The DHE Cell Line As a Model for Studying Rat Gastro-Intestinal Mucin Expression: Effects of Dexamethasone

Mucin; Rat; Intestine; Dexamethasone; Real-time RT-PCR

Introduction

The expression of mucin genes was evaluated in rat intestinal cell lines in order to establish an in vitro model for investigating the regulation of intestinal mucin expression in this species. Two rat intestinal cancer cell lines (DHE, LGA) and three non-tumoral rat intestinal cell lines (IEC6, IEC17, IEC18) were screened. The mRNA expression of rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC mucin genes was studied by semi-quantitative RT-PCR, real-time RT-PCR and Northern-blot analysis. Results were correlated with immunohistochemical expression of rat gastric and intestinal mucin proteins, and secretion of glycoconjugates was examined by enzyme- linked lectin assay. We showed that mRNA of rMuc1 and rMuc2 were constitutively expressed in all IEC cell populations but periodic acid Schiff staining of these cells did not reveal the presence of glycoproteins. DHE cells expressed rMuc1-5AC mRNA and LGA expressed the same mucins but the level of rMuc4 was much lower. Mucin mRNA expression also differed in relation with the length of cultivation. Immunocytochemical studies revealed the presence of gastric and intestinal mucins in the two tumoral cell lines. Functional experiments showed that bethanechol, A23187 and PMA stimulated release of glycoconjugates in DHE but not in LGA cells. Treatment of DHE cells with dexamethasone (10^sup -7^ mol/l) enhanced rMuc2 mRNA but decreased rMuc1 and rMuc5AC mRNA. Real-time RT-PCR showed that the expression of rMuc1 and rMuc5AC genes was reduced by more than tenfold after 24 h. The increased expression of rMuc2 gene was confirmed by Northern blot analysis. In conclusion, DHE cells provide a valuable cellular model for research on rat mucin secretion and expression.

Abbreviations. BSA Bovine serum albumine. – DMEM Dulbecco’s modified Eagle’s minimal essential medium. – ELLA Enzyme-linked lectin assay. FBS Fetal bovine serum. – OPD o-Phenylenediamine dihydrochloride tablets. – PAS Periodic acid Schiff. – PS Penicillin/ streptomycin. – WGA Wheat germ agglutinin.

Mucins are high molecular weight glycoproteins with wide biological functions, including physicochemical protection from acids, enzymes, toxins and mutagens, adhesion modulation, signal transduction, and regulation of cell growth (Moniaux et al., 2001; Porchet et al., 1999). The protein backbone of human mucins is encoded by a family of MUC genes, and it has been demonstrated that each mucin has a characteristic tissue distribution (Audie et al., 1993; Ho et al., 1995; Reis et al., 1997). In the intestine, MUC2, MUC3 and MUC4 are the prominent mucins (Allen et al., 1998). MUC2 is the main secretory mucin and is confined to goblet cells while the transmembrane mucins MUC3 and MUC4 are detectable both in goblet cells and in cnlcrocytes. MUC1 is also expressed at low levels in the intestinal epithelium. Like in human, the major mucins produced in rodent intestine are rMuc2, rMuc3 and rMuc4 (Carraway et al., 2002; Khatri et al., 2001; Rossi et al., 1996). They are orthologs of human MUC2, MUC3 and MUC4 mucins, respectively (Desseyn et al., 2002; Khatri et al., 2001).

Alterations in intestinal mucin expression are associated with diseases such as inflammatory bowel diseases, adenoma and carcinoma (Bara et al., 2003; Decaens et al., 1988). De novo expression of the gastric mucin MUC5AC is thus described as an early marker of human and rat carcinogenesis. This gastric mucin is expressed in human colorectal adenocarcinomas (Sylvester et al., 2001) and in rat colon during dimethyhydrazine or methyl-N’-nitroso-guanidine (MNNG)- induced carcinogenesis (Bara et al., 2003; Decaens et al., 1988). In contrast, a decrease of MUC2 expression was found in poorly differentiated human colorectal tumor (Sylvester et al., 2001). Alteration of the expression of MUC5AC and MUC2 may thus have some implication in colorectal carcinogenesis and it would be very interesting to explore the regulation of the expression of mucins.

Elucidation of the regulation of gastro-intestinal mucin secretion and expression requires both cellular and animal models. Thus, several attractive in vivo and ex vivo rat intestinal preparations have been used to identify neuro-immuno-endocrine and luminal agents that regulate intestinal mucus secretion (Akiba et al., 2000; Barcelo et al., 2000; Claustre et al., 2002; Moore et al., 1993; Plaisancie et al., 1998; Shimotoyodome et al., 2000; Specian and Neutra, 1982). However, these models are limited by the life duration of the preparation that hardly exceeds one or at most few hours. A rat cell line will thus provide a useful and complementary tool for studying intestinal mucin expression. Hence, we investigated several cell lines derived from rat intestinal tract for their expression of the secretory mucins rMuc2 and rMuc5AC but also of other rMuc genes known to be expressed in the intestinal tract. We also tested the ability of these cells to conduct baseline and regulated glycoconjugate secretion.

The DHE cell line was then used to study the effect of dexamethasone on the expression of mucins. Indeed, although glucocorticoids are one of the major drugs in management of inflammatory bowel diseases (Rizzello et al., 2003; Scribano and Prantera, 2003), little is known about the influence of these drugs on intestinal mucin expression. This study was conducted in order to determine if dexamethasone could modulate mucin gene expression and thereby improve protection. The restoration of a functional mucus gel is indeed a prerequisite for a healthy mucosa.

We provide evidence that DHE and LGA, two rat intestinal cell lines, express gastro-intestinal mucins. In DHE cells, bethanechol, A23187 and PMA induce mucin glycoconjugate secretion, thus implying a regulated secretion. We also demonstrate that dexamethasone differentially modulates the expression of mucins in these cells. Interestingly, dexamethasone increases rMuc2 expression and secretion.

DHE cells thus provide a reliable tool for the study of regulation of rat gastrointestinal mucin expression and secretion.

Materials and methods

Materials

Dulbecco’s modified Eagle’s minimal essential medium (DMEM), penicillin/streptomycin (PS), trypsin, Trizol, RT-PCR reagents and enzymes, distilled RNase-free water, and Random Primers DNA Labeling System were obtained from Invitrogen (Cergy Pontoise, France). Culture flasks and plates were from Becton Dickinson Labware (Franklin Lakes, NJ, USA). pGEM-T Easy vector, restriction endonuclease EcoR I and E. coli strain JM 109 were provided by Promega (Madison, WI, USA). Qiaprep Spin miniprep Kit was obtained from Qiagen (Hilden, Germany). The FastStart DNA Master SYBR Green I kit was from Roche diagnostics (Meylan, France). Fetal bovine serum (FBS), bovine serum albumin (BSA), porcine gastric mucin, PBS, insulin, bethanechol, phorbol 12-myristate 13-acetate (PMA), calcium ionophore A23187, dexamethasone, neutral buffered formaldehyde, avidin peroxidase conjugate, biotinylated goat anti-mouse IgG, o- phenylenediamine dihydrochloride tablets (OPD), and primers for PCR were provided by Sigma (Saint Louis, MO, USA). Biotinylated wheat germ agglutinin (WGA), biotinylated goat anti-mouse antibody, biotinylated goat anti-rabbit antibody, avidin/biotinylated peroxidase complex (Vectastain Elite ABC reagent), and 3,3′- diaminobenzidine solution were provided by Vector laboratories (Burlingame, CA, USA). Microtiter plates (NUNC-Immunoplate) were obtained from POLYLABO (Paul Block & Cie., Strasbourg, France).

