Goblet Cell Line Research Articles

Effects of Zinc Oxide Nanoparticles on Model Systems of the Intestinal Barrier.

Zinc oxide nanoparticles (ZnO NP) are often used in the food sector, among others, because of their advantageous properties. As part of the human food chain, they are inevitably taken up orally. The debate on the toxicity of orally ingested ZnO NP continues due to incomplete data. Therefore, the aim of our study was to examine the effects of two differently sized ZnO NP (<50 nm and <100 nm primary particle size; 123–614 µmol/L) on two model systems of the intestinal barrier. Differentiated Caco-2 enterocytes were grown on Transwell inserts in monoculture and also in coculture with the mucus-producing goblet cell line HT29-MTX. Although no comprehensive mucus layer was detectable in the coculture, cellular zinc uptake was clearly lower after a 24-h treatment with ZnO NP than in monocultured cells. ZnO NP showed no influence on the permeability, metabolic activity, cytoskeleton and cell nuclei. The transepithelial electrical resistance was significantly increased in the coculture model after treatment with ≥307 µmol/L ZnO NP. Only small zinc amounts (0.07–0.65 µg/mL) reached the basolateral area. Our results reveal that the cells of an intact intestinal barrier interact with ZnO NP but do not suffer serious damage.

Topical administration of ambroxol eye drops augments tear secretion in rabbits.

To investigate if topical administration of ambroxol promotes tear secretion and to compare with Diquas ophthalmic eye drop. Two consecutive studies were conducted using sixteen (32 eyes) New Zealand White rabbits. The first study compared the efficacy of ambroxol hydrochloride (0.05%, 0.2%, and 1.0%) on tear and mucin secretion when administered twice daily. Tear secretion was assessed by Schirmer test I and mucin production by conjunctival impression cytology and PAS stain. The second study compared 0.2% ambroxol hydrochloride with Diquas. A human goblet cell line and human conjunctival tissue were used to test the effect of ambroxol hydrochloride on the expression of aquaporin 5 (AQP5) and MUC5AC, using reverse transcriptase-quantitative polymerase chain reaction and immunoblotting. All three concentrations of ambroxol hydrochloride demonstrated significant efficacy on tear stimulation within 2 weeks of treatment and total mucin component appeared increased. When administered topically twice daily, 0.2% ambroxol hydrochloride was more effective in augmenting tear secretion than Diquas. With 24 h of treatment, 5 μM of ambroxol hydrochloride upregulated AQP5 and MU5AC mRNA and MUC5AC protein in a goblet cell line. When tested on preserved human conjunctiva tissue, a trend of increased production of MUC5AC protein was seen (P = 0.26). Ambroxol is effective in augmenting tear secretion at the ocular surface in rabbits. With actions desirable of a candidate dry eye drug, further investigation of ambroxol and related compounds is warranted to explore their value toward clinical application.

Hairy Region Concentrate of Pectin Strongly Stimulates Mucin Secretion in HT29-MTX Cells, but to a Lessor Degree in Rat Small Intestine

Pectin enhances mucin secretion in the rat small intestine. However, what structural features of pectin to stimulate mucin secretion remain unclear. The study aimed to clarify active constituents of pectin using a human goblet cell line, HT29-MTX. Various pectins at 100 mg/L commonly stimulated MUC5AC secretion, irrespective of their differences in molecular size, plant origin and degree of methoxylation, whereas other dietary fiber materials at 100 mg/L did not show any effects, except fucoidan. Hairy region concentrate (HRC) and its further fractions (F1-F3) were prepared by polygalacturonase treatment of citrus pectin and successive anion exchange chromatography. Neutral sugars, such as galactose and arabinose were enriched in these fractions. HRC and F1-F3 at 30 mg/L significantly increased MUC5AC secretion, which were 3 times more potent compared with a starting material (citrus pectin). Further, a dose-dependent study showed that F1 significantly increased MUC5AC secretion from at 0.3 mg/L, much stronger than that of mucin-secretagogue lipopolysaccharides. Rats consumed 5% apple pectin diet showed significant increases of luminal mucin contents and Muc2 expression in the small intestine, while the luminal mucin contents in rats consumed 1.5% HRC diet were increased by 24% compared to those in rats consumed control diet, but the difference did not reach significant. Thus, HRC is supposed to be active constituents of mucin-secretory effect of pectin in vitro. At present, however, the effect of HRC has not been verified in vivo.

