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1.  Coupling of Airway Ciliary Activity and Mucin Secretion to Mechanical Stresses by Purinergic Signaling 
Respiratory physiology & neurobiology  2008;163(1-3):208-213.
The mucociliary clearance system is comprised of three components, ion transport activities controlling the height of airway surface liquid (ASL), mucin secretion, and ciliary activity. These activities in humans are controlled principally by local agonists, extracellular nucleotides and nucleosides released from the epithelium. Importantly, mechanical stresses stimulate goblet cell mucin secretion, ciliary beating, and Cl− and fluid secretion through mechanically-induced nucleotide release. Emerging evidence also implicates co-secretion of nucleotides and mucin from goblet cells as a source of extracellular agonist. At rest, ATP is released onto airway surfaces at ∼370 fmoles/min cm2, but only ∼3% of released ATP is recovered in ASL. Secreted UTP meets with a similar fate. A wide variety of hydrolytic and trans-phosphorylating ecto-enzymes convert the triphosphate nucleotides into ADP, AMP, and adenosine, UDP, UMP, and uridine. Of these, ATP, adenosine, UTP, and UDP act as agonists at apical P2Y2 (ATP, UTP), P2Y6 (UDP), and A2B (adenosine) receptors on ciliated and/or goblet cells to regulate mucociliary clearance.
doi:10.1016/j.resp.2008.05.015
PMCID: PMC2583098  PMID: 18635403
2.  Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways 
ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)–associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling–promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca2+ chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca2+-dependent vesicular mechanisms not associated with mucin granule secretion.
doi:10.1165/rcmb.2012-0493OC
PMCID: PMC3931099  PMID: 23763446
human bronchial epithelia; supernatant of mucopurulent material from human CF airways; high performance liquid chromatography; well-differentiated vesicular nucleotide transporter
3.  IL-33 stimulates CXCL8/IL-8 secretion in goblet cells but not normally differentiated airway cells 
Background
IL-13, a helper T-cell type2 (Th2) cytokine transforms cultured airway epithelial cells to goblet cells and this is not inhibited by corticosteroids. IL-33 stimulates Th2 cytokines and is highly expressed in airways of persons with asthma. The effect of IL-33 on goblet cell differentiation and cytokine secretion has not been described.
Objective
We examined the effect of IL-33 on CXCL8/IL-8 secretion from goblet or normally differentiated human bronchial epithelial (NHBE) cells and signaling pathways associated with IL-33 activation in these cells.
Methods
NHBE cells were grown to goblet or normally differentiated ciliated cell phenotype at air-liquid interface in the presence or absence of IL-13. After 14 days, differentiated cells were exposed to IL-33 for 24 hours.
Results
CXCL8/IL-8 secretion into the apical (air) side of the goblet cells was greater than from normally differentiated cells (p<0.01) and IL-33 stimulated apical CXCL8/IL-8 release from goblet cells but not from normally differentiated cells (p<0.01). IL-33 increased ERK 1/2 phosphorylation in goblet cells (p<0.05) and PD98059, a MAPK/ERK kinase inhibitor attenuated IL-33 stimulated CXCL8/IL-8 secretion from goblet cells (p<0.001). IL-13 induced ST2 mRNA (p<0.02) and membrane bound ST2 protein expression on the apical side surface of goblet cells compared with normally differentiated cells, and neutralization with anti-ST2R antibody attenuated IL-33-induced apical CXCL8/IL-8 secretion from goblet cells (p<0.02).
Conclusions and Clinical Relevance
Goblet cells secrete CXCL8/IL-8 and this is increased by IL-33 through ST2R-ERK pathway suggesting a mechanism for enhanced airway inflammation in the asthmatic airway with goblet cell metaplasia.
doi:10.1111/cea.12283
PMCID: PMC3982145  PMID: 24479526
airway; asthma; goblet cell hyperplasia; IL-13; mucin; normal human bronchial epithelial cell; Th2 cytokine
4.  Interleukin-13–Induced Mucous Metaplasia Increases Susceptibility of Human Airway Epithelium to Rhinovirus Infection 
Infection of airway epithelium by rhinovirus is the most common cause of asthma exacerbations. Even in mild asthma, airway epithelium exhibits mucous metaplasia, which increases with increasing severity of the disease. We previously showed that squamous cultures of human airway epithelium manifest rhinoviral infection at levels many times higher than in well-differentiated cultures of a mucociliary phenotype. Here we tested the hypothesis that mucous metaplasia is also associated with increased levels of rhinoviral infection. Mucous metaplasia was induced with IL-13, which doubled the numbers of goblet cells. In both control (mucociliary) and IL-13– treated (mucous metaplastic) cultures, goblet cells were preferentially infected by rhinovirus. IL-13 doubled the numbers of infected cells by increasing the numbers of infected goblet cells. Furthermore, IL-13 increased both the maturity of goblet cells and the probability that a goblet cell would be infected. The infection of cells other than goblet cells was unaltered by IL-13. Treatment with IL-13 did not alter the levels of rhinovirus receptor ICAM-1, nor did the proliferative effects of IL-13 enhance infection, because rhinovirus did not colocalize with dividing cells. However, the induction of mucous metaplasia caused changes in the apical membrane structure, notably a marked decrease in overall ciliation, and an increase in the overall flatness of the apical surface. We conclude that mucous metaplasia in asthma increases the susceptibility of airway epithelium to infection by rhinovirus because of changes in the overall architecture of the apical surface.
doi:10.1165/rcmb.2009-0244OC
PMCID: PMC2993086  PMID: 20081054
asthma; interleukin-13; rhinovirus; respiratory tract infection; goblet cells
5.  Physiological Regulation of ATP Release at the Apical Surface of Human Airway Epithelia* 
The Journal of biological chemistry  2006;281(32):22992-23002.
Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin (~7 μm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1–10 nM. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of ~250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached ~1 μM independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intra-cellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.
doi:10.1074/jbc.M603019200
PMCID: PMC2924190  PMID: 16754672
6.  TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells 
eLife  2013;2:e00658.
Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca2+-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca2+ signal. ATP-induced MUC5AC secretion depended strongly on Ca2+ influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca2+ entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca2+ and by inhibition of the Na+/Ca2+ exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na+ entry to promote Ca2+ uptake via an NCX to trigger MUC5AC secretion.
DOI: http://dx.doi.org/10.7554/eLife.00658.001
eLife digest
Goblet cells are specialized cells that produce proteins called mucins, which combine with water, salt and other proteins to form mucus, the slippery fluid that protects the respiratory and digestive tracts from bacteria, viruses and other pathogens. However, a defect in the production of one particular type of mucin—Mucin 5AC—can result in diseases such as cystic fibrosis, chronic obstructive pulmonary disease and Crohn’s disease, so there is a clear need to understand the production of mucus in detail.
Before they are secreted, the mucins are packaged inside granules in the goblet cells. When a certain extracellular signal arrives at a goblet cell, these granules move through the cell, fuse with the cell membrane and release the mucins, which then expand their volume by a factor of up to a 1000. Calcium ions (Ca2+) have a critical role in the signal that leads to the secretion of mucins, but many details about the signalling and secretion processes are poorly understood.
Now, Mitrovic et al. have used genetic methods to study 7343 gene products in goblet cells derived from a human colon. They identified 16 new proteins that are involved in the secretion of Mucin 5AC, including a channel protein called TRPM5. This protein is activated when the concentration of Ca2+ inside the cell increases, and its activation allows sodium (Na+) ions to enter the cells. These intracellular Na+ ions are then exchanged for Ca2+ ions from outside the cell, and these Ca2+ ions then couple to the molecular machinery that is responsible for the secretion of the mucins.
By using electrophysiological and Ca2+ imaging approaches, Mitrovic et al. were able to visualize and measure TRPM5-mediated Na+ currents and the subsequent Ca2+ uptake by the cells, and confirmed that extracellular Ca2+ ions were responsible for stimulating the secretion of mucins. The next step is to determine how the other 15 genes are involved in mucin secretion and, in the longer term, explore how these insights might be translated into treatments for cystic fibrosis and other conditions associated with defective mucus secretion.
DOI: http://dx.doi.org/10.7554/eLife.00658.002
doi:10.7554/eLife.00658
PMCID: PMC3667631  PMID: 23741618
Mucin5AC; TRPM5; Secretion; Human
7.  P2 purinoceptor regulation of mucin release by airway goblet cells in primary culture. 
British Journal of Pharmacology  1991;103(1):1053-1056.
1. The effects of adenine analogues on mucin release by airway goblet cells have been examined in a hamster primary tracheal epithelial cell culture. 2. Adenosine, a P1 receptor agonist, had no effect on mucin release even at 2 mM, while ATP, a P2 receptor agonist, stimulated mucin release in a dose-dependent fashion with an apparent EC50 of 20 microM. The relative potency order among adenine nucleotides was ATP greater than ADP greater than AMP = adenosine. 3. ATP gamma S, a non-hydrolyzable analogue of ATP, was equipotent with ATP in stimulating mucin release. The potency order among some ATP analogues was ATP greater than 2-methylthio ATP greater than alpha, beta-methylene ATP greater than beta, gamma-methylene ATP. Reactive blue 2, a putative P2 gamma-purinoceptor antagonist, did not block the ATP-induced mucin release. 4. The present results indicate that mucin release by airway goblet cells is stimulated by extracellular ATP via P2 receptor-mediated mechanism. We suggest that this mechanism may be important in the physiological regulation of airway goblet cell mucin release in vivo.
PMCID: PMC1908084  PMID: 1878744
8.  ATP induced MUC5AC release from human airways in vitro. 
Mediators of Inflammation  2000;9(6):277-284.
BACKGROUND: Chronic airway diseases are often associated with marked mucus production, however, little is known about the regulation of secretory activity by locally released endogenous mediators. AIM: This investigation was performed to determine the release of MUC5AC mucin from human bronchial preparations using the purinergic agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP). METHODS: Immunohistochemical and immunoradiometric assays (IRMA) were used to detect the MUC5AC mucin. Immunohistochemical analysis were performed using individual 1-13 M1 and 21 M1 MAbs recognizing a recombinant M1 mucin partially encoded by the MUC5AC gene. IRMA measurments were performed using a mixture of eight anti-M1 mucin MAbs (PM8), which included both 1-13 M1 and 21 M1 MAbs. Lysozyme and protein were also measured in the biological fluids derived from human bronchial preparations obtained from patients who had undergone surgery for lung carcinoma. RESULTS: The anti-M1 monoclonal antibodies labelled epithelial goblet cells. After challenge of human bronchial preparations with ATP, the goblet cells exhibited less staining. In contrast, UTP did not alter the immunolabelling of goblet cells. MUC5AC mucin in the bronchial fluids derived from ATP-challenged preparations was increased while UTP had no effect on release. ATP did not alter either the quantities of lysozyme or protein detected in the biological fluids. CONCLUSION: These results suggest that ATP may regulate epithelial goblet cell secretion of MUC5AC mucin from human airways in vitro.
