Mucin gene expression in biliary epithelial cells

Background/Aims: In recent years considerable advances have been made in our knowledge of human mucin genes. Although analysis of their genomic organization is still in progress, the pattern of their expression in different human mucosae is now fairly well established. However, little is known about their expression in the biliary tree. In this study we determined the pattern of expression of the different human mucin genes in gallbladder biliary epithelial cells, intrahepatic bile ducts and liver.Methods: Two complementary methods were used: Northern-blot and in situ hybridization analyses. The experiments were performed with eight probes corresponding to MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6 and MUC7.Results: Our results revealed a strong mRNA expression of MUC3, MUC6 and MUC5B, a week expression of MUC1, MUC5AC and MUC2, and no expression of MUC4 and MUC7. Surprisingly, MUC3, which was the gene which was most expressed in the biliary tree, was also found in hepatocytes, suggesting another function for the MUC3 protein than that of a secreted mucin.Conclusions: We conclude that MUC3, MUC6 and MUC5B were the main mucin genes expressed in biliary epithelial cells.     

Mucin gene expression in intestinal epithelial cells in Crohn's disease

BACKGROUND: Crohn's disease (CD) is a chronic relapsing inflammatory bowel disease of unknown origin. It is characterised by chronic mucosal ulcerations which affect any part of the intestine but most commonly are found in the ileum and proximal colon. AIMS: Studies were undertaken to provide information regarding cell specific expression of mucin genes in the ileum of patients with CD. PATIENTS AND METHODS: Expression of mucin genes was analysed in the ileal mucosa of patients with CD and controls by in situ hybridisation and immunohistochemistry. RESULTS: In healthy ileal mucosa, patients with CD showed a pattern identical to normal controls with main expression of MUC2 and MUC3, lesser expression of MUC1 and MUC4, and no expression of MUC5AC, MUC5B, MUC6, or MUC7. In the involved mucosa, the pattern was somewhat comparable although heterogeneous to that observed in healthy ileal mucosa. Importantly, a particular mucin gene expression pattern was observed in ileal mucosa close to the ulcer margins in ulcer associated cell lineage, with the appearance of MUC5AC and MUC6 mRNAs and peptides, which are normally restricted to the stomach (MUC5AC and MUC6) and duodenum (MUC6), and disappearance of MUC2. CONCLUSIONS: Our results suggest that gel forming mucins (more particularly MUC5AC and MUC6) may have a role in epithelial wound healing after mucosal injury in inflammatory bowel diseases in addition to mucosal protection.     

Mucin gene expression in human embryonic and fetal intestine

Background—The intestinal epithelium is coveredby a continuous layer of mucus which is secreted by well differentiatedepithelial cells. Disregulation of the expression of mucins has beenreported to have possible implications in the neoplastic process which affects intestinal mucosae. It is well known that preneoplastic andneoplastic tissues can express fetal phenotypic characteristics.
Aims—To assess whether the expression of mucingenes in the intestinal tract is linked to the stage of cellulardifferentiation and tissue development, by studying the expression ofsix mucin genes in human fetal small intestine and colon, and alsoadult tissues.
Methods—In situ hybridisation was used to studymRNA expression of MUC2, MUC3, MUC4, MUC5B, MUC5AC, and MUC6 in 32 human embryos and fetuses (6.5-27 weeks gestation). Normal adultmucosae were used as controls.
Results—Three mucin genes, MUC2, MUC4, andMUC5AC, were differently expressed in fetal intestine compared withexpression in normal adults.
Conclusion—These differences in mucin geneexpression suggest a possible regulatory role for these products inintestinal epithelial cell differentiation.

