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 (IFNgammaR-/-), 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-gamma, TNF, and IL-4.

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).

Cloning and characterization of mouse intestinal MUC3 mucin: 3' sequence contains epidermal-growth-factor-like domains.

Mucin glycoproteins are a heterogeneous family of high-molecular-mass, heavily glycosylated proteins differentially expressed in epithelial tissue of the gastrointestinal, reproductive and respiratory tracts. We report here the cloning of a mouse caecal mucin (MCM). Amino acid analysis of purified MCM revealed a high content of serine (10.8%) and threonine (25.1%). Antibodies against deglycosylated MCM were prepared for immunohistochemical analysis and for screening a mouse caecal cDNA library. Immunohistochemical analysis showed strong staining of goblet cells and patchy staining of surface columnar cells in the duodenum, small intestine, caecum, colon and rectum. Screening of a mouse caecal cDNA library yielded clones containing tandem repeats of 18 bp with two predominant peptide sequences of TTTADV and TTTVVV. The tandem repeat domain is followed by 1137 bp of non-repetitive sequence and 521 bp of 3' untranslated sequence prior to the poly(A) tail. Two cysteine-rich regions lie within the 3' non-repetitive domain. The arrangement of the cysteines within these regions corresponds to epidermal growth factor-like domains. Following the second cysteine-rich region is a stretch of 19 hydrophobic amino acids which may act as a transmembrane domain or allow for interaction with hydrophobic molecules. Northern blot analysis indicates the mRNA is approximately 13.5 kb with greatest expression in the caecum and lesser amounts in the colon and small intestine. No MCM message is found in mouse stomach, trachea, lung, kidney, oesophagus or pancreas. In situ hybridization studies show that MCM message is expressed at the tips of villi in the intestine and in the upper crypts and surface cells of the caecum and colon. Chromosomal analysis assigns this gene to mouse chromosome 5 in a region of conserved linkage with human chromosome 7, the location of the human MUC3 gene. We conclude that we have identified a mouse caecal mucin which represents the mouse homologue of human MUC3. The mouse MUC3 cDNA sequence suggests that it is a novel non-polymerizing mucin which may participate in membrane or intermolecular interactions through its 3' non-repetitive region.

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.

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.

Mouse gastric mucin: cloning and chromosomal localization.

Mucins protect gastric epithelium by maintaining a favourable pH gradient and preventing autodigestion. The purpose of this study was to clone a mouse gastric mucin which would provide a foundation for analysis of mucin gene regulation. Mucin was purified from the glandular portion of gastric specimens and deglycosylated by HF solvolysis. Antibodies against native and deglycosylated mouse gastric mucin (MGM) were raised in chickens. Screening of a mouse stomach cDNA library with the anti-(deglycosylated MGM) antibody yielded partial clones containing a 48 bp tandem repeat and 768 bp of non-repetitive sequence. The 16-amino-acid tandem repeat has a consensus sequence of QTSSPNTGKTSTISTT with 25% serine and 38% threonine. The MGM tandem repeat sequence bears no similarity to previously identified mucins. The MGM non-repetitive region shares sequence similarity with human MUC5AC and, to a lesser extent, human MUC2 and rat intestinal mucin. Northern blot analysis reveals a polydisperse message beginning at 13.5 kb in mouse stomach with no expression in oesophagus, trachea, small intestine, large intestine, caecum, lung or kidney. Immunoreactivity of antibodies against deglycosylated MGM and against a synthetic MGM tandem repeat peptide was restricted to superficial mucous cells, antral glands and Brunner's glands in the pyloric-duodenal region. DNA analysis shows that MGM recognizes mouse and rat DNA but not hamster, rabbit or human DNA. The MGM gene maps to a site on mouse chromosome 7 homologous to the location of a human secretory mucin gene cluster on human chromosome 11p15. Due to sequence similarity and predominant expression in the stomach, the MGM gene may be considered a MUC5AC homologue and named Muc5ac.

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 Brunner's glands of the duodenum. CONCLUSIONS: MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.

Mucin gene expression in normal, preneoplastic, and neoplastic human gastric epithelium.

Mucins synthesized by malignant cells may contribute (via decreased cellular adhesion and immune recognition) to cancer invasion and metastases. Human mucins are derived from a heterogeneous family of genes, labeled MUC1-6. Our aim was to determine the pattern of mucin gene expression in normal, preneoplastic (intestinal metaplasia), and malignant gastric specimens. Probes and antibodies for specific mucin tandem repeat sequences were used for RNA and immunohistochemical analysis. Normal stomach mucosa was characterized by expression of MUC1, MUC5, and MUC6 mRNA and immunoreactive protein, without MUC2, MUC3, and MUC4 gene expression. In contrast, high levels of MUC2 and MUC3 mucin mRNA and immunoreactive protein were found in specimens with intestinal metaplasia. Gastric cancers exhibited markedly altered secretory mucin mRNA levels compared with adjacent normal mucosa, with decreased levels of MUC5 and MUC6 mRNA and increased levels of MUC3 and MUC4 mRNA. Overall, immunoreactive MUC1 mucin was detected in 72% of 33 gastric cancers, and secretory mucin core peptides were expressed in 34% (MUC2), 45% (MUC3), 19% (MUC5), and 57% (MUC6) of these specimens. Coexpression of multiple (three or more) mucin core proteins occurred in 15 of 25 (60%) advanced (stages III and IV) cancers compared with 1 of 8 (12.5%) early (stages I and II) cancers (P < 0.048). We conclude that human gastric epithelium has a unique mucin gene pattern, which becomes markedly altered in preneoplastic and neoplastic specimens. Increased mucin gene heterogeneity in gastric adenocarcinomas is associated with advanced cancer stage.