PMCID: PMC1781772  PMID: 11213911
9.  INTESTINAL DIFFERENTIATION IN METAPLASTIC, NON-GOBLET COLUMNAR EPITHELIUM IN THE ESOPHAGUS 
Barrett's esophagus (BE) is defined by the presence of metaplastic esophageal columnar epithelium with goblet cells within endoscopically recognizable areas of the esophagus. However, some carcinomas in BE, or from the GEJ region, develop within mucosa devoid of goblet cells. However, the biological properties, pathogenesis, and risk of malignancy of metaplastic, esophageal non-goblet columnar epithelium, is, essentially, unknown. In this study, 89 patients with metaplastic esophageal columnar epithelium were evaluated immunohistochemically for markers of intestinal differentiation, such as MUC2, DAS-1, Villin, and CDX2, a marker of gastric differentiation (MUC5AC), and Ki67, a marker of cell proliferation. Of the 89 patients, 59 had columnar metaplasia with goblet cells (BE), which were further separated into low density goblet cell and high density goblet cell groups based on the percentage of crypts with goblet cells, and 30 patients had columnar metaplasia of the esophagus without goblet cells. As controls, gastric biopsies from 19 age and sex matched patients without esophageal or gastric pathology were used. The rate of positivity of the markers and the location of Ki67 staining was evaluated only in non-goblet columnar epithelium from all patient groups. Patients with metaplastic esophageal columnar epithelium without goblet cells showed positivity for MUC5AC, MUC2, DAS-1, Villin, and CDX2 in 100%, 0%, 30%, 70%, and 43% of cases, respectively. 17% of cases showed aberrant surface Ki67 positivity. These values were significantly higher than gastric controls, which showed absence of staining for all markers except MUC5AC (100%). In patients with metaplastic esophageal columnar epithelium with goblet cells (BE) a significant increased rate of staining was observed for all markers, except MUC5AC. In addition, both MUC2 and surface Ki67 staining were significantly increased in BE patients with high density goblet cells versus those with low-density goblet cells. In a separate analysis in which metaplastic esophageal non-goblet epithelium was evaluated in areas of mucosa devoid of goblet cells compared to areas of mucosa with goblet cells, from patients who had goblet cells elsewhere in the mucosa (N=59), no significant differences were observed with regard to the percentage of cases that stained with any of the markers in the non-goblet epithelium in areas devoid of goblet cells, similar to the patient group with metaplastic esophageal epithelium without goblet cells (N=30). Similar to above, in all cases, expression of intestinal markers increased in areas of mucosa adjacent to goblet cells. This study provides evidence that metaplastic esophageal columnar epithelium without goblet cells shows phenotypic evidence of intestinal differentiation and supports the theory that squamous epithelium converts initially to non-goblet columnar epithelium prior to goblet cell metaplasia. Further prospective studies are needed to evaluate the pathogenetic sequence, natural history, and risk of malignancy of metaplastic esophageal non-goblet epithelium.
doi:10.1097/PAS.0b013e31819f57e9
PMCID: PMC2807916  PMID: 19363439
10.  Chronic IL9 and IL-13 Exposure Leads to an Altered Differentiation of Ciliated Cells in a Well-Differentiated Paediatric Bronchial Epithelial Cell Model 
PLoS ONE  2013;8(5):e61023.
Asthma is a chronic inflammatory disease characterised by airways remodelling. In mouse models IL-9 and IL-13 have been implicated in airways remodelling including mucus hypersecretion and goblet cell hyperplasia. Their role, especially that of IL-9, has been much less studied in authentic human ex vivo models of the bronchial epithelium from normal and asthmatic children. We assessed the effects of IL-9, IL-13 and an IL-9/IL-13 combination, during differentiation of bronchial epithelial cells from normal (n = 6) and asthmatic (n = 8) children. Cultures were analysed for morphological markers and factors associated with altered differentiation (MUC5AC, SPDEF and MMP-7). IL-9, IL-9/IL-13 combination and IL-13 stimulated bronchial epithelial cells from normal children had fewer ciliated cells [14.8% (SD 8.9), p = 0.048, 12.4 (SD 6.1), p = 0.016 and 7.3% (SD 6.6), p = 0.031] respectively compared with unstimulated [(21.4% (SD 9.6)]. IL-9 stimulation had no effect on goblet cell number in either group whereas IL-9/IL-13 combination and IL-13 significantly increased goblet cell number [24.8% (SD 8.8), p = 0.02), 32.9% (SD 8.6), p = 0.007] compared with unstimulated normal bronchial cells [(18.6% (SD 6.2)]. All stimulations increased MUC5AC mRNA in bronchial epithelial cells from normal children and increased MUC5AC mucin secretion. MMP-7 localisation was dysregulated in normal bronchial epithelium stimulated with Th2 cytokines which resembled the unstimulated bronchial epithelium of asthmatic children. All stimulations resulted in a significant reduction in transepithelial electrical resistance values over time suggesting a role in altered tight junction formation. We conclude that IL-9 does not increase goblet cell numbers in bronchial epithelial cell cultures from normal or asthmatic children. IL-9 and IL-13 alone and in combination, reduce ciliated cell numbers and transepithelial electrical resistance during differentiation of normal epithelium, which clinically could inhibit mucociliary clearance and drive an altered repair mechanism. This suggests an alternative role for IL-9 in airways remodelling and reaffirms IL-9 as a potential therapeutic target.
doi:10.1371/journal.pone.0061023
PMCID: PMC3650011  PMID: 23671562
11.  Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress and Spontaneous Inflammation Resembling Ulcerative Colitis 
PLoS Medicine  2008;5(3):e54.
Background
MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.
Methods and Findings
By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1β, TNF-α, and IFN-γ was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-γ, TNF-α, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.
Conclusions
Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.
Michael McGuckin and colleagues identify two mutations that cause aberrant mucin oligomerization in mice. The resulting phenotype, including endoplasmic reticulum stress, resembles clinical and pathologic features of human ulcerative colitis.