Keywords:mucin genes; mucins; intestine; differentiation; human fetus

     

Mucin gene expression in Barrett's oesophagus: an in situ hybridisation and immunohistochemical study

BACKGROUND AND AIMS: Mucin genes are expressed in a site specific manner throughout the gastrointestinal tract. Little is known about the expression pattern in the oesophagus. In this study we have investigated MUC gene expression in both the normal oesophagus and specialised intestinal metaplasia (Barrett's oesophagus). PATIENTS: Archived paraffin embedded material from eight specimens of normal oesophagus, 18 Barrett's oesophagus, eight gastric metaplasia, six high grade dysplasia, and six cases of adenocarcinoma were examined for expression of the mucin genes MUC1-6. METHODS: Mucin mRNA was detected by in situ hybridisation using [(35)S] dATP labelled oligonucleotide probes. Mucin core protein was detected by immunohistochemistry. RESULTS: Normal oesophagus expressed MUC5B in the submucosal glands and MUC1 and MUC4 in the stratified squamous epithelium. Barrett's oesophagus strongly expressed MUC5AC and MUC3 in the superficial columnar epithelium, MUC2 in the goblet cells, and MUC6 in the glands. In high grade dysplasia and adenocarcinoma there was downregulation of MUC2, MUC3, MUC5AC, and MUC6, but upregulation of MUC1 and MUC4 in half of the specimens examined. CONCLUSIONS: Normal oesophagus and Barrett's oesophagus have a novel pattern of mucin gene expression. Barrett's oesophagus expressed the mucins associated with normal gastric epithelium and normal intestinal epithelium. While most mucin genes were downregulated in severely dysplastic and neoplastic tissues, there was upregulation of the membrane bound mucins MUC1 and MUC4. This may prove useful in detecting early signs of progression to adenocarcinoma of the oesophagus.     

Altered Mucin Gene Expression in Stone-Containing Intrahepatic Bile Ducts and Cholangiocarcinomas

Neoplastic transformation of epithelial cells is commonly associated with alterations in the synthesis and structures of mucin. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Clinically, hepatolithiasis has been regarded as a risk factor for cholangiocarcinoma. The aims of this study were to determine the possible alteration of mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas and to try to predict whether or not hepatolithiasis has a predisposition to development of cholangiocarcinoma. In situ hybridization with DIG-tailed oligonucleotides was performed on sections of paraffin-embedded tissues of stone-containing intrahepatic bile ducts, cholangiocarcinomas, and normal controls to identify the expression of MUC2, MUC3, MUC4, MUC5B, and MUC5AC in nonneoplastic and neoplastic biliary epithelium. The findings showed that (1) while multiple diverse mucin genes were expressed in the biliary epithelium, MUC3 and MUC5B mRNA were the main mucin genes expressed in the biliary epithelium of stone-containing intrahepatic bile ducts and normal controls; (2) absent or decreased expression of MUC2, MUC3, and MUC5B of mRNA was found in cholangiocarcinomas in contrast to nonneoplastic biliary epithelium; and (3) increased expression of MUC4 and MU5AC of mRNA was found in cholangiocarcinomas and the biliary epithelium, especially for dysplastic cells of stone-containing intrahepatic bile ducts compared with normal controls. In this study, using in situ hybridization we demonstrated that neoplastic transformation of the biliary epithelium is accompained by alterations in mucin gene expression, the altered mucin gene expression in dysplastic cells of stone-containing intrahepatic bile ducts may reflect a higher potential for malignant transformation in these cells, and it could be a precursor of cholangiocarcinoma in the presence of hepatolithiasis.     

Inhibition of gastric mucin synthesis by Helicobacter pylori

BACKGROUND & AIMS: Mucins are high-molecular-weight glycoproteins that protect the gastric epithelium. Previous data suggested that gastric surface-type mucin is decreased in Helicobacter pylori-infected patients and restored after eradication of the infection. Our aim was to determine the effect of H. pylori on mucin synthesis in cultured gastric epithelial cells. METHODS: Mucin synthesis was measured by labeling with [(3)H]glucosamine and size-exclusion chromatography. Expression of MUC5AC and MUC1 mucin protein antigens was quantitated by Western blot analysis. RESULTS: Mucin synthesis was inhibited more than 80% when KATO III cells were incubated with H. pylori, with no effect on mucin secretion or degradation. Inhibition was rapid (4 hours), partially reversible, dependent on concentration of bacteria, and associated with the insoluble membrane fraction. H. pylori decreased levels of MUC5AC and MUC1 mucins. MUC1 inhibition was half-maximal by 4 hours and partially reversed by 24 hours, but the decrease in MUC5AC was less rapid and not reversible within 24 hours. CONCLUSIONS: H. pylori inhibits total mucin synthesis in vitro and decreases the expression of MUC5AC and MUC1. A decrease in gastric mucin synthesis in vivo may disrupt the protective surface mucin layer.     