Rat gastrointestinal biopsies

Gastric and colonic biopsies were obtained from male Wistar rats (250-350 g), purchased from Le Centre d’Elevage DePRe (Saint Doulchard, France). Rats were anaesthetized with sodium pentobarbilal (50 mg/kg, IP). Biopsies (2-3 mm width) were snap frozen in liquid nitrogen for mRNA extraction.

Cell culture

The cell lines DHE and LGA were a generous gift of Professor F. Martin (INSERM U517, Dijon, France). The DHE cells were derived from a mucinous colonic carcinoma induced by dimethylhydrazine in BD-IX rat. The LGA cell line was obtained from an intestinal carcinoma induced by N-methyl-N’-nitro-N-nitrosoguanidine in Lewis rat. DHE and LGA were grown in DMEM supplemented with 10% FBS and 100 mg/ml PS. The intestinal epithelial cell lines (IEC6, IEC17, IEC18) were all established from newborn rats, and their standard culture conditions were described previously (Quaroni and Hochman, 1996). IEC-6 cells (rat enterocyte-like cells) were derived from the entire small intestine, IEC-17 cells were from the proximal small intestine, and IEC-18 cells were from the ileum. They were obtained from American Type Culture Collection (Manassas, VA). Cells were maintained in DMEM supplemented with 10% FBS, 100 mg/ml PS, and 0.1 U/ml insulin.

Cell lines were grown in plastic 25-cm^sup 2^ culture flasks and maintained at 37C in a 5% CO2 atmosphere within a humidified incubator. Medium was replaced every two days. Cells were routinely treated with trypsin at confluent densities, splitted and seeded at a density of 10^sup 6^ cells/5 ml (25 cm^sup 2^ area).

To compare mucin mRNA expression in the different cell lines, RNA was isolated when cells reached 90% confluency. State of confluency was monitored microscopically. Histochemical an\alysis of mucins was also done in the state of near confluency.

To assess the influence of the length of cultivation on mucin mRNA expression, cells (DHE, LGA, IEC17) were seeded at identical densities (10^sup 6^ cells/5 ml) in 25-cm^sup 2^ flasks and kept in culture up to 18 days post-confluency. Medium was replaced every two days. RNA was isolated at pre-confluency and at various days post- confluency.

To study glycoconjugate secretion, DHE and LGA cells were seeded in 12-well culture plates at a density of 5 10^sup 5^ cells/well in 2ml of medium. The cells were given fresh medium every two days. Experiments were performed 3 days after reaching confluency for DHE and 9 days after confluency for LGA. The experimental protocol for study of the effects of putative secretagogues was the following: 24 h before the addition of the secretagogue, the culture medium was replaced by serum-free medium in order to starve the cells from serum and to eliminate any interference from extraneous proteins or hormones on the glycoconjugate assay. Then, the wells were washed twice with PBS (37C). The incubation medium (1 ml DMEM with PS) with or without the agent to be tested was added in each well. The cells were stimulated for 15 to 60 min with bethanechol, and for 30 min with A23187 and PMA. The incubation was performed at 37C in a humidified atmosphere. The incubation medium was then collected, frozen and stored at -20C for subsequent determination of the released glycoproteins (ng/ 10^sup 6^ cells). Cells were then processed with trypsin and the cell number per well was determined. All experiments were performed at least three times in triplicate.

To assess the effect of dexamethasone on mucin gene expression, DHE cells were also seeded in 12-well culture plates. Three-day post- confluent DHE cells were washed twice with PBS and incubated with serum-free medium for 24 h. Then cells were incubated for 1 to 48 h with serum-free medium containing or not 10^sup -7^ mol/l dexamethasone. The medium was collected to determine the release of glycoconjugate. Cells were treated with trypsin and total RNA was isolated.

RNA extraction

Tissue samples (100 mg) were homogenized in Trizol reagent with an ultra-turrax dispersing instrument (Labo-moderne, Paris, France) at room temperature. LGA and DHE cells were placed in Trizol reagent. Total RNA was extracted according to the manufacturer’s guidelines. Thereafter, RNA was dissolved in 20 l of distilled RNase- free water, and stored at -80C until further analysis. RNA concentration and purity were determined measuring absorbance at 260 and 280 nm (GeneQuant, Pharmacia, Uppsala, Sweden).

RT-PCR for evaluation of rMuc and cyclophilin mRNA expression

Total RNA (2 g) was reverse transcribed to cDNA by 1 l (200 U) M- MLV (Moloney- Murine Leukemia Virus) reverse transcriptase, 1 l 10mM dNTP, 3 l 20 M pd(N)6 random hexamer, 2 l 100 mM dithiothreitol, 4 l 5 buffer and sterile water in a 200-l reaction volume. The reaction mixture was incubated at 25C for 10 min, at 37C for 50 min and at 70C for 15 min.

Mucin cDNA rMuc1-rMuc5AC were amplified by PCR using primer sequences previously published or designed with the assistance of computer software (Table 1). The housekeeping gene cyclophilin was amplified as a reference gene.

The PCR reaction mixture (50 l final volume) contained 18.5 l distilled RNase-free water, 5 l 10 Taq polymerase buffer, 1.5 l 50 mM MgCl^sub 2^, 1 l 10 mM dNTP, 2 l of both primers (12.5 M each), 2 l RT reaction mixture and 20 l diluted Taq polymerase (1.25 U Taq 1:80 in water). The reaction mixtures were overlaid with 40 l mineral oil and incubated in a thermal cycler (Thermojet, Equibio, Belgium) at 94C for 2 min and then for 34 cycles under the following conditions (denaturation, annealing, extension): 94C for 30 s, 60C for 1 min, and 72C for 1 min. The reaction was terminated by a final extension at 72C for 15 min. This number of cycles was chosen in order to fall into the exponential phase of amplification. The PCR conditions were found to amplify the cDNA molecules in a linear fashion when serial dilutions of cDNA were amplified using primers for rat cyclophilin, rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC. The identity of PCR products was confirmed by sequencing (BIOFIDAL, Vaulx en Velin, France).

Table 1. Primers for semi-quantitative and real-time PCR.