Fenretinide favorably affects mucins (MUC5AC/MUC5B) and fatty acid imbalance in a manner mimicking CFTR-induced correction

Cystic fibrosis (CF) is the most common genetic disease in Caucasians. CF is manifested by abnormal accumulation of mucus in the lungs, which serves as fertile ground for the growth of microorganisms leading to recurrent infections and ultimately, lung failure. Mucus in CF patients consists of DNA from dead neutrophils as well as mucins produced by goblet cells. MUC5AC mucin leads to pathological plugging of the airways whereas MUC5B has a protective role against bacterial infection. Therefore, decreasing the level of MUC5AC while maintaining MUC5B intact would in principle be a desirable mucoregulatory treatment outcome.Fenretinide prevented the lipopolysaccharide-induced increase of MUC5AC gene expression, without affecting the level of MUC5B, in a lung goblet cell line. Additionally, fenretinide treatment reversed the pro-inflammatory imbalance of fatty acids by increasing docosahexaenoic acid and decreasing the levels of arachidonic acid in a lung epithelial cell line and primary leukocytes derived from CF patients. Furthermore, for the first time we also demonstrate the effect of fenretinide on multiple unsaturated fatty acids, as well as differential effects on the levels of long- compared to very-long-chain saturated fatty acids which are important substrates of complex phospholipids. Finally, we demonstrate that pre-treating mice with fenretinide in a chronic model of P. aeruginosa lung infection efficiently decreases the accumulation of mucus. These findings suggest that fenretinide may offer a new approach to therapeutic modulation of pathological mucus production in CF.

In Vitro Studies on Zinc Binding and Buffering by Intestinal Mucins.

The investigation of luminal factors influencing zinc availability and accessibility in the intestine is of great interest when analyzing parameters regulating intestinal zinc resorption. Of note, intestinal mucins were suggested to play a beneficial role in the luminal availability of zinc. Their exact zinc binding properties, however, remain unknown and the impact of these glycoproteins on human intestinal zinc resorption has not been investigated in detail. Thus, the aim of this study is to elucidate the impact of intestinal mucins on luminal uptake of zinc into enterocytes and its transfer into the blood. In the present study, in vitro zinc binding properties of mucins were analyzed using commercially available porcine mucins and secreted mucins of the goblet cell line HT-29-MTX. The molecular zinc binding capacity and average zinc binding affinity of these glycoproteins demonstrates that mucins contain multiple zinc-binding sites with biologically relevant affinity within one mucin molecule. Zinc uptake into the enterocyte cell line Caco-2 was impaired by zinc-depleted mucins. Yet this does not represent their form in the intestinal lumen in vivo under zinc adequate conditions. In fact, zinc-uptake studies into enterocytes in the presence of mucins with differing degree of zinc saturation revealed zinc buffering by these glycoproteins, indicating that mucin-bound zinc is still available for the cells. Finally, the impact of mucins on zinc resorption using three-dimensional cultures was studied comparing the zinc transfer of a Caco-2/HT-29-MTX co-culture and conventional Caco-2 monoculture. Here, the mucin secreting co-cultures yielded higher fractional zinc resorption and elevated zinc transport rates, suggesting that intestinal mucins facilitate the zinc uptake into enterocytes and act as a zinc delivery system for the intestinal epithelium.

