Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis

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.     

Intestinal secretory cell ER stress and inflammation

Data from animal models and human inflammatory bowel diseases have implicated the ER (endoplasmic reticulum) stress pathway in intestinal inflammation. We have characterized the development of inflammation in Winnie mice in which ER stress arises due to a single missense mutation in the MUC2 mucin produced by intestinal goblet cells. This model has allowed us to explore the genesis of inflammation ensuing from a single gene polymorphism affecting secretory cells. In these mice, a proportion of MUC2 misfolds during biosynthesis, leading to ER stress and activation of the unfolded protein response. Winnie mice develop spontaneous complex progressive inflammation that is most severe in the distal colon. Inflammation involves TH1, TH2 and TH17 T-cells, with a progressive development of a TH17-dominated response, but also involves innate immunity, in a pattern not dissimilar to human colitis. Experimental inhibition of tolerance in this model severely exacerbates colitis, demonstrating active effective suppression of inflammation. Even though the misfolding of MUC2 is a consequence of an inherited mutation, as inflammation develops, the molecular markers of ER stress increase further and goblet cell pathology becomes worse, suggesting that inflammation itself exacerbates ER stress.     

TNF-alpha in the regulation of MUC5AC secretion: some aspects of cytokine-induced mucin hypersecretion on the in vitro model

TNF-alpha has been implicated in the aetiology of otitis media with effusion (OME), where goblet cells proliferate in a modified respiratory epithelium, leading to the accumulation of a mucin-rich effusion in the middle-ear cleft. The MUC5AC mucin gene product has been identified as a component of these effusions. Here we have used the HT29-MTX goblet cell line, which secretes MUC5AC mucin, as a model to study the effect of TNF-alpha on goblet cells. MUC5AC mucin was identified and quantitated with a monoclonal antibody NCL-HGM-45M1. TNF-alpha stimulates MUC5AC mucin secretion in a dose-dependent manner, with 20 ng/ml producing maximal stimulation. Both pre-confluent and confluent cells showed peak stimulation after 7 h, however the pre-confluent cells showed twice the level of mucin hypersecretion. These results suggest that TNF-alpha stimulation of mucin secretion could play an important role in the early acute phase of the development of OME. This hypersecretion of mucin could then lead to the failure of the mucociliary clearance system, resulting in the accumulation of a mucin-rich effusion in the middle ear and the movement to a more chronic phase of the disease.     

The cationic C-terminus of rat Muc2 facilitates dimer formation post translationally and is subsequently removed by furin.

Earlier immunolocalization experiments showed that the extreme cationic C-terminus of the rat intestinal mucin Muc2 (RMC) was present at the base of intestinal goblet cells in the vicinity of ER and golgi compartments, but was not found with the rest of the mucin in apical storage granules. This prompted us to investigate the possibility that an early proteolytic cleavage reaction occurs post-translationally. A plasmid pRMC, encoding the C-terminal 534 amino acids of the mucin, was expressed in COS-7 cells and was shown to undergo cleavage at an R-T-R-R sequence located within the C-terminal 14 amino acids. Cleavage did not occur with the construct RMCfH, a furin site-mutated (A-T-A-A) counterpart of pRMCH (poly His6 tagged RMC). Addition of a furin inhibitor to COS-7 cell incubations also prevented cleavage of RMC and RMCH products. 35S pulse-chase kinetic experiments revealed that a truncated mutant lacking the C-terminal 14 amino acids (pRMCDeltaCT) forms faulty (doublet) dimers in the ER. These were not secreted as efficiently as the normal dimer of wild-type (pRMC) constructs. Thus the cationic C-terminus of rMuc2 apppears to facilitate the correct formation of normal Muc2 domain dimers.     

LPS up-regulates mucin and cytokine mRNA expression and stimulates mucin and cytokine secretion in goblet cells

Bacterial inflammation in mucosa is accompanied by morphological and proliferative changes in goblet cells and mucin hypersecretion. Main stimulators of bacterial inflammation are bacterial lipopolysaccharides (LPS). In vitro investigation of the LPS effect on the molecular processes in goblet cells, using the human mucin-secreting goblet cell line HT29-MTX, showed the following results. LPS up-regulated mucin and cytokine mRNA expression and secretion in goblet cells in a concentration and time-dependent manner, with a maximum output at an LPS concentration of 100 ng/ml. LPS (100 ng/ml) increased mRNA expression of MUC5AC (2.4x), MUC5B (2.1x), and IL-8 (2.3x) and stimulated secretion of mucins (MUC5AC up to 39%, MUC5B up to 31%) and the inflammatory cytokine IL-8 (up to 10x). A significant correlation was found between the LPS-induced IL-8 secretion and secretion of mucins. These results suggest: (1) goblet cells, responding to the direct stimulation of bacterial LPS by two inflammatory-related processes such as production and secretion of the gel-forming mucins and the inflammatory cytokine IL-8, can be considered as an important part of mucosal immunity and (2) LPS- induced goblet cell mucin secretion can occur partly via IL-8-dependent pathway.     

