Secretion Of Mucin Glycoproteins Research Articles

Middle Ear Response of Muc5ac and Muc5b Mucins to Nontypeable Haemophilus influenzae.

Chronic otitis media with effusion is characterized by middle ear secretion of mucin glycoproteins, predominantly MUC5B; MUC5AC, the other secretory mucin studied frequently, has also been identified in the middle ear. Emerging evidence suggests a dichotomous role for these mucins in innate immune responses. We hypothesized that MUC5AC is an acute responder and MUC5B is expressed at later time points, reflecting a chronic situation. To determine middle ear regulation of MUC5B and MUC5AC following in vitro bacterial and cytokine exposure. An in vitro cell-based model of mucin gene regulation was conducted in a basic science laboratory at a tertiary pediatric hospital. The study was conducted from July 1, 2014, to June 30, 2015; data analysis was performed in July 2015. Nontypeable Haemophilus influenzae (NTHi) lysates were generated and used to stimulate mouse middle ear epithelial cells (mMEECs) for 2 hours during 3 weeks. Real-time quantitative polymerase chain reaction, luciferase assays, Western blot assay, and immunofluorescence techniques were performed to determine Muc5ac and Muc5b expression over time, Cxcl2 chemokine response, and nuclear factor-κB activation. Luciferase reporter assays were performed to evaluate specific promoter responses after NTHi exposure. Nontypeable H influenzae lysates (200 μg/mL) drove differential mucin gene activation, with Muc5ac being induced up to 2.04 fold at 24 hours and 2.79 fold at 96 hours (P < .05) and Muc5b being induced only at more long-term points: 1.61 fold at 96 hours, 1.41 fold at 1 week, and 1.53 fold at 3 weeks (P < .05). Although NTHi lysates induced robust, early nuclear factor-κB nuclear translocation with nuclear factor-κB-dependent induction of Cxlc2 expression, the lysates had minimal to no effect on Muc5ac and Muc5b promoter activity. However, in contrast to NTHi lysates, CXCL2 induced significant transcription of both Muc5b and Muc5ac as early as 24 hours. Nontypeable H influenzae lysates activate differential mucin gene activation in mMEECs. Although Muc5ac is an early response mucin gene, Muc5b appears to react as a chronic response mucin.

Salivary gland homeostasis is maintained through acinar cell self-duplication.

Current dogma suggests that salivary gland homeostasis is stem cell dependent. However, the extent of stem cell contribution to salivary gland maintenance has not been determined. We investigated acinar cell replacement during homeostasis, growth, and regeneration, using an inducible CreER(T2) expressed under the control of the Mist1 gene locus. Genetic labeling, followed by a chase period, showed that acinar cell replacement is not driven by the differentiation of unlabeled stem cells. Analysis using R26(Brainbow2.1) reporter revealed continued proliferation and clonal expansion of terminally differentiated acinar cells in all major salivary glands. Induced injury also demonstrated the regenerative potential of pre-labeled acinar cells. Our results support a revised model for salivary gland homeostasis based predominantly on self-duplication of acinar cells, rather than on differentiation of stem cells. The proliferative capacity of differentiated acinar cells may prove critical in the implementation of cell-based strategies to restore the salivary glands.

The eicosanoid, 15-(S)-HETE, stimulates secretion of mucin-like glycoprotein by the corneal epithelium.

The eicosanoid, 15-(S)-hydroxyeicosa-5Z, 8Z-11Z, 13E-tetraenoic acid (15-(S)-HETE), is known to stimulate production of mucin glycoprotein by airway epithelium. This study investigated the effect of 15-(S)-HETE on the mucin glycoprotein secretion by the corneal epithelium. To determine the effect of dose, corneas of anesthetized New Zealand White rabbits were treated with 50, 500, or 5,000 nM 15-(S)-HETE in artificial tears for 120 minutes. To determine the time to onset of the response, corneas were treated with 500 or 1,000 nM 15-(S)-HETE in balanced salt solution for periods ranging from 5 to 120 minutes. Corneas were fixed for electron microscopy in fixative containing 0.5% cetylpyridinium chloride (CPC) to stabilize the layer of mucin-like glycoprotein on the corneal surface. The mucin layer thickness was measured by image analysis of electron micrographs. The layer of CPC-fixed mucin-like glycoprotein on the surface of control corneas was 0.46 +/- 0.04 microm thick. After treatment with 15-(S)-HETE, the thickness of the mucin layer increased to 0.64 +/- 0.1 microm at 50 or 5,000 nM HETE and as much as 1.02 +/- 0.2 microm in response to 500 nM HETE. Mucin thickness reached a statistical maximum of 0.59 +/- 0.1 microm after only 5 minutes of exposure to 500 or 1,000 nM HETE. Exposure of the cornea to 15-(S)-HETE causes a rapid-onset increase in the thickness of a layer of mucin-like glycoprotein on the surface of the corneal epithelium. This supports previous reports that corneal epithelial cells produce mucin and suggests that treatment with topical 15-(S)-HETE may be effective in treating ocular surface mucin deficiency in dry eye syndrome.

