for disease among subjects who were heterozygous and those who were homozygous for the minor allele of this SNP were 6.8 (95% confidence interval [CI], 3.9 to 12.0) and 20.8 (95% CI, 3.8 to 113.7), respectively, for familial interstitial pneumonia and 9.0 (95% CI, 6.2 to 13.1) and 21.8 (95% CI, 5.1 to 93.5), respectively, for idiopathic pulmonary fibrosis. MUC5B expression in the lung was 14.1 times as high in subjects who had idiopathic pulmonary fibrosis as in those who did not (P<0.001). The variant allele of rs35705950 was associated with up-regulation in MUC5B expression in the lung in unaffected subjects (expression was 37.4 times as high as in unaffected subjects homozygous for the wild-type allele, P<0.001). MUC5B protein was expressed in lesions of idiopathic pulmonary fibrosis. CONCLUSIONS A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis. Our findings suggest that dysregulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis. (Funded by the National Heart, Lung, and Blood Institute and others.).}, number={16}, journal={NEW ENGLAND JOURNAL OF MEDICINE}, author={Seibold, Max A. and Wise, Anastasia L. and Speer, Marcy C. and Steele, Mark P. and Brown, Kevin K. and Loyd, James E. and Fingerlin, Tasha E. and Zhang, Weiming and Gudmundsson, Gunnar and Groshong, Steve D. and et al.}, year={2011}, month={Apr}, pages={1503–1512} } @article{raiford_park_lin_fang_crews_adler_2011, title={Mucin granule-associated proteins in human bronchial epithelial cells: the airway goblet cell "granulome"}, volume={12}, ISSN={["1465-993X"]}, DOI={10.1186/1465-9921-12-118}, abstractNote={Abstract}, journal={RESPIRATORY RESEARCH}, author={Raiford, Kimberly L. and Park, Joungjoa and Lin, Ko-Wei and Fang, Shijing and Crews, Anne L. and Adler, Kenneth B.}, year={2011}, month={Sep} } @misc{green_crews_park_fang_adler_2011, title={Regulation of mucin secretion and inflammation in asthma: A role for MARCKS protein?}, volume={1810}, ISSN={["0304-4165"]}, DOI={10.1016/j.bbagen.2011.01.009}, abstractNote={A major characteristic of asthmatic airways is an increase in mucin (the glycoprotein component of mucus) producing and secreting cells, which leads to increased mucin release that further clogs constricted airways and contributes markedly to airway obstruction and, in the most severe cases, to status asthmaticus. Asthmatic airways show both a hyperplasia and metaplasia of goblet cells, mucin-producing cells in the epithelium; hyperplasia refers to enhanced numbers of goblet cells in larger airways, while metaplasia refers to the appearance of these cells in smaller airways where they normally are not seen. With the number of mucin-producing and secreting cells increased, there is a coincident hypersecretion of mucin which characterizes asthma. On a cellular level, a major regulator of airway mucin secretion in both in vitro and in vivo studies has been shown to be MARCKS (myristoylated alanine-rich C kinase substrate) protein, a ubiquitous substrate of protein kinase C (PKC). In this review, properties of MARCKS and how the protein may regulate mucin secretion at a cellular level will be discussed. In addition, the roles of MARCKS in airway inflammation related to both influx of inflammatory cells into the lung and release of granules containing inflammatory mediators by these cells will be explored. This article is part of a Special Issue entitled: Biochemistry of Asthma.}, number={11}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS}, author={Green, Teresa D. and Crews, Anne L. and Park, Joungjoa and Fang, Shijing and Adler, Kenneth B.}, year={2011}, month={Nov}, pages={1110–1113} } @article{lin_fang_park_crews_adler_2010, title={MARCKS and Related Chaperones Bind to Unconventional Myosin V Isoforms in Airway Epithelial Cells}, volume={43}, ISSN={["1535-4989"]}, DOI={10.1165/rcmb.2010-0016rc}, abstractNote={We have shown previously that myristoylated alanine-rich C kinase substrate (MARCKS) is a key regulatory molecule in the process of mucin secretion by airway epithelial cells, and that part of the secretory mechanism involves intracellular associations of MARCKS with specific chaperones: heat shock protein 70 (Hsp70) and cysteine string protein (CSP). Here, we report that MARCKS also interacts with unconventional myosin isoforms within these cells, and further molecular interactions between MARCKS and these chaperones/cytoskeletal proteins are elucidated. Primary human bronchial epithelial cells and the HBE1 cell line both expressed myosin V and VI proteins, and both MARCKS and CSP were shown to bind to myosin V, specifically Va and Vc. This binding was enhanced by exposing the cells to phorbol-12-myristate-13-acetate, an activator of protein kinase C and stimulator of mucin secretion. Binding of MARCKS, Hsp70, and CSP was further investigated by His-tagged pull down assays of purified recombinant proteins and multiple transfections of HBE1 cells with fusion proteins (MARCKS-HA; Flag-Hsp70; c-Myc-CSP) and immunoprecipitation. The results showed that MARCKS binds directly to Hsp70, and that Hsp70 binds directly to CSP, but that MARCKS binding to CSP appears to require the presence of Hsp70. Interrelated binding(s) of MARCKS, chaperones, and unconventional myosin isoforms may be integral to the mucin secretion process.}, number={2}, journal={AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY}, author={Lin, Ko-Wei and Fang, Shijing and Park, Joungjoa and Crews, Anne L. and Adler, Kenneth B.