Editors' Summary
Background.
Inflammatory bowel diseases (IBD) are common disorders in which parts of the digestive tract become inflamed. The two main types of IBD are Crohn's disease, which mainly affects the small bowel, and ulcerative colitis (UC), which mainly affects the large bowel (colon). Both types tend to run in families and usually develop between 15 and 35 years old. Their symptoms include diarrhea, abdominal cramps, and unintentional weight loss. These symptoms can vary in severity, can be chronic (persistent) or intermittent, and may start gradually or suddenly. There is no cure for IBD (except removal of the affected part of the digestive tract), but drugs that modulate the immune system (for example, corticosteroids) or that inhibit “proinflammatory cytokines” (proteins made by the immune system that stimulate inflammation) can sometimes help.
Why Was This Study Done?
Although the clinical and pathological (disease-associated) features of Crohn's disease and UC are somewhat different, both disorders are probably caused by an immune system imbalance. Normally, the immune system protects the body from potentially harmful microbes in the gut but does not react to the many harmless bacteria that live there or to the food that passes along the digestive tract. In IBD, the immune system becomes overactive for unknown reasons, and lymphocytes (immune system cells) accumulate in the lining of the bowel and cause inflammation. In this study, the researchers use a technique called random mutagenesis (the random introduction of small changes, called mutations, into the genes of an organism using a chemical that damages DNA) to develop two mouse models that resemble human UC and that throw new light on to how this disorder develops.
What Did the Researchers Do and Find?
The researchers establish two mutant mouse strains—Winnie and Eeyore mice—that develop mild spontaneous inflammation of the colon and chronic diarrhea and that have more proinflammatory cytokines and more lymphocytes in their colons than normal mice. 25% and 40% of the Winnie and Eeyore mice, respectively, have severe clinical signs of colitis by 1 year of age. Both strains have a mutation in the Muc2 gene, which codes for MUC2 mucin, the main protein in mucus. This viscous substance (which coats the inside of the intestine) is produced by and stored in intestinal “goblet” cells. Mucus helps to maintain the intestine's immunological balance but is depleted in UC. The researchers show that the manufacture and assembly of Muc2 molecules is abnormal in Winnie and Eeyore mice, that less mucin is stored in their goblet cells than in normal mice, and that their intestinal mucus barrier is reduced. In addition, an incompletely assembled version of the molecule, called Muc2 precursor, accumulates in the endoplasmic reticulum (ER; the cellular apparatus that prepares newly manufactured proteins for release) of goblet cells, leading to overload with abnormal protein and causing a state of cellular distress known as the “ER stress response.” Finally, the researchers report that MUC2 precursor also accumulates in the goblet cells of people with UC and that even the noninflamed intestinal tissue of these patients shows signs of ER stress.
What Do These Findings Mean?
These findings indicate that mucin abnormalities and ER stress can initiate colitis in mice. Results from animal studies do not always reflect what happens in people, but these findings, together with those from the small study in humans, suggest that ER stress-related mucin depletion could be a component in the development of human colitis. The results do not identify the genetic changes and/or environmental factors that might trigger ER stress in human colitis, but suggest that once initiated, ER stress might interfere with MUC2 production, which would lead to a diminished mucus barrier, expose the lining of the intestine to more toxins and foreign substances, and trigger local mucosal inflammation. The release of inflammatory cytokines would then damage the intestine's lining and exacerbate ER stress, thus setting up a cycle of intestinal damage and inflammation. Clinical studies to look for genetic changes and environmental factors capable of triggering ER stress and for ER-stress related changes in human UC should now be undertaken to test this hypothesis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050054.
The MedlinePlus Encyclopedia has pages on Crohn's disease and on ulcerative colitis (in English and Spanish)
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on Crohn's disease and ulcerative colitis
Information and support for patients with inflammatory bowel disease and their caregivers is provided by the Crohn's and Colitis Foundation of America and by the UK National Association for Colitis and Crohn's Disease
Wikipedia has pages on mucins and on mucus (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050054
PMCID: PMC2270292  PMID: 18318598
12.  Aldose Reductase Inhibition Prevents Metaplasia of Airway Epithelial Cells 
PLoS ONE  2010;5(12):e14440.
Background
Goblet cell metaplasia that causes mucus hypersecretion and obstruction in the airway lumen could be life threatening in asthma and chronic obstructive pulmonary disease patients. Inflammatory cytokines such as IL-13 mediate the transformation of airway ciliary epithelial cells to mucin-secreting goblet cells in acute as well as chronic airway inflammatory diseases. However, no effective and specific pharmacologic treatment is currently available. Here, we investigated the mechanisms by which aldose reductase (AR) regulates the mucus cell metaplasia in vitro and in vivo.
Methodology/Findings
Metaplasia in primary human small airway epithelial cells (SAEC) was induced by a Th2 cytokine, IL-13, without or with AR inhibitor, fidarestat. After 48 h of incubation with IL-13 a large number of SAEC were transformed into goblet cells as determined by periodic acid-schiff (PAS)-staining and immunohistochemistry using antibodies against Mucin5AC. Further, IL-13 significantly increased the expression of Mucin5AC at mRNA and protein levels. These changes were significantly prevented by treatment of the SAEC with AR inhibitor. AR inhibition also decreased IL-13-induced expression of Muc5AC, Muc5B, and SPDEF, and phosphorylation of JAK-1, ERK1/2 and STAT-6. In a mouse model of ragweed pollen extract (RWE)-induced allergic asthma treatment with fidarestat prevented the expression of IL-13, phosphorylation of STAT-6 and transformation of epithelial cells to goblet cells in the lung. Additionally, while the AR-null mice were resistant, wild-type mice showed goblet cell metaplasia after challenge with RWE.
