ARTICLE |
Correspondence to: Nicole Porchet, Unité 377 INSERM, Place de Verdun, 59045 Lille Cedex, France. E-mail: jpa@lille.inserm.fr
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Summary |
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Studies were undertaken to provide information regarding cell-specific expression of mucin genes in stomach and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of eight mucin genes (MUC14, MUC5AC, MUC5B, MUC6, MUC7) in stomach of 13 human embryos and fetuses (827 weeks' gestation), comparing these with normal, metaplastic, and neoplastic adult tissues. These investigations have demonstrated that MUC1, MUC4, MUC5AC, MUC5B, and MUC6 are already expressed in the embryonic stomach at 8 weeks of gestation. MUC3 mRNA expression can be observed from 10.5 weeks of gestation. MUC2 is expressed at later stages, concomitant with mucous gland cytodifferentiation. Normal adult stomach is characterized by strong expression of MUC1, MUC5AC, and MUC6, less prominent MUC2, and sporadic MUC3 and MUC4, without MUC5B and MUC7. Intestinal metaplasia is characterized by an intestinal-type pattern with MUC2 and MUC3 mRNA expression. Gastric carcinomas exhibit altered mucin gene expression patterns with disappearance of MUC5AC and MUC6 mRNAs in some tumor glands, abnormal expression of MUC2, and reappearance of MUC5B mRNAs. In conclusion, we have observed that patterns of mucin gene expression in embryonic and fetal stomach could show similarities with some gastric carcinomas in adults. Differences in mucin gene expression in developmental, metaplastic, and neoplastic stomach compared to normal adult stomach suggest a possible regulatory role for their products in gastric epithelial cell proliferation and differentiation.
(J Histochem Cytochem 48:16571665, 2000)
Key Words: human mucin genes, stomach, development, differentiation, gastric carcinomas, in situ hybridization
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Introduction |
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The gastrointestinal tract and accessory digestive glands (liver, gallbladder, and pancreas) have a common epithelial origin. Surface epithelium and glands of stomach, intestine, liver, biliary tree, and pancreas arise from the entoblastic layer lining the primitive gut (
To date, at least eight mucin genes (MUC1MUC4, MUC5AC, MUC5B, and MUC6MUC7) have been identified and characterized from various normal or carcinoma tissues and epithelial cell-derived cell lines (
In this study, we examined the expression of all known mucin genes (MUC1MUC4, MUC5AC, MUC5B, and MUC67) in the stomach of 13 human embryos and fetuses (aged 827 weeks of gestation) and correlated this with gene expression in normal, metaplastic, and neoplastic tissues in adults. Our results demonstrated that patterns of mucin gene expression in embryonic and fetal stomach may show similarities with some gastric carcinomas in adults.
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Materials and Methods |
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Tissues
Stomach (cardia, fundus, and antrum) was obtained from four human embryos and nine human fetuses after spontaneous or therapeutic abortion with the approval of the local ethical committees. The specimens ranged in age from 8 to 27 weeks of gestation, dated from the last menstruation (data obtained from clinical records and confirmed by foot and crownrump length). There was no evidence of congenital anomalies in the digestive tract of any of the specimens.
Samples of normal mucosa consisted of biopsy specimens taken from five patients undergoing endoscopy for epigastralgia. Biopsies were histologically normal and were used as controls. Additional specimens of macroscopically normal and tumor mucosae were obtained from five patients undergoing gastrectomy for gastric carcinoma.
Samples were obtained within 30 min of removal to avoid mRNA degradation and tissue lysis. They were immediately immersed in either fresh 4% paraformaldehyde or 10% phosphate-buffered formalin for in situ hybridization and further embedded in paraffin. Three-µm-thick sections were cut, mounted on gelatin-covered slides, and stored at 4C until used. Serial sections were systematically stained with hematoxylineosinsafran and astra blue for histological analysis.
Probes
In situ hybridization was performed using eight 35S-labeled oligonucleotide probes corresponding to each tandem repeat domain of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7 as described in earlier studies (
In Situ Hybridization
The hybridization steps were as described previously (
The following controls were performed: (a) tissue sections treated with 50 µg/ml ribonuclease A (Boehringer Mannheim); (b) tissue sections treated with a large excess of unlabeled oligonucleotide identical to or distinct from the 35S-labeled-probe; and (c) adult and fetal tissue sections were tested in parallel under the same conditions.
The intensity of the hybridization signal was scored semiquantitatively by two independent observers (MPB, LD) as: -, absent; +, weak (visible at magnification x200); ++, moderate (visible at magnification x100); +++, strong (visible at magnification x40).
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Results |
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Hybridization data are summarized in Table 1.
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Embryos and Fetuses
Mucin gene mRNAs were detected in all human embryonic and fetal specimens analyzed, from 8 to 27 weeks after gestation.
