| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
The epithelial surface of the respiratory tract is coated with a protective film of mucus secreted by epithelial goblet and submucosal gland cells. Histology of the airway mucosa and composition of secretions during the second trimester of fetal life are known to differ from the normal adult in that these secretions show similarities with those of hypersecretory disorders. To provide information regarding cell-specific expression of mucin genes and their relation to developmental patterns of epithelial cytodifferentiation, we studied the expression of eight different mucin genes (MUC1-MUC4, MUC5AC, MUC5B, MUC6, MUC7) in human embryonic and fetal respiratory tract using in situ hybridization. These investigations demonstrated that MUC4 is the earliest gene expressed in the foregut at 6.5 wk, followed by MUC1 and MUC2 from 9.5 wk of gestation in trachea, bronchi, epithelial tubules, and terminal sacs before epithelial cytodifferentiation. In contrast, MUC5AC, MUC5B, and MUC7 are expressed at later gestational ages concomitant with epithelial cytodifferentiation. During this developmental stage, MUC1 and MUC4 mRNAs are located in goblet and ciliated cells, whereas MUC2 mRNAs are located in basal and goblet cells. MUC5AC expression is confined to goblet cells. In the submucosal glands, MUC2 mRNAs are located in both mucous and serous cells, whereas MUC5B and MUC7 mRNAs are expressed in mucous and in serous cells, respectively. These data suggest distinct developmental roles for MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7 in the elongation, branching, and epithelial cytodifferentiation of the respiratory tract during ontogenesis. Distinct patterns of mucin gene expression are also likely to play an important role in regulating appropriate epithelial cell proliferation and cytodifferentiation in adult airway mucosa as it is indicated by aberrant expression in hypersecretory disorders.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
The epithelial surface of the respiratory tract is coated with mucus secreted by goblet cells in the surface epithelium and by mucous and serous cells in the submucosal glands. Respiratory mucus protects the lower airways and alveoli from dehydration and damage from inhaled particles, pathogens, and chemical irritants (1, 2). The primitive lung is a small epithelial tubule originating as a diverticulum from the foregut. Subsequently, the respiratory epithelium grows and undergoes a striking multiplication of branches by sequential budding and segmentation and progressive elongation that ultimately generates airways and distal alveoli. As branching proceeds, the lung epithelium undergoes cytodifferentiation. In fetal respiratory mucosae, secretion of mucus begins by 13 wk of gestation (3), although the biologic role of fetal derived mucus in utero remains largely unknown. Histology of the airway mucosa and composition of secretions during the second trimester of fetal life differ from that in normal adults, and instead show similarities with those of chronic bronchitis, bronchiectasis, asthma, and cystic fibrosis (2).
Mucus properties can be attributed largely to its constituent mucin O-glycoproteins that exhibit high density and
viscoelasticity. Our understanding of the structure of mucins has advanced considerably with the isolation and characterization of cDNA clones encoding the large mucin peptide backbones. To date, nine mucin genes (MUC1-MUC4, MUC5AC, MUC5B, MUC6-MUC8) have been identified (8, 9). Three of these genes
MUC4, MUC5AC, and
MUC5Bhave been cloned in our laboratory from cDNA
libraries prepared from adult human tracheobronchial mucosa (10). Using in situ hybridization, we have previously demonstrated that MUC4, MUC5AC, and MUC5B
are differentially expressed in specific cell lineages in the
airways (13). Other investigations using Northern blot, reverse transcriptase-polymerase chain reaction (RT-PCR),
and in situ hybridization have indicated that MUC1, MUC2,
MUC3, MUC7, and MUC8 may also contribute to the biosynthesis of airway mucus (14).
The aim of this study was to characterize mucin gene expression during human fetal airway development to correlate this with gene expression in normal and diseased tissue in adults. Using in situ hybridization, we examined the expression of eight mucin genes (MUC1-MUC4, MUC5AC, MUC5B, MUC6, MUC7) in 13 human embryos and fetuses (aged 6.5 to 27 wk of gestation) and demonstrated that patterns of epithelial gene expression correlated more closely with hypersecretory respiratory disorders than with normal respiratory mucosae. A potential role for this differential mucin gene expression during fetal airway development and in the pathogenesis of hypersecretory respiratory disorders is discussed.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Tissues
Trachea, bronchus, and lung were obtained from three human embryos and 10 human fetuses after spontaneous or therapeutic abortion with local ethical committee approval. The specimens ranged in age from 6.5 to 27 wk of gestation, dated from the last menstruation (data obtained from clinical records and confirmed by foot and crown- rump length). There was no evidence of congenital anomalies in the respiratory tract of any of the specimens. Several tissue samples were obtained from a majority of the fetuses, allowing us to examine developmental mucin gene expression in the different levels of the respiratory tree. Samples were taken within 30 min of removal to avoid messenger RNA (mRNA) degradation and tissue lysis.
Specimens of normal adult respiratory mucosae were used as controls. Four specimens (trachea [n = 2] and bronchi [n = 2]) were obtained from two organ donors without evidence of inflammatory or neoplastic disease. Additional specimens (bronchi [n = 3] and lung [n = 2]) were obtained from histologically normal tissue adjacent to a region of carcinoma from five patients.