Protease Activated Receptor‐2 Mediates Giardia‐Induced Disruptions of the Intestinal Mucus Barrier

BackgroundGiardia duodenalis has been associated with disruptions of the mucus layers of the small and large intestines, in part via Giardia's cysteine proteases. Disruption of the intestinal mucus barrier has been implicated in a variety of intestinal disorders, but mechanisms currently remain unclear.HypothesisGoblet cell function and mucus disruptions are modulated by activation of protease activated receptor 2 (PAR2), which is highly expressed through the gastrointestinal tract and is known to regulate mucus production in the airway and stomach. Giardia's cysteine proteases cleave and activate PAR2 on intestinal goblet cells.MethodsMUC2 mucin and PAR gene expression were assessed in the human colonic epithelial cell line LS174T using quantitative PCR (qPCR). LS174T were pre‐treated with the PAR2 antagonist p2Pal‐218S pepducin, and incubated with Giardia trophozoites or the PAR1 and PAR2 agonist peptides TFLLRN and 2fLIGRLO, respectively. Ability of Giardia to cleave PAR1 and PAR2 at the N‐terminal domain was determined using CHO cells transformed with N‐luciferase tagged PAR1 or PAR2. Following incubation with Giardia, culture supernatant was collected, reacted with luciferin, and luminescence was measured. Wild‐type and PAR2 deficient mice were infected with Giardia trophozoites. Thickness of the mucus layer was measured by fluorescently staining mucus with fluorescein‐coupled WGA, and expression of Muc2 and Muc5ac genes were determined in the colon and jejunum via qPCR.ResultsLS174T cells express functional and responsive PAR2, but little or no PAR1. Treatment of cells with 2fLIGRLO PAR2 agonist, but not TFLLRN PAR1 agonist, increased MUC2 gene expression. Incubation of cells with Giardia trophozoites increased MUC2 mRNA production, and this increase was abolished by pre‐treatment of cells with a PAR2 antagonist. Giardia also caused decreased PAR2 expression in LS174T. Giardia NF and Giardia GSM are both able to cleave PAR1 and PAR2 at the N terminal domain. Cleavage by NF was more efficient than GSM, and PAR1 was cleaved more extensively than PAR2 by both strains. Giardia infection increased Muc2 and Muc5ac expression in the colon and increased Muc5ac expression in the jejunum of wild‐type mice compared to uninfected controls. In PAR2−/− mice, Giardia infection reduced expression of Muc5ac in the jejunum, and had no impact on either gene in the colon. Giardia infection caused thinning of the mucus layer in wild‐type mice. In contrast, infection induced thickening of the mucus layer in PAR2−/− mice.ConclusionsUsing a model of Giardia infection, the findings demonstrate that PAR2 plays a significant role in mucin gene regulation and function in mice and in a human colonic goblet cell line, and that Giardia is capable of cleaving both PAR1 and PAR2 receptors with strain‐specific efficiencies.Support or Funding InformationCrohn's Colitis Canada (CCC), NSERCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High MUC2 Mucin Expression and Misfolding Induce Cellular Stress, Reactive Oxygen Production, and Apoptosis in Goblet Cells

MUC2 mucin is a large glycoprotein produced by goblet cells that forms the protective mucus blanket overlying the intestinal epithelium as the first line of innate host defense. High MUC2 production in inflammatory bowel disease and infectious colitis depletes goblet cells and the mucus layer by an unknown mechanism. Herein, we analyzed the effect of high MUC2 biosynthesis on endoplasmic reticulum (ER) stress and apoptosis in goblet cells using a high MUC2-producing human goblet cell line (HT29-H) and an HT29-H clone (HT29-L) silenced for MUC2 expression by lentivirus-mediated shRNA. Goblet cell ER stress and apoptosis were quantified during early onset of dextran sulfate sodium-induced colitis in C57BL/6 and Math1M1GFP mice. Compared with HT29-L and MUC2 nonproducing Caco-2 cells, high MUC2-producing HT29-H cells had significantly increased ER stress and apoptosis after treatment with ER stress-inducing agents. Apoptosis was driven by increased misfolded MUC2 that triggered elevated levels of reactive oxygen species. Correcting MUC2 folding and inhibiting reactive oxygen species alleviated ER stress and rescued cells from apoptosis. During early-onset colitis, mucus hypersecretion caused severe ER stress and apoptosis of goblet cells that preceded absorptive epithelial cell damage. Thus, in gastrointestinal inflammation, high MUC2 biosynthesis and goblet cell apoptosis lead to a dysfunctional epithelial barrier. Enhancing MUC2 folding may help alleviate goblet cell depletion and maintain mucosal integrity.

