Laurie L. Shekels

Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression

BACKGROUND & AIMS Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunners glands of the duodenum. CONCLUSIONS MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.

Selective induction of mucin-3 by hypoxia in intestinal epithelia.

Epithelial cells line mucosal surfaces (e.g., lung, intestine) and critically function as a semipermeable barrier to the outside world. Mucosal organs are highly vascular with extensive metabolic demands, and for this reason, are particularly susceptible to diminished blood flow and resultant tissue hypoxia. Here, we pursue the hypothesis that intestinal barrier function is regulated in a protective manner by hypoxia responsive genes. We demonstrate by PCR confirmation of microarray data and by avidin blotting of immunoprecipitated human Mucin 3 (MUC3), that surface MUC3 expression is induced in T84 intestinal epithelial cells following exposure to hypoxia. MUC3 RNA is minimally detectable while surface protein expression is absent under baseline normoxic conditions. There is a robust induction in both the mRNA (first evident by 8 h) and protein expression, first observed and maximally expressed following 24 h hypoxia. This is followed by a subsequent decline in protein expression, which remains well above baseline at 48 h of hypoxia. Further, we demonstrate that this induction of MUC3 protein is associated with a transient increase in the barrier restorative peptide, intestinal trefoil factor (ITF). ITF not only colocalizes with MUC3, by confocal microscopy, to the apical surface of T84 cells following exposure to hypoxia, but is also found, by co‐immunoprecipitation, to be physically associated with MUC3, following 24 h of hypoxia. In exploration of the mechanism of hypoxic regulation of mucin 3 expression, we demonstrated by luciferase assay that the full‐length promoter for mouse Mucin 3 (Muc3) is hypoxia‐responsive with a 5.08 ± 1.76‐fold induction following 24 h of hypoxia. Furthermore, analysis of both the human (MUC3A) and mouse (Muc3) promoters revealed potential HIF‐1 binding sites which were shown by chromatin immunoprecipitation to bind the pivotal hypoxia‐regulating transcription factor HIF‐1α. Taken together, these studies implicate the HIF‐1α mediated hypoxic induced expression of mucin 3 and associated ITF in the maintenance of intestinal barrier function under hypoxic conditions. J. Cell. Biochem. 99: 1616–1627, 2006.

The Adherent Gastric Mucous Layer Is Composed of Alternating Layers of MUC5AC and MUC6 Mucin Proteins

Mucin-type glycoproteins are the major structural proteins in gastric mucus. Stomach mucin proteins include MUC5AC, synthesized by surface foveolar or pit cells, and MUC6, synthesized by neck and gland cells. The aim of this study was to determine the spatial distribution of these mucin proteins within the extracellular mucous coat. Double-labeling immunoflourescence/confocal microscopy was used in histologically normal surgical resection specimens. Intralumenal mucin within antral glands consisted entirely of MUC6 protein. Intralumenal mucin within the gland isthmus region consisted of an irregular mixture of MUC5AC and MUC6. The mucous layer on the gastric surface consisted primarily of MUC5AC extending in layered sheets with MUC6 protein layered in between. The laminated appearance of the surface mucus was present in both H. pylori-infected and noninfected specimens. These data indicate that MUC5AC and MUC6 proteins remain segregated within the mucous gel in a laminated linear arrangement. The physical stratification of mucin proteins may confer increased strength to the mucous layer or represent independent and redundant protection.

Coordinated Muc2 and Muc3 Mucin Gene Expression in Trichinella spiralis Infection in Wild-Type and Cytokine-Deficient Mice

Mucin hypersecretion is an important component of the immune response to gastrointestinal nematode infection. Two discrete types of mucin proteins exist in the mouse intestine, secretory Muc2 and membrane-bound Muc3. We examined Muc2 and Muc3 expression in wild-type mice and mice lacking gamma interferon receptor (IFNγR−/−), tumor necrosis factor receptor 1 (TNFR1−/−) and interleukin 4 (IL4−/−) infected with Trichinella spiralis. Infected wild-type mice demonstrated significant goblet cell hyperplasia and increased mucin glycoprotein. In situ hybridization showed this was accompanied by increases in Muc2 and Muc3 mRNA. Total intestinal mucin protein and Muc2 and Muc3 mRNA levels were also significantly increased in cytokine-deficient mice. These data demonstrate the coordinated up-regulation of two types of mucin genes in response to T. spiralis infection and may form the basis of an innate mucosal response independent of IFN-γ, TNF, and IL-4.

Tauroursodeoxycholic acid protects in vitro models of human colonic cancer cells from cytotoxic effects of hydrophobic bile acids.