Each specimen was immediately immersed either in fresh 4% paraformaldehyde or 10% phosphate-buffered formalin for in situ hybridization, and further embedded in paraffin. Three-micrometer-thick sections were cut, mounted on gelatin-covered slides, and stored at 4°C until used. Serial sections were systematically stained with hematoxylin-eosin-safran and astra blue for histologic analysis.
Probes
In situ hybridization was performed using seven 35S-labeled oligonucleotide probes corresponding to each tandem repeat domain of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 as described in previous studies (13, 21). One additional 48-mer oligonucleotide antisense probe corresponding to MUC7 (22) was synthesized (Eurogentec, Liège, Belgium). Nucleotide sequences of oligonucleotide probes are given in Table 1.
|
In situ Hybridization
The hybridization steps were as described in detail by Audié and colleagues (13). Briefly, tissue sections were deparaffinized, rehydrated, incubated with 2 µg/ml proteinase K (Boehringer Mannheim, Meylan, Germany) for 15 min, and fixed again in 4% paraformaldehyde in phosphate-buffered saline for 15 min. Sections were then immersed in 0.1 M triethanolamine (Sigma, L'Isle d'Abeau Chesnes, France) containing 0.25% acetic anhydride for 10 min. Sections were prehybridized in 4× standard sodium phosphate ethylenediamine tetraacetic acid (SSPE), 1× Denhardt's buffer for 45 min, and hybridized overnight at 42°C in 20 to 100 µl of a 4× SSPE solution containing 50% formamide (vol/vol), 0.1% N-lauroylsarcosine (wt/vol), 1.2 M sodium phosphate (pH 7.2), 1× Denhardt's buffer, 3 mg/ ml yeast transfer RNA, 20 mM dithiothreitol, and 7.5 × 103 dpm/µl of 35S-labeled oligonucleotide. After posthybridization washes, slides were dipped in LM-1 emulsion (Amersham, Les Ulis, France), developed 2 to 3 wk after exposure, and finally counterstained with methyl green pyronin (Sigma).
The following controls were performed: (1) fetal tissue sections treated with 50 µg/ml RNase A (Boehringer Mannheim); (2) fetal tissue sections treated with a large excess of unlabeled oligonucleotide identical to or distinct from the 35S-labeled probe; and (3) adult and fetal tissue sections tested in parallel under the same conditions.
The intensity of the hybridization signal was scored
semiquantitatively by two independent observers (M.P.B.,
L.D.) as follows: 
, absent; +, weak (visible at magnification ×200); ++, moderate (visible at magnification ×100);
+++, strong (visible at magnification ×40).
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Mucin gene mRNAs were detected in all human embryonic and fetal specimens analyzed, as early as 6.5 wk after gestation. Hybridization data are summarized in Table 2.
|
MUC1. MUC1 mRNAs were first detected in trachea, developing bronchi, epithelial tubules, and terminal sacs between 9 and 10 wk of gestation (Figures 1a and 1b). Although MUC1 mRNAs were present in all surface epithelial cells, the labeling was weak and located at the apical pole of these epithelial cells. In the pseudostratified epithelium of the trachea and main bronchi, the labeling was weak and distributed throughout the epithelium with a predominance in the upper third (Figures 1b and 1c). Submucosal glands were unlabeled. A gradient in the labeling intensity was observed along the respiratory tract after 18 wk of gestation, with a progressively weaker signal in larger airways to bronchioles to developing alveoli. Similarly, in adult airways, weak labeling was detected in trachea, bronchi, and bronchioles, with the MUC1 probe hybridizing to both ciliated and goblet surface epithelial cells. As in fetal tissues, adult submucosal glands remained unlabeled.
|
MUC2. MUC2 mRNAs were first detected in trachea, developing bronchi, epithelial tubules, and terminal sacs between 9 to 10 wk of gestation (Figures 2a, 2b, and 2c). The labeling was located in all surface epithelial cells and was distributed throughout the cytoplasm up to 13 wk of gestation. After this time, the labeling was confined to basal cells of the pseudostratified epithelium (Figure 2d). After 23 wk of gestation, although the most intense signal was confined to basal cells, additional epithelial cells tentatively identified as immature suprabasal cells were also labeled. By 18 wk of gestation, a weak signal could also be detected in the submucosal glands. In adult airways, MUC2 mRNAs were present in goblet, basal, and suprabasal cells of the surface epithelium; however, only some goblet cells were labeled (Figure 2e). A weak signal was also observed in mucous cells and in some serous cells of the submucosal glands (Figure 2f). The MUC2 probe showed similar hybridization in both fetal and adult tissues, with a progressively weaker signal from trachea and larger bronchi, to small bronchi and proximal bronchioles, to distal bronchioles and terminal sacs or alveoli.