Lactic Acid Bacteria May Impact Intestinal Barrier Function by Modulating Goblet Cells.

ScopeLactic acid bacteria (LAB) are recognized to promote gastrointestinal health by mechanisms that are not fully understood. LABs might modulate the mucus and thereby enhance intestinal barrier function. Herein, we investigate effects of different LAB strains and species on goblet cell genes involved in mucus synthesis.Methods and resultsGene expression profiles of goblet‐cell‐associated products (mucin MUC2, trefoil factor 3, resistin‐like molecule β, carbohydrate sulfotransferase 5, and galactose‐3‐O‐sulfotransferase 2) induced by LAB or their derived conditioned medium in human goblet cell line LS174T are studied. Effects of LAB on gene transcription are assessed with or without exposure to TNF‐α, IL‐13, or the mucus damaging agent tunicamycin. LAB do impact the related genes in a species‐ and strain‐specific fashion and their effects are different in the presence of the cytokines and tunicamycin. Bioactive factors secreted by some strains are also found to regulate goblet cell‐related genes.ConclusionOur findings provide novel insights in differences in modulatory efficacy on mucus genes between LAB species and strains. This study further unravels direct interactions between LAB and intestinal goblet cells, and highlights the importance of rationally selecting appropriate LAB candidates to achieve specific benefits in the gut.

Oxyresveratrol-containing Ramulus mori ethanol extract attenuates acute colitis by suppressing inflammation and increasing mucin secretion

Control of inflammation and mucin expression is important in managing inflammatory bowel disease (IBD). This study investigated the effects of an ethanol extract of Ramulus mori containing oxyresveratrol (ERMO) on inflammation and intestinal mucin production. The anti-inflammatory effect of ERMO was measured both in vitro using lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cell line, and in vivo with a dextran sodium sulfate (DSS)-induced mouse colitis model; its mucin-promoting activity was measured in LS 174T goblet cell line. We found that ERMO significantly reduced inflammatory mediators both cells and mice. In the LS 174T goblet cells, ERMO significantly increased MUC2 and TFF3 mRNA expression in a dose-dependent manner compared with negative control cells. ERMO regenerated intestinal mucus layer and showed disease-alleviating effects in DSS-induced mouse colitis model. In conclusion, ERMO significantly attenuates acute colitis by suppressing inflammation and stimulating mucin production.

Heat-labile enterotoxin of Escherichia coli promotes intestinal colonization of Salmonella enterica.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of infantile and travellers' diarrhoea, which poses a serious health burden, especially in developing countries. In addition, ETEC bacteria are a major cause of illness and death in neonatal and recently weaned pigs. The production of a heat-labile enterotoxin (LT) promotes the colonization and pathogenicity of ETEC and may exacerbate co-infections with other enteric pathogens such as Salmonella enterica. We showed that the intraintestinal presence of LT dramatically increased the intestinal Salmonella Typhimurium load in experimentally inoculated pigs. This could not be explained by direct alteration of the invasion or survival capacity of Salmonella in enterocytes, in vitro. However, we demonstrated that LT affects the enteric mucus layer composition in a mucus-secreting goblet cell line by significantly decreasing the expression of mucin 4. The current results show that LT alters the intestinal mucus composition and aggravates a Salmonella Typhimurium infection, which may result in the exacerbation of the diarrhoeal illness.

Giardia duodenalis Depletes Goblet Cell Mucins and Degrades MUC2, Facilitating Bacterial Translocation