Conclusions
The results show that exposure of SAEC to IL-13 caused goblet cell metaplasia, which was significantly prevented by AR inhibition. Administration of fidarestat to mice prevented RWE-induced goblet cell metaplasia and AR null mice were largely resistant to allergen induced changes in the lung. Thus our results indicate that AR inhibitors such as fidarestat could be developed as therapeutic agents to prevent goblet cell metaplasia in asthma and related pathologies.
doi:10.1371/journal.pone.0014440
PMCID: PMC3010981  PMID: 21203431
13.  A3 Adenosine Receptor Signaling Contributes to Airway Mucin Secretion after Allergen Challenge 
Mucin hypersecretion is a prominent feature of obstructive airway diseases such as asthma. Clara cells conditionally produce mucin in response to inflammatory signals in a process termed mucous metaplasia. This can be followed by mucin secretion stimulated by various signaling molecules. The cellular and molecular mechanisms that regulate mucin production and secretion are not well understood. Adenosine is a signaling nucleoside that has been implicated in airway diseases in which mucus obstruction is prominent. Furthermore, the A3 adenosine receptor (A3AR) is upregulated in mucin-producing goblet cells of the airway, thereby implicating it in processes involved in mucous cell biology. Here we use genetic approaches to investigate the contribution of A3AR signaling to mucus production and secretion in a mouse model of allergen-induced pulmonary disease. We found that the degree of mucin production in response to allergen is similar in wild-type and A3AR-deficient mice, and that overexpression of this receptor in Clara cells neither induces mucin production itself, nor enhances mucin production in response to allergen challenge. Collectively, these experiments demonstrate that the A3AR is neither necessary nor sufficient for mucous cell metaplasia. In contrast to the lack of effect on mucin production, agonist-induced mucin secretion was increased in goblet cells overexpressing the A3AR, and was absent in A3AR-deficient mice. Thus, the A3AR contributes to mucin secretion in allergen-induced metaplasia. Signaling through this receptor may contribute to mucus airway obstruction seen in pulmonary disorders in which adenosine levels are elevated.
doi:10.1165/rcmb.2006-0060OC
PMCID: PMC2643274  PMID: 16763221
mucin; mucous cell metaplasia; secretion; adenosine receptors; allergic lung disease
14.  The pathogenesis of duodenal gastric metaplasia: the role of local goblet cell transformation 
Gut  2000;46(5):632-638.
BACKGROUND AND AIMS—Gastric metaplasia is frequently seen in biopsies of the duodenal cap, particularly when inflamed or ulcerated. In its initial manifestation small patches of gastric foveolar cells appear near the tip of a villus. These cells contain periodic acid-Schiff (PAS) positive neutral mucins in contrast with the alcian blue (AB) positive acidic mucins within duodenal goblet cells. Previous investigations have suggested that these PAS positive cells originate either in Brunner's gland ducts or at the base of duodenal crypts and migrate in distinct streams to the upper villus. To investigate the origin of gastric metaplasia in superficial patches, we used the PAS/AB stain to distinguish between neutral and acidic mucins and in addition specific antibodies to immunolocalise foveolar cell mucin MUC5AC, the foveolar cell secretory product, gastric trefoil factor (TFF1), the mature goblet cell mucin MUC2, and MUC2 core antigen.
RESULTS—Cells in focal patches of gastric metaplasia contained secretory granules of both gastric and goblet cell phenotypes. MUC5AC and TFF1 were present as expected in gastric foveolar cells but in addition, MUC2 core antigen, normally present only in the Golgi of intestinal goblet cells, was expressed in secretory granules. Goblet cells in the vicinity of metaplastic patches also expressed both gastric and intestinal antigens. MUC5AC/MUC2 containing goblet cells were most common near the villus tip but were also seen at the base of crypts. Where crypts and Brunner's gland ducts merged they were always seen on the crypt side of the junction. Goblet cells were the only cells to express gastric antigens in these areas. In advanced metaplastic lesions, dual phenotype goblet cells were less evident and fewer cells expressed intestinal mucin antigens.
CONCLUSIONS—We suggest that goblet cells that express both intestinal and gastric antigens may represent local precursors of gastric metaplasia undergoing a transition to foveolar-like cells of mixed phenotype at the site of early metaplastic patches. As metaplasia becomes more widespread, a more pure gastric phenotype emerges. This progression is likely to be controlled by local inflammatory signals.


Keywords: gastric metaplasia; goblet cells; mucin
doi:10.1136/gut.46.5.632
PMCID: PMC1727926  PMID: 10764705
15.  Advances in Mucous Cell Metaplasia 
Mucous cell metaplasia is induced in response to harmful insults and provides front-line protection to clear the airway of toxic substances and cellular debris. In chronic airway diseases mucous metaplasia persists and results in airway obstruction and contributes significantly to morbidity and mortality. Mucus hypersecretion involves increased expression of mucin genes, and increased mucin production and release. The past decade has seen significant advances in our understanding of the molecular mechanisms by which these events occur. Inflammation stimulates epidermal growth factor receptor activation and IL-13 to induce both Clara and ciliated cells to transition into goblet cells through the coordinated actions of FoxA2, TTF-1, SPDEF, and GABAAR. Ultimately, these steps lead to up-regulation of MUC5AC expression, and increased mucin in goblet cell granules that fuse to the plasma membrane through actions of MARCKS, SNAREs, and Munc proteins. Blockade of mucus in exacerbations of asthma and chronic obstructive pulmonary disease may affect morbidity. Development of new therapies to target mucus production and secretion are now possible given the advances in our understanding of molecular mechanisms of mucous metaplasia. We now have a greater incentive to focus on inhibition of mucus as a therapy for chronic airway diseases.
doi:10.1165/rcmb.2009-0151TR
PMCID: PMC2830403  PMID: 19520914
mucus; goblet cell; airway epithelium; asthma; COPD
16.  Regulation of mucin secretion and inflammation in asthma; A role for MARCKS protein? 
Biochimica et biophysica acta  2011;1810(11):1110-1113.