}, year={2010}, month={Aug}, pages={131–136} } @article{foster_adler_crews_potts_fischer_voynow_2010, title={MARCKS-related peptide modulates in vivo the secretion of airway Muc5ac (Retracted article. See vol. 309, pg. L 750, 2015)}, volume={299}, ISSN={["1522-1504"]}, DOI={10.1152/ajplung.00067.2010}, abstractNote={ In a mouse model of neutrophil elastase-induced bronchitis that exhibits goblet cell metaplasia and inflammation, we investigated the effects of intratracheal instillation of the MANS peptide, a peptide identical to the NH2 terminus of the myristoylated alanine-rich C kinase substrate (MARCKS) on mucin protein airway secretion, inflammation, and airway reactivity. To induce mucus cell metaplasia in the airways, male BALB/c mice were treated repetitively with the serine protease, neutrophil elastase, on days 1, 4, and 7. On day 11, when goblet cell metaplasia was fully developed and profiles of proinflammatory cytokines were maximal, the animals were exposed to aerosolized methacholine after intratracheal instillation of MANS or a missense control peptide (RNS). MANS, but not RNS, attenuated the methacholine-stimulated secretion of the major respiratory mucin protein, Muc5ac (50% reduction). Concurrently, elastase-induced proinflammatory cytokines typically recovered in bronchoalveolar lavage (BAL), including KC, IL-1β, IL-6, MCP-1, and TNFα, were reduced by the MANS peptide (mean levels decreased 50–60%). Secondary to the effects of MANS on mucin secretion and inflammation, mechanical lung function by forced oscillation technique was characterized with respect to airway reactivity in response to cumulative aerosol stimulation with serotonin. The MANS peptide was also found to effectively attenuate airway hyperresponsiveness to serotonin in this airway hypersecretory model. Collectively, these findings support the concept that even in airway epithelia remodeled with goblet cell metaplasia and in a state of mucin hypersecretion, exogenous attenuation of function of MARCKS protein via the MANS peptide decreases airway mucin secretion, inflammation, and hyperreactivity. }, number={3}, journal={AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY}, author={Foster, W. Michael and Adler, Kenneth B. and Crews, Anne L. and Potts, Erin N. and Fischer, Bernard M. and Voynow, Judith A.}, year={2010}, month={Sep}, pages={L345–L352} } @article{crews_potts_voynow_fischer_bumgardner_adler_foster_2008, title={A MARCKS-related peptide attenuates both mucin hypersecretion and inflammatory cell infiltration in an elastase model of chronic bronchitis.}, volume={177}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Crews, A. L. and Potts, E. and Voynow, J. A. and Fischer, B. M. and Bumgardner, S. and Adler, K. B. and Foster, W. M.}, year={2008}, pages={A332} } @article{wang_adler_slade_church_webb_chu_jinwright_crews_kraft_2008, title={A MARCKS-related peptide inhibits MUC5AC expression in airway epithelial cells isolated from asthmatic subjects.}, volume={177}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Wang, Y. and Adler, K. and Slade, D. J. and Church, T. D. and Webb, R. F. and Chu, H. W. and Jinwright, D. and Crews, A. and Kraft, M.}, year={2008}, pages={A814} } @article{park_crews_alper_schwartz_adler_2008, title={A potential role for airway epithelial cell mucin genes in fibrogenesis.}, volume={177}, journal={American Journal of Respiratory and Critical Care Medicine}, author={Park, J. J. and Crews, A. L. and Alper, S. and Schwartz, D. A. and Adler, K. B.}, year={2008}, pages={A727} } @article{park_fang_crews_lin_adler_2008, title={MARCKS regulation of mucin secretion by airway epithelial cells in vitro: interactions with chaperone proteins.}, volume={102}, journal={American Journal of Respiratory Cell and Molecular Biology}, author={Park, J. J. and Fang, S. and Crews, A. L. and Lin, K.-W. and Adler, K. B.}, year={2008}, pages={949–955} } @article{park_fang_crews_lin_adler_2008, title={MARCKS regulation of mucin secretion by airway epithelium in vitro - Interaction with chaperones}, volume={39}, DOI={10.1165/rcm6.2007-0139OC}, number={1}, journal={American Journal of Respiratory Cell and Molecular Biology}, author={Park, J. and Fang, S. J. and Crews, A. L. and Lin, K. W. and Adler, K. B.}, year={2008}, pages={68–76} } @article{kraft_adler_ingram_crews_atkinson_cairns_krause_chu_2008, title={Mycoplasma pneumoniae increases airway epithelial cell MUC5AC expression in asthma.}, volume={34}, journal={European Respiratory Journal}, author={Kraft, M. and Adler, K. B. and Ingram, J. L. and Crews, A. L. and Atkinson, T. P. and Cairns, C. B. and Krause, D. C. and Chu, H. W.}, year={2008}, pages={43–46} } @article{kraft_adler_ingram_crews_atkinson_cairns_krause_chu_2008, title={Mycoplasma pneumoniae induces airway epithelial cell expression of MUC5AC in asthma}, volume={31}, ISSN={["1399-3003"]}, DOI={10.1183/09031936.00103307}, abstractNote={As excess mucin expression can contribute to the exacerbation of asthma, the present authors hypothesised that Mycoplasma pneumoniae significantly induces MUC5AC (the major airway mucin) expression in airway epithelial cells isolated directly from asthmatic subjects. A total of 11 subjects with asthma and six normal controls underwent bronchoscopy with airway brushing. Epithelial cells were cultured at an air–liquid interface and incubated with and without M. pneumoniae for 48 h, and in the presence and absence of nuclear factor (NF)-κB and a toll-like receptor (TLR)2 inhibitor. Quantitative PCR was performed for MUC5AC and TLR2 mRNA. MUC5AC protein and total protein were determined by ELISA. M. pneumoniae exposure significantly increased MUC5AC mRNA and protein expression after 48 h in epithelial cells isolated from asthmatic, but not from normal control subjects, at all concentrations as compared to unexposed cells. TLR2 mRNA expression was significantly increased in asthmatic epithelial cells at 4 h compared with unexposed cells. NF-κB and TLR2 inhibition reduced MUC5AC expression to the level of the unexposed control in both groups. Mycoplasma pneumoniae exposure significantly increased MUC5AC mRNA and protein expression preferentially in airway epithelial cells isolated from asthmatic subjects. The toll-like receptor 2 pathway may be involved in this process.}, number={1}, journal={EUROPEAN RESPIRATORY JOURNAL}, author={Kraft, M. and Adler, K. B. and Ingram, J. L. and Crews, A. L. and Atkinson, T. P. and Cairns, C. B. and Krause, D. C. and Chu, H. W.