Mucin gene expression in gallbladder epithelium with black pigment stone ascertained by in situ hybridization

Black pigment stones are usually found in patients with liver cirrhosis or hemolytic disease. Mucoglycoproteins are present in a significant amount in black pigment stones and contribute to the matrix of gallstones. Epithelium of stone-containing gallbladders contains much more mucin than those without stones. In this study, we try to determine by in situ hybridization the mucin gene expression in black stone-containing gallbladders and try to find the diversity of mucin gene expression in gallbladders containing black pigment stones and those without stones. In situ hybridization with DIG-tailed oligonucleotides was performed on sections of paraffin-embedded tissues of gallbladders with black pigment stones (n = 10) and those without stones (n = 6) to identify the expression of MUC1, MUC2, MUC3, MUC4, MUC5B and MUC6 in gallbladder epithelium. The findings showed that (1) mRNA expression of MUC1, MUC3, MUC5B and MUC6 were found in all gallbladders with black pigment stones, while they were expressed in 33.3%, 83.3%, 83.3% and 66.7% respectively in those without stones. They were expressed more strongly and extensively in gallbladders with stones when compared to those without stones. (2) MUC2 and MUC4 labeling were absent in gallbladders without stones, while they were present in 20% and 60% of gallbladders with black pigment stones, respectively. We conclude that MUC3, MUC5B and MUC6 were the main mucin gene expression in either gallbladder with or without stones. Altered mucin gene expression occurred in gallbladders with black pigment stones, such as the presence of MUC2 and MUC4 and increased expression of MUC1, MUC3, MUC5B and MUC6 in black stone-containing gallbladders. The higher incidence and stronger labeling intensity of mucin gene expression of MUC2, MUC3, MUC5B and MUC6 in black stone-containing gallbladder may reflect abundant mucin content in these gallbladders. Increased expression of MUC2 and MUC4 in black stone-containing gallbladder epithelium indicated that intestinal metaplasia and altered mucin genes could occur in diseased gallbladders.     

MUC1 expression in human breast cancer cells is altered by the factors affecting cell proliferation

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. In the present study, we have investigated if the factors affecting cells proliferation could influence MUC1 mucin biosynthesis and shedding from cell surface into the culture medium in two human breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ER-). Using MCF-7 line we found that estradiol at a concentration of 10(-7) M increased [3H]glucosamine incorporation into mucin in cell lysate approximately twofold in comparison with control cultures, and a similar increase was observed in the culture medium. The selective estrogen receptor modulator, tamoxifen (at concentrations of 10(-6) M and 10(-5) M) had a little inhibitory effect. MDA-MB-231 cells in culture were stimulated with phorbol ester PMA, the protein kinase C activator. We noted that PMA greatly stimulated MUC1 synthesis and its shedding to culture medium and that this effect was abolished by protein kinase C specific inhibitor--bisindolylmaleimide.     

Abnormalities in mucin gene expression in Crohn's disease

Alterations in the structure and/or quantity of mucins could alter the barrier function of mucus and play a role in initiating and maintaining mucosal inflammation in Crohn's disease. To investigate the hypothesis of a mucin gene defect in Crohn's disease, we analyzed the expression of the different mucin genes in the ileal mucosa of patients with Crohn's disease and controls. mRNA expression levels were assessed by a quantitative dot blot analysis and compared (i) between healthy and involved ileal mucosa of patients with Crohn's disease and (ii) between healthy mucosa of patients with Crohn's disease and controls. Expression of the different mucin genes was heterogeneous among controls and patients with Crohn's disease, except for MUC6 in controls. Nevertheless, MUC1 mRNA expression was significantly decreased in the involved ileal mucosa of patients with Crohn's disease when compared to the healthy mucosa (p = 0.02). Moreover, the expression levels of MUC3, MUC4, and MUC5B were significantly lower in both healthy and involved ileal mucosa of patients with Crohn's disease compared to controls (p < or = 0.05). The decrease of expression levels of some mucin genes (more particularly MUC3, MUC4, and MUC5B) in both healthy and involved ileal mucosa suggests a primary or very early mucosal defect of these genes in CD.     