Biological properties and expression of mucins in 5-fluorouracil resistant HT29 human colon cancer cells.

We have previously reported that HT29 human colon cancer cells selected by adaptation to 5-fluorouracil (5FU) (HT29-5FU cells) express increased levels of a major intestinal mucin MUC2 mRNA compared with parental HT29 cells. In this study, we examined in detail the changes in synthesis and secretion of mucin that occur in these cells and accompanying changes in the expression of cancer associated mucin related carbohydrate antigens and cell lineage associated biochemical markers. We further investigated their relationship to biological properties of cells. Northern blot analysis revealed a markedly increased level of MUC2 mRNA but no significant change in the mRNA levels of other mucins in HT29-5FU cells compared with parental HT29 cells. Labeling with radiolabeled precursors demonstrated increased synthesis and secretion of mucin glycoproteins by HT29-5FU cells. Immunoblot analysis showed a higher expression of mucin associated carbohydrate antigens such as T, Tn, sialyl Tn, sialyl Lea, sialyl Lex and non-O-acetylated sialic acid concomitant with significant increases in the expression of goblet cell lineage marker, MUC2 apomucin and a panepithelial cell marker, carcinoembryonic antigen. HT29-5FU cells showed significantly higher adhesion to E-selectin and to matrigel and in vitro invasive properties and significantly increased liver colonization capacity in nude mice following splenic vein injection. Nude mouse xenograft tumors produced by HT29-5FU cells showed a greater degree of differentiation, consisting of mucin secreting glands than those produced by parental HT29 cells. These results indicate that predominantly colonic type mucin, MUC2, has been selectively induced in HT29-5FU cells and that altered regulation of mucin genes associated with altered synthesis and secretion of mucin glycoproteins and the degree of differentiation in cancer cells may be responsible for the altered biological properties of these cells.

Cystic Fibrosis: A Multiple Exocrinopathy Caused by Dysfunctions in a Multifunctional Transport Protein

Cystic fibrosis (CF) is a lethal, autosomal recessive genetic disease caused by mutations in the CF gene that encodes a protein called CFTR (1,2). According to the latest available statistics from the CF Foundation (CFF Patient Registry Annual Data Report, August 1995), approximately 20,000 children and young adults in North America (excluding Canada) have been diagnosed with CF. This disease presents as a multiple exocrinopathy resulting in abnormalities in many exocrine tissues including the airways, lung, pancreas, liver, intestine, vas deferens, and sweat gland/duct (3,4). Mutations in the CFTR gene result in a plethora of clinical symptoms including thick, dehydrated airway mucus, chronic, recurrent, and tenacious lung infections with bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus, early-onset lung inflammation specific to infiltration by neutrophils, pancreatic insufficiency, bile duct obstruction, intestinal obstruction (meconium ileus), infertility in males, high sweat Cl, nasal polyp formation, and chronic sinusitis (3,4). The most common mutation is a deletion in a single phenylalanine residue at position 508 in the CFTR protein, the so-called DF508 mutation. This mutation accounts for more than 50% of the mutations isolated from CF patients worldwide; however, over 700 separate mutations in the CFTR gene have been identified as of late May 1997 by the CF Genetic Analysis Consortium. Mortality in CF patients is related to pulmonary disease (1– 4). The respiratory epithelium is covered by a periciliary fluid layer or airway surface liquid (ASL) that is produced in combination by columnar epithelial ion and water transport, secretion of mucin glycoproteins by goblet cells, and secretions from the submucosal glands (5). The composition, volume, and depth of this fluid layer are critical for the normal beating of the cilia that cover the apical surface of the airway epithelium (5). Atop the ASL lies the layer of airway mucus that is also produced from the secretions of the cells listed above (5). Secretion and absorption of fluid and electrolytes across the respiratory epithelium play critical roles in controlling the amount of mucus hydration and the depth of the periciliary layer (5). For example, in the mammalian fetus, secretory mechanisms predominate and the airways are filled completely with fluid (6). Shortly before, during, and immediately following birth, the absorptive pathways are activated and the secretory mechanisms are down-regulated, leading to a massive absorption of the lung fluid appropriate for air breathing (6). In CF, where Cl secretion is defective and Na absorption hyperactivated, the secreted mucus is too viscous and sticky in the airway, and it clogs the airways with such tenacity that conducting airways become obstructed (1–5). Moreover, the depth and composition of the ASL is affected such that ciliary beat is compromised, the mucus layer invades the ASL, and the micro-environment may become more ideal for bacterial infection. Treatment of the disease continues to improve mainly because of pancreatic enzyme replacement, nutritional management, and more potent and selective antibiotics (7). The median age for survival continues to increase (up to 31 years); however, therapies that correct the basic cellular defects that underlie these clinical phenotypes remain elusive (7).