Cyclosporine has a direct effect on the differentiation of a mucin-secreting cell line

Cyclosporine is a potent immunosuppressant used in the treatment of ulcerative colitis and keratoconjunctivitis sicca. Neither the etiologies of these diseases nor the mechanism by which cyclosporine exerts its therapeutic effect is well understood. Since both diseases are linked by a common decrease in mucin-filled goblet cells, this study tests a hypothesis that cyclosporine acts directly on goblet cells to promote their differentiation and production of secretory mucins. The HT29-18N2 human colon adenocarcinoma cell line, which is capable of forming monolayers of well-differentiated goblet cells, was used as a model system. Cyclosporine induced a dose-dependent increase in intracellular mucin stores. A 2-week exposure to 1 microM cyclosporine resulted in an average increase in mucin volume of 94%. This increase resulted from both a higher percentage of cells with mucin stores and an increased volume of mucin per cell. PSC-833, a nonimmunosuppressive analog of cyclosporine, also increased mucin production. The intracellular accumulation of mucin was not a result of reduced secretion, since the time required for the release of pulse-radiolabeled glycoproteins was similar for both control and cyclosporine-treated monolayers. The effect of cyclosporine was not mediated by the drug's previously documented abilities to decrease cellular proliferation rates, inhibit calmodulin, antagonize prolactin receptor binding, or modulate prostaglandin production.     

Secretory glycoconjugates of a mucin-synthesizing human colonic adenocarcinoma cell line. Analysis using double labeling with lectins

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.     

ATP induced MUC5AC release from human airways in vitro

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.     

Fast renewal of the distal colonic mucus layers by the surface goblet cells as measured by in vivo labeling of mucin glycoproteins

The enormous bacterial load and mechanical forces in colon create a special requirement for protection of the epithelium. In the distal colon, this problem is largely solved by separation of the bacteria from the epithelium by a firmly attached inner mucus layer. In addition, an outer mucus layer entraps bacteria to be cleared by distal transport. The mucus layers contain a network of Muc2 mucins as the main structural component. Here, the renewal rate of the inner protective mucus layer was studied as well as the production and secretion of Muc2 mucin in the distal colon. This was performed by intraperitoneal injection of N-azidoacetyl-galactosamine (GalNAz) that was in vivo incorporated during biosynthesis of O-glycosylated glycoproteins. The only gel-forming mucin produced in the colon is the Muc2 mucin and as it carries numerous O-glycans, the granulae of the goblet cells producing Muc2 mucin were intensely stained. The GalNAz-labeled glycoproteins were first observed in the Golgi apparatus of most cells. Goblet cells in the luminal surface epithelium had the fastest biosynthesis of Muc2 and secreted material already three hours after labeling. This secreted GalNAz-labeled Muc2 mucin formed the inner mucus layer. The goblet cells along the crypt epithelium accumulated labeled mucin vesicles for a longer period and secretion of labeled Muc2 mucin was first observed after 6 to 8 h. This study reveals a fast turnover (1 h) of the inner mucus layer in the distal colon mediated by goblet cells of the luminal surface epithelium.     

Correlative histochemical study providing evidence for the dual nature of human colorectal cancer mucin

Summary Luminal secretions within colorectal cancers have been assumed to be the counterpart of normal goblet cell mucin. The aim of this study was to establish whether secretory material within colorectal cancers may in fact be traced to different lineages: goblet cells and columnar cells. The distribution of the apomucins MUC1 and MUC2, non-O-acetylated sialic acid and the carbohydrate structures sialosyl Tn, Tn, Lewis^x, sialosyl Lewis^x and Lewis^y was studied in normal colorectal mucosa and colorectal cancer specimens using standard histochemical techniques. Unmasking of MUC1 and MUC2 was achieved using periodic acid and saponification-neuraminidase-periodic acid pretreatment respectively. Within normal and malignant epithelium, correlations and/or co-localization could be demonstrated for goblet cells with MUC2, non-O-acetylated sialic acid, sialosyl Tn, Tn (Golgi region) and sialosyl Lewis^x, and for columnar cells with MUC1, Lewis^x, sialosyl Le^x, Tn (cytoplasm) and Lewis^y (UEA-1). The goblet cell spectrum was associated with mucin-like (type I) luminal secretions within cancers, whereas the columnar cell spectrum characterized non-mucin-like (type II) secretions and intracytoplasmic lumina. These data indicate that colorectal cancer mucin can be broadly separated into two types: secretory mucin linked to cells of goblet lineage and up-regulated membrane-associated mucin of presumed columnar cell origin. This revised version was published online in November 2006 with corrections to the Cover Date.     