白细胞介素-13对人支气管上皮细胞SPDEF表达的影响及SPDEF在哮喘气道黏液高分泌中的作用

目的探讨白细胞介素(IL)-13对人支气管上皮细胞SPDEF表达的影响及SPDEF在哮喘气道黏液高分泌中的作用。方法将人原代支气管上皮细胞(NHBE细胞)分为对照组、IL-13组及IL-13+SPDEF siRNA组。培养28天后,收集NHBE细胞并提取总RNA,采用实时荧光定量聚合酶链反应检测NHBE细胞SPDEF、粘蛋白MUC5AC及MUC5B mRNA的表达水平,流式细胞术检测MUC5AC阳性细胞数量和免疫荧光强度,免疫荧光染色检测粘蛋白MUC5AC和MUC5B的表达水平。结果IL-13组NHBE细胞SPDEF和MUC5AC mRNA的表达水平、MUC5AC阳性细胞数量和免疫荧光强度均明显高于对照组(P<0.001),而MUC5B mRNA的表达水平明显低于对照组(P<0.05);IL-13+SPDEF siRNA组NHBE细胞SPDEF和MUC5AC mRNA的表达水平、MUC5AC阳性细胞数量和免疫荧光强度均明显低于IL-13组(P<0.001);IL-13+SPDEF siRNA组MUC5B mRNA的表达水平明显低于对照组(P<0.05)。IL-13组NHBE细胞MUC5AC的表达水平明显高于对照组和IL-13+SPDEF siRNA组,而MUC5B的表达水平低于对照组。结论IL-13可能通过诱导人支气管上皮细胞SPDEF上调,促进气道粘蛋白MUC5AC的高表达,引起哮喘气道黏液高分泌。     

Mucin gene expression in bile of patients with and without gallstone disease, collected by endoscopic retrograde cholangiography

AIM： To investigate the pattern of mucin expression and concentration in bile obtained during endoscopic retrograde cholangiography （ERC） in relation to gallstone disease.METHODS： Bile samples obtained at ERC from 29 consecutive patients, 17 with and 12 without gallstone disease were evaluated for mucin content by gel filtration on a Sepharose CL-4B column. Dot blot analysis for bile mucin apoproteins was performed with antibodies to Mucin 1 （MUC1）, MUC2, MUC3, MUC5AC, MUC5B and MUC6. Staining intensity score （0-3） was used as a measure of antigen expression.RESULTS： MUCl, MUC2, MUC3, MUCSAC, MUC5B and MUC6 were demonstrated in 34.4%, 34.4%, 51.7%, 51.7%, 55.1% and 27.5% of bile samples, respectively.The staining intensity scores were 0.62 ± 0.94, 0.58 ± 0.90, 0.79 ± 0.97, 1.06 ± 1.22, 1.20 ± 2.26 and 0.41 ± 0.73, respectively. Mean mucin concentration measured in bile by the Sepharose CL-4B method was 22.8 ± 24.0 mg/mL （range 3.4-89.0 mg/mL）. Mean protein concentration was 8.1 ± 4.8 mg/mL （range 1.7-23.2 mg/mL）.CONCLUSION： High levels of MUC3, MUC5AC and MUC5B are expressed in bile aspirated during ERC examination. A specific pattern of mucin gene expression or change in mucin concentration was not found in gallstone disease.     

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.     

