K. Chul Kim

Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice.

MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1−/− mice with Muc1+/+ littermates following intranasal instillation of PA or flagellin. Compared with Muc1+/+ mice, Muc1−/− mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-α and KC in bronchoalveolar lavage fluid, higher levels of TNF-α in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.

Airway mucus: its components and function

The airway surface liquid (ASL), often referred to as mucus, is a thin layer of fluid covering the luminal surface of the airway. The major function of mucus is to protect the lung through mucociliary clearance against foreign particles and chemicals entering the lung. The mucus is comprised of water, ions, and various kinds of macromolecules some of which possess the protective functions such as anti-microbial, anti-protease, and anti-oxidant activity. Mucus glycoproteins or mucins are mainly responsible for the viscoelastic property of mucus, which is crucial for the effective mucociliary clearance. There are at least eight mucin genes identified in the human airways, which will potentially generate various kinds of mucin molecules. At present, neither the exact structures of mucin proteins nor their regulation are understood although it seems likely that different types of mucins are involved in different functions and might also be associated with certain airway diseases. The fact that mucins are tightly associated with various macromolecules present in ASL seems to suggest that the defensive role of ASL is determined not only by these individual components but rather by a combination of these components. Collectively, mucins in ASL may be compared to aircraft carriers carrying various types of weapons in defense of airborne enemies.

Muc1 Cell Surface Mucin Attenuates Epithelial Inflammation in Response to a Common Mucosal Pathogen

Helicobacter pylori infection of the gastric mucosa causes an active-chronic inflammation that is strongly linked to the development of duodenal and gastric ulcers and stomach cancer. However, greater than 80% of individuals infected with H. pylori are asymptomatic beyond histologic inflammation, and it is unknown what factors influence the incidence and character of bacterial-associated gastritis and related disorders. Because previous studies demonstrated that the Muc1 epithelial glycoprotein inhibited inflammation during acute lung infection by Pseudomonas aeruginosa, we asked whether Muc1 might also counter-regulate gastric inflammation in response to H. pylori infection. Muc1−/− mice displayed increased bacterial colonization of the stomach and greater TNF-α and keratinocyte chemoattractant transcript levels compared with Muc1+/+ mice after experimental H. pylori infection. Knockdown of Muc1 expression in AGS human gastric epithelial cells by RNA interference was associated with increased phosphorylation of IκBα, augmented activation and nuclear translocation of NF-κB, and enhanced production of interleulin-8 compared with Muc1-expressing cells. Conversely, Muc1 overexpression was correlated with decreased NF-κB activation, reduced interleulin-8 production, and diminished IκB kinase β (IKKβ)/IKKγ coimmunoprecipitation compared with cells expressing Muc1 endogenously. Cotransfection of AGS cells with Muc1 plus IKKβ, but not a catalytically inactive IKKβ mutant, reversed the Muc1 inhibitory effect. Finally, Muc1 formed a coimmunoprecipitation complex with IKKγ but not with IKKβ. These results are consistent with the hypothesis that Muc1 binds to IKKγ, thereby inhibiting formation of the catalytically active IKK complex and blocking the ability of H. pylori to stimulate IκBα phosphorylation, NF-κB activation, and downstream inflammatory responses.

Nicotine Primarily Suppresses Lung Th2 but Not Goblet Cell and Muscle Cell Responses to Allergens

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C4; and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.

MUC1 Mucin : A Peacemaker in the Lung

MUC1 is a membrane-tethered mucin expressed on the surface of epithelial cells lining mucosal surfaces. Recent studies have begun to elucidate the physiologic function of MUC1 in the airways, pointing to an antiinflammatory role that is initiated late in the course of bacterial infection and is mediated through inhibition of TLR signaling. These new findings have great potential for clinical applications in controlling excessive and prolonged lung inflammation. This review briefly summarizes the protein structural features of MUC1 relevant to its function, the discovery of its antiinflammatory properties, and potential directions for future avenues of study.

Membrane-Tethered MUC1 Mucin Is Phosphorylated by Epidermal Growth Factor Receptor in Airway Epithelial Cells and Associates with TLR5 To Inhibit Recruitment of MyD88

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-κB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-κB or MAPK activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-α induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.

Analysis of the proteome of human airway epithelial secretions

BackgroundAirway surface liquid, often referred to as mucus, is a thin layer of fluid covering the luminal surface that plays an important defensive role against foreign particles and chemicals entering the lungs. Airway mucus contains various macromolecules, the most abundant being mucin glycoproteins, which contribute to its defensive function. Airway epithelial cells cultured in vitro secrete mucins and nonmucin proteins from their apical surface that mimics mucus production in vivo. The current study was undertaken to identify the polypeptide constituents of human airway epithelial cell secretions to gain a better understanding of the protein composition of respiratory mucus.ResultsFifty-five proteins were identified in the high molecular weight fraction of apical secretions collected from in vitro cultures of well-differentiated primary human airway epithelial cells and isolated under physiological conditions. Among these were MUC1, MUC4, MUC5B, and MUC16 mucins. By proteomic analysis, the nonmucin proteins could be classified as inflammatory, anti-inflammatory, anti-oxidative, and/or anti-microbial.ConclusionsBecause the majority of the nonmucin proteins possess molecular weights less than that selected for analysis, it is theoretically possible that they may associate with the high molecular weight and negatively charged mucins to form a highly ordered structural organization that is likely to be important for maintaining the proper defensive function of airway mucus.