|
MUC4. MUC4 mRNAs were found in all embryonic and fetal specimens analyzed and were detected as early as 6.5 wk after gestation in the embryonic foregut (Figures 3a and 3b). Between 8 and 12 wk, MUC4 mRNAs were present only in trachea and larger bronchi. After 12 wk, MUC4 mRNAs were progressively identified in small bronchi and bronchioles, where labeling was continuous and homogeneously throughout the epithelium. In undifferentiated surface epithelial cells, labeling was located throughout the cytoplasm, although hybridization predominated somewhat in the apical zone of epithelial cells. Labeling was located in goblet and ciliated cells in the upper two-thirds of the pseudostratified epithelium, where basal cells were weakly labeled (Figure 3c). Terminal sacs and alveoli remained unlabeled. A gradient in the labeling intensity was observed, with a progressively weaker signal from the surface epithelium to the submucosal glands, which were unlabeled (Figure 3c). Moreover, the MUC4 probe showed a gradient in the labeling intensity with stronger labeling in larger airways than in bronchioles, with no labeling in terminal sacs or alveoli (Figure 3d). The expression pattern of the MUC4 gene remained identical from 13 wk of gestation to adulthood.
|
MUC5AC. MUC5AC mRNAs were expressed at a moderate level from the 13th wk of gestation in epithelial buds of segmental bronchi but not in the originating bronchus (Figure 4a). MUC5AC mRNAs were inconsistently located in the surface epithelium of trachea and bronchi but never in bronchioles or terminal sacs. When present, MUC5AC mRNA expression was restricted to epithelial folds and gland ducts (Figures 4b and 4c). Occasional rare surface epithelial cells, probably goblet cells, were also labeled. In adults, MUC5AC mRNAs were observed in all goblet cells of the surface epithelium and in gland ducts. Submucosal glands remained unlabeled in adults.
|
MUC5B. MUC5B mRNAs were also detected after 13 wk of gestation, when the labeling was weak and diffuse in bronchial epithelium. From the 18th week, labeling of moderate intensity was largely restricted to mucous glands (Figure 4d), with a weak signal also present in rare epithelial cells, epithelial folds, and gland ducts, in a similar fashion to MUC5AC. In addition, hybridization increased from the surface epithelium to the submucosal glands (Figures 4d and 4e). In adult airways, MUC5B mRNAs were located essentially in the mucous cells of submucosal glands and in gland ducts. Serous cells were unlabeled. Weak labeling was also detected in the surface epithelium of trachea and bronchi, whereas bronchioles were unlabeled.
MUC7. MUC7 mRNAs were detected only from the 23rd wk of gestation in trachea and bronchi, where labeling was located only in occasional cells in the submucosal glands (Figure 4f). In adults, labeling was only present in serous cells as mucous cells of submucosal glands and epithelial cells of the surface epithelium remained unlabeled.
MUC3 and MUC6. MUC3 and MUC6 mRNAs were not detected in trachea, bronchi, bronchioles, terminal sacs, or alveoli in any of the samples examined.
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Mucus hypersecretion is a common feature of many airway diseases. Mucus plugs in airways contribute significantly to the morbidity and mortality associated with chronic bronchitis, asthma, and cystic fibrosis (23). Moreover, histochemical studies have described abnormalities in mucin glycosylation in airway diseases with an increased amount of intracellular acidic (sulfated) mucins (2, 7). Similarly, abnormalities of mucin-type glycoproteins have been described in lung carcinomas (24), although the molecular basis for such differences remains unknown. Therefore, there is an intense interest in understanding the molecular and cellular mechanisms that regulate mucin production and how these are altered in disease states.
Epithelial renewal is a fundamental process required for the maintenance of normal epithelia. Aberrant regulation of cell division may lead to pathologic states, such as neoplasia. Moreover, regenerating epithelia of mature adult tissues display phenotypic characteristics that are essentially similar to those during fetal development (3, 5). This study aimed to provide information regarding the spatiotemporal regulation of mucin gene expression during human fetal lung development. Specifically, we used in situ hybridization to analyze the expression of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6, and MUC7 in the respiratory tract of 13 human embryos and fetuses, comparing this with normal adults. To our knowledge, the only other developmental study of respiratory mucin gene expression was performed by Chambers and colleagues (25), who analyzed MUC1 and MUC2 gene expression in four fetuses aged between 12 and 19 wk of gestation.
Little information is available about the relationship between the expression of the mucin genes and the level of cytodifferentiation of human airway epithelial cells. In cultured tracheal epithelial cells, expression of MUC1 and MUC5AC is coordinately regulated with mucous differentiation (26, 27). Both genes are expressed at low levels in undifferentiated cultures but are strongly induced during mucous cell differentiation. Furthermore, neither mucin gene is expressed in retinoid-deficient cultures that undergo squamous rather than mucous cell differentiation. Contradictory results have also been reported for the expression of MUC2. For example, using monkey tracheobronchial epithelial cells, An and coworkers (28) demonstrated that MUC2 expression is downregulated by retinoids in vitro, thereby suggesting that the expression of this gene does not necessarily coincide with mucous cell differentiation. In contrast, Guzman and colleagues (29) demonstrated recently that retinoids induce both MUC2 and MUC5AC mRNA levels in vitro in normal human tracheobronchial epithelial cells.