Constitutive Mucin Secretion Linked to CFTR Expression

Mucus plugging is a hallmark of cystic fibrosis, but the link between the defective gene product, the cystic fibrosis transmembrane conductance regulator, and the abnormal mucus phenotype is unclear. To demonstrate CFTR involvement in mucin glycoprotein secretion in epithelial cells, a retroviral vector was used to overexpress CFTR in gallbladder epithelial cells, and constitutive mucin labeling and secretion were monitored. Achievement of high-level vector expression was confirmed by transduction with marker genes. Cells transduced with vectors carrying CFTR cDNA showed 5-fold increased expression of CFTR by Western blotting. Mucin labeling and secretion were 4-fold elevated in transduced cells. These results suggest constitutive mucin synthesis and secretion in gallbladder epithelial cells are regulated by CFTR.

Transdifferentiation of outgrowth cells and cultured epithelial cells from swine trachea.

The morphologic and functional properties of explant out-growth cells and epithelial cells isolated from swine trachea epithelium by proteolysis were examined. A mixed population of ciliated, serous, and basal cells, obtained from out-growths, from proteolysis of trachea epithelium, and from unattached explants in organ culture, all yielded cell cultures that werecomposed almost entirely of mucus-secreting cells. When the cells were grown in primary or secondary culture on a modified collagen matrix in supplemented HAM:DMEM (1:1) medium they expressed a mucus-secreting phenotype with numerous mucus granules at various stages of maturation and incorporated [3H]GlcN and 35SO4 into secreied mucin glycoproteins. Results obtained in these studies suggest that extensive transdifferentiation of ciliated and serous cells to mucus-secreting-cells occurs after the release and during subsequent attachment and culture. Ciliated cells containing mucus granules were seen in various stages of cilia resorption. Basal cells containing mucus granules were also frequently observed. The number of mucus-secreting cells and the synthesis of mucin glycoproteins increased dramatically with time of attachment and culture, whereas cell proliferation, population doubling time of 72 h, and incorporation of [3H]-thymidine into DNA increased much more slowly. The number of mucus-secreting cells correlated closely with the level of secretion of mucin glycoproteins. Taken collectively, these studies help to elucidate the transdifferentiation process, which dramatically increases the number of mucus-secreting cells after disruption and release of epithelial cells from swine tracheobronchial epithelium. A similar mechanism involving disruption of the extracellular matrix may be involved in the stimulation of hypersecretion of mucus and mucin glycoproteins by chemical and infections irritants.

