TY - JOUR
AU - Porchet, Nicole
AB - Summary Alterations in the structure and/or quantity of mucins could alter the barrier function of mucus and play a role in initiating and maintaining mucosal inflammation in Crohn's disease. To investigate the hypothesis of a mucin gene defect in Crohn's disease, we analyzed the expression of the different mucin genes in the ileal mucosa of patients with Crohn's disease and controls. mRNA expression levels were assessed by a quantitative dot blot analysis and compared (i) between healthy and involved ileal mucosa of patients with Crohn's disease and (ii) between healthy mucosa of patients with Crohn's disease and controls. Expression of the different mucin genes was heterogeneous among controls and patients with Crohn's disease, except for MUC6 in controls. Nevertheless, MUCI mRNA expression was significantly decreased in the involved ileal mucosa of patients with Crohn's disease when compared to the healthy mucosa (p = 0.02). Moreover, the expression levels of MUC3, MUC4, and MUC5B were significantly lower in both healthy and involved ileal mucosa of patients with Crohn's disease compared to controls (p ≤ 0.05). The decrease of expression levels of some mucin genes (more particularly MUC3, MUC4, and MUC5B) in both healthy and involved ileal mucosa suggests a primary or very early mucosal defect of these genes in CD. Mucin genes, Mucins, Crohn's disease, Expression Introduction Crohn's disease (CD) is a chronic relapsing inflammatory bowel disease of unknown origin. There is ample evidence that disease initiation and pathogenesis is multifactorial [for review, see (1)]. The most frequently cited theory is that, through a genetic predisposition, this pathological condition arises from an abnormal mucosal response to unknown luminal antigens. An infectious agent or a dietary antigen may gain access to the lamina propria, perhaps through an abnormally permeable epithelium, and initiate an inflammatory response (1). Thus, constituents that contribute to mucosal integrity must be fundamental in preventing the activation of the intestinal immune system by luminal factors. The intestinal epithelium is covered by a continuous layer of mucus, which has lubricative and protective functions. Mucus provides a physical barrier between the underlying epithelium and aggressive agents present in the gastrointestinal tract lumen, such as digestive enzymes, alimentary particles, pathogenic microorganisms and the toxins they produce (2,3). Most of the mucus properties have been attributed to mucins. To date, nine human epithelial mucin genes have been identified: MUCI (4,–6), MUC2 (7), MUC3 (8), MUC4 (9), MUC5AC (10,11), MUC5B (10,12), MUC6 (13), MUC7 (14), and very recently, an additional partial cDNA pAM1, which could be referred as MUC8 (15,16). MUC1, MUC3, MUC4, MUC7, and MUC8 have been mapped respectively to chromosome 1q21 (17), 7q22 (18), 3q29 (19), 4q13-q21 (20), 12q24.3 (16). MUC2, MUC5AC, MUC5B, MUC6 have been mapped to chromosome 11p15.5 (13,21,22). Knowledge of the expression pattern of the mucin genes is increasing. These are expressed in a highly tissue- and cell-specific manner suggesting distinct roles for each of them. MUC2, MUC3, and MUC4 are the prominent mucin genes expressed in small intestine and colon (23,–26). Since alterations in the structure and/or quantity of mucins could alter the barrier function of mucus and play a role in initiating and maintaining mucosal inflammation, mucin glycoproteins have been extensively analyzed in inflammatory bowel diseases. Changes included a depletion of mucins in ulcerative colitis (UC), while mucosecretion was preserved in CD (27,28), histochemical changes with an increase in the relative amount of sialomucins in colonic mucosa in CD and UC (29,30), an increase in O-acetylated sialomucins in the involved terminal ileum in CD apparently correlated with the severity of disease (31), alterations in binding of a variety of lectins, and antibodies prepared against intestinal mucins (32,33). One purified fraction of colonic mucins was found to be reduced in patients with UC and their unaffected monozygotic twins, but not in CD (34,–36). However, other investigators were unable to confirm this finding (37). Most of changes in mucins were related to alterations in the glycosylated portion of mucins. It is not known, however, whether they reflect abnormalities in specific apomucins. To investigate the hypothesis of a mucin gene expression defect in CD, we analyzed the expression of the different mucin genes in the ileal mucosa of patients with