In this study, we have demonstrated that mucin gene expression is subjected to profound differential regulation during human fetal lung development from 6.5 to 27 wk of gestation. MUC4 is the first mucin gene to be expressed, initially occurring in the foregut before organogenesis. MUC1 and MUC2 are expressed from 9.5 wk of gestation in trachea, bronchi, epithelial tubules, and terminal sacs. At this stage of gestation, the tracheal epithelium is poorly differentiated, with ciliated cells only appearing between 11 and 12 wk and secretory cells appearing by 13 wk. These cells develop at later stages of gestation in bronchi as differentiation proceeds peripherally through the tissue (30, 31). Thus, MUC1, MUC2, and MUC4 are expressed in fetal lung before epithelial cytodifferentiation into ciliated or secretory cells. In contrast, MUC5AC and MUC5B expression is initiated concomitantly with epithelial cell differentiation around 13 wk of gestation. In particular, this expression is associated with developing mucous glands until 25 to 27 wk of gestation (3, 31). In addition, MUC5B is detected in mucous acini as soon as they form. Finally, the late onset of MUC7 expression is confined to serous cells at 23 wk of gestation and appears to be associated with the cytodifferentiation of this cell type.
Our results concerning MUC1 and MUC5AC are in accordance with in vitro observations (26, 27). Even though the expression of MUC1 and MUC5AC is tightly regulated, MUC1 is often detected earlier than MUC5AC during cytodifferentiation (27), a feature confirmed in the present study. Similarly, MUC1 mRNA and protein expression during mouse embryogenesis demonstrated that this correlates with morphologic differentiation before the epithelium acquires a functional activity (32). Studies in other organ systems have suggested a role for MUC1 in maintaining epithelial polarity and morphology, and in establishing stage-specific cell recognition (33, 34). All of these observations strongly suggest a role for MUC1 in regulating epithelial cytodifferentiation.
Although MUC4 has been isolated from a tracheobronchial mucosal cDNA library (10, 11), virtually nothing is known about MUC4 expression during lung development. In addition to MUC1 and MUC3, MUC4 expression differs from MUC2, MUC5AC, MUC5B, and MUC6, as the former are expressed in both mucus-secreting and non-mucus-secreting cells in adult airway epithelia. For example, MUC1 and MUC4 are expressed in both ciliated and secretory cells. Interestingly, MUC4 mRNAs are expressed at high levels in squamous cell carcinomas, which contrasts with an absence of MUC1 mRNAs in this type of lung carcinoma (18).
In this study, particular attention was paid to the localization of mucin mRNAs in the pseudostratified epithelium of proximal airways. In fetuses, as in adults, MUC1 and MUC4 mRNAs are essentially confined to the upper part of the epithelium, that is, in mature epithelial cells. Although MUC1 and MUC4 expression is acquired early during embryonic life, these genes are expressed at a very low level in poorly differentiated cells, such as basal cells. These observations suggest that several distinct functions may be played by these apomucins.
MUC2 expression in the developing lung is quite different from that observed in normal adult respiratory mucosae. We have shown that MUC2 mRNAs are present by 9.5 wk in undifferentiated epithelial cells. After this time, labeling is confined to basal cells until 23 wk of gestation. In fetuses older than 23 wk, as in adults, MUC2 is strongly expressed in epithelial goblet, basal, and suprabasal cells, as well as in submucosal glands. Therefore, at this gestational age, the fetal respiratory epithelium is fully differentiated and closely resembles adult respiratory tissue (30). Similarly, we have previously demonstrated in fetal intestine that MUC2 is expressed in undifferentiated crypt epithelial cells at early stages of tissue development and acquires an adult pattern of gene expression at 23 wk of gestation, where it is expressed in all cells belonging to the mucus-secreting cell lineages (35).
Interestingly, only a subpopulation of goblet cells expressed MUC2, possibly reflecting cells at varying stages of mucin synthesis as previously suggested (15). Alternatively, distinct subpopulations of goblet cells may exist in the respiratory tract, as has been demonstrated for the intestine (36, 37).
MUC2 is not a prominent mucin in respiratory secretions (38). Consequently, several studies have suggested a role for MUC2 in the pathogenesis of inflammatory airway disorders (14, 39, 40). Moreover, transcriptional activation of MUC2 by Pseudomonas aeruginosa lipopolysaccharide may play a role in the pathogenesis of cystic fibrosis (41). Mucus-secreting cells have been reported to influence the function of the pluripotential stem cells or basal cells in fetuses and in adults after mucosal injury (3, 30). Induction of MUC2 gene expression in inflammatory airway disorders may therefore act as a putative positive regulator of epithelial cell proliferation and mucous cell differentiation.
MUC5AC expression in the developing respiratory tract is of particular interest because this gene product is reported to be a major component of respiratory secretions (38, 42). Moreover, cytokine-mediated induction of MUC5AC gene expression correlates well with mucin hypersecretion (43). This study demonstrated that MUC5AC is primarily expressed in epithelial goblet cells in adults, confirming previous results from this laboratory (13). In contrast, fetal MUC5AC expression initiates at 13 wk of gestation, when it is associated primarily with the development of glandular ducts. Each gland forms from surface epithelial folds, where basal cells multiply and form a cellular bud that grows into the juxtaposed mesenchyme as a solid cylinder while maintaining continuity with the surface epithelium (3, 44). MUC5AC expression is therefore initiated during this developmental phase, when it may also play a potential role in regulating the formation of glandular ducts and glands. It would be interesting to explore this putative function for MUC5AC in an in vitro system of branching or duct morphogenesis using embryonic lung.