Muscarinic cholinergic receptor subtypes in rat sublingual glands

Mucin glycoprotein secretion by rat sublingual glands is regulated primarily by muscarinic cholinergic receptors. Studies were conducted to identify muscarinic receptor subtypes in whole glands as well as in isolated acinar structures. In radioligand binding studies, we used subtype-selective antagonists in competition studies to initially determine receptor subtype heterogeneity. In membranes from whole glands, both pirenzepine and methoctramine displayed two affinity sites (M1 and M3) of nearly equal proportions. In contrast, acinar membranes contained a 1:2 and 2:1 ratio of M1 to M3 sites for pirenzepine and methoctramine, respectively. In all cases, p-fluoro-hexahydro-siladifenidol and 4-diphenylacetoxy-N-methylpiperidine each bound to a single class of binding sites. Northern analysis using oligonucleotide probes specific for the 5' ends of the translated regions of m1 through m5 receptors detected only m1 and m3 subtypes in poly(A)+ RNA from whole glands. We also used antisera specific for each receptor subtype to immunoprecipitate solubilized receptors from membrane preparations. Only m1 (51.7 and 64.9%) and m3 (48.3 and 34.7%) subtypes were found consistently in membranes from whole sublingual glands and isolated acini, respectively. Studies with other exocrine glands generally described the predominance of m3 receptors, and m1 receptors, if present, were presumably associated with contaminating neural structures. Our results therefore demonstrate that mucous acini from rat sublingual glands contain abundant amounts of both m1 and m3 receptors.

The effect of 16,16-dimethyl prostaglandin E 2 on proliferation of an intestinal goblet cell line and its synthesis and secretion of mucin glycoproteins

The effect of 16,16′-dimethyl prostaglandin E 2 (dmPGE 2) on the human colonic adenocarcinoma derived mucus-secreting goblet cell line HT29-18N2 was investigated. The proliferation rate of HT29-18N2 was increased by exposure to 10 or 100 μM dmPGE 2. Exposure to 10 or 100 μM dmPGE 2 caused a significant decrease in the rate of radiolabeled glucosamine incorporation into newly synthesized glycoproteins during an 8 or 24 h exposure. At concentrations as low as 1 μM, dmPGE 2 accelerated the secretion of mucin glycoproteins as assessed by the release of newly synthesized radiolabeled glycoproteins, a mucin-specific enzyme-linked immunoassay and a whole-mount immunofluorescence assay. A 1 h exposure to dmPGE 2 did not, however, result in a morphometrically detectable decrease in intracellular mucous granule stores or elicit any other readily detectable morphological change. The experimental results suggest elevated levels of PGs may contribute to the previously recognized decreases in intracellular mucin stores and shifts in the types of mucins species present at sites of mucosal inflammation in ulcerative colitis patients.

Regulation of the synthesis of mucin glycoproteins in swine trachea explants.

Swine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm2 per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 microM glucosamine. A higher concentration of 35SO4, 1000 microM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 microgram/ml increased the rate of secretion twofold, whereas 0.1 to 100 micrograms/ml of hydrocortisone and 0.1 to 100 micrograms/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10(-5) M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10(-9) M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats. Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2 M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistinguishable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio of N-acetylgalactosamine, N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations.

Arachidonic acid stimulates mucin secretion in prairie dog gallbladder.

Mucin glycoprotein secretion from prairie dog gallbladder explants was studied in 24-h organ culture using [3H]glucosamine as a precursor. Indomethacin caused a reversible dose-dependent inhibition of both mucin release (50% inhibition between 10(-6) and 10(-5) M indomethacin) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) release (66% inhibition at 10(-7) M). Sepharose 4B chromatography of secreted [3H]-glucosamine-labeled glycoproteins revealed that indomethacin inhibited release of a high-molecular (greater than 10(6) daltons) mucin-type glycoprotein. Addition of sodium arachidonate (10(-4) M) to organ culture medium caused an approximate two- to fivefold increase (P less than 0.02) in mucin release compared with control and an increase in the secretory component of total glycoprotein synthesis from 37.5% in control explants to 75.2% (P less than 0.01). The stimulatory effect of arachidonate on mucin secretion was blocked by indomethacin. We incubated explants for 1 h with [14C]arachidonate and studied prostaglandin and thromboxane products in medium by high-performance liquid chromatography. The only conversion product identified was 6-keto-PGF1 alpha, the stable breakdown product of prostacyclin. Release of gallbladder mucin is significantly inhibited by indomethacin and increased by arachidonate. Mucin secretion may be regulated in part by endogenous levels of prostacyclin, the major cyclooxygenase product in prairie dog gallbladder epithelium.