Giardia duodenalis, a cosmopolitan diarrheagenic parasite of the small intestine, leads to post‐infectious irritable bowel syndrome (PI‐IBS) and extraintestinal complications via unclear mechanisms. The effects of Giardia on the protective mucus layers of the intestine are unknown.ObjectivesC57BL/6 (WT) and Muc2‐/‐ mice, human goblet cell line LS174T, and purified human mucin were exposed to Giardia trophozoites or Giardia's secreted product to assess the effects of Giardia on MUC2 and goblet cell mucins.ResultsInfected Muc2‐/‐ mice had higher parasitic loads and inhibition of weight gain versus WT mice. Goblet cell mucin (stained with PAS/AB and WGA) was depleted in the small intestine and colon of infected WT mice. Bacterial translocation to the liver and spleen was also increased in infected mice. Immunofluorescence staining of MUC2 was reduced in human goblet cells in vitro after exposure to Giardia trophozoites. In addition, a co‐incubation of Giardia's secreted products with purified human mucin degraded MUC2.ConclusionsMUC2 mucin is protective in giardiasis, but Giardia is able to overcome this barrier. While Giardia colonizes the upper small intestine, it induces mucin depletion in goblet cells throughout the small intestine and colon. Mucin depletion and degradation break down the protective mucus barrier, facilitating bacterial translocation. These mechanisms may contribute to PI‐IBS. Supported by HPI NSERC CREATE, NSERC Discovery and Queen Elizabeth II Master's Scholarship

HCLCA1 DNA Vaccine Suppresses Cell Hyperplasia and Mucin Expression of Goblet Cells in vitro

Background: Excessive airway mucus secretion is a remarkable trait of asthma. Mucus overproduction mainly resulted from an increase in goblet cell numbers, which causes considerable damage to health. However, effective therapeutic treatments are still lacking for mucus hypersecretion. Human calcium-activated chloride channel 1 (hCLCA1) has been identified to be predominantly responsible for mucus hypersecretion. Objectives: In this study, we investigated the effects of an hCLCA1 DNA vaccine on the control of mucus production and goblet cell proliferation using an in vitro model goblet cell line (NCI-H292). Methods: The effect of the hCLCA1 DNA vaccine on cell viability and proliferative activity of NCI-H292/hCLCA1 was analyzed by electron microscopy, MTT assay, and flow cytometry. Expression of mucins and MUC5AC, a major member of the mucin gene family in airway goblet cells, was assessed under hCLCA1 DNA vaccine challenges by periodic acid-Schiff staining, quantitative real-time PCR and Western blot, respectively, and the expression profile of granulocyte-macrophage colony-stimulating factor (GM-CSF), a critical cytokine in airway inflammation, was also examined by real-time PCR and immunocytochemistry. Results: Results showed that hCLCA1 overexpression caused high cell proliferation and mucin expression, whereas the hCLCA1 DNA vaccine could effectively reverse these abnormal effects. In addition, GM-CSF expression was highly induced by hCLCA1 overexpression and efficiently suppressed by hCLCA1 DNA vaccine. Conclusions: These results illustrate that the hCLCA1 DNA vaccine effectively inhibits cell hyperplasia and mucin gene expression of goblet cells, suggesting that the hCLCA1 DNA vaccine has potential value in the treatment of human asthma.

TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells

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 Ca(2+)-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 Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion. DOI:http://dx.doi.org/10.7554/eLife.00658.001.

High MUC2 production in goblet cells caused ER stress and susceptibility to apoptosis

MUC2 produced by goblet cells is the gel forming mucin in mucus that protects the intestinal epithelium against harmful substances and the development of tumors. In this study, we investigated the role of MUC2 in colonic epithelial cell apoptosis, a process essential in preventing tumor formation. A high MUC2‐producing colonic goblet cell line (HT29‐H) and a clone silenced for MUC2 (HT29‐L) by lentivirus‐shRNA were used. Cells were exposed to apoptosis‐inducing agents and apoptosis quantified by caspase 3 and Poly (ADP‐ribose) polymerase cleavage, DNA fragmentation and lactate dehydrogenase (LDH) release. Expression of endoplasmic reticulum (ER) stress markers was evaluated to monitor ER activities as well as expression of genes involved in MUC2 biosynthesis. HT29‐H was found to be highly susceptible to apoptosis as compared to HT29‐L cells. Over expression of MUC2 in HT29‐H cells caused increased expression of ER stress markers and decreased MUC2 folding genes as compared to HT29‐L or parental wild‐type cells. Our findings indicate that MUC2 induces apoptosis in an ER stress‐dependent manner and strongly suggests that it may be a useful therapeutic agent in colorectal carcinomas. Supported by CIHR.

