Tumor cell MUC1 and CD43 are glycosylated differently with sialyl-Lewis a and x epitopes and show variable interactions with E-selectin under physiological flow conditions

The mucins secreted from the colon carcinoma cell line COLO 205 have the MUC1 and CD43 (leukosialin) as core proteins, where both carry sialyl-Lewis a and MUC1 sialyl-Lewis x epitopes. The adhesion of E-selectin expressing CHO cells to the coated mucins was analyzed in a flow system revealing that the MUC1 mucin adhered better than the CD43 mucin. One reason could be their different glycosylation, a difference that was explored by analyzing the biosynthesis of MUC1 and CD43 in COLO 205 cells. Both the MUC1 and CD43 mucins became sialyl-Lewis a reactive, but after different times as revealed by pulse-chase studies. However, only MUC1 became sialyl-Lewis x reactive. These differences suggest that MUC1 and CD43 are synthesized in different compartments of the cell. It was also observed that the mucins from colon carcinoma patients had MUC1-type mucins that carried both sialyl-Lewis a and x epitopes and CD43-type sialyl-Lewis a mucins with only low levels of sialyl-Lewis x epitopes. One could hypothesize that colon carcinoma derived MUC1 is decorated with potent E-selectin epitopes, and that this could be one of several reasons for the involvement of MUC1 in cancer development.     

Dual oxidase 2 generated reactive oxygen species selectively mediate the induction of mucins by epidermal growth factor in enterocytes

Dual oxidase 2 enzyme is a member of the reactive oxygen species-generating cell membrane NADPH oxidases involved in mucosal innate immunity. It is not known if the biological activity of dual oxidase 2 is mediated by direct bacterial killing by reactive oxygen species produced by the enzyme or by the same reactive oxygen species acting as second messengers that stimulate novel gene expression. To uncover the role of reactive oxygen species and dual oxidases as signaling molecules, we have dissected the pathway triggered by epidermal growth factor to induce mucins, the principal protective components of gastrointestinal mucus. We show that dual oxidase 2 is essential for selective epidermal growth factor induction of the transmembrane MUC3 and the secreted gel-forming MUC5AC mucins. Reactive oxygen species generated by dual oxidase 2 stabilize tyrosine phosphorylation of epidermal growth factor receptor and induce MUC3 and MUC5AC through persistent activation of extracellular signal-regulated kinases 1/2–protein kinase C. Knocking down dual oxidase 2 by selective RNA targeting (siRNA) reduced epidermal growth factor receptor phosphorylation, and MUC3 and MUC5AC gene expression. Extracellular reactive oxygen species produced by dual oxidase 2, upon stimulation by epidermal growth factor, stabilize epidermal growth factor receptor phosphorylation and activate extracellular signal-regulated kinases 1/2–protein kinase C which induce MUC5AC and MUC3. Extracellular reactive oxygen species produced by dual oxidase 2 that are known to directly kill bacteria, also contribute to the maintenance of the epidermal growth factor-amplification loop, which induces mucins. These data suggest a new function of dual oxidase 2 protein in the luminal protection of the gastrointestinal tract through the induction of mucin expression by growth factors.     

MUC genes: a superfamily of genes? Towards a functional classification of human apomucins

The MUC genes encode epithelial mucins. Eight different human genes have been well characterized, and two others identified more recently. Among them, a family of four genes, expressed in the respiratory and digestive tracts, is clustered to chromosome 11p15.5; and these genes encode gel-forming mucins which are structurally related to the superfamily of cystine-knot growth factors. A second group is composed of three independent genes encoding various isoforms of mucins including membrane-bound mucins associated to carcinomas. In this second group, MUC3 and MUC4 encode large apomucins containing EGF-like domains.     

Identification of MUC1 proteolytic cleavage sites in vivo

Mucins are high molecular weight glycoproteins that provide a protective layer on epithelial surfaces and are involved in cell-cell interactions, signaling, and metastasis. The identification of several membrane-tethered mucins, including MUC1, MUC3, MUC4, and MUC12, has incited interest in the processing of these mucins and the mechanisms that govern their release from the cell surface. MUC1 consists of an extracellular subunit and a membrane-associated subunit. The two moieties are produced from a single precursor polypeptide by an early proteolytic cleavage event but remain associated throughout intracellular processing and transport to the cell surface. We identified the MUC1 proteolytic cleavage site and showed it to be identical in pancreas and colon cell lines and not to be influenced by the presence of heavily glycosylated tandem repeats. The MUC1 cleavage site shows homology with sequences in other cell-surface-associated proteins and may represent a common mechanism for processing of these molecules.     