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.
doi:10.1016/j.bbagen.2011.01.009
PMCID: PMC3097255  PMID: 21281703
17.  Proteinases of Pseudomonas aeruginosa evoke mucin release by tracheal epithelium. 
Journal of Clinical Investigation  1984;74(5):1669-1678.
We have determined the potential of exoproducts from pathogenic bacteria to stimulate the release of high molecular weight mucins from goblet cells of airway epithelium in a rabbit tracheal explant system. Culture supernatants from proteolytic strains of Pseudomonas aeruginosa and Serratia marcescens, but not supernatants from a number of non-proteolytic strains, released mucins from goblet cells. Highly purified elastase and alkaline proteinase from P. aeruginosa stimulated goblet cell mucin release in a dose-dependent fashion. Lipopolysaccharide, exotoxin A, and alginate of P. aeruginosa did not possess mucin release properties. Proteolytic activity was required for mucin release by P. aeruginosa elastase, but such release in goblet cells was not mediated by cyclic AMP. Morphologic studies suggested rapid release of mucins from goblet cells was response to elastase by a process resembling apocrine secretion. Several nonbacterial proteinases mimicked the effect of Pseudomonas proteases. These studies provide support for the hypothesis that bacterial and other play a role in the pathogenesis of mucus hypersecretion in acute and chronic lung infections.
Images
PMCID: PMC425344  PMID: 6568227
18.  Conjunctival Goblet Cell Secretion Stimulated by Leukotrienes Is Reduced by Resolvins D1 and E1 To Promote Resolution of Inflammation 
The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT1 and CysLT2, were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB4, LTC4, LTD4, and LTE4, as well as PGD2, stimulated goblet cell secretion in rat goblet cells. LTD4 and LTE4 increased the intracellular Ca2+ concentration ([Ca2+]i), and LTD4 activated ERK1/2. The CysLT1 receptor antagonist MK571 significantly decreased LTD4-stimulated rat goblet cell secretion and the increase in [Ca2+]i. Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD4-stimulated goblet cell secretion in cultured rat goblet cells. LTD4-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD4- and LTE4-stimulated increases in [Ca2+]i, as well as LTD4 activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD4 using the CysLT1 receptor. Stimulated secretion is terminated by preventing the increase in [Ca2+]i and activation of ERK1/2 by RvD1 and RvE1.
doi:10.4049/jimmunol.1000833
PMCID: PMC3532815  PMID: 21357260
19.  PKC and Ca2+Effect of Protein kinase C and Ca2+ on p42/p44 MAPK, Pyk2, and Src Activation in Rat Conjunctival Goblet Cells 
Experimental eye research  2007;85(6):836-844.
Conjunctival goblet cells synthesize and secrete mucins onto the ocular surface to lubricate it and protect it from bacterial infections. Mucin secretion is under neural control, and cholinergic agonists released from parasympathetic nerves are major stimuli of this secretion. The signal transduction pathways these agonists use to stimulate secretion involve activating protein kinase C (PKC) and increasing intracellular [Ca2+] to activate the non-receptor kinases Pyk2 and p60Src (Src) to transactivate the EGF receptor. Transactivation of the EGF receptor activates a kinase cascade culminating in the activation of p42/p44 MAPK (MAPK) and ultimately that leads to secretion of high molecular weight glycocongujates (HMWGC), including mucins. To further examine the roles of PKC and Ca2+ in the activation of MAPK, Pyk2, and Src in mucin secretion, rat conjunctival pieces and cultured goblet cells were incubated with the PKC activator phorbol myristate acid (PMA), the cholinergic agonist carbachol, or the calcium ionophore, ionomycin for varying times. Conjunctival pieces were preincubated with PKC inhibitors 10 mins prior to addition of carbachol (10−4 M) for 10 min. The amount of phosphorylated (activated) MAPK, Pyk2 and Src was determined by western blotting techniques using antibodies specific to the phosphorylated forms of each kinase. PMA significantly increased the activation of MAPK, Pyk2, and Src in a time and concentration-dependent manner. PMA-stimulated MAPK activity was completely inhibited by the EGF receptor inhibitor AG1478 (10−7 M). Carbachol-stimulated MAPK activity was inhibited by three PKC inhibitors, calphostin C, chelethyrine, and staurosporine. Ionomycin (10−6 M)-stimulated MAPK activity was inhibited 66% by AG1478 (10−7 M). Ionomycin also significantly increased Pyk2 and Src in time dependent manner. PKC and ionomycin also activated p42/p44 MAPL, Pyk2, and Src in cultured conjunctival goblet cells. We conclude that PKC and intracellular Ca2+ activate Pyk2 and Src and phosphorylated the EGF receptor leading to stimulation of MAPK in conjunctival goblet cells.
doi:10.1016/j.exer.2007.08.019
PMCID: PMC2277506  PMID: 17919561
goblet cells; signal transduction; MAPK; mucin secretion
20.  Defective goblet cell exocytosis contributes to murine cystic fibrosis–associated intestinal disease 
The Journal of Clinical Investigation  2015;125(3):1056-1068.