Ten l of the PCR products were separated by clectrophoresis on a 2% agarose gel and stained with ethidium bromide. For semi- quantitative analysis of rMuc1, rMuc2, rMuc3, rMuc4 and rMuc5AC mRNA expression, gels were visualized with the “Image System” (Quantum Appligene, Pleasanton, CA, USA) and densitometrically analyzed with scion image version 4.0.2. Each series of experiments was performed in triplicate. Representative gels are presented.

cDNA probes

cDNA of rMuc1, rMuc2, rMuc5AC and rat cyclophilin were obtained by RT-PCR using RNA extracted from fresh rat colon or stomach and subcloned into pGEM-T Easy vector, using E. coli strain JM 109 as a host bacterium. Transformed bacteria were identified through bluewhite colour selection. Plasmids were purified using the Qiaprep Spin Miniprep Kit according to the manufacturer’s protocol and then quantified by spectrophotometry. Extracted plasmid DNA was digested with restriction endonuclease EcoR I and then visualized on a 1% agarose gel. For Northern blot analysis, rMuc2 and rat cyclophilin DNA probes were labeled with [α-^sup 32^P]dCTP using a Random Primers DNA Labeling System.

Real-time PCR

All PCRs were performed using the real-time fluorescence detection method (Roche Diagnostics, Meylan, France) using the LightCycler System with a FastStart DNA Master SYBR Green I kit. A reaction mixture containing the following components was prepared: 5.5 l water, 0.5 l MgCl^sub 2^ (25 mM), 1 l of forward and of reverse primer (12.5 M), and 2 l LightCycler Fast Start DNA Master SYBR Green I Mix. The reaction mixture was distributed into precooled capillaries and diluted (1/10) reverse-transcribed total RNA or purified and quantified cloned plasmid DNA for mucin (construction of a standard curve) in a volume of 10 l was added as PCR template. The primer sequences for rMuc1, rMuc5AC and rat cyclophilin are described in Table 1. The cycling conditions were as follows: initial denaturation at 95C for 10 min, followed by 40 amplification cycles of 95C for 15s, 60C for 16s, and 72C for 30 s. After cycling, melting curves of the PCR products were acquired by cooling and maintaining samples at 65C for 15 s and then by a stepwise increase of the temperature from 65 to 98C. Cloned plasmid DNA for each sample was used to generate a standard curve. (2 10^sup -4^-2 10^sup 3^ attomoles).

Northern blot analysis

Ten micrograms of denatured (heating shock) RNA per assay were separated on 1% agarose/formaldchyde denaturating electrophoresis gels and then blotted onto nylon membranes. Blots were prehybridized in buffer containing 0.9 mol/l NaCl, 1% SDS, 0.2% ficoll and 50% formamide at 65C for 3 h in a volume of 10 ml and hybridized overnight at 65C with 10^sup 7^ cpm/membrane [α-^sup 32^P]dCTP- labeled probe (rMuc2, rat cyclophilin) in prehybridization solution. Blots were then washed in 2 sodium chloride sodium citrate (0.3 mol/ l NaCI, 0.03 mol/l sodium citrate)/2% SDS and finally autoradiographied overnight. Hybridization signals were then quantified with PhosphoImager (Molecular Dynamics, USA). The autoradiograms were analyzed with Scion image version 4.0.2 software.

Histochemical and immunohistochemical procedures

Cells (3 10^sup 4^/well) were cultured in eight-well chamber slides (Costar, Cambridge, MA). They were then fixed with 4% neutral buffered formaldehyde, and mucus glycoproteins were visualized by periodic acid Schiff (PAS) staining. For mucin immuno- histochcmistry, cells were fixed with 95% ethanol for 5 minutes, and rinsed in PBS. Next, cells were rinsed with a “blocking solution” consisting of 10% BSA in PBS and incubated in this solution for 30 minutes. Cells were then incubated with mouse monoclonal antibody Mab 589M (anti-rat Muc5AC mucin), or with rabbit antiserum 45C (anti- rat colonic mucin) (Nollet et al., 2002; Plaisancie et al., 1998). The MAb 589M has been shown to immunoreact with the product of human MUC5AC cDNA (D4(vWF)-like domain) (Nollet et al., 2002). Primary antibody dilutions were as follows: 589M (1:200) and 45C (1:100). Primary antibody incubation lasted 60 minutes, after which the slides were rinsed five times with blocking solution. Slides were then sequentially exposed to biotinylated goat anti-mouse secondary antibody or to biotinylated goat anti-rabbit antibody for 30 minutes, to avidin/biotinylated pcroxidase complex (Vectastain Elite ABC reagent), and to 3,3′-diaminobenzidinc solution. The slides were then cleared and mounted.

Enzyme linked lectin assay

The amount of high-molecular-weight glycoconjugate (an index of mucin secretion) was determined by enzyme-linked lectin assay (ELLA) with biotinylated WGA, known to react with specific carbohydrates present in sugars on mucins synthesized, stored, and secreted by goblet cells (Campo et al., 1998).

At time of analysis, serial dilutions of the incubation medium (1:5, 1:10 and 1:20) were plated in duplicate on 96-well microtiter plates. Glycoprotein content of samples was determined from standard curves prepared from purified rat intestinal mucin obtained from CsCl-gradient purified material (Plaisancie et al., 1998). All assays were performed in duplicate. Briefly, wells of a microtiter plate were coated with 100 l of purified mucins or with sample diluted in sodium carbonate buffer (0.5 M, pH 9.6) and then incubated overnight at 4C. On the following day, the microplate was washed four times with PBS containing 0.1% Tween (PBS-Tween, pH 7). The remaining binding sites in the wells were blocked by addition of 250l of PBS-Tweenbovine albumin (0.2 g albumin in 100 ml PBS-Tween) (PBS-Tween-BA) for 1 h at 37C, and the plate was washed again. At this stage the wells were incubated with 100 l of biotinylated WGA (5 g/ml) in PBS-Tween-BA for 1 h at 37C. The wells were then washed, 100 l of avidin-peroxidase conjugate were added and allowed to bind for 1 h at room temperature. The plate was washed five times. One hundred microliters of OPD solution were then added to each well and the color was allowed to develop in the dark for 5-10 min. The reaction was stopped by adding 25 l of 3 M sulfuric acid to each well. The absorbance was read at 492 nm on a micro-ELISA plate reader. Samples of stimulants were also measured for checking the absence of interference in the assay. The amount of glycoprotein secreted in the incubation medium was expressed as nanograms of mucin per 10^sup 6^ cells and the results were given as percent of controls.

Table 2. Mucin mRNA expression in three non-transformed intestinal and in two cancer intestinal cell lines and in rat gastric and colonic biopsies, based on RT-PCR analysis.