Bile acids have been implicated as tumor promoters that enhance epithelial proliferation and the development of colonic tumors. This study investigated the effects of bile acids on the growth of in vitro models of human colonic epithelial cells. Cell lines with varying degrees of differentiation (Caco2, HT29, LS174T, and Lovo) were studied. Cell viability and number were measured by a tetrazolium (MTT) spectrophotometric assay. Enhanced cell growth was not observed with any bile acid over the range 10 nmol/L to 2.5 mmol/L. Cytotoxicity was consistently observed at concentrations of unconjugated bile acids greater than 0.1 mmol/L. The bile acid concentration at which 50% growth inhibition occurred was similar for all cell lines and increased in the following order: deoxycholic acid = chenodeoxycholic acid < taurodeoxycholic acid < ursodeoxycholic acid < taurochenodeoxycholic acid < cholic acid < tauroursodeoxycholic acid. Coincubation of tauroursodeoxycholic acid (TUDC) with taurodeoxycholic acid (TDC) or taurochenodeoxycholic acid (TDCD) reversed the short-term (30-minute) cytotoxicity and release of glycoprotein induced by TDC or TCDC regardless of differentiation status. In contrast, TUDC did not reverse the cytotoxicity of deoxycholic acid. Unconjugated ursodeoxycholic acid did not alter short-term cytotoxicity of any bile acid. These data indicate that bile acids do not stimulate cell growth in undifferentiated or differentiated colon cancer cell lines, in contrast to normal colonic epithelium in vivo. Bile acid cytotoxicity correlates with the relative hydrophobicity of the bile acid. Because tauroursodeoxycholic acid alters the cytotoxicity of hydrophobic bile acids in vitro, further understanding of bile acid interactions in the colon may have important implications in altering tumor promotion.

Bile acid-induced alterations of mucin production in differentiated human colon cancer cell lines

Damage to the gastrointestinal tract mucous layer may render underlying cells susceptible to intraluminal toxins or carcinogens. Our aim was to determine the effect of bile acids on mucin, the primary constituent of mucous. Differentiated Caco-2 and HT29 cells were used as models of human colonic epithelial cells. Mucin was measured by [3H]-glucosamine labeling. Short term (30 min) incubations with 1-5 mM unconjugated bile acids or taurodeoxycholic acid induced mucin release relative to bile acid hydrophobicity. Longer incubations were cytotoxic. Long term (7 days) incubation at nontoxic concentrations (0.1 mM) of deoxycholic acid (DC) decreased total mucin by 36 +/- 2% (SEM, P = 0.0003) in differentiated HT29 cells and by 57.2 +/- 2% (P < 0.05) in Caco-2 cells. Tauroursodeoxycholic acid (TUDC) or ursodeoxycholic acid (0.1-0.5 mM) did not alter mucin levels. Simultaneous incubation of 0.1 mM DC and 0.1-0.5 mM TUDC or 2.5 mM TDC and TUDC did not change mucin levels. Differentiated HT29 and Caco-2 cells contained high levels of intestinal mucin MUC3 mRNA while undifferentiated HT29 cells did not possess a MUC3 message. Deoxycholic acid (0.1 mM) did not alter the MUC3 mRNA level. Neither cell type showed detectable expression of intestinal MUC2 or gastric MUC6. Thus, cytotoxic concentrations of bile acids induce mucin release, presumably due to detergent effects. Nontoxic concentrations of DC reduce mucin levels in differentiated enterocyte-like cells, which can be prevented by coincubation with TUDC. The bile acid-induced alterations in mucin production by enterocytes observed in vitro may influence intestinal cytoprotection in vivo.

Analysis of the ACP1 gene product : classification as an FMN phosphatase

The relationship between the ACP1 gene product, an 18kDa acid phosphatase (E.C. 3.1.3.2) postulated to function as a protein tyrosyl phosphatase, and the cellular flavin mononucleotide (FMN) phosphatase has been examined in vitro and by using cultured Chinese hamster ovary (CHO) cells. Kinetic analysis indicated that at pH 6 the acid phosphatase utilized a variety of phosphate monoesters as substrates. While small molecules such as FMN were effectively utilized as substrates (kcat/Km = 7.3 x 10(3) s-1M-1), the tyrosyl phosphorylated form of the adipocyte lipid binding protein was a relatively poor substrate (kcat/Km = 1.7 x 10(-1) s-1M-1) suggesting a role for the phosphatase in flavin metabolism. Fractionation of CHO cell extracts revealed that 90% of the FMN phosphatase activity was soluble and that all of the soluble activity eluted from a Sephadex G-75 column with the acid phosphatase. All of the soluble FMN phosphatase activity was inhibited by immunospecific antibodies directed against the bovine heart ACP1 gene product. These results suggest that the ACP1 gene product functions cellularly not as a protein tyrosyl phosphatase but as a soluble FMN phosphatase.

