Caco-2 and LS174T cell lines provide different models for studying mucin expression in colon cancer

To compare the differences in MUC2 and MUC5AC mRNA among four colon cancer cell lines and to identify the best in vitro models for studying mucin expression, quantitative real-time polymerase chain reaction was used to measure the expression of MUC2 and MUC5AC mRNA in Caco-2, HT29, LoVo, and LS174T cell lines. The levels of MUC2 mRNA expression in the four colon cancer cell lines ranked in order of mRNA abundance were: LS174T > LoVo > HT-29 > Caco-2. In contrast to MUC2, the abundances of MUC5AC mRNA were in the order: Caco-2 > HT-29 > LS174T > LoVo. Caco-2 (highest level of MUC5AC mRNA) and LS174T (highest level of MUC2 mRNA) were used to investigate the phenotypes. Morphologically, Caco-2 cells were larger with low electron density mucus-storing vacuoles, many cell surface microvilli, and no obvious intercellular spaces between cells, compared to LS174T cells. The proliferative and invasive capacities of LS174T cells were significantly higher than those of Caco-2 cells. Caco-2 and LS174T cells provide excellent in vitro models for studying mucin expression in colon cancer.     

Production of MUC1 and MUC2 mucins by human tumor cell lines.

A mucus secreting, clonal derivative (HT29-SB) of the human colonic adenocarcinoma cell line HT29, and the LS174T colon cancer cell line, secrete mucin into the culture medium as a viscoelastic gel. Mab BC2, which defines a peptide epitope present in the variable number of tandem repeats (VNTR) of the MUC1 core protein, reacted with this material after deglycosylation. Two high molecular weight bands were detected in TFMSA treated gel-formed mucin from HT29-SB and LS174T by western blotting (Mr 580 kDa and 420 kDa). A similar pattern of reactivity was seen with the culture supernatants from HT29-SB, the ovarian tumor cell line COLO-316, and the breast cancer cell line MCF-7. Mab CCP58 (anti-MUC2 VNTR) reacted with a 580 kDa band in gel-formed mucin produced by LS174T, but was not reactive with mucin produced by the other cell lines. The findings indicate that human colonic cell lines, in addition to breast and ovarian cell lines, may both express and secrete the MUC1 protein core, and that the LS174T cell line expresses and secretes both the MUC1 and MUC2 core proteins.     

The short chain fatty acid, butyrate, stimulates MUC2 mucin production in the human colon cancer cell line, LS174T

The short fatty acid, butyrate, which is produced by intestinal anaerobic bacteria in the colon, has inhibitory activity on histone deacetylases (HDACs). Treatment of the human colon cancer cell line, LS174T, with 1-2 mM sodium butyrate stimulated MUC2 mucin production, as determined by histological PAS staining of carbohydrate chains of mucin, and confirmed at the protein and mRNA levels by immunoblotting with anti-MUC2 antibody and real-time RT-PCR, respectively. Increases in acetylated histone H3 in the LS174T cells treated with butyrate suggest inhibition of HDACs in these cells. Butyrate-stimulated MUC2 production in the LS174T cells was inhibited by the MEK inhibitor, U0126, implicating the involvement of extracellular signal-regulated kinase (ERK) cascades in this process. Proliferation of the LS174T cells was inhibited by butyrate treatment. Although apoptotic nuclear DNA fragmentation could not be detected, cell-cycle arrest at the G0/G1 phase in the butyrate-treated cells was demonstrated by flow cytometry. Thus butyrate, an HDAC inhibitor, inhibits proliferation of LS174T cells but stimulates MUC2 production in individual cells.     