}, year={2008}, month={Jan}, pages={43–46} } @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} } @article{green_eckert_sharief_crews_adler_jones_2007, title={A peptide against the N-terminus of MARCKS protein attenuates leukocyte migration.}, volume={175}, journal={404nOtfound}, author={Green, T. D. and Eckert, B. S. and Sharief, Y. and Crews, A. L. and Adler, K. B. and Jones, S. L.}, year={2007}, pages={A915} } @article{park_takashi_crews_miller-larsson_adler_2007, title={Anti-inflammatory effects of budesonide and formoterol in primary cultures of human airway epithelial cells.}, volume={175}, journal={404nOtfound}, author={Park, J. J. and Takashi, S. and Crews, A. L. and Miller-Larsson, A. and Adler, K. B.}, year={2007}, pages={A178} } @article{kraft_nijira lugogo_adler_crews_moss_stalls_church_slade_beaver_chu_2007, title={Mycoplasma pneumoniae increases airway epithelial cell MUC5AC expression in asthma.}, volume={175}, journal={404nOtfound}, author={Kraft, M. and Nijira Lugogo, H. W. and Adler, K. B. and Crews, A. L. and Moss, T. A. and Stalls, M. A. and Church, T. D. and Slade, D. J. and Beaver, D. M. and Chu, H. W.}, year={2007}, pages={A324} } @article{park_crews_lampe_fang_park_adler_2007, title={Protein kinase C delta regulates airway mucin secretion via phosphorylation of MARCKS protein}, volume={171}, ISSN={["1525-2191"]}, DOI={10.2353/ajpath.2007.070318}, abstractNote={Mucin hypersecretion is a major pathological feature of many respiratory diseases, yet cellular mechanisms regulating secretion of mucin have not been fully elucidated. Previously, we reported that mucin hypersecretion induced by human neutrophil elastase involves activation of protein kinase C (PKC), specifically the δ-isoform (PKCδ). Here, we further investigated the role of PKCδ in mucin hypersecretion using both primary human bronchial epithelial cells and the human bronchial epithelial 1 cell line as in vitro model systems. Phorbol-12-myristate-13-acetate (PMA)-induced mucin hypersecretion was significantly attenuated by rottlerin, a PKCδ-selective inhibitor. Rottlerin also reduced PMA- or human neutrophil elastase-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in these cells. Both secretion and MARCKS phosphorylation were significantly enhanced by the PKCδ activator bryostatin 1. A dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) transfected into human bronchial epithelial 1 cells significantly attenuated both PMA-induced mucin secretion and phosphorylation of MARCKS, whereas transfection of a wild-type construct increased PKCδ and enhanced mucin secretion and MARCKS phosphorylation. Similar transfections of a dominant-negative or wild-type PKCε construct did not affect either mucin secretion or MARCKS phosphorylation. The results suggest that PKCδ plays an important role in mucin secretion by airway epithelium via regulation of MARCKS phosphorylation. Mucin hypersecretion is a major pathological feature of many respiratory diseases, yet cellular mechanisms regulating secretion of mucin have not been fully elucidated. Previously, we reported that mucin hypersecretion induced by human neutrophil elastase involves activation of protein kinase C (PKC), specifically the δ-isoform (PKCδ). Here, we further investigated the role of PKCδ in mucin hypersecretion using both primary human bronchial epithelial cells and the human bronchial epithelial 1 cell line as in vitro model systems. Phorbol-12-myristate-13-acetate (PMA)-induced mucin hypersecretion was significantly attenuated by rottlerin, a PKCδ-selective inhibitor. Rottlerin also reduced PMA- or human neutrophil elastase-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in these cells. Both secretion and MARCKS phosphorylation were significantly enhanced by the PKCδ activator bryostatin 1. A dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) transfected into human bronchial epithelial 1 cells significantly attenuated both PMA-induced mucin secretion and phosphorylation of MARCKS, whereas transfection of a wild-type construct increased PKCδ and enhanced mucin secretion and MARCKS phosphorylation. Similar transfections of a dominant-negative or wild-type PKCε construct did not affect either mucin secretion or MARCKS phosphorylation. The results suggest that PKCδ plays an important role in mucin secretion by airway epithelium via regulation of MARCKS phosphorylation. Mucus produced by epithelium of respiratory, gastrointestinal, and reproductive tracts provides a barrier between the external environment and cellular components of the epithelial layer. Mucins, the glycoprotein component of mucus, constitute a family of large, highly glycosylated macromolecules that impart physical (aggregation, viscosity, viscoelasticity, and lubrication) and biological (protection) properties to mucus (reviewed in Ref. 1Rose MC Mucins: structure, function, and role in pulmonary diseases.Am J Physiol. 1992; 263: L413-L429PubMed Google Scholar). Airway mucus is an integral component of the mucociliary clearance system in the trachea and bronchi and thus serves to protect the lower airways and alveoli from impingement of particulate matter and pathogens. However, mucin secretion is abnormally augmented in disease states, such as chronic bronchitis, asthma, and cystic fibrosis, increasing morbidity and mortality in these patients (reviewed in Refs. 1Rose MC Mucins: structure, function, and role in pulmonary diseases.Am J Physiol. 1992; 263: L413-L429PubMed Google Scholar and 2Rogers DF Barnes PJ Treatment of airway mucus hypersecretion.Ann Med. 2006; 38: 116-125Crossref PubMed Scopus (172) Google Scholar). Mucin hypersecretion is potentiated by many pathophysiological mediators, such as bacterial proteinases and endotoxin, adenine and guanine nucleotides, cytokines, inflammatory mediators, and eicosanoids (reviewed in Ref. 3Adler KB Li Y Airway epithelium and mucus: intracellular signaling pathways for gene expression and secretion.Am J Respir Cell Mol Biol. 2001; 25: 397-400Crossref PubMed Scopus (50) Google Scholar). Intracellular mechanisms and signaling molecules involved in the secretory process have not been fully elucidated.Protein kinase C (PKC) is a serine/threonine kinase involved in various exocytotic events in different cell types, including secretion of mucin,4Abdullah LH Bundy JT Ehre C Davis CW Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA.Am J Physiol Lung Cell Mol Physiol. 