Cystic fibrosis (CF) intestinal disease is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator–mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation.
doi:10.1172/JCI73193
PMCID: PMC4362271  PMID: 25642775
21.  Presence of EGF growth factor ligands and their effects on cultured rat conjunctival goblet cell proliferation 
Experimental eye research  2007;86(2):322-334.
The amount of mucin on the ocular surface is regulated by the rate of mucin synthesis, mucin secretion, and the number of goblet cells. We have previously shown that cholinergic agonists are potent stimuli of mucin secretion. In contrast, there have been no studies on the control of goblet cell proliferation. In this study we investigate the presence of the EGF family of growth factors and their receptors in rat conjunctiva and cultured rat conjunctival goblet cells as well as their effects on activation of signaling intermediates and goblet cell proliferation. Rat conjunctival goblet cells were grown in organ culture and identified as goblet cells by their morphology and positive staining for the lectin UEA-1 and cytokeratin 7. In the rat conjunctiva, the presence of the EGF family members epidermal growth factor (EGF), transforming growth factor α (TGF-α), heparin binding EGF (HB-EGF), and heregulin was determined by RT-PCR. The receptors for these ligands, EGF receptor (EGFR), erbB2, erbB3, and erbB4 were detected in both rat conjunctiva and goblet cells by Western blot analysis. Immunofluorescence microscopy of conjunctival tissue determined that EGFR was present as punctate staining in the cytoplasm of conjunctival goblet cells while ErbB2 was present in the basolateral and lateral membranes of goblet cells. ErbB3 was localized to the cytosol of rat conjunctival goblet cells. In cultured goblet cells, EGFR and ErbB2 were present in the perinuclear area of the cells. ErbB3 was widely distributed throughout the cytoplasm of the cells. ErbB4 was not detected in either the conjunctiva or goblet cells by immunofluorescence microscopy. Using a multiplex assay system we measured phosphorylation (activation) of p44/p42 mitogen-activated protein kinase (MAPK), also known as ERK, Jun N-terminal kinase (JNK), p38 MAPK and AKT (also known as protein kinase B), molecules known to be activated by EGF receptor members. EGF, TGF-α and HB-EGF activated the signaling intermediate proteins whereas heregulin did not. No EGF family member significantly activated AKT. Consistent with these findings, EGF, TGF-α and HB-EGF each stimulated goblet cell proliferation as measured by WST-1 assay or immunofluorescence microscopy using an antibody against Ki-67, a protein expressed in dividing cells. Heregulin did not cause goblet cell proliferation. We conclude that multiple members of the EGF family, EGF, TGF-α and HB-EGF, and heregulin are present with three of the four erbB receptor subtypes. EGF, TGF-α and HB-EGF all stimulated the activation of signaling intermediates and caused goblet cell proliferation.
doi:10.1016/j.exer.2007.11.004
PMCID: PMC2547492  PMID: 18155194
goblet cell; proliferation; secretion; conjunctiva
22.  Complement C3a Regulates Muc5ac Expression by Airway Clara Cells Independently of Th2 Responses 
Rationale: The factors that control the secretion of epithelial mucins are essential to understanding obstructive airway diseases such as asthma. Although the complement anaphylatoxin C3a and its receptor have been shown to promote many features of allergic lung inflammation, the contribution to mucin expression has not been elucidated.
Objectives: To determine if the C3a receptor with its ligand regulates airway epithelial mucin production.
Methods: Mice deficient in the C3a receptor were examined in a model of allergic airway disease for the presence of goblet cells and the gel-forming secreted mucin Muc5ac.
Measurements and Main Results: Lungs from antigen-challenged C3a receptor–deficient mice revealed a dramatic decrease in goblet cells and Muc5ac compared with challenged wild-type control animals. These differences were dependent on C3a binding to its receptor since intranasal challenge with C3a induced the formation of goblet cells only in wild-type but not C3a receptor–deficient mice. Increased numbers of goblet cells were also found in C3a-stimulated RAG-1–deficient mice demonstrating a mechanism independent of T lymphocytes and Th2 cytokines, mediators which have been shown to regulate mucin expression. A direct physiological role for C3a in these models was further demonstrated in cultures of airway epithelial Clara cells, which not only express the C3a receptor but also produce Muc5ac in response to C3a.
Conclusions: These studies identify a novel C3a receptor–dependent mechanism in the development of airway epithelial goblet cells and regulation of Muc5ac production and implicate C3a as a mediator of airway obstruction in asthma.
doi:10.1164/rccm.200701-049OC
PMCID: PMC2176092  PMID: 17400733
T cells; allergy; lung; inflammation
23.  Metaplastic Esophageal Columnar Epithelium Without Goblet Cells Shows DNA Content Abnormalities Similar to Goblet Cell—Containing Epithelium 
OBJECTIVES
The mucosa of patients with columnar-lined esophagus recognized on endoscopy usually shows epithelium with and without goblet cells. Columnar epithelium with goblet cells (“Barrett’s esophagus”) is generally believed to represent a premalignant lesion and has been shown to contain DNA abnormalities. However, the biological properties of non-goblet columnar epithelium remain unknown. The purpose of this study was to determine the DNA content properties of non-goblet epithelium in patients with metaplastic columnar epithelium of the esophagus.
METHODS
Mucosal biopsies of the esophagus from 68 patients with columnar metaplasia of the esophagus (22 without goblet cells and 46 with goblet cells) and 19 patients with normal gastric mucosa (controls) were histologically evaluated for the density of goblet cells. The latter group was divided into low-density, high-density, and very high—density goblet cell subgroups. Tissue sections of non-goblet epithelium and goblet cell epithelium (where present) were evaluated by image cytometry, and high-fi delity DNA histograms were created to indicate the G0/G1 peak DNA index (DI), DNA content heterogeneity index (HI), and the percentage of cells with DNA exceeding 5N (5N-EC). G0/G1 peaks with DI >1.1 were considered aneuploid.