The human intestinal cell lines Caco-2 and LS174T as models to study cell-type specific mucin expression

Mucin expression was studied during proliferation and differentiation of the enterocyte-like Caco-2 and goblet cell-like LS174T cell lines. Caco-2 cells express mRNAs of MUC1, MUC3, MUC4 and MUC5A/C whereas MUC2 and MUC6 mRNAs are virtually absent. Furthermore, MUC3 mRNA is expressed in a differentiation dependent manner, as is the case for enterocytes. Concomitantly MUC3 protein precursor (550 kDa) was detected in Caco-2 cells. In LS174T cells mucin mRNAs of MUC1, MUC2 and MUC6 are constitutively expressed at high levels, whereas MUC3, MUC4 and MUC5A/C mRNAs are present at low levels. At the protein level LS174T cells express the goblet cell specific mucin protein precursors MUC2, MUC5A/C and MUC6 with apparent molecular masses of about 600 kDa, 470/500 kDa and 400 kDa respectively. MUC3 protein is not detectable. Furthermore, human gallbladder mucin protein (470 kDa precursor), of which the gene has not yet been identified, is expressed in LS174T cells. In addition, synthesis and secretion of the goblet cell specific mature MUC2, MUC5A/C and human gallbladder mucin was demonstrated in LS174T cells. It is concluded that Caco-2 and LS174T cell lines provide excellentin vitro models to elucidate the cell-type specific mechanisms responsible for mucin expression.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - DMEM Dulbecco's modified Eagle's medium - EMEM Eagle's minimum essential medium - Endo-H endo--N-acetylglucosaminidase H - HGBM human gallbladder mucin - dpc days past confluence - PBS phosphate buffered saline     

Epithelial inflammation response induced by Shigella flexneri depends on mucin gene expression

The protective effects of different mucin gene profiles on gut protection were assessed by the evaluation of TNFalpha production by intestinal epithelial cells infected by Shigella flexneri. Three HT-29 cell lines were used: HT29-G(-) (enterocyte-like cells, secreting no mucins), HT29-FU (highly expressing MUC2 and MUC4) and HT29-MTX (highly expressing MUC3 and MUC5AC). These cells were infected either by an invasive (M90T) or the control isogenic (BS176) strains of S. flexneri, and TNFalpha mRNA production was quantified by competitive PCR. In the HT29-G(-) cells, M90T induced an increased production of TNFalpha mRNA compared to BS176, giving a TNFalpha ratio of 5.6 +/- 3.3. In contrast, similar levels of TNFalpha mRNA were detected in HT29-FU and HT29-MTX cells stimulated with either M90T or BS176, giving ratios of 1.4 +/- 1.3 and 1.0 +/- 0.1, respectively. The results suggest that mucin genes have abilities to protect epithelial cells against S. flexneri. Furthermore, the difference in the TNFalpha ratio between the HT29-FU and HT29-MTX cells suggests distinct protective effects for these two mucin-secreting epithelial cells.     

Muc17 protects intestinal epithelial cells from enteroinvasive E. coli infection by promoting epithelial barrier integrity

The membrane-bound mucin MUC17 (mouse homolog Muc3) is highly expressed on the apical surface of intestinal epithelia and is thought to play a role in epithelial restitution and protection. Therefore, we hypothesized that MUC17 has a role in protection of the intestinal mucosa against luminal pathogens. Human intestinal cell lines were transfected by electroporation (Caco-2 and HT 29/19A) and by retroviral expression vector (LS174T, a cell line with high levels of MUC17 expression) using MUC17 siRNA. Transepithelial electrical resistance, permeability, tight-junction protein expression, adhesion, and invasion in response to enteroinvasive Escherichia coli (EIEC) were measured in all cell lines. In some experiments, the effect of the addition of exogenous purified crude mucin or recombinant Muc3 cysteine-rich domain protein (Muc3 CRD1-L-CRD2) as preventative or protective treatment was tested. Reduction of endogenous MUC17 is associated with increased permeability, inducible nitric oxide synthase and cyclooxygenase 2 induction, and enhanced bacterial invasion in response to EIEC exposure. Bacterial adhesion is not affected. Exogenous mucin (Muc3) and recombinant Muc3CRD treatment had a small but significant effect in attenuating the effects of EIEC infection. In conclusion, these data suggest that both native and exogenous MUC17 play a role in attachment and invasion of EIEC in colonic cell lines and in maintaining epithelial barrier function.     