2003; 285: L149-L160PubMed Google Scholar, 5Plaisancie P Ducroc R El Homsi M Tsocas A Guilmeau S Zoghbi S Thibaudeau O Bado A Luminal leptin activates mucin-secreting goblet cells in the large bowel.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G805-G812Crossref PubMed Scopus (59) Google Scholar insulin,6Yaney GC Fairbanks JM Deeney JT Korchak HM Tornheim K Corkey BE Potentiation of insulin secretion by phorbol esters is mediated by PKC-alpha and nPKC isoforms.Am J Physiol Endocrinol Metab. 2002; 283: E880-E888Crossref PubMed Scopus (34) Google Scholar neurotransmitters,7Shoji-Kasai Y Itakura M Kataoka M Yamamori S Takahashi M Protein kinase C-mediated translocation of secretory vesicles to plasma membrane and enhancement of neurotransmitter release from PC12 cells.Eur J Neurosci. 2002; 15: 1390-1394Crossref PubMed Scopus (50) Google Scholar and platelet dense granules.8Murugappan S Tuluc F Dorsam RT Shankar H Kunapuli SP Differential role of protein kinase C delta isoform in agonist-induced dense granule secretion in human platelets.J Biol Chem. 2004; 279: 2360-2367Crossref PubMed Scopus (92) Google Scholar Previously, we demonstrated that mucin secretion in airway epithelial cells is regulated by PKC via phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS).9Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (150) Google Scholar, 10Singer M Martin LD Vargaftig BB Park J Gruber AD Li Y Adler KB A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma.Nat Med. 2004; 10: 193-196Crossref PubMed Scopus (147) Google Scholar In addition, we demonstrated that mucin hypersecretion in human airway epithelial cells in vitro in response to human neutrophil elastase (HNE) appears to be mediated by the δ-isoform of PKC (PKCδ).11Park JA He F Martin LD Li Y Chorley BN Adler KB Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C{delta}-mediated mechanism.Am J Pathol. 2005; 167: 651-661Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Not surprisingly, PKCδ, a novel PKC isoform, has a strong affinity for MARCKS and can phosphorylate MARCKS both in vitro and in vivo.12Fujise A Mizuno K Ueda Y Osada S Hirai S Takayanagi A Shimizu N Owada MK Nakajima H Ohno S Specificity of the high affinity interaction of protein kinase C with a physiological substrate, myristoylated alanine-rich protein kinase C substrate.J Biol Chem. 1994; 269: 31642-31648Abstract Full Text PDF PubMed Google Scholar, 13Herget T Oehrlein SA Pappin DJ Rozengurt E Parker PJ The myristoylated alanine-rich C-kinase substrate (MARCKS) is sequentially phosphorylated by conventional, novel and atypical isotypes of protein kinase C.Eur J Biochem. 1995; 233: 448-457Crossref PubMed Scopus (79) Google Scholar, 14Cabell CH Verghese GM Rankl NB Burns DJ Blackshear PJ MARCKS phosphorylation by individual protein kinase C isozymes in insect Sf9 cells.Proc Assoc Am Physicians. 1996; 108: 37-46PubMed Google Scholar Increasing evidence suggests that PKCδ mediates exocytotic secretion in several different cell types.4Abdullah LH Bundy JT Ehre C Davis CW Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA.Am J Physiol Lung Cell Mol Physiol. 2003; 285: L149-L160PubMed Google Scholar, 8Murugappan S Tuluc F Dorsam RT Shankar H Kunapuli SP Differential role of protein kinase C delta isoform in agonist-induced dense granule secretion in human platelets.J Biol Chem. 2004; 279: 2360-2367Crossref PubMed Scopus (92) Google Scholar, 15Ishikawa T Iwasaki E Kanatani K Sugino F Kaneko Y Obara K Nakayama K Involvement of novel protein kinase C isoforms in carbachol-stimulated insulin secretion from rat pancreatic islets.Life Sci. 2005; 77: 462-469Crossref PubMed Scopus (12) Google Scholar, 16Leitges M Gimborn K Elis W Kalesnikoff J Hughes MR Krystal G Huber M Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation.Mol Cell Biol. 2002; 22: 3970-3980Crossref PubMed Scopus (115) Google Scholar, 17Cho SH Woo CH Yoon SB Kim JH Protein kinase Cdelta functions downstream of Ca2+ mobilization in FcepsilonRI signaling to degranulation in mast cells.J Allergy Clin Immunol. 2004; 114: 1085-1092Abstract Full Text Full Text PDF PubMed Scopus (55) Google ScholarHere, we further elucidate the role of PKCδ in the mucin secretory pathway in human airway epithelial cells in vitro. The mucin secretory response and phosphorylation of MARCKS were assessed after exposure of well differentiated normal human bronchial epithelial (NHBE) cells to phorbol-12-myristate-13-acetate (PMA), a general PKC activator, or bryostatin 1, a PKCδ/ε activator. In addition, we used the papilloma virus-transformed human bronchial epithelial 1 (HBE1) cell line for molecular manipulations. A dominant-negative PKCδ construct (K376R) transfected into HBE1 cells attenuated PMA-stimulated mucin secretion and MARCKS phosphorylation, whereas a similar dominant-negative PKCε construct was without effect. The results indicate that PKCδ is a key isoform regulating airway mucin secretion, and the mechanism of its action appears to involve phosphorylation of MARCKS protein.Materials and MethodsCulture of Bronchial Epithelial CellsPrimary NHBE cells purchased from Cambrex Bioscience (Walkersville, MD) were expanded and maintained in a humidified air/5% CO2 incubator as described previously.9Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (150) Google Scholar, 11Park JA He F Martin LD Li Y Chorley BN Adler KB Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C{delta}-mediated mechanism.Am J Pathol. 