Expression profiles of MUC mucins and trefoil factor family (TFF) peptides in the intrahepatic biliary system: physiological distribution and pathological significance

Mucin secreted by mucosal epithelial cells plays a role in the protection of the mucosal surface and also is involved in pathological processes. So far, MUC1-4, 5AC, 5B, 6-8, 11-13 and 15-17 genes coding the backbone mucin core protein have been identified in humans. Their diverse physiological distribution and pathological alterations have been reported. Trefoil factor family (TFF) peptides are mucin-associated molecules co-expressed with MUC mucins and involved in the maintenance of mucosal barrier and the biological behavior of epithelial and carcinoma cells. Intrahepatic biliary system is a route linking the bile canaliculi and the extrahepatic bile duct for the excretion of bile synthesized by hepatocytes. Biliary epithelial cells line in the intrahepatic biliary system, secreting mucin and other molecules involved in the maintenance and regulation of the system. In this review, the latest information regarding properties, expression profiles and regulation of MUC mucins and TFF peptides in the intrahepatic biliary system is summarized. In particular, we focus on the expression profiles and their significance of MUC mucins in developmental and normal livers, various hepatobiliary diseases and intrahepatic cholangiocarcinoma.     

Mucin (Muc) expression during pancreatic cancer progression in spontaneous mouse model: potential implications for diagnosis and therapy

Pancreatic cancer (PC) is a lethal malignancy primarily driven by activated Kras mutations and characterized by the deregulation of several genes including mucins. Previous studies on mucins have identified their significant role in both benign and malignant human diseases including PC progression and metastasis. However, the initiation of MUC expression during PC remains unknown because of lack of early stage tumor tissues from PC patients. In the present study, we have evaluated stage specific expression patterns of mucins during mouse PC progression in (KrasG12D;Pdx1-Cre (KC)) murine PC model from pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDAC) by immunohistochemistry and quantitative real-time PCR. In agreement with previous studies on human PC, we observed a progressive increase in the expression of mucins particularly Muc1, Muc4 and Muc5AC in the pancreas of KC (as early as PanIN I) mice with advancement of PanIN lesions and PDAC both at mRNA and protein levels. Additionally, mucin expression correlated with the increased expression of inflammatory cytokines IFN-γ (p?相似文献   

Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis or tumor invasion. To clarify the relationship of the expression patterns of mucins in human neoplasms with their biological behavior, we examined the expression profiles of MUC1, MUC2, and MUC4 mucins in various human neoplasms using immunohistochemistry and in situ hybridization, and compared them with clinicopathologic factors including outcome of the patients. MUC1 or MUC4 expression is related with the aggressive behavior of human neoplasms and a poor outcome of the patients. In contrast, MUC2 expression tends to be related with the indolent behavior of human neoplasms and a favorable outcome of the patients, although indolent pancreatobiliary neoplasms sometimes show invasive growth with MUC1 expression in the invasive areas. The expression of MUC2 mucin in indolent pancreatobiliary neoplasms coincided with expression of MUC2 mRNA. Our recent studies to clarify the MUC2 gene regulation mechanism disclosed that DNA methylation and histone modification in the 5' flanking region of the MUC2 promoter may play an important role. Further studies of the epigenetics also in MUC1 and MUC4 gene expression may be needed to understand the relationship between the expression of mucins in human neoplasms with their biological behavior.     

Mucins in the pathogenesis of breast cancer: implications in diagnosis, prognosis and therapy

Mucins are high molecular weight, multifunctional glycoproteins comprised of two structural classes-the large transmembrane mucins and the gel-forming or secreted mucins. The primary function of mucins is to protect and lubricate the luminal surfaces of epithelium-lined ducts in the human body. Recent studies have identified a differential expression of both membrane bound (MUC1, MUC4 and MUC16) and secreted mucins (MUC2, MUC5AC, MUC5B and MUC6) in breast cancer tissues when compared with the non-neoplastic breast tissues. Functional studies have also uncovered many unique roles of mucins during the progression of breast cancer, which include modulation in proliferative, invasive and metastatic potential of tumor cells. Mucins function through many unique domains that can form complex association with various signaling molecules including growth factor receptors and intercellular adhesion molecules. While there is growing information about mucins in various malignancies including breast cancer, no focused review is there on the expression and functional roles of mucins in breast cancer. In this present review, we have discussed the differential expression and functional roles of mucins in breast cancer. The potential of mucins as diagnostic and prognostic markers and as therapeutic targets in breast cancer have also been discussed.     