nPKCepsilon, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells

Airway goblet cell mucin secretion is controlled by agonist activation of P2Y(2) purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP(3)), diacylglycerol, Ca(2+) and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKCalpha, nPKCdelta, nPKCepsilon, and nPKCeta; of these, only nPKCdelta translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149-L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKCalpha, nPKCdelta, and nPKCeta had the same levels of ATPgammaS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKCepsilon (14.6 and 23.5%, for ATPgammaS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKCepsilon exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPgammaS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y(2)-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKCdelta knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKCepsilon KO mice relative to its WT littermates. We conclude that nPKCepsilon is the effector isoform downstream of P2Y(2)-R activation in the goblet cell secretory response. The translocation of nPKCdelta observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology.     

Ultrastructural localization of epithelial mucin core proteins in colorectal tissues.

Mucins are high molecular weight glycoproteins with a variety of postulated biological functions, including physicochemical protection from toxins and mutagens, adhesion modulation, signal transduction, and regulation of cell growth. Mucins are widely and differentially expressed in the gastrointestinal tract. To date, studies of cellular expression have relied on light microscopy using in situ hybridization and immunohistochemistry. Although informative, it has been difficult with these techniques to ascertain exactly which cell types are producing a given mucin. We studied expression of MUC1, MUC2, and MUC4 apomucins in a series of normal colon biopsies using a combination of immunoelectron microscopy and light microscopy. MUC1 mucin was localized to both goblet and columnar cells, where it was seen in secretory vesicles, microvilli, and in cytoplasmic remnants in goblet cell thecae. MUC2 expression was restricted to goblet cells, in which reactivity was concentrated in the rough endoplasmic reticulum (RER). MUC4 expression was seen in both columnar and goblet cells, localized to the RER. The inability to detect MUC2 and MUC4 apomucins in the Golgi complex and the mature mucous gel probably represents masking of peptide epitopes following O-glycosylation. This study has helped clarify lineage-specific mucin synthesis in the normal colon.     

Calcium signaling in human airway goblet cells following purinergic activation

Despite the general importance of Ca(2+) signaling in signal transduction, and of goblet cell mucin hypersecretion in inflammatory pulmonary diseases, measurement of airway goblet cell intracellular Ca(2+) (Ca(i)(2+)) has not been reported. In this article, we describe the results of experiments measuring Ca(i)(2+) in primary cultures of human bronchial goblet cells after stimulation with the purinergic agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and phorbol 12-myristate 13-acetate (PMA). Ca(2+) signaling in human goblet cells after purinergic stimulation follows the classic paradigm of a Ca(i)(2+) transient from a basal activity of 110 nM to a peak response of 260.1 +/- 41.2 nM within 2 min, followed by a long superbasal plateau (155.3 +/- 0.2 nM) between 10 and 15 min. The rise in Ca(i)(2+) appears to result from a mobilization of intracellular stores, because the transient was nearly abolished by inhibition of PLC with the phosphatidylinositol-specific PLC inhibitor U-73122, and it was not affected significantly by removal of extracellular Ca(2+). Loading goblet cells with BAPTA inhibited the ATPgammaS-induced Ca(2+) transient by 86.0 +/- 13.1%, relative to control. Finally, in contrast to the massive effects of high doses of PMA (300 nM) on mucin secretion from goblet cells, phorbol ester stimulated a small (27.1 +/- 7% of the ATPgammaS control peak), brief rise in Ca(i)(2+). This diminutive signal likely denotes a local Ca(2+) gradient, which may be associated with the mucin granule exocytotic process.     

Cleavage in the GDPH sequence of the C-terminal cysteine-rich part of the human MUC5AC mucin