2005; 167: 651-661Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Cells from at least three separate donors were used in these studies. Passage 31 or 32 of human papilloma virus-transformed human bronchial epithelial cells (HBE1)18Yankaskas JR Haizlip JE Conrad M Koval D Lazarowski E Paradiso AM Rinehart Jr, CA Sarkadi B Schlegel R Boucher RC Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype.Am J Physiol. 1993; 264: C1219-C1230PubMed Google Scholar was seeded and maintained as described previously. Transient transfection of HBE1 cells was performed after 10 days of culture in air-liquid interface.Exposure of Cells to Inhibitors or SecretagoguesWell differentiated NHBE cells were exposed to test agents both apically and basolaterally for 15 minutes (unless otherwise indicated). Transfected HBE1 cells were exposed to PMA (EMD Biosciences, La Jolla, CA) applied apically only. Cells were preincubated with the PKCδ-selective inhibitor, rottlerin (EMD Biosciences) for 20 minutes before PMA exposure. Initial stock solutions of rottlerin or PMA were prepared in dimethyl sulfoxide, kept at −20°C, and diluted in growth medium directly before use. When cells were exposed to PMA in the presence or absence of rottlerin, PMA was “spiked” into each well at the indicated concentration.Measurement of Mucin SecretionMucin was collected both at baseline and after treatments as described previously.9Li Y Martin LD Spizz G Adler KB MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.J Biol Chem. 2001; 276: 40982-40990Crossref PubMed Scopus (150) Google Scholar Baseline mucin secretion was used to normalize well-to-well variation. After baseline mucin samples were collected, cells were rested overnight and exposed to test reagents the next day for indicated periods of time. After each treatment period, secreted mucin was collected as the baseline sample and quantified by sandwich enzyme-linked immunosorbent assay using the 17Q2 antibody (Covance Research Products, Berkeley, CA), a monoclonal antibody that reacts specifically with a carbohydrate epitope on human airway mucins.19Lin H Carlson DM St George JA Plopper CG Wu R An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates.Am J Respir Cell Mol Biol. 1989; 1: 41-48Crossref PubMed Scopus (63) Google Scholar The 17Q2 antibody was purified using an ImmunoPure(G) IgG purification kit (Pierce Biotechnology, Rockford, IL) following the manufacturer's protocol and then conjugated with alkaline phosphatase (EMD Biosciences). To account for variability between cultures and experiments, levels of mucin secretion were reported as percentage of the nontreated control. Actual values for mucin released by control cell cultures in these experiments ranged from 40 to 70 ng/ml in NHBE cells and 7 to 10 ng/ml in HBE1 cells.Subcellular Localization of PKC IsoformsActivation of PKCδ was assessed by subcellular fractionation following the protocol described by Kajstura et al20Kajstura J Cigola E Malhotra A Li P Cheng W Meggs LG Anversa P Angiotensin II induces apoptosis of adult ventricular myocytes in vitro.J Mol Cell Cardiol. 1997; 29: 859-870Abstract Full Text PDF PubMed Scopus (375) Google Scholar and subsequent Western blot analysis using a PKCδ-specific antibody (Cell Signaling Technology, Inc., Danvers, MA). Briefly, cells were washed with cold PBS and scraped into lysis buffer [20 mmol/L Tris-Cl (pH 7.5), 1 mmol/L ethylenediamine tetraacetic acid, 100 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, 1% (v/v) protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma, St. Louis, MO)]. The lysate was then sonicated and pelleted at 20,000 × g (Eppendorf 5417 centrifuge) for 40 minutes. The supernatant was collected and kept as the cytosolic fraction at −80°C until used. The remaining pellet was resuspended in lysis buffer containing 1% Triton X-100, sonicated, and centrifuged at 20,000 × g for 40 minutes. The supernatant membrane fraction was stored at −80°C until analyzed by Western blot.Western Blot AnalysisTotal MARCKS, phosphorylated MARCKS, PKCδ, and PKCε protein levels were measured via Western blot. The protein concentrations of cell lysates were quantified by a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Sample lysates were prepared by boiling in 2× SDS sample buffer [125 mmol/L Tris-Cl (pH 6.8), 25% glycerol, 4% SDS, 10% β-mercaptoethanol, and 0.04% bromphenol blue] for 10 minutes. Sample lysates (30 to 60 μg) were loaded on 10 or 12% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane (Schleicher & Schuell BioScience, Inc., Keene, NH) following electrophoresis. Polyvinylidene difluoride membranes were blocked with 5% nonfat milk and then probed with an appropriate dilution of primary antibody followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies. Chemiluminescent detection was performed using ECL detection reagents (GE Health care Life Sciences, Piscataway, NJ) following the manufacturer's protocol. Amounts of specific proteins in bands were quantified using Labworks image acquisition and analysis software 4.0. (Ultra Violet Products, Ltd., Upland, CA).Antibodies against α-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and E-cadherin (BD Biosciences, San Jose, CA) were used as loading controls for cytosolic and membrane fractions, respectively. Phosphorylated MARCKS (at serine 152/156) was detected with a specific antibody (Cell Signaling Technology, Inc.). After detection, the membrane was stripped in 62.5 mmol/L Tris-Cl (pH 6.5), 10% SDS, and 100 mmol/L β-mercaptoethanol for 10 minutes at room temperature and reprobed with a monoclonal antibody against total MARCKS protein (clone no. 2F12; Upstate, Charlottesville, VA) to verify equal loading.Transient Transfection of PKC ConstructsTransient transfection of vectors overexpressing wild-type or dominant-negative PKCδ and PKCε in HBE1 cells was performed using the FuGene 6 transfection reagent (Roche Applied Science, Indianapolis, IN) following the manufacturer's