Bronchial secretions from normal individuals and patients with chronic bronchitis contain two major apoproteinsMUC5AC and an unknown apoprotein, possibly
MUC5B (38, 42, 45). MUC5B is expressed from 13 wk of gestation in epithelium folds in a similar fashion to MUC5AC.
After 13 wk it is also expressed in gland ducts and in mucous gland cells, with an increased gradient from the surface epithelium to the glands.
MUC7 has been reported to be expressed in trachea and in salivary glands containing mucous acinar cells by Northern blot and RT-PCR analyses (19, 22). Immunohistochemistry using monoclonal antibodies developed to a carbohydrate-containing epitope or a synthetic peptide of MG2 (MUC7) localized MUC7 to discrete mucous acini in submandibular and labial glands (46) and subpopulations of serous cells (47), respectively. This study is the first to localize MUC7 mRNA transcripts in the human respiratory tract, where hybridization is located in serous acini and serous cells (immature and mature) forming crescent shapes encompassing the mucous acini of submucosal glands. During early development, these glands consist only of pure mucous acini (3, 31) that express MUC5B. Although lysozyme-secreting cells can be detected from the 16th week, the first typical serous cells are found only after 25 to 27 wk (3).
This study has also confirmed previous reports that MUC3 and MUC6 mRNAs are not detectable in the respiratory tract (13, 14, 48).
Taken together, our results demonstrate distinct regulation of MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7 during elongation, branching, and epithelial cytodifferentiation of the developing respiratory tract, as well as in proliferative zones in adult epithelium. To date, very little information is available regarding specific promoters that regulate mucin genes (except for MUC1). The fact that three of the six mucin genes that are subject to developmental regulation during the formation of the respiratory tract (i.e., MUC2, MUC5AC, and MUC5B) are clustered on chromosome 11p15.5 (49) is of particular interest. In the future, isolation of promoters and functional mapping studies, as well as using transgenic mice, should bring new insights about regulatory elements directing appropriate spatial and temporal patterns of mucin gene expression.
Finally, there is evidence that mucins play diverse roles in addition to protective and lubricating functions. More particularly, mucins may be implicated in the progression of human carcinomas and in promoting tumor cell metastasis, resulting in a poor prognosis for the patients (50). Studies performed by Northern blot, slot blot, and RT- PCR analyses have shown that the expression of MUC1, MUC3, and MUC4 genes, but not MUC2, is markedly altered in lung carcinomas (14, 18, 51). Well-differentiated lung adenocarcinomas exhibit the largest increase in MUC1, MUC3, and MUC4 mRNA levels, whereas lung squamous cell, adenosquamous, and large-cell carcinomas are characterized by increased levels of MUC4 only. In contrast, using slot blot analysis with specific antisense oligonucleotides, Yu and colleagues (52) reported an overexpression of MUC1, MUC2, MUC3, MUC4, MUC5AC, and MUC5B. However, this overexpression did not correlate with tumor or nodal stage, histology, or pathological differentiation grade, except for MUC5AC and MUC5B expression, which correlated with early postoperative relapse and metastasis. These data need to be confirmed at the cellular level by in situ hybridization, which is currently being performed in our laboratory. Moreover, given the clear cell-specific pattern of mucin gene expression, such a study should bring new insights into the histogenesis of lung neoplasia.
| |
Footnotes |
|---|
Abbreviations: messenger RNA, mRNA; reverse transcriptase-polymerase chain reaction, RT-PCR.
(Received in original form December 2, 1997 and in revised form April 20, 1998).
Acknowledgments: This work was supported by grants from the Association pour la Recherche sur le Cancer, the Comité du Nord de la Ligue Nationale contre le Cancer, and the CH&U de Lille (PHRC 1994, contracts number 96/ 09.29/9595 and number 96/38/9713). The authors are indebted to Dr. Tor Savidge for help in improving the style of the paper, and thank Marie-Claire Dieu and Pascal Mathon for excellent technical assistance.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
1.
Rose, M. C..
1992.
Mucins: structure, function, and role in pulmonary diseases.
Am. J. Physiol.
263:
L413-L429
2. Rogers, D. F.. 1994. Airway goblet cells: responsive and adaptable front-line defenders. Eur. Respir. J. 5: 93-104 .
3. Jeffery, P. K., D. Gaillard, and S. Moret. 1992. Human airway secretory cells during development and in mature airway epithelium. Eur. Respir. J. 7: 1690-1706 .
4. Reid, L.. 1954. Pathology of chronic bronchitis. Lancet i: 275-278 .
5. Gaillard, D., S. Moret, A. Lallemand, and S. Girod. 1990. Histochemistry of glycoconjugates and lysozyme in the human fetal trachea. Eur. Respir. J. 10: 258 .
6. Lamblin, G., J.-P. Aubert, J.-M. Perini, A. Klein, N. Porchet, P. Degand, and P. Roussel. 1992. Human respiratory mucins. Eur. Respir. J. 5: 247-256 [Abstract].