ELISA for rMuc2 and rMuc5AC mucins

rMuc5AC and rMuc2 mucin secretion from DHE cells were measured by an ELISA method using the 45M1 and H-300 primary monoclonal antibodies (Santa Cruz Biotechnology, CA, USA), respectively. Samples of incubation medium (1:5, 1:10 and 1:20) were incubated for 24 hours at 37C in 96-well plates. Plates were then washed three times with PBS containing 0.1% Tween (PBS-Tween) and blocked with 2% bovine serum albumin in PBS-Tween for one hour. They were then washed again and incubated with 50 l of mouse monoclonal antibody (1:100) for 1 hour. The wells were incubated with 100 l of biotinylated goat anti-mouse antibody IgG conjugate (1:10,000) for 1 hour. After three washes, 100 l of avidin-peroxidase conjugate were added and plates were processed as described above. Porcine gastric mucin, previously shown to react strongly with anti-human gastric monoclonal 45M1 antibody (Hutton et al., 1998), was treated in the same way to obtain a MUC5AC mucin standard curve. rMuc2 content of samples was determined from standard curves prepared from purified rat intestinal mucin. The results were given as percent of controls.

Statistical analysis

Data were compared using repeated-measures ANOVA, followed by U- test of Mann-Whitney when appropriate or Mann-Whitney test alone for single comparisons. Differences with P < 0.05 were considered significant. Data were analyzed by using Statview 4.57 for Windows (Abacus concept, Berkeley, CA, USA) and are presented as mean SEM.

Results

Mucin mRNA expression in cell lines and rat colonie tissue

To identify cell lines that express rat mucin genes normally detected in gastro-intestinal epithelium, we evaluated mRNA expression of rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC in three non- transformed rat intestinal cell lines (IEC6, IEC17, IEC18), in two intestinal cancer cell lines (DHE, LGA) and in rat gastric and colonie tissues. In cell lines, expression of mucin genes was analyzed at 90% confluence by RT-PCR using primers presented in Table 1.

The non-transformed cell lines (IEC6, IEC17, IEC18) expressed the membrane-associated mucin rMuc1 and the intestinal secretory mucin rMuc2, and to a lesser extent rMuc4 and rMuc5AC. Results are summarized in Table 2. The two cancer cell lines also expressed the membrane-associated mucin rMuc1. In LGA cells, the transcripts for rMuc2, rMuc3, rMuc4 were at very low or not detectable levels. On the contrary, the rMuc2 gene encoding the intestinal secretory mucin and the rMuc3 gene encoding a membrane mucin were clearly expressed in DHE cells. A very low expression of the membrane mucin-encoding gene rMuc4 was also detected (Table 2). A similar mucin gene expression pattern was found in rat colonie tissue, but the signal intensity of mucin PCR fragments was much stronger than in the DHE cell line. Additionally, an aberrant (non-intestinal type) expression of the rMuc5AC gene encoding a gastric secretory mucin was detected in the LGA and DHE cell lines.

Table 3. Influence of the duration of cultivation on mucin mRNA expression in the IEC17, LGA and DHE cell lines based on RT-PCR analysis.

Influence of the length of cultivation on rat mucin mRNA expression

Because the length of cell cultivation has been identified as an important factor of the MUC gene expression in several human intestinal cell lines (Cornberg et al., 1999), we addressed this possibility in IEC17 cells and in the two cancer cell lines (Table 3). In the IEC17 cells, the expression of none of the mucin genes showed a significant change in relation to the length of cultivation. In LGA and DHE cells, rMuc1 mRNA expression also was not modified all along the experiment. In contrast, the transcripts for rMuc2, rMuc3 and rMuc4 increased in DHE cells during the first 3 days after confluency and then remained at a similar level up to day 18 after confluency.

In LGA cells, the transcripts for rMuc2 and the transcripts for rMuc3 and rMuc5AC reached the maximal level at 6 and 9 days post- confluency, respectively, and then remained at a similar level up to day 18 after confluency. In this cell line, rMuc4 mRNA appeared at a low or not detectable level even at late confluency.

Mucin protein expression and glycoconjugate secretion in DHE and LGA cells

To explore whether mucin mRNA expression correlates with protein expression, histochemistry and immunohistochemistry analyses were performed on IEC17, LGA and DHE cells. In IEC17 cells, the absence of mucus glycoproteins was demonstrated by the absence of staining observed with periodic acid Schiff (not shown). In contrast, DHE cells showed PAS staining. Immunocytochemical analysis with an anti- rat colonic mucin antiserum (45C) and an anti-MUC5AC Mab (589M) showed that DHE cells stained positive in a pattern similar to PAS staining (Fig. 1). LGA cells also showed positive staining with PAS and reacted with both anti-MUC5AC antibody and anti-rat colonie mucin antiserum (not shown).

Figure 2a shows the basal time-related secretion of glycoconjugates by LGA and DHE cells. In the DHE cell line, the addition of 10^sup -3^ M bethanechol to the incubation medium induced a significant increase in glycoconjugate secretion with a peak occurring 30 min after addition of the secretagogue (Fig. 2b). DHE cells were also exposed for 30 min to PMA and A23187, which are also known to produce mucin secretion from human goblet cell lines (Forstner et al., 1994; Klinkspoor et al., 1999; McCool et al., 1990). As shown in Figure 2c, PMA (2 10^sup -6^ M) and A23187 (4 10^sup -5^ M) induced glycoprotein release from DHE cells. In contrast, none of these agents (bethanechol, A23187, PMA) produced glycoprotein discharge in LGA (Fig. 2d).

Effect of dexamethasone on mucin mRNA expression and on the secretion of mucins by DHE cells

Glucocorticoids exert biological effects on a variety of cells, among which mucus cells (Finnie et al., 1996; Kai et al., 1996; Tanaka et al., 2001). Accordingly, the effect of dexamethasone on the expression of the mRNA of mucins was evaluated on DHE. The cells (3 days after confluency) were treated with dexamethasone (10^sup – 7^ mol/l) for 24 h, at which time they were harvested and RNA was isolated and studied by semi-quantitative RT-PCR. As shown in Figure 3a, dexamethasone treatment induced an increase in rMuc2 mRNA level and attenuated rMuc1 and rMuc5 AC mRNA. In contrast, dexamethasone did not modify the expression of rMuc3 and rMuc4 (Fig. 3a). A time- course study was then performed on the effect of dexamethasone on expression of the three mutins rMuc1, rMuc2 and rMucSAC. Dexamethasone decreased rMuc1 and rMuc5AC mRNA levels as soon as 24 h and 6 h, respectively. The rMuc2 mRN A level raised after 6 h of exposure to dexamethasone, and this effect was clearly maintained until 24 h (Fig. 3b).

Fig. 1. Histochemistry and immunohistochemistry on DHE cells. A. PAS staining of DHE cells for identification of mucin glycoconjugates. Cells were cultured in eight-well chamber slides. They were then fixed with formalin and mucin glycoconjugates were visualized by periodic acid Schiff staining. B-E. Immunocytochemical analysis for intestinal and gastric mucins. B. Antiserum 45C was used for rat intestinal mucin detection in DHE cells. Immunolabeling is evident as dark reaction product. C. Parallel control experiment giving negative immunorcaction by omitting the antiserum 45 C. D. The monoclonal antibody 589M was used for rat gastric mucin detection in DHE cells. Immunolabeling is evident as dark reaction product. E. Parallel control experiment giving negative immunoreaction by omitting the Mab 589M.