Experimental model of upper intestinal adenocarcinoma induced by N-methyl-N'-nitro-N-nitrosoguanidine in C57BL/6 mice.

The purpose of this study was to develop a model of gastrointestinal carcinogenesis using C57BL/6 mice. Treatment regimens consisted of one control group and 2 groups which received N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in drinking water: 50 micrograms/ml x 52 weeks and 100 micrograms/ml x 27 weeks. In addition, 2 protocols using adjuvant agents intended to increase tumor formation were used: MNNG (100 micrograms/ml) x 27 weeks + 0.2% taurocholic acid added to the diet from weeks 13-52, and MNNG (50 micrograms/ml) x 33 weeks+caerulein (10 micrograms/kg) subcutaneously 3 times/week from weeks 21-52. High-grade dysplasia was observed in the duodenum of 1/13 mice treated with MNNG (50 micrograms/ml). The combination of the latter and caerulein did not augment tumorigenesis. Mice treated with MNNG (100 micrograms/ml) frequently developed neoplasia in the duodenum and upper jejunum. Foci of low-grade and high-grade dysplasia alone were found in 3/12 (25%) mice; and intramucosal and invasive adenocarcinoma were found in 7/12 (58.3%) mice. The addition of taurocholic acid significantly increased the number and histological stages of the tumors (adenocarcinoma occurred in 100%, P = 0.03) and decreased the time for tumor formation.

Synthesis of phosphotyrosine-containing peptides and their use as substrates for protein tyrosine phosphatases.

Prior methods for the chemical synthesis of phosphotyrosine-containing peptides involved the incorporation of fully protected phosphoamino acids into the peptide chain or phosphorylation of free phenol side chains after peptide assembly is complete. The present work describes a novel and general methodology for the solid-phase synthesis of phosphopeptides, featuring direct incorporation of N alpha-(9-fluorenylmethyloxycarbonyl)-O-phospho-L-tyrosine (unprotected side chain). This technique obviated the formation of peptide byproducts containing tyrosine H-phosphonate, a previously unrecognized side reaction from literature phosphorylation/oxidation approaches. Phosphopeptides corresponding to the tyrosine phosphorylation site of adipocyte lipid binding protein were synthesized by the newer, preferred method. These peptides were purified and characterized by high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), amino acid analysis (AAA), fast atom bombardment mass spectrometry (FABMS), and 31P nuclear magnetic resonance (31P NMR). The synthetic peptides were tested as substrates for two distinct protein tyrosine phosphatases, rat brain protein tyrosine phosphatase (PTPase) and human acid phosphatase. Substrate specificity was measured at pH 6.0 and 37 degrees C, using a colorimetric assay for released inorganic phosphate. Kinetic analysis revealed that both the rat brain PTPase and the human adipocyte acid phosphatase catalyzed peptide dephosphorylation but with different rates and affinities. The rat brain PTPase displayed classical Michaelis-Menten kinetics, with Km's of 68 +/- 9 microM and 42 +/- 11 microM and kcat/Km values of 4.9 x 10(5) s-1 M-1 and 6.9 x 10(5) s-1 M-1 determined for phosphorylated peptides of lengths 4 and 10 residues, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.

In vitro phosphorylation of the adipocyte lipid-binding protein (p15) by the insulin receptor. Effects of fatty acid on receptor kinase and substrate phosphorylation.

Phosphorylation of the adipocyte lipid-binding protein (ALBP) isolated from 3T3-L1 cells has been studied in vitro utilizing the wheat germ agglutinin-purified 3T3-L1 adipocyte insulin receptor and the soluble kinase domain of the human insulin receptor. Following insulin-stimulated, ATP-dependent autophosphorylation of the wheat germ agglutinin-purified receptor beta-subunit, ALBP was phosphorylated exclusively on tyrosine 19 in the sequence Glu-Asn-Phe-Asp-Asp-Tyr19, analogous to the substrate phosphorylation consensus sequence observed for several tyrosyl kinases. The concentration of insulin necessary for half-maximal receptor autophosphorylation (KIR0.5) was identical to that necessary for half-maximal ALBP phosphorylation (KALBP0.5), 10 nM. Kinetic analysis indicated that stimulation of ALBP phosphorylation by insulin was attributable to a 5-fold increase in the Vmax (to 0.33 fmol/min/fmol insulin-binding sites) while the Km for ALBP was largely unaffected. By utilizing the soluble kinase domain of the human receptor beta-subunit, the presence of oleate bound to ALBP increased the kcat/Km greater than 3-fold. Oleate dramatically inhibited autophosphorylation of the 38-kDa fragment of the soluble receptor kinase in a concentration dependent fashion (I0.5 approximately 4 microM). The 48-kDa kinase exhibited much less sensitivity to the effects of oleate (I0.5 approximately 190 microM). The inhibition of autophosphorylation of the 48-kDa soluble kinase by oleate was reversed by adding saturating levels of ALBP. These results demonstrate that in vitro the murine adipocyte lipid-binding protein is phosphorylated on tyrosine 19 in an insulin-stimulated fashion by the insulin receptor and that the presence of a bound fatty acid on ALBP increases the affinity of insulin receptor for ALBP. Inhibition of insulin receptor kinase activity by unbound fatty acids suggests that the end products of the lipogenic pathway may feedback inhibit the tyrosyl kinase and that fatty acid-binding proteins have the potential to modulate such interaction.