7. Jeffery, P. K., and D. Li. 1997. Airway mucosa: secretory cells, mucus and mucin genes. Eur. Respir. J. 10: 1655-1662 [Abstract].
8.
Gendler, S.,
J. Taylor-Papadimitriou,
T. Duhig,
J. Rothbard, and
J. Burchell.
1988.
A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats.
J. Biol.
Chem.
263:
12820-12823
9.
Van Klinken, B. J.,
J. Dekker,
H. A. Buller, and
A. W. Einerhand.
1995.
Mucin gene structure and expression: protection vs. adhesion.
Am. J. Physiol.
269:
G613-G627
10. Aubert, J.-P., N. Porchet, M. Crépin, M. Duterque-Coquillaud, G. Vergnes, M. Mazzuca, B. Debuire, D. Petitprez, and P. Degand. 1991. Evidence for different human tracheobronchial peptides deduced from nucleotide cDNA sequences. Am. J. Respir. Cell Mol. Biol. 5: 178-185 .
11. Porchet, N., V. C. Nguyen, J. Dufossé, J.-P. Audié, V. Guyonnet-Dupérat, M. S. Gross, C. Denis, P. Degand, A. Bernheim, and J.-P. Aubert. 1991. Molecular cloning and chromosomal localization of a novel human tracheobronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs. Biochem. Biophys. Res. Commun. 175: 414-422 [Medline].
12. Guyonnet-Dupérat, V., J.-P. Audié, V. Debailleul, A. Laine, M.-P. Buisine, S. Zouitina, P. Pigny, P. Degand, J.-P. Aubert, and N. Porchet. 1995. Characterization of the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucin genes? Biochem. J. 305: 211-219 .
13. Audié, J.-P., A. Janin, N. Porchet, M.-C. Copin, B. Gosselin, and J.-P. Aubert. 1993. Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization. J. Histochem. Cytochem. 41: 1479-1485 [Abstract].
14.
Ho, S. B.,
G. A. Niehans,
C. Lyftogt,
P. S. Yan,
D. L. Cherwitz,
E. T. Gum,
R. Dahiya, and
Y. S. Kim.
1993.
Heterogeneity of mucin gene expression
in normal and neoplastic tissues.
Cancer Res.
53:
641-651
15. Dohrman, A., T. Tsuda, E. Escudier, M. Cardone, B. Jany, J. Gum, Y. Kim, and C. Basbaum. 1994. Distribution of lysozyme and mucin (MUC2 and MUC3) mRNA in human bronchus. Exp. Lung Res. 20: 367-380 [Medline].
16.
Guzman, K.,
T. E. Gray,
J.-H. Yoon, and
P. Nettesheim.
1996.
Quantitation
of mucin RNA by PCR reveals induction of both MUC2 and MUC5AC
mRNA levels by retinoids.
Am. J. Physiol.
271:
L1023-L1028
17. Yu, C. J., P. C. Yang, J. Y. Shew, T. M. Hong, S. C. Yang, Y. C. Lee, L. N. Lee, K. T. Luh, and C. W. Wu. 1996. Mucin mRNA expression in lung adenocarcinoma cell lines and tissues. Oncology 53: 118-126 [Medline].
18. Nguyen, P. L., G. A. Niehans, D. L. Cherwitz, Y. S. Kim, and S. B. Ho. 1996. Membrane-bound (MUC1) and secretory (MUC2, MUC3, and MUC4) mucin gene expression in human lung cancer. Tumor Biol. 17: 176-192 .
19. Biesbrock, A. R., L. A. Bobek, and M. J. Levine. 1997. MUC7 gene expression and genetic polymorphism. Glycoconj. J 14: 415-422 [Medline].
20. Shankar, V., P. Pichan, R. L. Eddy, V. Tonk, N. Nowak, S. N. J. Sait, T. B. Shows, R. E. Schultz, G. Gotway, R. C. Elkins, M. S. Gilmore, and G. P. Sachdev. 1997. Chromosomal localization of a human mucin gene (MUC8) and cloning of the cDNA corresponding to the carboxy terminus. Am. J. Respir. Cell Mol. Biol. 16: 232-241 [Abstract].
21. Buisine, M.-P., A. Janin, V. Maunoury, J.-P. Audié, M.-P. Delescaut, M.-C. Copin, J.-F. Colombel, P. Degand, J.-P. Aubert, and N. Porchet. 1996. Aberrant expression of a human mucin gene (MUC5AC) in rectosigmoid villous adenoma. Gastroenterology 110: 84-91 [Medline].
22.
Bobek, L. A.,
H. Tsai,
A. R. Biesbrock, and
M. J. Levine.
1993.
Molecular
cloning, sequence, and specificity of expression of the gene encoding the
low molecular weight human salivary mucin (MUC7).
J. Biol. Chem.
268:
20563-20569
23. Kim, W. D.. 1997. Lung mucus: a clinician's view. Eur. Respir. J. 10: 1914-1917 [Abstract].
24. Kawai, T., M. Suzuki, K. Kase, and Y. Ozeki. 1993. Expression of carbohydrate antigens in human pulmonary adenocarcinoma. Cancer 72: 1581-1587 [Medline].