protocol. The pEGFP-N1 vectors containing a wild-type PKCδ cDNA21Mischak H Pierce JH Goodnight J Kazanietz MG Blumberg PM Mushinski JF Phorbol ester-induced myeloid differentiation is mediated by protein kinase C-alpha and -delta and not by protein kinase C-beta II, -epsilon, -zeta, and -eta.J Biol Chem. 1993; 268: 20110-20115Abstract Full Text PDF PubMed Google Scholar and a dominant-negative PKCδ mutant cDNA [lysine (AAG)→arginine (AGG) mutation, position 376]22Li L Lorenzo PS Bogi K Blumberg PM Yuspa SH Protein kinase Cdelta targets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector.Mol Cell Biol. 1999; 19: 8547-8558Crossref PubMed Google Scholar were generously provided by Dr. Arti Shukla (University of Vermont, Burlington, VT) and Dr. Peter Blumberg (National Cancer Institute, Bethesda, MD). The K376→R mutation in the ATP binding site of the catalytic domain has been demonstrated previously to inhibit PKCδ kinase activity.23Li W Yu JC Shin DY Pierce JH Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant: an inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity.J Biol Chem. 1995; 270: 8311-8318Crossref PubMed Scopus (89) Google Scholar Briefly, HBE1 cells grown in air/liquid interface were dissociated in versene solution (Invitrogen, Carlsbad, CA) and re-seeded in 12-well culture plates at a density of 1 × 105 cells/cm2. After overnight incubation, cells were transfected with the pEGFP-N1 vector alone or the pEGFP-N1 vector containing either a wild-type or dominant-negative PKCδ cDNA (K376R). Isotype controls for the dominant-negative PKCδ consisted of transient transfection of both wild-type PKCε as well as a catalytically inactive dominant-negative PKCε (K437R) construct-tagged with hemagglutin24Lee YJ Soh JW Jeoung DI Cho CK Jhon GJ Lee SJ Lee YS PKC epsilon -mediated ERK1/2 activation involved in radiation-induced cell death in NIH3T3 cells.Biochim Biophys Acta. 2003; 1593: 219-229Crossref PubMed Scopus (32) Google Scholar (PKCε constructs were kindly provided by Dr. Jae-Won Soh, University of Inha, Inha, Republic of Korea). Cells were subsequently cultured for 48 hours to allow for detectable protein expression. Transfection of PKCδ constructs was confirmed by fluorescent microscopy and assessment of expression of green fluorescence protein (GFP)-tagged PKCδ assessed by Western blot analysis using monoclonal antibodies against PKCδ or GFP (Cell Signaling Technology, Inc.). Transfection of PKCε was confirmed by Western blot analysis using PKCε and HA monoclonal antibodies (Covance Research Products).Cytotoxicity AssayAll treatments used were tested for cytotoxicity using a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI) according to the manufacturer's instructions. The results were expressed as the ratio of released lactate dehydrogenase to total lactate dehydrogenase. Released lactate dehydrogenase never exceeded 10% of total lactate dehydrogenase with any of the treatments (data not shown).Statistical AnalysisData were expressed as the ratio of treatment to the corresponding vehicle (dimethyl sulfoxide or media) control. Results were evaluated using one-way analysis of variance with Dunnett's test and a Bonferroni posttest correction for multiple comparisons.25Kleinbaum DG Kupper LL Muller KE Applied Regression Analysis and Other Multivariable Methods. PWS-Kent Publishing Co., Boston, MA1988Google Scholar A P value of less than 0.05 was considered significant.ResultsEffect of Rottlerin on PMA-Induced Mucin Secretion and MARCKS Phosphorylation in NHBE CellsTo determine whether PKCδ is an important regulatory molecule in mucin secretion, we investigated the effect of rottlerin, a PKCδ-selective inhibitor, on PMA-induced mucin secretion in well differentiated NHBE cells. As illustrated in Figure 1A, 100 nmol/L PMA provoked translocation of PKCδ from cytosol to membrane in these cells. Inhibition of PKCδ activity by pretreatment of cells with rottlerin (1 to 10 μmol/L) for 20 minutes significantly attenuated PMA-induced mucin secretion in a concentration-dependent manner (Figure 1B). Phosphorylation of MARCKS mediated by PMA in the presence or absence of 15 μmol/L rottlerin was analyzed by Western blot. HNE, previously shown to stimulate mucin secretion in NHBE cells via a rottlerin-inhibitable mechanism,11Park JA He F Martin LD Li Y Chorley BN Adler KB Human neutrophil elastase induces hypersecretion of mucin from well-differentiated human bronchial epithelial cells in vitro via a protein kinase C{delta}-mediated mechanism.Am J Pathol. 2005; 167: 651-661Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar was used as an additional control. As shown in Figure 1C, both PMA- and HNE-induced phosphorylation of MARCKS were decreased by pretreatment with rottlerin.Bryostatin 1 Provokes Mucin Secretion and MARCKS Phosphorylation in NHBE CellsBryostatin 1, a PKCδ/ε activator, was used to investigate further the relationship between PKCδ and mucin secretion in NHBE cells. A naturally occurring marine invertebrate-derived cyclic lactone, Bryostatin 1, causes rapid activation of PKCδ and prolonged protection of PKCδ against ubiquitination and proteolysis.26Szallasi Z Denning MF Smith CB Dlugosz AA Yuspa SH Pettit GR Blumberg PM Bryostatin 1 protects protein kinase C-delta from down-regulation in mouse keratinocytes in parallel with its inhibition of phorbol ester-induced differentiation.Mol Pharmacol. 1994; 46: 840-850PubMed Google Scholar, 27Heit I Wieser RJ Herget T Faust D Borchert-Stuhltrager M Oesch F Dietrich C Involvement of protein kinase Cdelta in contact-dependent inhibition of growth in human and murine fibroblasts.Oncogene. 