MUC5AC is the main gel-forming mucin expressed by goblet cells of the airways and stomach where it protects the underlying epithelia. We expressed the C-terminal cysteine-rich part of the human MUC5AC mucin in CHO-K1 cells (Chinese-hamster ovary K1 cells) where it formed disulfide-linked dimers in the ER (endoplasmic reticulum). After reducing the disulfide bonds of these dimers, not only the expected monomers were found, but also two smaller fragments, indicating that the protein was partially cleaved. The site of cleavage was located at an Asp-Pro bond situated in a GDPH (Gly-Asp-Pro-His) sequence found in the vWD4 (von Willebrand D4) domain. This sequence is also found in the human MUC2 mucin, previously shown to be cleaved at the same site by a slow, non-enzymatic process triggered by a pH below 6 [Lidell, Johansson and Hansson (2003) J. Biol. Chem. 278, 13944-13951]. In contrast with this, the cleavage of MUC5AC started already in the neutral ER. However, it continued and was slightly accelerated at a pH below 6.5, a pH found in the later parts of the secretory pathway. The cleavage generated a reactive group in the new C-terminus that could link the protein to a primary amine. No cleavage of MUC5AC has so far been reported. By using an antibody reacting with the C-terminal cleavage fragment, we could verify that the cleavage occurs in wild-type MUC5AC produced by HT-29 cells. The cleavage of MUC5AC and the generation of the reactive new C-terminus could contribute to the adherent and viscous mucus found at chronic lung diseases such as asthma and cystic fibrosis, characterized by mucus hypersecretion and lowered pH of the airways.     

Secretion of endogenous lectin by chicken intestinal goblet cells

The two lactose-binding lectins found in adult chicken intestine, chicken-lactose-lectin-1 (CLL-1) and chicken-lactose-lectin-11 (CLL- 11), were localized within the vesicles of the mucin-secreting goblet cells by indirect immunofluorescence and immunoperoxidase staining methods. Attention was concentrated on CLL-11 which is 200 time more abundant than CLL-1 in adult intestine. The localization of CLL-11 in secretory vesicles, combined with its demonstration on the intestinal epithelial surface by immune staining methods and by specific elution with lactose, suggested that at least a portion of the CLL-11 in the vesicles was secreted by the goblet cells and then became associated with the mucosal surface. In support of this, treatment of isolated intestinal strips with a cholinergic agent, bethanechol (10(-7 M) produced a small but significant increase in the amount of CLL-11 that could be eluted from their surface with lactose. Secretion of lectin may occur in conjunction with mucin because both are localized in the secretory vesicles and CLL-1 and CLL-11 apparently bind to purified chicken intestinal mucin, which is a potent inhibitor of their hemagglutination activities. The mucin is six orders of magnitude more potent than lactose as a hemagglutination inhibitor of CLL-1 or CLL-11 on a molar basis, and three orders of magnitude more potent when expressed per mole of hexose. These results suggest that CLL-11, and perhaps CLL-1, are secreted from the goblet cells along with mucin. They may function in the organization of mucin for secretion and/or in its association with the intestinal mucosal surface.     

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G_q-coupled P2Y₂ receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of - and -actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of - or -actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca²⁺-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis. lung; mucus; exocytosis     

Lectin histochemistry of 1,2-dimethylhydrazine-induced rat colon neoplasia

Lectins linked to fluorescein were used as carbohydrate probes to examine the goblet cell mucin and epithelial cell surface glycoconjugate alterations in an experimental rodent model of colonic neoplasia induced with parenteral 1,2-dimethylhydrazine dihydrochloride. Lectins derived from Triticum vulgare (WGA), Ricinus communis (RCA1), and Limulus polyphemus (LPA) showed reduced labeling of goblet cell mucin in these tumors, while binding with peanut lectin from Arachis hypogaea (PNA), a lectin ordinarily failing to bind to mucin in normal colon, was positive. In addition, RCA1 and LPA showed increased cell surface labeling of neoplastic epithelial cells. Finally, alterations were observed in lectin binding to "transitional" colonic mucosa adjacent to colonic tumors from carcinogen-treated rats. These findings indicate that significant alterations in both membrane and mucin glycoconjugates occur in colonic tumors and mucosa adjacent to tumors in a chemically induced experimental animal model of human colon cancer.     

Use of an improved zirconyl hematoxylin stain in the diagnosis of Barrett's esophagus

Barrett's esophagus is a precancerous condition characterized by replacement of the normal stratified squamous epithelium by a simple columnar epithelium with goblet cells that secrete an acidic mucin. As originally formulated, fresh solutions of zirconyl hematoxylin stain goblet cells poorly. An improved formula, quintupling the amount of oxidant, yields zirconyl hematoxylin solutions that stain goblet cells darkly even when fresh. The improved zirconyl hematoxylin can be used in place of alcian blue in the diagnosis of Barrett's esophagus. The ingredients of zirconyl hematoxylin are always readily available and are generally recognized as safe.     

Regulation of mucin secretion and inflammation in asthma: a role for MARCKS protein?

BACKGROUND: 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). GENERAL SIGNIFICANCE: In this review, properties of MARCKS and how the protein may regulate mucin secretion at a cellular level will be discussed. In addition, the roles of MARCKS in airway inflammation related to both influx of inflammatory cells into the lung and release of granules containing inflammatory mediators by these cells will be explored. This article is part of a Special Issue entitled: Biochemistry of Asthma.