Altered mucin core peptide expression in acute and chronic cholecystitis

Human mucin genes include membrane-bound mucins (MUC1, MUC3, MUC4) and secretory mucins (MUC2, MUC5AC, MUC5B, MUC6). Our aim was to determine mucin gene expression in human gallbladder cell lines, normal gallbladder from liver donors (N = 7) and surgical specimens with mild chronic cholecystitis (N = 29), chronic cholecystitis (N = 48), and acute and chronic cholecystitis (N = 27). MUC1 mRNA was ubiquitous; however, only rare MUC1 immunoreactivity was detected. MUC3, MUC5AC, MUC5B, and MUC6 mRNA were present in all gallbladder specimens and cell lines examined. Prominent MUC3, MUC5AC, MUC5B, and MUC6 immunoreactivity was present in 86–100% of normal gallbladders. The frequency of MUC5AC reactivity was decreased in specimens with acute cholecystitis (P < 0.05). In contrast, MUC2-reactivity was absent in normal gallbladder and present in 53.8% of acute cholecystitis specimens (P < 0.05). Surface epithelium is characterized by MUC3, MUC5AC, and MUC5B, whereas deeper mucosal folds display MUC5B and MUC6 immunoreactivity. Gallbladder epithelium demonstrates a unique and diverse pattern of mucin core proteins that becomes altered with increasing degrees of inflammation.

Mucin Gene Expression in the Rat Middle Ear: An Improved Method for Rna Harvest

Mucins are heavily glycosylated proteins characterized by high molecular weight and heterogeneous structure. Mucin genes are expressed in a tissue- or epithelium-specific manner. Although mucins are known to be important structural components of the mucociliary transport system that protects epithelium against invading microorganisms, very little is known about mucin gene expression unique to the middle ear. This study demonstrated that middle ear messenger RNA specifically hybridized with rat MUC2 and human MUC2 (SMUC-41) complementary DNA probes. MUC3 and MUC5AC mucin genes, dominantly expressed in rodent intestine and trachea, were not detected in the rat middle ears in this study. The middle ear MUC2 messenger RNA harvested by lavage was characterized by a single transcript — unlike its counterpart in intestine and airways, which is characterized by polydispersity — suggestive of a better method for RNA analysis. It was concluded that rat middle ears possess a MUC2 mucin gene or homologue of human MUC2 (SMUC-41).

Human intestinal MUC17 mucin augments intestinal cell restitution and enhances healing of experimental colitis

UNLABELLED The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and are thought to be cytoprotective. The extracellular regions of these mucins contain EGF-like Cys-rich segments (CRD1 and CRD2) connected by an intervening linker domain (L). The purpose of this study was to determine the functional activity of human MUC17 membrane-bound mucin. METHODS Endogenous MUC17 was inhibited in LS174T colon cells by stable transfection of a small hairpin RNA targeting MUC17 (LSsi cells). The effect of recombinant MUC17-CRD1-L-CRD2 protein on migration, apoptosis, and experimental colitis was determined. RESULTS Reduced MUC17 expression in LSsi cells was associated with visibly reduced cell aggregation, reduced cell-cell adherence, and reduced cell migration, but no change in tumorigenicity. LSsi cells also demonstrated a 3.7-fold increase in apoptosis rates compared with control cells following treatment with etoposide. Exposure of colonic cell lines to exogenous recombinant MUC17-CRD1-L-CRD2 protein significantly increased cell migration and inhibited apoptosis. As a marker of biologic activity, MUC17-CRD1-L-CRD2 proteins stimulate ERK phosphorylation in colonic cell lines; and inhibition of ERK phosphorylation reduced the anti-apoptosis and migratory effect of MUC17-CRD1-L-CRD2. Finally, mice treated with MUC17-CRD1-L-CRD2 protein given per rectum demonstrated accelerated healing in acetic acid and dextran sodium sulfate induced colitis in vivo. These data indicate that both native MUC17 and the exogenous recombinant cysteine-rich domain of MUC17 play a role in diverse cellular mechanisms related to cell restitution, and suggest a potential role for MUC17-CRD1-L-CRD2 recombinant protein in the treatment of mucosal inflammatory diseases.