2001; 20: 5143-5154Crossref PubMed Scopus (33) Google Scholar Bryostatin 1 interacts with the diacylglycerol binding site on PKC,28Mutter R Wills M Chemistry and clinical biology of the bryostatins.Bioorg Med Chem. 2000; 8: 1841-1860Crossref PubMed Scopus (179) Google Scholar but its complete mode of action has not been fully elucidated, and it can affect PKCs in a cell-type specific manner.29Szallasi Z Smith CB Pettit GR Blumberg PM Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts.J Biol Chem. 1994; 269: 2118-2124Abstract Full Text PDF PubMed Google Scholar As illustrated in Figure 2, exposure of NHBE cells to bryostatin 1 over a range of concentrations (10 to 1000 nmol/L) for 15 minutes resulted in translocation of PKCδ from cytosol to membrane in response to all concentrations tested (Figure 2A). Mucin secretion was also significantly increased by bryostatin 1, with maximal stimulation at 100 nmol/L (Figure 2B). As illustrated in Figure 2C, phosphorylation of MARCKS also was induced in these cells in response to bryostatin 1 (from 10 to 1000 nmol/L) with maximal phosphorylation at 100 nmol/L. None of these treatments induced cytotoxicity as measured by lactate dehydrogenase release assay (data not shown).Figure 2Effect of bryostatin 1, a PKCδ activator, on mucin secretion in well differentiated NHBE cells. NHBE cells were exposed to bryostatin 1 over a range of concentrations from 1 to 1000 nmol/L for 15 minutes. A: PKCδ translocates from cytosol to membrane in response to bryostatin 1. α-Tubulin and E-cadherin were used as controls for the cytosolic and membrane fractions, respectively. Blots are representative of three replicate experiments. B: Bryostatin 1 provokes mucin secretion by NHBE cells in a concentration-dependent manner. Significantly different from vehicle control: *P < 0.05; †P < 0.001; ‡P < 0.005. Data are presented as mean ± SEM (n = 4). C: Phosphorylation of MARCKS in NHBE cells is induced by bryostatin 1 in a concentration-dependent manner. Blots are representative of three replicate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PKC Activation Stimulates Mucin Secretion in HBE1 CellsTo investigate further a role for PKCδ as a regulator of mucin secretion using molecular manipulation of PKCδ activity, the HBE1 cell line was used. As illustrated in Figure 3, exposure of HBE1 cells to 500 nmol/L PMA for 15 minutes significantly increased mucin secretion (by ∼1.7-fold compared with medium vehicle control) and also induced phosphorylation of MARCKS in these cells.Figure 3HBE-1 cells secrete mucin in response to PKC activation. HBE-1 cells maintained in air/liquid interface were exposed to 100 or 500 nmol/L PMA for 15 minutes. Mucin secretion and phosphorylation of MARCKS were assessed by enzyme-linked immunosorbent assay and Western blot analysis, respectively. A: Mucin secretion is significantly enhanced by PMA at 500 nmol/L (but not 100 nmol/L) in HBE1 cells. *Significantly different from vehicle control (P < 0.05). Data are presented as mean ± SEM (n = 4). B: Phosphorylation of MARCKS is increased by exposure of HBE1 cells to 500 nmol/L PMA. Blots are representative of three replicate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PKCδ Appears to Regulate Mucin Secretion in Airway Epithelial CellsTransient transfection of the PKCδ and PKCε constructs into HBE1 cells was confirmed by fluorescent microscopy and Western blot analysis. After 48 hours of transfection, GFP expressed in the transfected cells was detected with a fluorescent microscope [Eclipse TE300 (Nikon, Tokyo, Japan) or Axiovert 35 (Zeiss, Welwyn Garden City, UK)] before PMA exposure (data not shown). After exposure to PMA, cells were lysed to detect expression of PKCδ protein fused with GFP via Western blot analysis using anti-PKCδ and -GFP antibodies (data not shown). Transfection of PKCε was confirmed by Western blot analysis using an antibody against PKCε and an HA tag. Transfection efficiency of all constructs and controls was about 20 to 25% as determined by quantification of GFP and X-gal assay for PKCδ and PKCε, respectively (data not shown).As illustrated in Figure 4A, mucin secretion by transfected HBE1 cells was stimulated by exposure to 500 nmol/L PMA. Transfection of HBE1 cells with the dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) resulted in significant reduction of PMA-induced mucin secretion (∼45%), whereas cells transfected with the wild-type PKCδ construct (pEGFP-N1/PKCδ) showed a significant enhancement of PMA-induced mucin secretion (∼40%) compared with control cells transfected with no DNA or empty vector (pEGFP-N1). In additional control studies, the effects of Bryostatin 1 on both mucin secretion and phosphorylation of MARCKS were markedly attenuated in cells transfected with the dnPKCδ construct (data not shown). PKCε constructs, either wild type or mutated, did not affect the secretory response to PMA. As shown in Figure 4B, phosphorylation of MARCKS in response to PMA was decreased in HBE1 cells transfected with the dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) but increased in cells transfected with the wild-type PKCδ construct (pEGFP-N1/PKCδ). PKCε constructs, either wild type or mutated, had no effect on PMA-induced MARCKS phosphorylation when transfected into HBE1 cells (Figure 4C).Figure 4Transient transfection of HBE1 cells with a dominant-negative PKCδ construct results in reduction of mucin hypersecretion. HBE1 cells were transiently transfected with empty vector (pEGFP-N1), a wild-type PKCδ construct (pEGFP-N1/PKCδ), or a dominant-negative construct (pEGFP-N1/PKCδK376R) using the FuGene 6 transfection reagent as described in Materials and Methods. As an additional control, a wild-type (pHACE/PKCε) and a dominant-negative PKCε construct (pHACE/PKCεK437R) were also transfected. A: After 48 hours transfection, cells were exposed to 500 nmol/L PMA (lanes 2 to 8) or vehicle control (lane 1) for 15 minutes, at which time media were collected and mucin secretion assessed by enzyme-linked immunosorbent assay. Data are significantly different from media control (*P < 0.05; **P < 0.001); significantly different from cells transfected with n}, number={6}, journal={AMERICAN JOURNAL OF PATHOLOGY}, author={Park, Jin-Ah and Crews, Anne L. and Lampe, William R. and Fang, Shijing and Park, Joungjoa and Adler, Kenneth B.