Quantitative analysis was then done by real-time RT-PCR amplification on samples obtained after a 24-h exposure to dcxamethasone. As shown in Figure 4, we measured a more than tenfold decrease of rMucl as well as rMucSAC mRNA expression in dexamethasone-treated cells compared with control cells. Northern blot hybridization of rMuc2 mRNA from DHE cells produced a band higher than 9 kb. As shown in Figure 5, the level of rMuc2 mRNA from dexamethasone-supplemented cells was markedly increased.

The overall release of glycoconjugate under the influence of dexamethasone was not altered when determined by ELLA (Fig. 6). In contrast, the specific determination of rMuc2 and rMuc5AC in incubation medium demonstrated that rMuc2 secretion was moderately increased after 48 h of stimulation (p < 0.05) whereas rMuc5AC release was significantly decreased at the same time.

Discussion

We initiated this study to identify a rat intestinal cell line that could provide a reliable model for elucidating the regulation of mucin gene expression in this species. There are two classes of mutins, membrane-bound and secretory, which differ in biological structure and cellular location. At least 17 distinct mucin genes from thehuman have been updated (Gipson, 2004; Moniaux et al., 2001). In rats, only five distinct mucin genes have been identified so far and designated as rMuc1-5AC. They are orthologs of human mutins MUC1, MUC2, MUC3, MUC4 and MUC5AC (Carraway et al., 2002; Inatomi et al., 1997; Khatri et al., 2001; Pemberton et al., 1992; Rossi et al., 1996). Of these, rMuc1, rMuc3 and rMuc4 encode membrane-bound mucins, and rMuc2 and rMucSAC encode secretory mucins. The data presented here showed the expression of rMucl mRNA in rat colon and in both normal and cancerous cell lines. This result is in agreement with the concept that in human, MUC1 is a panepithelial mucin gene expressed in different normal tissues and cancer cells (Gendler and Spicer, 1995).

The major mucin produced in the rodent intestine is Muc2, a goblet-cell product (Khatri et al., 1993). In agreement with this finding, the present study showed that rMuc2 mRNA was highly expressed in the rat large intestine. The transcript of rMuc2 was also detected in DHE and LGA cells and the level of this secreted mucin mRNA reached a steady state level 3 and 6 days after confluency in DHE and EGA, respectively. Surprisingly, mRNA of the goblet-cell mucin rMuc2 was also expressed albeit at low level in cell lines derived from rat crypt cells (IEC). IEC-6, IEC-17 and IEC- 18 have properties of stem cells (Quaroni and Hochman, 1996) and an attractive hypothesis that could explain the expression of rMuc2 mRNA in these cells is that, to some extent, they own characteristics of the four main differentiated intestinal cell types: enterocytes, goblet cells, entero-endocrine cells and paneth cells. This hypothesis is supported by the observation that, like entero-endocrine L cells, IEC-18 cells produce glucagon-like immunoreactive peptidcs (Brubaker and Vranic, 1987; Quaroni and Hochman, 1996). The expression of rMuc2 in IEC cells may also be reminiscent of human MUC2 expression in undifferentiated epithelial cells of the human embryonic and fetal intestine (Buisine et al., 1998).

In the rodent intestine, Muc3 is also a predominant mucin (Wang et al., 2002). In our study, rMuc3 mRNA was detected only in rat colon and in the two intestinal cancerous cell lines (DHE, EGA). The other membrane-bound mucin rMuc4 is normally expressed in a number of normal secretory epithelial tissues in the adult rat including small and large intestine, trachea, uterus, and lactating mammary gland (Price-Schiavi et al., 1998). In this study, we showed that rMuc4 was expressed by colonie tissue and DHE cells, and minimally expressed by the other cell lines investigated. Thus, the two tumoral cell lines tested (DHE and EGA) expressed the intestinal mucin-type rMucl, rMuc2, rMuc3 and to a lesser extend rMuc4.

In the human digestive tract, MUC5AC expression is strong in gastric mucosa but is never detectable in normal intestinal and colonic tissues (Audie et al., 1993). In agreement with this result, we found that rMuc5 AC mRNA was not detectable in rat normal colonic tissue. On the contrary, the two tumoral cell lines showed an aberrant expression of rMuc5AC mRNA that was confirmed at the protein level by immunohistochemistry. Our data are consistent with the numerous studies showing an alteration of mucin gene expression in neoplastic intestinal tissues (Seregni et al., 1997; Sylvester et al., 2001), among others an aberrant expression of the MUC5AC gastric mucin. In support of this view, it has been demonstrated that HT29-MTX, a cultured colon carcinoma cell line of mucin- secrcting phenotype, expressed high level of MUC5AC (Lesuffleur et al., 1993).

Fig. 2. Glycoconjugate secretion from DHE cells, a. Time- dependent basal secretion of glycoconjugates from LGA and DHE cells. Glycoconjugates were measured in the incubation medium by ELLA as described in Materials and methods. Post-confluent LGA and DHE cells were incubated for 15, 30 and 60 min in serum-free medium. b. Effects of bethanechol (10^sup -3^ M) on glycoconjugate secretion from DHE cells. Each point represents the mean ( SEM) of 3 experiments performed in triplicate. * p < 0.05 versus controls, c, d. Effect of bethanechol, PMA and A23187 on glycoconjugate release in DHE (c) and LGA (d) cells. The cells were incubated with hethanechol (10^sup -3^ M), A23187 (4 10^sup -5^ M) and PMA (2 10^sup -6^ M) at 37C for 30 min. Each point represents the mean (SEM) of 3 experiments performed in triplicate. * p < 0.05 versus controls.

Fig. 3. Effect of dexamethasone on mucin mRNA expression in the rat colonic cell line DHE. a. DHE cells were exposed to dcxamethasone (10^sup -7^ M) for 24 h. Total RNA was isolated and mucin mRNA levels were analysed by RT-PCR. Amplified PCR fragments were separated by electrophoresis on 2% agarose gels and stained with ethidium bromide. Images are representative of 3 separate experiments performed in triplicate. CT: untreated DHE cells after 24 h of culture. DEXA: DHE cells treated for 24 h with 10^sup -7^ M dcxamethasone. b. DHE cells were exposed to dexamethasone (10^sup – 7^ M) in the medium for 1 to 24 h. Total RNA was isolated at different time point from dexamethasone-stimulated (DEXA) and from control (CT) cells. Images are representative of 3 separate experiments performed in triplicate.