Sphingosine kinase 1 regulates mucin production via ERK phosphorylation

Our previous report showed that inhibition of sphingosine kinase (SphK) ameliorates eosinophilic inflammation and mucin production in a mouse asthmatic model. To clarify the role of SphK in airway mucin production, we utilized the mouse asthmatic model and found that both SphK and MUC5AC expression were increased and co-localized in airway epithelium. Next we cultured normal human bronchial epithelial cells in an air–liquid interface and treated with IL-13 to induce their differentiation into goblet cells. We found that SphK1 and MUC5AC expression was increased by IL-13 treatment at both protein and mRNA levels, whereas SphK2 expression was not changed. N,N-dimethylsphingosine (DMS), a potent SphK inhibitor, decreased MUC5AC expression up-regulated by IL-13 treatment. Furthermore, DMS inhibited IL-13-induced ERK1/2 phosphorylation but neither p38 MAPK nor STAT6 phosphorylation. These results suggest that SphK1 is involved in MUC5AC production induced by IL-13 upstream of ERK1/2 phosphorylation, and independent of STAT6 phosphorylation.     

Pseudomonas aeruginosa induces MUC5AC production via epidermal growth factor receptor.

Hypersecretory disease associated with Pseudomonas aeruginosa (PA) infections is characterised by increased goblet cells and increased mucin production. Recently, an epidermal growth factor receptor (EGFR) signalling cascade was shown to be a common pathway through which many stimuli induce mucin MUC5AC expression in airways by differentiation to a goblet cell phenotype. This study looked at whether PA products induce EGFR expression and activation and thus result in mucin MUC5AC production. Human airway epithelial (NCI-H292) cells were stimulated with PA culture supernatant (Sup). MUC5AC protein production, MUC5AC and EGFR messenger ribonucleic acid (mRNA) expression, and phosphorylated EGFR and phosphorylated p44/42 mitogen-activated protein kinase (MAPK) were all examined using enzyme-linked immunosorbent assay, by in situ hybridisation and by immunoblotting. PA Sup induced MUC5AC mRNA and subsequent protein expression, EGFR and p44/42 MAPK phosphorylation and EGFR mRNA expression. Induction of MUC5AC mRNA and protein expression and EGFR and p44/42 MAPK phosphorylation were inhibited completely by pretreatment with a selective EGFR tyrosine kinase inhibitor. Pretreatment with a selective inhibitor of MAPK kinase prevented MUC5AC production and p44/42 MAPK phosphorylation but not EGFR phosphorylation. The authors conclude that PA products induce mucin MUC5AC production in human airway epithelial cells via the expression and activation of epidermal growth factor receptor.     

Aberrant expression of MUC5AC and MUC6 gastric mucins and sialyl Tn antigen in intraepithelial neoplasms of the pancreas

BACKGROUND & AIMS: It has recently been suggested that infiltrating adenocarcinoma of the pancreas arises from histologically well-defined precursor ductal lesions called pancreatic intraepithelial neoplasia (PanIN-1A, -1B, -2, and -3). This study examined alterations in the pattern and the level of expression of several mucin genes (MUC1, MUC2, MUC5AC, and MUC6) and mucin-associated tumor antigens (Nd2 and sialyl Tn) in these precursor lesions. METHODS: We examined 139 PanINs and 68 infiltrating ductal adenocarcinomas of the pancreas by using immunohistochemistry and in situ hybridization methods. RESULTS: Overexpression of MUC1, a pan-epithelial mucin, and MUC6, a pyloric-gland mucin, and de novo expression of MUC5AC, a gastric foveolar mucin, was observed in all stages of PanINs and invasive ductal adenocarcinoma. In contrast, the expression of mucin-associated carbohydrate antigen, sialyl Tn, was markedly increased only in PanlN-3 and invasive ductal adenocarcinoma. In addition, a decrease in the expression of these mucin-associated peptide and carbohydrate antigens was correlated with the degree of differentiation of the tumor. CONCLUSIONS: Expression of both gastric-foveolar and pyloric-gland mucin in PanINs is an early event, whereas sialyl Tn expression is a late event in the recently defined progression model of pancreatic carcinogenesis. This altered mucin gene expression provides new insight into the role of cell lineage-associated metaplasia in pancreatic carcinogenesis.