RESULTS
Normal gastric controls showed a mean peak DI of 1.02±0.03 and an HI of 11.6±0.7. None of the controls revealed aneuploidy or 5N-EC. Patients with metaplastic columnar epithelium with goblet cells showed a DI of 1.15±0.12, HI of 18.2±2.1, mild aneuploidy in 54% of the cases, and 5N-EC in 15% of the cases, all of which were signifi cantly higher than in controls. Patients with metaplastic columnar epithelium without goblet cells showed DNA content results statistically similar to those of patients with metaplastic columnar epithelium with goblet cells, and also revealed signifi cantly higher values compared with those of controls. Furthermore, there were no signifi cant differences in any of the key DNA content abnormalities between non-goblet and goblet cell—containing epithelium in patients with metaplastic columnar epithelium with goblet cells, or between these two types of epithelium according to the density of goblet cells.
CONCLUSIONS
DNA content abnormalities occur with equal frequency and extent in metaplastic columnar epithelium of the esophagus without goblet cells compared with metaplastic columnar epithelium with goblet cells. These findings suggest that metaplastic non-goblet columnar epithelium of the esophagus may have neoplastic potential.
doi:10.1038/ajg.2009.85
PMCID: PMC2722438  PMID: 19293780
24.  Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores 
PLoS ONE  2015;10(5):e0127267.
Airway mucin secretion studies have focused on goblet cell responses to exogenous agonists almost to the exclusion of baseline mucin secretion (BLMS). In human bronchial epithelial cell cultures (HBECCs), maximal agonist-stimulated secretion exceeds baseline by ~3-fold as measured over hour-long periods, but mucin stores are discharged completely and require 24 h for full restoration. Hence, over 24 h, total baseline exceeds agonist-induced secretion by several-fold. Studies with HBECCs and mouse tracheas showed that BLMS is highly sensitive to mechanical stresses. Harvesting three consecutive 1 h baseline luminal incubations with HBECCs yielded equal rates of BLMS; however, lengthening the middle period to 72 h decreased the respective rate significantly, suggesting a stimulation of BLMS by the gentle washes of HBECC luminal surfaces. BLMS declined exponentially after washing HBECCs (t1/2 = 2.75 h), to rates approaching zero. HBECCs exposed to low perfusion rates exhibited spike-like increases in BLMS when flow was jumped 5-fold: BLMS increased >4 fold, then decreased within 5 min to a stable plateau at 1.5–2-fold over control. Higher flow jumps induced proportionally higher BLMS increases. Inducing mucous hyperplasia in HBECCs increased mucin production, BLMS and agonist-induced secretion. Mouse tracheal BLMS was ~6-fold higher during perfusion, than when flow was stopped. Munc13-2 null mouse tracheas, with their defect of accumulated cellular mucins, exhibited similar BLMS as WT, contrary to predictions of lower values. Graded mucous metaplasia induced in WT and Munc13-2 null tracheas with IL-13, caused proportional increases in BLMS, suggesting that naïve Munc13-2 mouse BLMS is elevated by increased mucin stores. We conclude that BLMS is, [i] a major component of mucin secretion in the lung, [ii] sustained by the mechanical activity of a dynamic lung, [iii] proportional to levels of mucin stores, and [iv] regulated differentially from agonist-induced mucin secretion.
doi:10.1371/journal.pone.0127267
PMCID: PMC4449158  PMID: 26024524
25.  Signaling pathways used by EGF to stimulate conjunctival goblet cell secretion 
Experimental eye research  2012;103:99-113.
The purpose of this study was to identify the signaling pathways that epidermal growth factor (EGF) uses to stimulate mucin secretion from cultured rat conjunctival goblet cells and to compare the pathways used by EGF with those used by the known secretagogue muscarinic, cholinergic agonists. To this end, goblet cells from rat conjunctiva were grown in culture using RPMI media. For immunofluorescence experiments, antibodies against EGF receptor (EGFR) and ERK 2 as well as muscarinic receptors (M1AchR, M2AchR, and M3AchR) were used, and the cells viewed by fluorescence microscopy. Intracellular [Ca2+] ([Ca2+]i) was measured using fura 2/AM. Glycoconjugate secretion was determined after cultured goblet cells were preincubated with inhibitors, and then stimulated with EGF or the cholinergic agonist carbachol (Cch). Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-I or ELISA for MUC5AC. In cultured goblet cells EGF stimulated an increase in [Ca2+]i in a concentration-dependent manner. EGF-stimulated increase in [Ca2+]i was blocked by inhibitors of the EGF receptor and removal of extracellular Ca2+. Inhibitors against the EGFR and ERK 1/2 blocked EGF-stimulated mucin secretion. In addition, cultured goblet cells expressed M1AchR, M2AchR, and M3AchRs. Cch-stimulated increase in [Ca2+]i was blocked by inhibitors for the M1AchRs, matrix metalloproteinases, and EGF receptors. Inhibitors against the EGF receptor and ERK 1/2 also blocked Cch-stimulated mucin secretion. We conclude that in conjunctival goblet cells, EGF itself increases [Ca2+]i and activates ERK 1/2 to stimulate mucin secretion. EGF-stimulated secretion is dependent on extracellular Ca2+. This mechanism of action is similar to cholinergic agonists that use muscarinic receptors to transactivate the EGF receptor, increase [Ca2+]i, and activate ERK 1/2 leading to an increase in mucin secretion.
doi:10.1016/j.exer.2012.08.010
PMCID: PMC3595052  PMID: 22975404
goblet cells; conjunctiva; epidermal growth factor; muscarinic receptors; secretion

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