PAR-2 activation increases human intestinal mucin secretion through EGFR transactivation

PAR-2 (protease-activated receptors-2) are G protein-coupled receptors whose action on mucin secretion by intestinal epithelial cells is still unknown. The aim of this study was to examine the effect of PAR-2 activation on mucin secretion in the human colonic goblet cell line HT29-Cl.16E and the intracellular pathways involved. We found that PAR-2 mRNA was constitutively expressed by HT29-Cl.16E cells as well as by isolated human normal colonocytes. The PAR-2-activating peptide SLIGKV-NH2 elicited rapid mucin secretion in HT29-Cl.16E, which was partially inhibited by calcium chelator BAPTA. Inhibitors of MAPK activation (PD98059) and EGFR tyrosine kinase activity (AG1478) abrogated PAR-2-induced ERK1/2 and EGFR tyrosine phosphorylation, respectively, and subsequent mucin secretion. Finally, PAR-2-induced EGFR transactivation was involved upstream of ERK1/2 activation. Our results show that the activation of PAR-2 expressed by human intestinal epithelial cells enhances mucin secretion, a component of the intestinal innate defence, via a pathway involving EGFR transactivation.

Metalloproteinase-9 (MMP-9) regulates intestinal goblet cell specific gene expression: Implications for the role of MMP-9 in the pathogenesis of colitis.

We recently demonstrated that MMP-9, a gelatinase upregulated during inflammatory bowel disease, is a critical mediator of tissue damage during colitis. MMP-9 null mice (MMP-9−/−) develop dramatically reduced inflammatory response to luminally administered colitic agents in the face of intact systemic immune response and inflammatory cell recruitment. In this study, we sought to address the mechanism underlying the resistance of MMP-9−/− mice to luminal insults. WT and MMP-9−/− mice were used as an in vivo model to study mucin expression and the role of MMP-9 in intestinal epithelial cell differentiation. The goblet cell line, HT29-cl.16E, was used for in vitro studies. MMP-9−/− mice demonstrated significantly increased goblet cells, mucin and MUC-2 compared to WT mice, suggesting altered epithelial cell differentiation in the absence of MMP-9. Temporal expression of MMP-9 and NICD, a transcription factor involved in intestinal cell fate determination, inversely correlated with MUC-2 expression during maturation of HT29-16E cells. We also demonstrate that MMP-9 activates Notch-1 in intestinal epithelial cells. MMP-9 also regulates goblet cell differentiation through changing the expression of transcription factors like KLF-4 and Elf-3. Finally, we demonstrate that MMP-9 overexpression inhibits the expression of goblet cellspecific protein, MUC-2, and under these conditions, adherence of Salmonella typhimurium is significantly increased. These data demonstrate that i) MMP-9 is involved in the regulation of MUC-2 expression; ii) upregulation of mucin and MUC-2 in MMP-9−/− mice may contribute to the resistance of these mice to luminally administered colitic agents; and iii) MMP-9 may regulate goblet cell function through activation of Notch-1, an evolutionarily conserved signaling pathway that plays a critical role in cell fate determination. These data uncover a novel function of MMP-9 in intestinal epithelial cells.

Matrix Metalloproteinase-9 Regulates MUC-2 Expression Through Its Effect on Goblet Cell Differentiation