Functional Heterogeneity of Colonic Adenocarcinoma Mucins for Inhibition of Entamoeba histolytica Adherence to Target Cells1

Mucins secreted from the gastrointestinal epithelium form the basis of the adherent mucus layer which is the host's first line of defense against invasion by Entamoeba histolytica. Galactose and N-acetyl-D-galactosamine residues of mucins specifically inhibit binding of the amebic 170 kDa heavy subunit Gal-lectin to target cells, an absolute prerequisite for pathogenesis. Herein we characterized the secretory mucins isolated from the human colon and from three human colonic adenocarcinoma cell lines: two with goblet cell-like (LS174T and T84) and one with absorptive cell-like morphology (Caco-2). By Northern blot analysis the intestinal mucin genes MUC2 and MUC3 were constitutively expressed by confluent LS174T and Caco-2 cells, whereas T84 cells only transcribed MUC2 and not MUC3 mRNA. ³H-glucosamine and ³H-threonine metabolically labeled proteins separated as high M_r mucins in the void (V_o > 10⁶ Da) of Sepharose-4B column chromatography and remained in the stacking gel of SDS-PAGE as depicted by fluorography. All mucin preparations contained high amounts of N-acetyl-glucosamine, galactose, N-acetyl-galactosamine, fucose and sialic acid, saccharides typical of the O-linked carbohydrate side chains. Mucin samples from the human colon and from LS174T and Caco-2 cells inhibited E. histolytica adherence to Chinese hamster ovary cells, whereas mucins from T84 cells did not. These results suggest that genetic heterogeneity and/or posttranslational modification in glycosylation of colonic mucins can affect specific epithelial barrier function against intestinal pathogens.     

Involvement of the MAP kinase ERK2 in MUC1 mucin signaling

MUC1 mucin is a receptor-like glycoprotein expressed abundantly in various cancer cell lines as well as in glandular secretory epithelial cells, including airway surface epithelial cells. The role of this cell surface mucin in the airway is not known. In an attempt to understand the signaling mechanism of MUC1 mucin, we established a stable cell line from COS-7 cells expressing a chimeric receptor consisting of the extracellular and transmembrane domains of CD8 and the cytoplasmic (CT) domain of MUC1 mucin (CD8/MUC1 cells). We previously observed that treatment of these cells with anti-CD8 antibody resulted in tyrosine phosphorylation of the CT domain of the chimera. Here we report that treatment of CD8/MUC1 cells with anti-CD8 resulted in activation of extracellular signal-regulated kinase (ERK) 2 as assessed by immunoblotting, kinase assay, and immunocytochemistry. The activation of ERK2 was completely blocked either by a dominant negative Ras mutant or in the presence of a mitogen-activated protein kinase kinase (MEK) inhibitor. We conclude that tyrosine phosphorylation of the CT domain of MUC1 mucin leads to activation of a mitogen-activated protein kinase pathway through the Ras-MEK-ERK2 pathway. Combined with the existing data by others, it is suggested that one of the roles of MUC1 mucin may be regulation of cell growth and differentiation via a common signaling pathway, namely the Grb2-Sos-Ras-MEK-ERK2 pathway.     

NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells.     

Somatostatin stimulates colonic MUC2 expression through SSTR5-Notch-Hes1 signaling pathway

Colonic mucus barrier is regarded as the first defense line against bacteria and antigens from directly attaching to the epithelium, which would further lead to intestinal inflammation activation and pathological conditions. As MUC2 mucin is the predominant component of the mucus, understanding the regulatory mechanisms of MUC2 is important for mucus barrier protection. Somatostatin (SST) has been found to play a role in colon protection through various manners. However, whether SST involves in colonic mucus barrier regulation is still unclear. The aim of this study is to investigate the effects and potential mechanisms of SST on colonic MUC2 expression and mucus secretion. In vivo study, exogenous somatostatin (octreotide) administration effectively stimulated mice colonic MUC2 expression and mucus secretion. In human goblet-like cell LS174T cells, SST exposure also significantly stimulated MUC2 expression and mucus secretion. Further studies indicated that SST receptor 5 (SSTR5) was significantly activated by SST, whereas specific SSTR5 siRNA transfection of LS174T cells significantly blocked SST-induced increase in MUC2 expression and mucus secretion. In addition, SSTR5 agonist L817,818 also upregulated MUC2 expression and mucus secretion in LS174T cells. Mechanistic studies further demonstrated that SST/SSTR5-mediated MUC2 upregulation was dependent on Notch-Hes1 pathway suppression by detecting notch intracellular domain (NICD) and Hes1 proteins. Taken together, our findings suggested that SST could participate in colonic mucus barrier regulation through SSTR5-Notch-Hes1-MUC2 signaling pathway. These findings provide a deep insight into the role of SST on colonic mucus regulation under physiological conditions.     