}, year={2007}, month={Dec}, pages={1822–1830} } @article{park_crews_adler_2007, title={Protein kinase C delta regulates airway mucin secretion via phosphorylation of MARCKS protein.}, volume={175}, journal={404nOtfound}, author={Park, J. A. and Crews, A. L. and Adler, K. B.}, year={2007}, pages={A752} } @article{raiford_lin_park_fang_crews_adler_2007, title={Proteomic analysis of mucin granule membrane-associated proteins in human airway epithelial cells: a mechanistic link between MARCKS and hClCA1?}, volume={175}, journal={404nOtfound}, author={Raiford, K. L. and Lin, K. W. and Park, J. and Fang, S. and Crews, A. L. and Adler, K. B.}, year={2007}, pages={A511} } @article{waterman_park_crews_adler_2007, title={The (S)?enantiomer of albuterol activates expression of iNOS via activation of NF-kB in primary cultures of normal human bronchial epithelial cells.}, volume={175}, journal={404nOtfound}, author={Waterman, J. T. and Park, J. A. and Crews, A. L. and Adler, K. B.}, year={2007}, pages={A178} } @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} } @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{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{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{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={The ability to create knockout and transgenic mice with phenotypes mimicking a variety of lung diseases has led to a large body of knowledge detailing the role of various gene products in the development of these diseases. Similarly, the use of well-differentiated human airway epithelial cell cultures has led to an understanding of precise signaling pathways regulating cellular functions such as mucus secretion, adhesion molecule and cytokine expression, and epithelial cell proliferation. The ability to combine these two powerful research approaches lies with creating an in vitro mouse tracheal epithelial (MTE) cell culture system. Here, we report the development of such a primary cell system that maintains morphologic and functional characteristics of the in vivo mouse airway epithelium. Specifically, epithelial cells dissociated from intact mouse tracheas are grown in air/liquid interface culture in defined media with or without serum. Under both conditions, Alcian blue/periodic acid-Schiff–positive mucous cells are observed. In contrast, ciliary development appears to require serum, suggesting that it may be possible to further manipulate this cell culture system to allow precise study of either mucous or ciliated cell development. This cell culture system has been examined to ensure its epithelial nature as indicated by Western blot analyses showing the culture findings to be positive for cytokeratin 5 expression. Using a mouse mucin 5ac-specific antibody to detect secreted protein by enzyme-linked immunosorbent assay, the cultures are found to secrete mucin constitutively and in a stimulated manner in response to known secretagogues (phorbol 12-myristate 13-acetate and 8-Br-cyclic guanosine monophosphate). Although a single trachea yields only 1 cm2 of differentiated culture, our preliminary studies indicate sufficient material can be obtained to perform gene array analyses of control and interleukin-13–exposed MTE cell cultures. Thus, we anticipate use of the MTE cell culture system not only to determine specific signaling pathways important to airway epithelial cell changes during lung disease, but by employing cells from knockout and transgenic mice, we expect to obtain an understanding of how expression of genes controlling these pathways is altered by genetic changes. In this manner, it should be possible to directly interface in vitro experimentation to define precise signaling pathways in airway epithelial cells with in vivo whole animal studies.}, 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{krovetz_helton_crews_horne_2000, title={C-terminal alternative splicing changes the gating properties of a human spinal cord calcium channel alpha 1A subunit}, volume={20}, DOI={10.1523/jneurosci.20-20-07564.2000}, abstractNote={The calcium channel α1Asubunit gene codes for proteins with diverse structure and function. This diversity may be important for fine tuning neurotransmitter release at central and peripheral synapses. The α1AC terminus, which serves a critical role in processing information from intracellular signaling molecules, is capable of undergoing extensive alternative splicing. The purpose of this study was to determine the extent to which C-terminal alternative splicing affects some of the fundamental biophysical properties of α1Asubunits. Specifically, the biophysical properties of two alternatively spliced α1Asubunits were compared. One variant was identical to an isoform identified previously in human brain, and the other was a novel isoform isolated from human spinal cord. The variants differed by two amino acids (NP) in the extracellular linker between transmembrane segments IVS3 and IVS4 and in two C-terminal regions encoded by exons 37 and 44. Expression inXenopusoocytes demonstrated that the two variants were similar with respect to current–voltage relationships and the voltage dependence of steady-state activation and inactivation. However, the rates of activation, inactivation, deactivation, and recovery from inactivation were all significantly slower for the spinal cord variant. A chimeric strategy demonstrated that the inclusion of the sequence encoded by exon 44 specifically affects the rate of inactivation. These findings demonstrate that C-terminal structural changes alone can influence the way in which α1Asubunits respond to a depolarizing stimulus and add to the developing picture of the C terminus as a critical domain in the regulation of Ca2+channel function.}, number={20}, journal={Journal of Neuroscience}, author={Krovetz, H. S. and Helton, T. D. and Crews, A. L. and Horne, W. A.}, year={2000}, pages={7564–7570} }