Intestinal goblet cells release mucin granules at a slow, baseline rate but also under stimulated conditions (McCool et al., 1995). DHE and LGA cell lines were thus exposed to compounds which are known to cause mucin secretion from human mucin-secreting cell lines (Bou-Hanna et al., 1994; Forstner, 1995; Forstner et al., 1993; Klinkspoor et al., 1996; Laboisse et al., 1996; McCool et al., 1990). We demonstrated that the cholinergic agonist bethanechol induced glycoconjugate release from DHE cells. This result suggests that this cell line has the intracellular equipment to couple cell surface muscarinic receptors to the distal process of mucin glycoprotein secretion. In our study, PMA also stimulated glycoconjugate release by DHE implicating a protein-kinase C- dependent pathway. Response to stimulation with the calcium ionophore A23187 suggests that calcium is also a modulator of mucin secretion in this cell line. This is in agreement with several data indicating that A23187 stimulates mucin secretion from several human cell lines (Forstner et al., 1993; McCool et al., 1990, 1995). These results suggest that, like in human goblet cell lines and in the rat ex vivo system (Barcelo et al., 2001), calcium entry is one of the mechanisms of the stimulus-secretion coupling process in DHE colonic cells. In contrast, no significant glycoconjugate secretion was observed after stimulation of LGA cells with these agents, indicating that expression of mucin genes can be independent of a mucin-secreting phenotype. This latter finding is consistent with a previous study showing that the transcripts of the secreted mucins MUC2 and MUC5AC were detectable in the entcrocyte-likc HT29G-(-) cells (Gouyer et al., 2001).

Fig. 4. Quantitative RT-PCR analysis ofrMucl and rMuc5AC mRNA in DHE cells. DHE cells were cultured with or without 10 7M dexamethasone for 24 h and total RNA was isolated and subjected to quantitative RT-PCR analysis. Cyclophilin was used as a reference gene for internal control. Results (means SEM) are representative of 3 separate experiments performed in triplicate.

Fig. 5. Effect of dexamethasone (10^sup -7^ M) on rMuc2 mRNA level in DHE cells. A. Autoradiogram of a Northern blot from DHE cells treated with dexamethasone (10^sup -7^ M) after hybridization with rMuc2 and ral cyclophilin cDNA probes. B. Analysis of the blot wilh scion image.

Fig. 6. Effect of dexamethasonc on release of glycoconjugates and mucins in DHE cells. DHE were incubated with dexamethasone (10^sup – 7^M). Glycoconjugates were measured in the incubation medium by ELLA (black bars). rMuc2 (grey bars) and rMuc5AC (white bars) were measured by ELISA as described in Materials and methods. Each point represents the mean (SEM) of 3 experiments performed in triplicate. * p < 0.05 versus controls.

The present study showed for the first time that dexamethasone induced an increase of the rMuc2 mucin mRNA level. A significant increase of the Muc2 protein was also found in incubation medium by ELISA. These results are in agreement with the previous finding of a rise in mucin synthesis in human colonie biopsies exposed to glucocorticoids (Finnie et al., 1996). It is recognized that corticosteroids are the primary treatment for patients with ulcerative colitis (Rizzello et al., 2003). Interestingly, several authors also demonstrated that the MUC2 level is significantly decreased in patients with ulcerative colitis compared to controls (Hinoda et al., 1998; Tytgat et al., 1996) and in a rat experimental colitis model (Renes et al., 2002a, b). Taken together, these data suggest that dexamethasone-induced MUC2 expression may be a protagonist in the therapeutic effect of corticosteroids. The present study also demonstrated that treatment of DHE cells with dexamethasone resulted in a dramatic decrease of rMuc1 and rMuc5AC mRNA levels. These data are consistent with those of Okazaki el al. (1998) showing that, in human stomach in vitro, dexamethasone markedly suppressed the expression of rMuc1 mRNA. Additionally, hydrocortisone exposure of rat gastric epithelial cells produced a decrease in the expression of rMucSAC mRNA (Tanaka el al., 2001). The level of the Muc5AC secretion by DHE also dropped substantially. It is noteworthy that, in this study, the expression of the genes of the two major secreted gastro-intestinal mucins rMuc2 and rMuc5AC were differently regulated by dexamethasone.

In summary, we demonstrated that rat DHE cells expressed a combination of rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC mucin genes. This profile of mucin gene expression is quite similar to that observed in rat gastrointestinal epithelium. Rat LGA cells also exp\ressed several gastrointestinal mucin genes but rMuc4 mRNA was only very weakly detected in these cells. Furthermore, we also provided evidence that glycoconjugate secretion from DHE cells was responsive to stimulation by bethanechol, PMA and calcium ionophore A23187. We have thus identified and characterized a cell line derived from intestinal tract tissue that will prove reliable in better understanding the regulation of rat mucin glycoprotein secretion and of rat mucin gene expression. The DHE cell line thus represents the first rat cell model in this area and already led to the demonstration that dexamethasone has an opposite effect on rMuc2 and rMucSAC expression and secretion.

References

Akiba, Y., Guth, P. H., Engel, E., Nastaskin, I., Kaunitz, J. D., 2000. Dynamic regulation of mucus gel thickness in rat duodenum. Am. J. Physiol. Gastrointest. Liver Physiol. 279, G437-447.

Allen, A., Hutton, D. A., Pearson, J. P., 1998. The MUC2 gene product: a human intestinal mucin. Int. J. Biochem. Cell Biol. 30, 797-801.

Audie, J. P., Janin, A., Porchet, N., Copin, M. C, Gosselin, B., Aubert, J. P., 1993. Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization. J. Histochem. Cytochem. 41,1479-1485.

Bara, J., Forgue-Lafitte, M. E., Maurin, N., Flejou, J. E, Zimber, A., 2003. Abnormal expression of gastric mucin in human and rat aberrant crypt foci during colon carcinogenesis. Tumour Biol. 24, 109-115.

Barcelo, A., Claustre, J., Abello, J., Chayvialle, J. A., Plaisancie, P, 2001. Selective involvement of calcium and calcium channels in stimulated mucin secretion from rat colon. Scand. J. Gastrocnlerol. 36, 1339 – 1343.

Barcelo, A., Claustre, X, Moro, F., Chayvialle, J. A., Cuber, J. C., Plaisancie, P., 2000. Mucin secretion is modulated by luminal factors in the isolated vascularly perfused rat colon. Gut 46, 218- 224.

Bou-Hanna, C, Berthon, B., Combettes, L., Claret, M., Laboisse, C. L., 1994. Role of calcium in carbachol- and neurotensin-induced mucin exocytosis in a human colonic goblet cell line and cross-talk with the cyclic AMP pathway. Biochem. J. 299, 579-585.

Brubaker, P. L., Vranic, M., 1987. Glucagon-like immunoreactive peptidcs in a rat ileal epithelial cell line (IEC-18). Endocr. Res. 13, 229-241.

Buisine, M. P., Devisme, L., Savidgc, T. C., Gespach, C., Gosselin, B., Porchet, N., Aubert, J. P., 1998. Mucin gene expression in human embryonic and fetal intestine. Gut 43, 519-524.

Campo, E., Condom, E., Palacin, A., Quesada, E., Cardesa, A., 1998. Lectin binding patterns in normal and neoplastic colonic mucosa. A study of Dolichos bifloriis agglulinin, peanut agglutinin, and wheat germ agglutinin. Dis. Colon Rectum 31, 892-899.