Up-regulation of mucin secretion in HT29-MTX cells by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-6

The pro-inflammatory cytokines IL-6 and TNF-alpha have been implicated in the pathogenesis of otitis media with effusion (OME). A disease 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 and MUC5B mucin gene products have been identified as components of these effusions. To determine the effect of IL-6 and TNF-alpha on MUC5AC and MUC5B secretion we have used HT29-MTX goblet cells, which secrete both types of mucins. MUC5AC and MUC5B mucin secretion was measured by an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody NCL-HGM-45M1 and polyclonal antiserum TEPA, respectively. Time response (0-72 hours) and dose response (1.5-150 ng/ml) studies were carried out. IL-6 and TNF-alpha stimulated MUC5AC and MUC5B mucin secretion in a time dependent manner, both in pre-confluent and post-confluent cells. IL-6 (15 ng/ml and 20 ng/ml) produced a low and prolonged stimulation of mucin secretion that persisted for 72 hours, with peak response at 24 hours after induction. The IL-6-mediated mucin secretion at 24 hours was concentration-dependent, with a maximal effect at 15 ng/ml. TNF-alpha (20 ng/ml) induced rapid stimulation of mucin secretion within the first 24 hours, with peak response at 7 hours after induction. IL-6 and TNF-alpha exposure significantly increased MUC5AC secretion, but not MUC5B secretion. Maximal levels of cytokine-induced mucin secretion were detected in pre-confluent cells that showed one and a half- and two-fold increases in MUC5AC secretion after IL-6 and TNF-alpha stimulation, respectively, in comparison with post-confluent cells. The results presented here suggest that IL-6 and TNF-alpha generate a differential up-regulation of mucin secretion and thus contribute to the expression of mucin genes in inflammatory responses.     

Leptin modulates the expression of secreted and membrane-associated mucins in colonic epithelial cells by targeting PKC, PI3K, and MAPK pathways

Mucins play an essential role in the protection and repair of gastrointestinal mucosa. We recently showed that luminal leptin strongly stimulated mucin secretion in vivo in rat colon. In the present study, we challenged the hypothesis that leptin may act directly on goblet cells to induce mucin expression in rat and human intestinal mucin-producing cells (DHE and HT29-MTX). The endoluminal effect of leptin was also studied in vivo in rat perfused colon model. The presence of leptin receptors was demonstrated in the two cell lines by Western blot and RT-PCR. In rat DHE cells, leptin (0.01-10 nmol/l, 60 min) dose dependently increased the secretion of mucins (210 +/- 3% of controls) and the expression of Muc2, Muc3, and Muc4 (twofold basal level) but not of Muc1 and Muc5AC. Luminal perfusion of leptin (60 min, 0.1-100 nmol/l) in rat colon also increased the mRNA level of Muc2, Muc3, and Muc4 but not of Muc1. In human HT29-MTX cells, leptin (0.01-10 nmol/l, 60 min) dose dependently enhanced MUC2, MUC5AC, and MUC4 mRNA levels. These effects were prevented by pretreatment of cells with the leptin mutein L39A/D40A/F41A, which acts as a receptor antagonist. Finally, pathway inhibition experiments suggest that leptin increased mucin expression by activating PKC-, phosphatidyl inositol 3-kinase-, and MAPK-dependent pathways but not the JAK/STAT pathway. In conclusion, leptin may contribute significantly to membrane-associated and secreted mucin production via a direct stimulation of colonic epithelial cells and the activation of leptin receptors. These data are consistent with a role for leptin in regulation of the intestinal barrier function.     