25. Chambers, J. A., M. A. Hollingworth, A. E. O. Trevize, and A. Harris. 1994. Developmental expression of mucin genes MUC1 and MUC2. J. Cell Sci. 107: 413-424 [Abstract].
26. Park, H., S. W. Hyun, and K. C. Kim. 1996. Expression of MUC1 mucin gene by hamster tracheal surface epithelial cells in primary culture. Am. J. Respir. Cell Mol. Biol. 15: 237-244 [Abstract].
27.
Guzman, K.,
T. Bader, and
P. Nettesheim.
1996.
Regulation of MUC5 and
MUC1 gene expression: correlation with airway mucous differentiation.
Am. J. Physiol.
270:
L846-L853
28. An, G., G. Luo, and R. Wu. 1994. Expression of MUC2 gene is down-regulated by vitamin A at the transcriptional level in vitro in tracheobronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 10: 546-551 [Abstract].
29. Guzman, K., T. E. Gray, J. H. Yoon, and P. Nettesheim. 1996. Quantitation of mucin RNA by PCR reveals induction of both MUC2 and MUC5AC mRNA levels by retinoids. Am. J. Physiol. 271: L1023-L1028 .
30. Gaillard, D. A., A. V. Lallement, A. F. Petit, and E. S. Puchelle. 1989. In vivo ciliogenesis in human fetal tracheal epithelium. Am. J. Anat. 185: 415-428 [Medline].
31. Askin, F. 1991. Respiratory tract disorders in the fetus and neonate. In Textbook of Fetal and Perinatal Pathology. J. S. Wigglesworth and D. B. Singer, editors. Blackwell Scientific Publications, Chicago. 643-688.
32. Braga, V. M. M., L. F. Pemberton, T. Duhig, and S. J. Gendler. 1992. Spatial and temporal expression of an epithelial mucin, Muc-1, during mouse development. Development 115: 427-437 [Abstract].
33. Gendler, S. J., A. P. Spicer, E. N. Lalani, T. Duhig, N. Peat, J. Burchell, L. Pemberton, M. Boshell, and J. Taylor-Papadimitriou. 1991. Structure and biology of a carcinoma-associated mucin, MUC1. Am. Rev. Respir. Dis. 144: S42-S47 [Medline].
34.
Parry, G.,
J. Li,
J. Stubbs,
M. J. Bissell,
C. Schmidhauser,
A. P. Spicer, and
S. J. Gendler.
1992.
Studies of Muc-1 mucin expression and polarity in the
mouse mammary gland demonstrate developmental regulation of Muc-1
glycosylation and establish the hormonal basis for mRNA expression.
J.
Cell Sci.
101:
191-199
35.
Buisine, M.-P.,
L. Devisme,
T. C. Savidge,
C. Gespach,
B. Gosselin,
N. Porchet, and
J.-P. Aubert.
1998.
Mucin gene expression in human embryonic
and foetal intestine.
Gut
43:
519-524
36.
Potten, C. S., and
M. Loeffler.
1990.
Stem cells: attributes, cycles, spirals,
pitfalls and uncertainties: lessons for and from the crypt.
Development
110:
1001-1020
37. Podolsky, D. K., D. A. Fournier, and K. E. Lynch. 1986. Human colonic goblet cells: demonstration of distinct subpopulations defined by mucin-specific monoclonal antibodies. J. Clin. Invest. 77: 1263-1271 .
38. Hovenberg, H. W., J. R. Davis, A. Herrmann, C.-J. Lindén, and I. Carlstedt. 1996. MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions. Glycoconj. J. 13: 839-847 [Medline].
39. Gerard, C., R. L. Eddy, and T. B. Shows. 1990. The core polypeptide of cystic fibrosis tracheal mucin contains a tandem repeat structure. J. Clin. Invest. 86: 1921-1927 .
40.
Levine, S. J.,
P. Larivée,
C. Logun,
C. W. Angus,
F. P. Ognibene, and
J. H. Shelhamer.
1995.
Tumor necrosis factor-
induces mucin hypersecretion
and MUC-2 gene expression by human airway epithelial cells.
Am. J. Respir.
Cell Mol. Biol.
12:
196-204
[Abstract].
41.
Li, J.-D.,
A. F. Dohrman,
M. Gallup,
S. Miyata,
J. R. Gum,
Y. S. Kim,
J. A. Nadel,
A. Prince, and
C. B. Basbaum.
1997.
Transcriptional activation of
mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis
of cystic fibrosis lung disease.
Proc. Natl. Acad. Sci. USA
94:
967-972
42. Hovenberg, H. W., J. R. Davis, and I. Carlstedt. 1996. Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells. Biochem. J. 318: 319-324 .
43. Temann, U.-A., B. Prasad, M. W. Gallup, C. Basbaum, S. B. Ho, R. A. Flavell, and J. A. Rankin. 1997. A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion. Am. J. Respir. Cell Mol. Biol. 16: 471-478 [Abstract].