CD and controls. mRNA expression levels were assessed by a quantitative dot blot analysis and compared (i) between healthy and involved ileal mucosa of patients with CD, and (ii) between healthy mucosa of patients with CD and controls. Patients and Methods Patients and Protocol Sixteen patients with CD (10 females, 6 males; mean age 30 years, range 18-67) were evaluated for mucin gene expression. The diagnosis of CD was established using defined criteria (38). Patients had pure ileal involvement (nine cases) or ileo-colonic CD (seven cases). They underwent surgery because of symptomatic stenosis (six cases), abscess or fistula (six cases), or treatment failure (four cases). Patients receiving immunosuppressive treatment were excluded from the study. If patients were under steroid therapy before surgery, the drug was tapered and discontinued within 4 weeks prior to resection. Six out of the 16 patients were current cigarette smokers. During the surgical procedure, an ileoscopy was systematically performed to assess macroscopically and histologically the integrity of the ileal mucosa 30 cm above the future anastomosis as previously described (39), and four ileal biopsies were systematically performed at 10 cm and 30 cm above the future anastomosis. Four biopsies were also obtained from the surgical specimens in the macroscopically ileal lesions. As controls, four ileal biopsy specimens were taken from 14 patients (11 females, 3 males; mean age 38 years, range 17-60) who underwent endoscopy for irritable bowel disease. No endoscopic lesions were found in these patients. Tissues Endoscopic and surgical biopsies were obtained directly from the operating room. Three biopsies were immediately snap-frozen in liquid nitrogen and stored at -80°C until RNA extraction. One biopsy was performed for histological analysis using hematoxylin-eosin and May-Grünwald-Giemsa stains. Histological Study Histological analyses were carried out blindly by two independent observers (NE and KG). Biopsies were scored for the presence of inflammatory lesions in a standardized way as previously described (40). This score included features of inflammatory changes such as the intensity of mononuclear and polymorphonuclear cell infiltration in the lamina propria, the interaction between inflammatory cells and the epithelium (cryptitis, crypt abscess), the features of epithelial cell damage, and structural changes. In addition, goblet cell hyperplasia (increase in number) and increased mucus content were recorded as being present or absent. The intensity of the inflammatory changes was graded from 1 to 13 corresponding respectively to absence and severe inflammatory changes. RNA Extraction All biopsy specimens taken during endoscopy in patients with CD and controls had a similar size and a mean weight of 4 ± 1 mg. Total RNA was extracted from biopsies using LiCl-Urea buffer according to the protocol described by Auffray and Rougeon (41). This method is known to be efficient at inhibiting nucleases and to give good yields especially from small amounts of tissue. Dot Blots Total RNAs were denatured in 50% desionised formamide (v/v), 18% formaldehyde (v/v), 0.02 mol/L MOPS, and transferred onto nylon Nytran plus membranes (Schleicher and Schuell, Ecquevilly, France) in 15 × SSC using a vacuum-driven dot blot apparatus (Stratagene, Montigny le Bretonneux, France). RNAs were fixed onto the membranes by ultraviolet crosslinking. Filters were prehybridized in 5 × SSPE, 2% SDS (w/v), 50% desionised formamide, 10 × Denhardt's, 100 mg/ml sheared herring sperm DNA (Boehringer Mannheim, Meylan) at 42°C for 2 h. Hybridization was performed in the same solution containing 10 × 106 cpm of 32P-labeled cDNA probes corresponding to β-actin, MUC1 (4), MUC2 (7), MUC3 (8), MUC4 (9), MUC5AC, MUC5B (10), MUC6 (13) at 42°C overnight. Filters were rinsed in 2 × SSPE, 0.1% SDS, and then washed twice in 0.1 × SSPE, 0.1% SDS at 65°C for 15 min. Following quantification, each membrane was stripped and probed again. Quantification of Mucin Gene mRNAs Filters were exposed for 3 days, and signal quantified by PhosphorImager 445SI (Molecular Dynamics, Inc., Sunnyvale, CA, U.S.A.). Quantification was determined using Image-QuaNT program (Molecular Dynamics) versus a dilution scale of a mixture of mucin gene and β-actin cDNAs. Because tissue concentrations of β-actin mRNAs do not change in the ileal mucosa of patients with CD (42), mucin gene mRNA quantification was calculated for each sample after it was normalized with corresponding β-actin mRNA quantification as previously described (25). Ratios MUC/β-actin were compared for each MUC probe. The validity of the method was checked using dilutions of quantified pure standard RNAs prepared from colonic mucosae and HT29-MTX cells as internal controls (43) (Fig. 1). Fig. 1. Open in new tabDownload slide Quantification of β-actin mRNAs. A: Data obtained with the dilution scale of a mixture of mucin gene and β-actin cDNAs hybridized with the β-actin probe. B: Quantification of β-actin mRNAs in dilutions of quantified pure standard RNAs prepared from colonic mucosa and HT29-MTX cells. Statistical Analysis Mucin gene expression was compared (i) between the healthy ileal mucosa taken 10 and 30 cm above the anastomosis and involved ileal mucosa of patients with CD, and (ii) between normal ileal mucosa of controls and healthy ileal mucosa of patients with CD. Statistical analyses were performed using the StatView 4.5 statistical program (Proxyd, Meylan, France). Comparisons were made with the nonparametric Wilcoxon Rank-T test for paired data and with the Mann-Whitney-U test for unpaired data. Data were expressed as median (range). Values were considered statistically significant when p ≤ 0.05. Ethical Considerations All patients gave their informed consent after approval from the ethical committee. Results Histological Study Controls All endoscopic biopsies performed in macroscopically healthy ileal mucosa from controls were histologically normal (score = 0/13). Fig. 2. Open in new tabDownload slide Mucin gene expression levels in patients with CD and controls. Data points represent the abundance of MUC mRNAs relative to β-actin mRNAs for individual patients (see Patients and Methods for description of mucin gene mRNA quantification). Horizontal bars indicate group median values. Patients with CD Ileal biopsies obtained in macroscopically unaffected areas from CD patients were normal (score = 0/13), except for one patient with a moderate inflammation (score = 4/13). A relative increase of the number of surface goblet cells (hyperplasia) or an increase in the amount of mucus content were not apparent in these biopsies. For the biopsies obtained from ileal lesions of patients with CD, the mean combined structural and inflammatory score was high (10/13, range 7-13). The lesions consisted mainly of an ulcer with adjacent mucosa showing features of inflammation with moderate epithelial damage (14/16 patients) and structural changes. In four cases, goblet cell hyperplasia was noted. Mucin Gene Expression The data are summarized in Fig. 1. Mucin gene mR-NAs were detected in all samples of patients with CD and controls, with higher levels for MUC1, MUC2, MUC3, and MUC4 compared to MUC5AC, MUC5B, and MUC6. A great heterogeneity in the expression level of the mucin genes was found in patients with CD and controls, except for MUC6 in controls. Involved Ileal Mucosa versus Healthy Ileal Mucosa of Patients with CD No difference in mucin gene mRNA levels was found between biopsies taken 10 cm and biopsies taken 30 cm above the anastomosis whatever the MUC probe (p > 0.05) (data not shown). Thus, the mean of the two values was used for next comparisons. The data are given in Table 1. There was no significant difference in the expression levels of MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 between healthy and involved ileal mucosa of patients with CD (p > 0.05). In contrast, the expression level of MUC1 was significantly lower in the involved ileal mucosa than in healthy ileal mucosa of patients with CD [0.130 (0.044-0.595) vs. 0.213 (0.036-0.694), p = 0.02]. No relation could be established between MUC1 expression levels and histological or clinical data including sex, age, disease location, and indication for surgery. Table 1. Comparison of mucin gene expression between the healthy and involved ileal mucosa of CD patients Open in new tab Table 1. Comparison of mucin gene expression between the healthy and involved ileal mucosa of CD patients Open in new tab Healthy Ileal Mucosa of Patients with CD versus Normal Ileal Mucosa of Controls The data are given in Table 2. In general, a trend of reduced mRNA expression levels of all mucin genes examined was observed, except for MUC1, between controls and healthy specimens from patients with Crohn's disease. However, there was no significant difference in the expression levels of MUC1, MUC2, MUC5AC, and MUC6 between healthy ileal mucosa of patients with CD and controls (p > 0.05). In contrast, we observed a significant decrease in the expression levels of MUC3 [0.192 (0.077-0.652) vs. 0.529 (0.286-1.246), p = 0.02], MUC4 [0.214 (0.012-0.788) vs. 0.427 (0.131-1.010), p = 0.05], and MUC5B [0.062 (0.004-0.291) vs. 0.131 (0.069-0.362) p = 0.002] in the healthy ileal mucosa of patients with CD when compared to controls. Yet, biopsies taken at 10 and 30 cm above the anastomosis biopsies were normal at histological examination. Patients who had low expression levels of MUC3 had also low expression levels of MUC4 and MUC5B. No relation could be established between the expression levels and histological or clinical data. Table 2. Comparison of mucin gene expression between normal ileal mucosa of controls and the healthy ileal mucosa of CD patients Open in new tab Table 2. Comparison of mucin gene expression between normal ileal mucosa of controls and the healthy ileal mucosa of CD patients Open in new tab Discussion In this study, we have used for the first time a quantitative method to assess the expression of the mucin genes in the ileum of patients with CD and controls. Although CD is most commonly found in the ileum and proximal colon, most of the studies on mucin glycoproteins have been carried out in colonic or rectal mucosa (28,29,30,32,–37). Only a small number of studies using histochemistry have referred to changes in the small intestine of patients with CD (29,31,44,45). All these changes, however, were related to alterations in the glycosylated portion of mucins and have not been linked to alterations in the expression of specific apomucins. Results concerning mucin gene expression in inflammatory bowel diseases are very scarce. MUC2 has been shown to be the most prominent colonic mucin expressed in UC (46), as it is in healthy colon (47). At the RNA level, normal significant expression of MUC2 and MUC4 with low levels of MUC3 and MUCI mRNAs have been found in UC (46,48). Recently, Weiss et al. have reported that MUC2 and MUC3 displayed a normal pattern in UC and CD (26). In the present study, mucin gene mRNAs were detected in all patients with higher levels for MUC2, MUC3, and MUC4. Our data are in accordance with previous Northern blot and in situ hybridization experiments on normal small intestine (7,8,23,26; and unpublished observations) and ileal mucosa from patients with CD (26) (unpublished observations). In addition, we found a great heterogeneity in the expression levels in patients with CD and controls, except for MUC6 in controls. Mucin genes are characterized by common structural features. These include a central domain consisting of tandem repeat units rich in serine and/or threonine residues flanked by non repetitive regions. These tandem repeat arrays result in a great polymorphism at the DNA level due to a large variation of the number of repeats (VNTR) per allele (6,19,49,50). VNTR polymorphism of these large size domains can be detected at the DNA level, but also at the RNA level (51). Thus, the intensity of each hybridization signal obtained with each mucin probe is directly influenced by the length of the mucin messenger analyzed. Interindividual polymorphism probably explains the heterogeneity in the expression levels we observed in controls and, at least in part, in patients with CD. Despite this heterogeneity, we have observed a defect in the expression levels of MUC1, MUC3, MUC4, and MUC5B in CD. We have demonstrated that amounts of MUC1 mRNAs were lower in the involved ileal mucosa than in healthy ileal mucosa of patients with CD. The first explanation that can be advanced is that the decrease of MUC1 mRNA expression in the involved mucosa may reflect epithelial lesions that likely result in a decrease of the relative number of cells expressing MUC1. This hypothesis, however, is contradicted by the fact that no significant difference was found for any of the other mucin genes, including MUC3, which is normally expressed in all surface epithelial cells (23,–26). This finding indicates that the expression level of MUC1 is really decreased in epithelial cells of the involved mucosa. More interestingly, we have demonstrated that the expression levels of MUC3, MUC4, and MUC5B were significantly lower in the healthy ileal mucosa of patients with CD when compared to controls. Likewise, the decrease of the expression levels of MUC3, MUC4, and MUC5B could not be due to a decrease in the number of epithelial cells expressing these genes, since careful histological examination of adjacent biopsies did not show any abnormality. These are in good agreement with the preliminary results of Lucas et al., who recently reported a decrease in the expression level of MUC5B but not in MUC2 in colonic specimens from patients