Carraway, K. L., Perez, A., Idris, N., Jepson, S., Arango, M., Komatsu, M., Haq, B., Price-Schiavi, S. A., Zhang, J., Carraway, C. A., 2002. Muc4/sialomucin complex, the intramembrane ErbB2 ligand, in cancer and epithclia: to protect and to survive. Prog. Nucleic Acid Res. Mol. Biol. 71, 149-185.

Cho, H. Y., 1999. Inflammatory and epithelial responses during the development of ozone-induced mucous cell metaplasia in the nasal epithelium of rats. Toxicol. Sci. 51, 135- 145.

Claustre, J., Toumi, F., Trompette, A., Jourdan, G., Guignard, H., Chayvialle, J. A., Plaisancie, P., 2002. Effects of peptides derived from dietary proteins on mucus secretion in rat jejunum. Am. J. Physiol. Gastrointest. Liver Physiol. 283, G521 -528.

Cornberg, M., Enss, M. L., Makkink, M. K., Beil, W., Bock, C. T., SohekKlocke, I., Mix, H., Hiller, W., Manns, M. P., Wagner, S., 1999. Variation of human mucingene expression in gastric cancer cell lines and gastric mucous cell primary cultures. Eur. J. Cell Biol. 78, 832-841.

Decaens, C., Gautier, R., Bara, J., Daher, R, Le Pendu, J., Burtin, P., 1988. A new mucin-associated oncofetal antigen, a marker of early carcinogenesis in rat colon. Cancer Res. 48, 1571- 1577.

Desseyn, J. L., Clavereau, I., Laine, A., 2002. Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4. Eur. J. Biochem. 269, 3150-3159.

Finnie, I. A., Campbell, B. J., Taylor, B. A., Milton, J. D., Sadek, S. K., Yu, L. G., Rhodes, J. M., 1996. Stimulation of colonie mucin synthesis by corticostcroids and nicotine. Clin. Sci. (Lond.) 91, 359-364.

Forstner, G., 1995. Signal transduction, packaging and secretion of mucins. Annu. Rev. Physiol. 57, 585-605.

Forstner, G., Zhang, Y., McCool, D., Forstner, J., 1993. Mucin secretion by T84 cells: stimulation by PKC, Ca^sup 2+^, and a protein kinase activated by Ca^sup 2+^ ionophore. Am. J. Physiol. 264, G1096-1102.

Forstner, G., Zhang, Y., McCool, D., Forstner, J., 1994. Regulation of mucin secretion in T84 adenocarcinoma cells by forskolin: relationship to Ca^sup 2+^ and PKC. Am. J. Physiol. 266, G606-612.

Gendler, S. J., Spicer, A. P., 1995. Epithelial mucin genes. Annu. Rev. Physiol. 57, 607-634.

Gipson, J. K., 2004. Distribution of mucins at the ocular surface. Exp. Eye Res. 78,379-388.

Gouyer, V., Wiede, A., Buisine, M. P., Dekeyser, S., Morcau, O., Lesuff leur, T., Hoffmann, W., H uet, G., 2001. Specific secretion of gelforming mucins and TFF pcptides in HT-29 cells of mucin- secreting phcnotype. Biochim. Biophys. Acta 1539, 71 -84.

Hinoda, Y., Akashi, H., Suwa, T., Itoh, F, Adachi, M., Endo, T., Satoh, M., Xing, P. X., Imai, K., 1998. Immunohistochemical detection of MUC2 mucin core protein in ulcerative colitis. J. Clin. Lab. Anal. 12, 150-153.

Ho, S. B., Shekels, L. L., Toribara, N. W., Kirn, Y. S., Lyftogt, C, Cherwitz, D. L., Nichans, G. A., 1995. Mucin gene expression in normal, preneoplastic, and neoplaslic human gastric epithelium. Cancer Res. 55, 2681-2690.

Hutton, D. A., Fogg, F. I, Kubba, H., Birchall, J. P., Pearson, J. P., 1998. Heterogeneity in the protein cores of mucins isolated from human middle ear effusions: evidence for expression of different mucin gene products. Glycoconj. J. 15, 283-291.

Inatomi, T., Tisdale, A. S., Zhan, Q., Spurr-Michaud, S., Gipson, I. K., 1997. Cloning of rat Muc5 AC mucin gene: comparison of its structure and tissue distribution to that of human and mouse homologues. Biochem. Biophys. Res. Commun. 236,789-797.

Kai, H., Yoshitake, K., Hisatsune, ?., Kido, T., Isohama, Y, Takahama, K., Miyata, T., 1996. Dexamethasone suppresses mucus production and MUC-2 and MUC-5ACgene expression by NCI-H292 cells. Am. J. Physiol. 271, L484-488.

Khatri, I. A., Forstner, G. G., Forstner, J. F., 1993. Suggestive evidence for two different mucin genes in rat intestine. Biochem. J. 294, 391-399.

Khatri, I. A., Ho, C, Spccian, R. D., Forstner, J. F., 2001. Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis. Am. J. Physiol. Gastrointest. Liver Physiol. 280, G1321-1330.

Klinkspoor, J. H., Mok, K. S., Van Klinken, B. J., Tytgat, G. N., Lee, S. P., Groen, A. K., 1999. Mucin secretion by the human colon cell line LS174T is regulated by bile salts. Glycobiology 9, 13-19.

Klinkspoor, J. H., Tytgat, G. R, Lee, S. P., Groen, A. K., 1996. Mechanism of bile salt-induced mucin secretion by cultured dog gallbladder epithelial cells. Biochem. J., 316, 873-877.

Laboisse, C, Jarry, A., Branka, J. E., Merlin, D., Bou-Hanna, C., Vallette, G., 1996. Recent aspects of the regulation of intestinal mucus secretion. Proc. Nutr. Soc., 55,259-264.

Lesuffleur, T, Porchet, R, Aubert, J. P., Swallow, D., Gum, J. R., Kirn, Y. S., Real, F. X., Zweibaum, ?., 1993. Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations. J. Cell Sci. 106, 771-783.

Lin, J., Ho, S., Shekels, L., Paparclla, M. M., Kim, Y., 1999. Mucin gene expression in the rat middle ear: an improved method for RNA harvest. Ann. Otol. Rhinol. Laryngol. 108, 762-768.

McCool, D. J., Forstner, J. R, Forstner, G.G., 1995. Regulated and unregulated pathways for MUC2 mucin secretion in human colonic LS180 adenocarcinoma cells are distinct. Biochem. J. 312, 125-133.

McCool, D. J., Marcon, M. A., Forstner, J. F., Forstner, G. G., 1990. The T84 human colonie adenocarcinoma cell line produces mucin in culture and releases it in response to various sccrctagogucs. Biochem. 1267, 491-500.

Moniaux, N., Escande, R, Porchel, N., Aubert, J. P., Batra, S. K., 2001. Structural organization and classification of the human mucin genes. Front. Biosci. 6, D1 192-1206.