Suppression of IL-8 production in gastric epithelial cells by MUC1 mucin and peroxisome proliferator-associated receptor-γ

MUC1 is a membrane-tethered mucin expressed on the apical surface of epithelial cells. Our previous report (Guang W, Ding H, Czinn SJ, Kim KC, Blanchard TG, Lillehoj EP. J Biol Chem 285: 20547-20557, 2010) demonstrated that expression of MUC1 in AGS gastric epithelial cells limits Helicobacter pylori infection and reduces bacterial-driven IL-8 production. In this study, we identified the peroxisome proliferator-associated receptor-γ (PPARγ) upstream of MUC1 in the anti-inflammatory pathway suppressing H. pylori- and phorbol 12-myristate 13-acetate (PMA)-stimulated IL-8 production. Treatment of AGS cells with H. pylori or PMA increased IL-8 levels in cell culture supernatants compared with cells treated with the respective vehicle controls. Prior small interfering (si)RNA-induced MUC1 silencing further increased H. pylori- and PMA-stimulated IL-8 levels compared with a negative control siRNA. MUC1-expressing AGS cells pretreated with the PPARγ agonist troglitazone (TGN) had reduced H. pylori- and PMA-stimulated IL-8 levels compared with cells treated with H. pylori or PMA alone. However, following MUC1 siRNA knockdown, no differences in IL-8 levels were seen between TGN/H. pylori and H. pylori-only cells or between TGN/PMA and PMA-only cells. Finally, TGN-treated AGS cells had increased Muc1 promoter activity, as measured using a Muc1-luciferase reporter gene, and greater MUC1 protein levels by Western blot analysis, compared with vehicle controls. These results support the hypothesis that PPARγ stimulates MUC1 expression by AGS cells, thereby attenuating H. pylori- and PMA-induced IL-8 production.

Membrane-Tethered MUC1 Mucin Counter-Regulates the Phagocytic Activity of Macrophages

MUC1 (MUC in human; Muc in animals) is a transmembrane mucin glycoprotein expressed in mucosal epithelial cells and hematopoietic cells. MUC1 is involved in the resolution of inflammation during airway Pseudomonas aeruginosa (Pa) infection by suppressing Toll-like receptor signaling in airway epithelial cells. Although alveolar macrophages are recognized as critical mediators of cell-mediated immunity against microorganisms inhaled into the airways, the role of MUC1 in regulating their response is unknown. The aims of this study were to determine whether macrophages express MUC1, and, if so, whether MUC1 expression might be associated with macrophage M0/M1/M2 differentiation or phagocytic activity. Human and mouse MUC1/Muc1 expression was drastically up-regulated in classically activated (M1) macrophages compared with nonactivated (M0) and alternatively activated (M2) macrophages. M1 polarization and Pa stimulation each increased MUC1 ectodomain shedding from the macrophage surface in a TNF-α-converting enzyme-dependent manner. MUC1/Muc1 deficiency in M0 macrophages increased adhesion and phagocytosis of Pa and Escherichia coli compared with MUC1/Muc1-expressing cells, and attenuation of phagocytosis by MUC1 was augmented after polarization into M1 macrophages compared with M0 macrophages. Finally, MUC1/Muc1 deficiency in macrophages increased reactive oxygen species production and TNF-α release in response to Pa compared with MUC1/Muc1-sufficient cells. These results indicate that MUC1/Muc1 expression by macrophages is predominantly in the M1 subtype, and that MUC1/Muc1 expression in these cells decreases their phagocytic activity in an antiinflammatory manner.

MUC1 Mucin Interacts with Calcium-Modulating Cyclophilin Ligand

MUC1 is an integral membrane glycoprotein expressed on epithelial and hematopoietic cells with a COOH-terminus (CT) that mediates intracellular signal transduction. To better understand MUC1-dependent signaling, we searched for proteins binding to its CT using the yeast two-hybrid system with the MUC1 CT as bait and a human epithelial cell cDNA library as prey. Of the six positive clones identified, all encoded calcium-modulating cyclophilin ligand (CAML). The MUC1 CT interacted with CAML in transformed yeast cells as revealed by growth on selective media and in situ X-alpha-galactosidase activity. Binding of the MUC1 CT to CAML in human epithelial cells was confirmed by reciprocal coimmunoprecipitations, confocal microscopy, protein crosslinking, and coupled transcription/translation analyses. By deletion mutagenesis, the NH(2)-terminus of CAML was responsible for binding to the MUC1 CT. Finally, transfection of cells with plasmids encoding MUC1 and CAML increased intracellular calcium levels compared with cells transfected with either plasmid alone, suggesting a possible biological significance of the MUC1-CAML interaction.