Transcriptional activation of the MUC2 gene by p53

MUC2 is one of the major components of mucins that provide a protective barrier between epithelial surfaces and the gut lumen. We investigated possible alterations of MUC2 gene expression by p53 and p21(Sdi1/Waf1/Cip1) in a human colon cancer cell line, DLD-1, establishing subclones in which a tetracycline-regulatable promoter controls exogenous p53 and p21 expression. MUC2 mRNA more significantly increased in response to p53 than to p21. Unexpectedly, MUC2 expression was also induced in human osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next performed a reporter assay to test the direct regulation of MUC2 gene expression by p53. Deletion and mutagenesis of the MUC2 promoter region showed that it contains two sites for transactivation by p53. Furthermore, an electrophoretic mobility shift assay indicated that p53 binds to those elements. We analyzed MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray, but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These results suggest that p53 directly activates the MUC2 gene in many cell types.     

In vivo glycosylation of mucin tandem repeats.

The biochemical and biophysical properties of mucins are largely determined by extensive O-glycosylation of serine- and threonine-rich tandem repeat (TR) domains. In a number of human diseases aberrant O-glycosylation is associated with variations in the properties of the cell surface-associated and secreted mucins. To evaluate in vivo the O-glycosylation of mucin TR domains, we generated recombinant chimeric mucins with TR sequences from MUC2, MUC4, MUC5AC, or MUC5B, which were substituted for the native TRs of epitope-tagged MUC1 protein (MUC1F). These hybrid mucins were extensively O-glycosylated and showed the expected association with the cell surface and release into culture media. The presence of different TR domains within the chimeric mucins appears to have limited influence on their posttranslational processing. Alterations in glycosylation were detailed by fast atom bombardment mass spectrometry and reactivity with antibodies against particular blood-group and tumor-associated carbohydrate antigens. Future applications of these chimeras will include investigations of mucin posttranslational modification in the context of disease.     

The oligomerization of a family of four genetically clustered human gastrointestinal mucins

Mucins are synthesized and secreted by many epithelia. They are complex glycoproteins that offer cytoprotection. In their functional configuration, mucins form oligomers by a biosynthetic process that is poorly understood. A family of four human gastrointestinal mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) is clustered to chromosome 11p15.5. To study oligomerization of these related mucins, we performed metabolic labeling experiments with [35S]amino acids in LS174T cells, and isolated mucin precursors by specific immunoprecipitations that were analyzed on SDS-PAGE. Each of the precursors of MUC2, MUC5AC, MUC5B, and MUC6 formed a single species of disulfide-linked homo-oligomer within 1 h after pulse labeling. Based on apparent molecular masses, these oligomeric precursors were most likely dimers. Inhibition of vesicular RER-to-Golgi transport, with brefeldin A and CCCP, did not affect the dimerization of MUC2 precursors, localizing dimerization to the RER. O-Glycosylation of MUC2 followed dimerization. Inhibition of N- glycosylation by tunicamycin retarded, but did not inhibit, dimerization, indicating that N-glycans play a role in efficient dimerization of MUC2 precursors. Based on sequence homology, the ability of MUC2, MUC5AC, MUC5B and MUC6 to dimerize most likely resides in their C-terminal domains. Thus, the RER-localized dimerization of secretory mucins likely proceeds by similar mechanisms, which is an essential step in the formation of the human gastrointestinal mucus- gels.      

A novel role for IkappaB kinase (IKK) alpha and IKKbeta in ERK-dependent up-regulation of MUC5AC mucin transcription by Streptococcus pneumoniae

Epithelial cells represent the first line of host innate defense against invading microbes by elaborating a range of molecules involved in pathogen clearance. In particular, epithelial mucins facilitate the mucociliary clearance by physically trapping inhaled microbes. Up-regulation of mucin production thus represents an important host innate defense response against invading microbes. How mucin is induced in upper respiratory Streptococcus pneumoniae infections is unknown. In this study, we show that pneumolysin is required for up-regulation of MUC5AC mucin via TLR4-dependent activation of ERK in human epithelial cells in vitro and in mice in vivo. Interestingly, a "second wave" of ERK activation appears to be important in mediating MUC5AC induction. Moreover, IkappaB kinase (IKK) alpha and IKKbeta are distinctly involved in MUC5AC induction via an ERK1-dependent, but IkappaBalpha-p65- and p100-p52-independent, mechanism, thereby revealing novel roles for IKKs in mediating up-regulation of MUC5AC mucin by S. pneumoniae.     

Evaluation of MUC6 mucin tandem repeats

The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.