with CD (52). However, in general, a trend of reduced mRNA expression levels of all mucin genes examined was observed, except for MUC1, between controls and healthy specimens from patients with Crohn's disease. Therefore, all secretory mucin genes seem to be down-regulated in patients with CD, although statistical significance could be obtained only with MUC3, MUC4, and MUC5B. Moreover, questions may be raised about the meaning of the MUC5B gene expression decrease in the ileum, since MUC5B is expressed at a low level in the ileal mucosa of both patients with CD and controls. The decrease in the expression levels of MUC1, MUC3, MUC4, and MUC5B we observed likely reflects a decrease in the number of the corresponding mRNAs, but it may also be relative to a reduced number of tandem repeats. An example exists in the literature concerning MUC1 (50). MUC1 encodes a polymorphic transmembrane molecule containing a large extracellular domain that is primarily composed of a 20 amino-acid tandem repeat varying in number from approximately 20—100 repeats (4,–6,49). Recently, a MUCI protein devoid of tandem repeat has been characterized in human breast cancer tissue (50). Strong evidence has been recently provided for the presence of susceptibility loci for both CD and ulcerative colitis on chromosomes 3, 7, and 12 (53). The region of potential linkage on chromosome 7 implicates promising candidate genes. Of these genes is MUC3 located on chromosome 7q22 (18). Moreover, MUC4 is located on chromosome 3 region q29 (19). These data bring additional arguments in favor of the involvement of these two genes in susceptibility for CD. Whatever the causes of their decreased expression levels, MUC1, MUC3, MUC4, and MUC5B seem to be implicated in CD. Our findings taken together suggest that the decrease of MUC1 expression in CD is a secondary phenomenon, since it was observed only the involved ileal mucosa. In contrast, the decrease of mucin gene expression levels (more particularly MUC3, MUC4, and MUC5B) in both healthy and involved ileal mucosa suggests a primary or very early mucosal defect of these genes in CD. However, due to the relatively small sample size, additional confirmatory studies in larger numbers of patients will be required to confirm this conclusion. Abnormal expression of several mucin genes normally expressed in the ileum is likely to have important functional consequences. Although mucins, within mucus, are implicated in protection, lubrication, and formation of a selective barrier, a large amount of data suggest that they may play other roles, particularly in disease states. MUC1, MUC3, and MUC4 differ from the four mucin genes located on chromosome 11p15.5 (i.e., MUC2, MUC5AC, MUC5B, and MUC6) by their expression pattern, since they are expressed not only in mucussecreting cells but also in nonmucus-secreting cells, suggesting additional functions for them (23,–26). There is strong evidence in the literature that MUC1 is able to prevent cell adhesion and immune recognition (54,–59). Cells expressing MUC1 are resistant to natural killer and cytotoxic T-cell killing (54,57). It has been demonstrated that MUC1 molecules in which most of the repeat domain is deleted loose their antiadhesive properties (50). Specific functions of the other mucin genes remain to be established. Nevertheless, several reports, based on the worse prognosis of mucin-producing tumors, suggest that they also may be involved in cell-cell interactions and in modulation of the immune system [for review, see (60)]. However that may be, alterations in the expression and/or in the structure of the mucin genes are likely to result in an abnormal composition of mucus. Alteration in the mucus barrier could result in penetration of factors either as microbial or alimentary antigens, substances with direct immune activity or proinflammatory activity that promote or perpetuate inflammation in CD. Acknowledgment This work was supported by grants from the Association F. Aupetit, the Association pour la Recherche sur le Cancer, INSERM (CRI 4U004B), and the CHRU de Lille (PHRC 1994, contracts 96/09.29/9595 and 96/38/9713). We thank F. Zerimech for statistical analyses. References 1. Sartor RB. 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TI - Abnormalities in Mucin Gene Expression in Crohn's Disease
JO - Inflammatory Bowel Diseases
DO - 10.1097/00054725-199902000-00004
DA - 1999-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/abnormalities-in-mucin-gene-expression-in-crohn-s-disease-JuXuqLHigZ
SP - 24
EP - 32
VL - 5
IS - 1
DP - DeepDyve
ER -