We recently demonstrated that epithelial-derived matrix metalloproteinase (MMP) 9 up-regulated during inflammatory bowel disease is a critical mediator of tissue damage during colitis. MMP-9 null mice (MMP-9(-/-)) develop dramatically reduced inflammatory response to luminally administered colitic agents in the face of intact systemic immune response and inflammatory cell recruitment, suggesting protected epithelial barrier in these mice. In this study, we sought to address the role and mechanism by which MMP-9 influences barrier protective function. Wild-type and MMP-9(-/-) mice were used for in vivo studies, and the goblet cell line HT29-cl.16E and the enterocyte cell line Caco2-BBE were used for in vitro studies. Compared with wild-type mice, MMP-9(-/-) mice had an increased number of goblet cells and MUC-2 expression. In addition, KLF-4 and Elf-3, transcription factors involved in terminal differentiation of goblet cells were up-regulated, whereas notch intracellular domain (NICD; activated Notch-1) was down-regulated in MMP-9(-/-) mice. These findings suggest altered epithelial cell differentiation in MMP-9(-/-) mice. Temporal expression of MMP-9 inversely correlated with MUC-2 expression during maturation of goblet cells. MMP-9 over expression inhibited goblet cell differentiation in vitro. Conversely, MMP-9 gene silencing in Caco2-BBE cells resulted in a change in their phenotype toward goblet cells. Finally, MMP-9 over expression or silencing in goblet cells increased or decreased Salmonella typhimurium adherence, respectively. MMP-9 regulates goblet cell differentiation in colon. The effect of MMP-9 on goblet cells could contribute to alteration in mucosal defense leading to inflammation. Together, our data uncover a novel function of MMP-9 in intestinal epithelial cells.

Methods for Detecting Internalized, FM 1-43 Stained Particles in Epithelial Cells and Monolayers

The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles after washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that 1), the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2), low density clusters exhibited significantly more internalized particles per cell than either filter-grown monolayers or high density clusters; 3), ATP stimulation significantly increased the number of internalized particles in all preparations; and 4), the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells.

An algorithm for detecting internalized, FM 1–43 stained membrane in epithelia demonstrates greater membrane internalization in low density cell clusters vs high density monolayers

The membrane dye FM 1–43 rapidly stains a cell’s extracellular membrane including any membrane exocytosed, a property used to quantify exocytosis in neurons. In epithelia, fluctuations in basal and lateral intracellular space staining render FM 1–43 unreliable as a marker of exocytosis. However, membrane retrieved in the presence of FM 1–43 retains the dye, and washout of external stain reveals punctate, fluorescent particles. We developed an algorithm to quantify the retained fluorescent puncta in cells using images acquired with serial sectioning, and tested the algorithm on both coverslip-grown cell clusters and filter-grown intact monolayers. Serial sectioning was performed with epifluorescence microscopy, and a subset of experiments used the fixable variant of FM 1–43 to allow serial sectioning of the same preparation using both epifluorescence and confocal microscopy. Tests using an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that: 1) the algorithm identified the same number of internalized particles for a set condition with either epifluorescence or confocal microscopy; 2) low density clusters with an average of 5 cells exhibited significantly more internalized particles per cell (316 ± 31) than filter grown monolayers (9 ± 3) or high density clusters with an average of 120 cells (8 ± 3); and 3) ATP stimulation significantly increased the number of internalized particles in all preparations. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells. (CFF)

Butyrate specifically modulates MUC gene expression in intestinal epithelial goblet cells deprived of glucose.

The mucus layer covering the gastrointestinal mucosa is considered the first line of defense against aggressions arising from the luminal content. It is mainly composed of high molecular weight glycoproteins called mucins. Butyrate, a short-chain fatty acid produced during carbohydrate fermentation, has been shown to increase mucin secretion. The aim of this study was to test 1) whether butyrate regulates the expression of various MUC genes, which are coding for protein backbones of mucins, and 2) whether this effect depends on butyrate status as the major energy source of colonocytes. Butyrate was provided at the apical side of human polarized colonic goblet cell line HT29-Cl.16E in glucose-rich or glucose-deprived medium. In glucose-rich medium, butyrate significantly increased MUC3 and MUC5B expression (1.6-fold basal level for both genes), tended to decrease MUC5AC expression, and had no effect on MUC2 expression. In glucose-deprived medium, i.e., when butyrate was the only energy source available, MUC3 and MUC5B increase persisted, whereas MUC5AC expression was significantly enhanced (3.7-fold basal level) and MUC2 expression was strikingly increased (23-fold basal level). Together, our findings show that butyrate is able to upregulate colonic mucins at the transcriptional level and even better when it is the major energy source of the cells. Thus the metabolism of butyrate in colonocytes is closely linked to some of its gene-regulating effects. The distinct effects of butyrate according to the different MUC genes could influence the composition and properties of the mucus gel and thus its protective function.