MUC5AC. MUC5AC mRNAs were detected in all gastric specimens analyzed from 8 weeks of gestation. The labeling was largely restricted to the region of stomach destined to become the antrum. In the pseudostratified epithelium, the labeling was of moderate intensity, with a marked predominance in the upper third (Fig 1A and Fig 1B). At 10.5 weeks, the signal remained stronger in epithelium of antrum, but epithelium of fundus was also labeled (Fig 1C). After 10.5 weeks of gestation, the labeling was homogeneous in gastric epithelium, irrespective of the region. Signal of moderate intensity was observed in all epithelial cells of the surface and pits (Fig 1D). After 18 weeks, developing glands could be easily distinguished from the crypts but remained unlabeled with the MUC5AC probe.
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MUC6. MUC6 mRNAs were detected from 8 weeks of gestation in the embryonic stomach. The labeling was weak and homogeneous throughout the epithelium, irrespective of the region, until 10.5 weeks of gestation (Fig 1E). At 12 weeks, labeling was restricted to epithelial folds. After this time, MUC6 mRNAs were observed only in developing cardial and antral glands (Fig 1F).
MUC1. MUC1 mRNAs were detected from 8 weeks of gestation. The labeling was weak but homogeneously distributed throughout the cytoplasm of all epithelial cells of the surface and pits (Fig 1G). A weak signal was also observed in developing cardial and antral glands, whereas fundic glands remained unlabeled. The hybridization pattern remained identical from 8 weeks to 27 weeks.
MUC5B. MUC5B mRNAs were first detected at 8 weeks of gestation in the embryonic stomach, where the labeling was weak and homogeneous throughout the epithelium (Fig 1H and Fig 1I). After this time, MUC5B mRNAs were inconsistently detected in stomach. When present, MUC5B expression was observed in the surface epithelium and epithelial folds (Fig 1J and Fig 1K). At 27 weeks the signal in fundus was distributed in epithelial cells of the surface and pits, whereas it was restricted to epithelial folds and mucous glands in antrum.
MUC4. MUC4 mRNAs were detected from 8 weeks of gestation in the embryonic stomach, where the labeling was weak or moderate and heterogeneous throughout the epithelium (Fig 1L). After Week 12, MUC4 mRNAs were inconsistently found in stomach. When present, the labeling was homogeneous or heterogeneous, depending on the specimen and irrespective of the region.
MUC3. MUC3 mRNAs were first detected at 10.5 weeks of gestation in the stomach of one of the two fetal specimens of this age. The labeling was weak and was distributed throughout the epithelium, with a stronger signal in the region of stomach destined to become the antrum. After this time, MUC3 mRNAs were occasionally detected in stomach, where the labeling was limited to most of the surface epithelial cells.
MUC2. MUC2 mRNAs were detected only from Week 26 of gestation onward in fetal stomach, where the labeling was weak and limited to antral glands. MUC2 mRNAs were not detected in surface epithelium of stomach and fundic glands at any gestational age.
MUC7. MUC7 was not detected in embryonic or fetal stomach by in situ hybridization.
Adults
Normal Controls.
Strong labeling was detected with the MUC5AC probe in epithelial cells of the surface and pits and in mucous neck cells, irrespective of the region (cardia, fundus, antrum) (Fig 2A). Strong labeling was also detected with the MUC6 probe, but the signal was restricted to neck cells and to cardial and antral glands (Fig 2B). A weak but homogenous signal was also observed with the MUC1 probe in epithelial cells of the surface and pits and in mucous glands. MUC3 and MUC4 mRNAs were inconsistently found in stomach, where the labeling was limited to the majority of surface epithelial cells. A weak signal was detected with the MUC2 probe in cardial and antral glands. Fundic glands remained unlabeled, whatever the probe. MUC5B and MUC7 mRNAs were never detected in normal adult stomach.
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Intestinal Metaplasia. Intestinal metaplasia was observed in stomach of three out of the five patients with gastric carcinoma, in macroscopically normal mucosa. It was classified on the basis of histological criteria as complete intestinal metaplasia (small intestine phenotype), with non-mucin-secreting columnar cells and goblet cells. The MUC2 probe hybridized strongly to the perinuclear region of goblet cells (Fig 2C). The MUC3 probe labeled all epithelial cells homogeneously. The signal was present in both goblet and columnar cells. MUC4 mRNAs were detected in one case, in goblet and columnar cells. MUC1, MUC5AC, MUC5B, MUC6, and MUC7 were not expressed in intestinal metaplasia.