Moore, B. A., Sharkey, K. A., Mantle, M., 1993. Neural mediation of cholera toxin-induced mucin secretion in the rat small intestine. Am. J. Physiol. 265, G1050-1056.

Nollet, S., Forgue-Lafitte, M. E., Kirkham, P., Bara, J., 2002. Mapping of two new epitopes on the apomucin encoded by MUC5AC gene: expression in normal G1 tract and colon tumors. Int. J. Cancer 20, 336-343.

Okazaki, K., Chiba, T., Hajiro, K., 1998. Downregulation of gastric mucin gene expression and its biosynthesis by dexamethasone in the human. J. Clin. Gastroenterol. 27, Suppl. 1. S91 -96.

Pemberton, L., Taylor-Papadimitriou, J., Gendler, S. J., 1992. Antibodies to the cytoplasmic domain of the MUC1 mucin show conservation throughout mammals. Biochem. Biophys. Res. Commun. 185, 167-175.

Plaisancie, P., Barcelo, A., Moro, R, Claustre, J., Chayviallc, J. A., Cuber, J. C., 199H. Effects of neurotransmitters, gut hormones, and inflammatory mediators on mucus discharge in rat colon. Am. J. Physiol. 275, G1073-1084.

Porchet, N., Buisinc, M. P., Desseyn, J. L., Moniaux, N.. Nollet, S., Degand, P., Pigny, P., Van Seuningen, 1., Laine, A., Aubert, J. P., 1999. [MUC genes: a superfamily of genes? Towards a functional classification of human apomucins]. (French). J. Soc. Biol. 193,85- 99.

Price-Schiavi, S. A., Meiler. D., Jing, X., Merritt, J., Carvajal, M. E., Tseng, S. C., Carraway, K. L., 1998. Sialomucin complex at the rat ocular surface: a new model for ocular surface protection. Biochem.J. 335, 457-463.

Quaroni, A., Hochman, J., 1996. Development of intestinal cell culture models for drug transport and metabolism studies. Adv. Drug Deliv. Rev. 22, 3-52.

Reis, C. A., David, L., Nielscn, P. A., Clausen, H., Mirgorodskaya, K., Rocpstorff, P., Sobrinho-Simoes, M., 1997. Immunohistochcmical study of MUC5AC expression in human gastric carcinomas using a novel monoclonal antibody. Int. J. Cancer 74, 112- 121.

Renes, J. B., Boshuizen, J. A., Van Nispen, D. J., Bulsing, N. P., Buller, H. A., Dekker, J., Einerhand, A. W., 2002a. Alterations in Muc2 biosynthesis and secretion during dextran sulfate sodium- induced colitis. Am. J. Physiol. Gastrointest. Liver Physiol. 282, G382-389.

Renes, I. B., Verburg, M., Van Nispen, D. J., Taminiau, J. A., Buller, H. A., Dekker, J., Einerhand, A. W., 2002b. Epithelial proliferation, cell death, and gene expression in experimental colitis: alterations in carbonic anhydrase I, mucin MUC2, and trefoil factor 3 expression. Int. J. Colorectal. Dis. 17, 317-326.

Rizzello, R, Gionchetti, P., Venturi, A., Campicri, M., 2003. Review article: medical treatment of severe ulcerative colitis. Aliment. Pharmacol. Ther. 17,7-10.

Rossi, E. A., McNeer, R. R., Price-Schiavi, S. A., Van den Brande, J. M., Komatsu, M., Thompson, J. R, Carraway, C. A., Fregien, N. L., Carraway, K. L., 1996. Sialomucin complex, a heterodimeric glycoprotein complex. Expression as a soluble, secretable form in lactating mammary gland and colon. J. Biol. Chem. 271,33476-33485.

Scribano, M., Prantera, C., 2003. Review article: medical treatment of moderate to severe Crohn’s disease. Aliment. Pharmacol. Ther. 17, 23-30.

Seregni, E., Botti, C., Massaron, S., Lombatdo, C., Capobianco, A., Bogni, A., Bombardieri, E., 1997. Structure, function and gene expression of epithelial mucins. Tumori 83, 625-632.

Shimotoyodome, A., Meguro, S., Hase, T., Tokimitsu, I., Sakata, T., 2000. Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 125, 525-531.

Specian, R. D., Neutra, M. R., 1982. Regulation of intestinal goblet cell secretion. I. Role of parasympathetic stimulation. Am. J. Physiol. 242, G 370-379.

Sylvester, P. A., Myerscough, N., Warren, B. R, Carlstedt, I., Corfield, A. P., Durdey, P., Thomas, M. G., 2001. Differential expression of the chromosome 11 mucin genes in coloreclal cancer. J. Pathol. 195,327-335.

Tanaka, T., Kobayashi, T., Sunaga, K., Tani, S., 2001. Effect of glucocorticoid on expression of rat MUC5AC mRNA in rat gastric mucosa in vivo and in vitro. Biol. Pharm. Bull. 24, 634-637.

Tei, M., Spurr-Michaud, S. J., Tisdale, A. S., Gipson, 1. K., 2000. Vitamin A deficiency alters the expression of mucin genes by the rat ocular surface epithelium. Invest. Ophthalmol. Vis. 41, 82- 88.

Tytgat, K. M., van der Wal, J. W., Einerhand, A. W., Buller, H. A., Dekker, J., 1996. Quantitative analysis of MUC2 synthesis in ulcerative colitis. Biochem. Biophys. Res. Commun. 224, 397-405.

Wang, R., Khatri, I. A., Forstner, J. F., 2002. C-terminal domain of rodent intestinal mucin Muc3 is proteolytically cleaved in the endoplasmic reticulum to generate extracellular and membrane components. Biochem. J. 366, 623-631.

Wu, K., Fregien, N., Carraway, K. L., 1994. Molecular cloning and sequencing of the mucin subunit of a heterodimeric, bifunctional cell surface glycoprotein complex of ascites rat mammary adcnocarcinoma cells. J. Biol. Chem. 269, 11950-11955.

Aurlien Trompette(a), Carine Blanchard(a), Sandra Zoghbi(a), Jacques Bara(b), Jean Claustre(a), Grard Jourdan(a), Jean Alain Chayvialle(a), Pascale Plaisanci1)a,c

a INSERM U45, IFR62, Facult de mdecine Lannec, Lyon, France

b INSERM U482, Hpital Saint-Antoine, Btiment Kourilsky, Paris, France

c Laboratoire d’Ecologie et de Physiologie du Systme Digestif, INRA, Centre de Recherche de Jouy-en-Josas, Jouy-en-Josas cedex, France

Received February 6, 2004

Received in revised version April 29, 2004

Accepted May 14, 2004

1) Corresponding author: Dr. Pascale Plaisanci, INSERM U45, IFR62, Facult de mdecine Lannec, 7 rue Guillaume Paradin, F-69372 Lyon Cedex 8, France, e-mail: plaisancie@lyon.inserm.fr, Fax: +334 78778780.

Copyright Urban & Fischer Verlag Aug 2004