Suppression of mucin 2 enhances the proliferation and invasion of LS174T human colorectal cancer cells

Altered expression of MUC2 (mucin 2) is related to tumour development in colorectal cancer. Colorectal mucinous carcinomas are positive for MUC2 expression, whereas MUC2 is down-regulated in non-mucinous adenocarcinomas. In the present study, we down-regulated MUC2 expression by RNAi (RNA interference) and investigated the in vitro and in vivo effects on the proliferation and invasion/migration potential of the LS174T human colorectal cancer cells. The LS174T cell line is a goblet-cell-like colorectal cancer cell line that continuously produces high levels of MUC2. Inhibition of MUC2 expression in vitro by transfection of LS174T cells with the recombinant plasmid pcDNA6.2-GW/EmGFP-miR-MUC2 led to the production of a stably transfected MUC2-RNAi LS174T cell line. The proliferation and invasion/migration of MUC2-RNAi cells in vitro were significantly higher than those in control cells, as assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], colony formation and transwell assays. Subcutaneous injection of MUC2-RNAi LS174T cells into nude mice resulted in the development of subcutaneous tumours visible to the naked eye after 1 week. The growth rate of tumours derived from MUC2-RNAi LS174T cells was greater than that of tumours derived from control cells. Ki67 and matrix metalloproteinase-9 proteins were detected by immunohistochemistry in the xenografts. The expression levels of these proteins were higher in the MUC2-RNAi-derived xenografts than in xenografts derived from control cells. Although the role of MUC2 in colorectal tumorigenesis is not fully understood, these results strongly suggest a relationship between the proliferation and invasion of LS174T cells and the expression of MUC2.     

Inhibition of Escherichia coli O157:H7 attachment by interactions between lactic acid bacteria and intestinal epithelial cells

The intestinal epithelial cell (IEC) layer of the intestinal tract makes direct contact with a number of microbiota communities, including bacteria known to have deleterious health effects. IECs possess innate protective strategies against pathogenic challenge, which primarily involve the formation of a physicochemical barrier. Intestinal tract mucins are principal components of the mucus layer on epithelial surfaces, and perform a protective function against microbial damage. However, little is currently known regarding the interactions between probiotics/pathogens and epithelial cell mucins. The principal objective of this study was to determine the effects of Lactobacillus on the upregulation of MUC2 mucin and the subsequent inhibition of E. coli O157:H7 attachment to epithelial cells. In the current study, the attachment of E. coli O157:H7 to HT-29 intestinal epithelial cells was inhibited significantly by L. acidophilus A4 and its cell extracts. It is also important to note that the expression of MUC2 mucin was increased as the result of the addition of L. acidophilus A4 cell extracts (10.0 mg/ml), which also induced a significant reduction in the degree to which E. coli O157:H7 attached to epithelial cells. In addition, the mRNA levels of IL-8, IL-1beta, and TNF-alpha in HT-29 cells were significantly induced by treatment with L. acidophilus A4 extracts. These results indicate that MUC2 mucin and cytokines are important regulatory factors in the immune systems of the gut, and that selected lactobacilli may be able to induce the upregulation of MUC2 mucin and specific cytokines, thereby inhibiting the attachment of E. coli O157:H7.     

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.     

Numb modulates intestinal epithelial cells toward goblet cell phenotype by inhibiting the Notch signaling pathway

Numb was originally identified as an important cell fate determinant that is asymmetrically inherited during mitosis and controls the fate of sibling cells by inhibiting the Notch signaling pathway in neural tissue. The small intestinal epithelium originates from the division of stem cells that reside in the crypt, which further differentiate into goblet cells, absorptive cells, paneth cells, and enteroendocrine cells. However, Numb's involvement in the differentiation process of intestinal epithelium is largely unknown. In the present study, we confirm that both the Numb mRNA and protein isoforms are expressed in adult mouse intestinal mucosa. Numb protein is ubiquitously expressed throughout the crypt–villus axis of the small intestinal epithelium and is mainly localized to the cytoplasmic membrane. Down-regulation of endogenous Numb using RNA interference in cultured intestinal LS174T cells increased Notch signaling, leading to the up-regulation of Hes1 and the down-regulation of Hath1. Knockdown of Numb alleviated MUC2 protein expression and led to loss of the goblet cell phenotype in LS174Tl cells. Our results provide the first evidence that Numb, an important cell fate determinant, modulates intestinal epithelial cells towards the goblet cell phenotype by inhibiting the Notch signaling pathway.