Gastric Carcinomas. Mucin gene expression was analyzed in five gastric adenocarcinomas, four of which were well-differentiated. The hybridization pattern varied greatly among the specimens and within tumor glands of a given specimen (Table 1). MUC1 and MUC4 mRNAs were detected in all specimens analyzed (Fig 2D). MUC3 mRNAs were detected in most of the specimens (4/5). MUC5AC mRNAs were detected in association with MUC6 mRNAs in 4/5 gastric adenocarcinomas, and MUC2 mRNAs in 3/5 gastric adenocarcinomas (Fig 2E). MUC5B mRNAs were also detected in 3/5 specimens (Fig 2F). In 2/3 cases, MUC5B mRNA expression was associated with MUC5AC in the same tumor glands. In contrast, within a given specimen, there was not any overlap between cellular localization of MUC2 mRNA expression and MUC5AC or MUC5B mRNA expression.
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Discussion |
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Gastric mucins are widely assumed to play important cytoprotective roles. They protect the epithelium against acid and pepsin in the gastric juice and against exogenous damaging agents (e.g., pathogens, drugs), and against mechanical damage (
Major changes in the normal pattern of mucin expression, quantitatively and/or qualitatively, have been described in various preneoplastic and neoplastic tissues. There is evidence that mucins play diverse roles in addition to protective functions. More particularly, mucins may be implicated in progression of human carcinomas and in promotion of tumor cell metastasis (
Transformed cells frequently transcribe genes that are expressed during embryonic and fetal stages. A number of mucin carbohydrate antigens, including blood group antigens Tn, T, and sialyl-Tn antigens, have been demonstrated to be "oncofetal antigens" in stomach (
In this study, we have characterized mucin gene expression during human fetal development of the stomach and compared this with gene expression in normal, metaplastic, and neoplastic tissues in adults. Using in situ hybridization, we have examined the expression of all presently characterized mucin genes (MUC1MUC4, MUC5AC, MUC5B, MUC67) in 13 human embryos and fetuses (aged 827 weeks of gestation). To our knowledge, the only studies describing developmental expression of gastric mucin genes were performed by
Synthesis of mucus begins late in fetal stomach by 12 weeks of gestation, some 2 weeks after intestinal mucosae (
In this study, we have confirmed that MUC5AC is, along with MUC6, the major mucin gene expressed in adult stomach, in which MUC5AC is expressed in all epithelial cells of surface and pits, whereas MUC6 is expressed in mucous neck cells and in cardial and antral cells (
MUC6 expression in the developing stomach is quite different from that observed in normal adult gastric mucosae. We have shown that MUC6 mRNAs are present first in all surface epithelial cells. After this time, MUC6 expression is restricted to epithelial folds and then to the developing mucous glands. This observation suggests that undifferentiated epithelial cells of the surface epithelium have the capacity to express MUC6 long before some of them migrate down into the mesenchyme, acquiring their cytodifferentiation into mucous gland cells (
MUC5B mRNAs are absent from normal adult stomach. Interestingly, we have shown that MUC5B is expressed in embryonic and fetal stomach from 8 to 27 weeks of gestation, in both antrum and fundus. Therefore, MUC5B expression is developmentally regulated during gastric ontogenesis. The persistence of MUC5B mRNA expression until near term is not surprising because differentiation of gastric mucosae is very slow compared to that of intestinal mucosae, complete maturation being attained only 34 years after birth (
Intestinal metaplasia is frequently associated with gastric carcinoma (
Very few data are available regarding mucin gene expression in preneoplastic and neoplastic gastric epithelium. In the present study, we have shown that gastric carcinomas exhibit marked altered patterns of mucin gene expression compared with normal mucosa, with increased levels of MUC2, MUC3, and MUC4 mRNAs and decreased levels of MUC5AC and MUC6 mRNAs in some tumor glands. These findings are in accordance with those obtained by
In summary, these investigations have demonstrated that MUC1, MUC4, MUC5AC, MUC5B, and MUC6 are already expressed in the embryonic stomach at 8 weeks of gestation. In contrast, MUC2 is expressed at later stages, concomitant with mucous gland cytodifferentiation. Intestinal metaplasia is characterized by an intestine-type pattern, with MUC2, MUC3, and sometimes MUC4 mRNA expression. Gastric carcinomas exhibit altered mucin gene expression patterns, with disappearance of MUC5AC and MUC6 mRNAs in some tumor glands, overexpression of MUC2, and reappearance of MUC5B mRNAs. Promoter sequences of some of the mucin genes (MUC1, MUC2, MUC5AC, and MUC5B) are now available (
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Acknowledgments |
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Supported by grants from the Association pour la Recherche sur le Cancer (contract no. 9643), the EC Concerted Action: Mucins in inflammatory disease, and the CHRU de Lille (contract no. 96/29/9595).
We thank Marie-Claire Dieu for excellent technical assistance and ICARE Multimédi@, particularly Gérard Espouy, for color prints.
Received for publication February 21, 2000; accepted June 14, 2000.
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