44. Engelhardt, J. F., H. Schlossberg, J. R. Yankaskas, and L. Dudus. 1995. Progenitor cells of the adult human airway involved in submucosal gland development. Development 121: 2031-2046 [Abstract].
45. Thornton, D. J., P. L. Devine, C. Hanski, M. Howard, and J. K. Sheehan. 1994. Identification of two major populations of mucins in respiratory secretions. Am. J. Respir. Crit. Care Med. 150: 823-832 [Abstract].
46. Cohen, R. E., A. Aguirre, M. E. Neiders, M. J. Levine, P. C. Jones, M. S. Reddy, and J. G. Haar. 1991. Immunochemistry and immunogenicity of low molecular weight human salivary mucin. Arch. Oral. Biol. 36: 347-356 [Medline].
47.
Nielsen, P. A.,
E. P. Bennett,
H. H. Wandall,
M. H. Therkildsen,
J. Hannibal, and
H. Clausen.
1997.
Identification of a major human high molecular
weight salivary mucin (MG1) as tracheobronchial mucin MUC5B.
Glycobiology
7:
413-419
48.
Toribara, N. W.,
A. M. Roberton,
S. B. Ho,
W. L. Kuo,
J. W. Hicks,
J. R. Gum,
J. C. Byrd,
B. Siddiki, and
Y. S. Kim.
1993.
Human gastric mucin.
J.
Biol. Chem.
268:
5879-5885
49. Pigny, P., V. Guyonnet-Dupérat, A. S. Hill, W. S. Pratt, S. Galiègue-Zouitina, M. Collyn, D'Hooghe, A. Laine, I. Van-Seuningen, P. Degand, J. R. Gum, Y. S. Kim, D. M. Swallow, J.-P. Aubert, and N. Porchet. 1996. Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes. Genomics 38: 340-352 [Medline].
50. Ho, S. B., and Y. S. Kim. 1995. Do mucins promote tumor cell metastasis? Intern. J. Oncol. 7: 913-926 .
51. Yu, C. J., P. C. Yang, J. Y. Shew, T. M. Hong, S. C. Yang, Y. C. Lee, L. N. Lee, K. T. Luh, and C. W. Wu. 1996. Mucin mRNA expression in lung adenocarcinoma cell lines and tissues. Oncology 53: 118-126 .
52. Yu, C. J., P. C. Yang, C. T. Shun, Y. C. Lee, S. H. Kuo, and K. T. Luh. 1996. Overexpression of MUC5 genes is associated with early post-operative metastasis in non-small-cell lung cancer. Int. J. Cancer 69: 457-465 [Medline].
This article has been cited by other articles:
 |
|
 |
|
  P. Chaturvedi, A. P. Singh, and S. K. Batra Structure, evolution, and biology of the MUC4 mucin FASEB J, April 1, 2008; 22(4): 966 - 981. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Kreda, S. F. Okada, C. A. van Heusden, W. O'Neal, S. Gabriel, L. Abdullah, C. W. Davis, R. C. Boucher, and E. R. Lazarowski Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells J. Physiol., October 1, 2007; 584(1): 245 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Jonckheere, A. Vincent, M. Perrais, M.-P. Ducourouble, A. K.-v. Male, J.-P. Aubert, P. Pigny, K. L. Carraway, J.-N. Freund, I. B. Renes, et al. The Human Mucin MUC4 Is Transcriptionally Regulated by Caudal-related Homeobox, Hepatocyte Nuclear Factors, Forkhead Box A, and GATA Endodermal Transcription Factors in Epithelial Cancer Cells J. Biol. Chem., August 3, 2007; 282(31): 22638 - 22650. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Chaturvedi, A. P. Singh, N. Moniaux, S. Senapati, S. Chakraborty, J. L. Meza, and S. K. Batra MUC4 Mucin Potentiates Pancreatic Tumor Cell Proliferation, Survival, and Invasive Properties and Interferes with Its Interaction to Extracellular Matrix Proteins Mol. Cancer Res., April 1, 2007; 5(4): 309 - 320. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tamada, H. Shibahara, M. Higashi, M. Goto, S. K. Batra, K. Imai, and S. Yonezawa MUC4 Is a Novel Prognostic Factor of Extrahepatic Bile Duct Carcinoma. Clin. Cancer Res., July 15, 2006; 12(14): 4257 - 4264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Rose and J. A. Voynow Respiratory Tract Mucin Genes and Mucin Glycoproteins in Health and Disease Physiol Rev, January 1, 2006; 86(1): 245 - 278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Saitou, M Goto, M Horinouchi, S Tamada, K Nagata, T Hamada, M Osako, S Takao, S K Batra, T Aikou, et al. MUC4 expression is a novel prognostic factor in patients with invasive ductal carcinoma of the pancreas J. Clin. Pathol., August 1, 2005; 58(8): 845 - 852. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Gray, P. Nettesheim, C. Loftin, J.-S. Koo, J. Bonner, S. Peddada, and R. Langenbach Interleukin-1{beta}-Induced Mucin Production in Human Airway Epithelium Is Mediated by Cyclooxygenase-2, Prostaglandin E2 Receptors, and Cyclic AMP-Protein Kinase A Signaling Mol. Pharmacol., August 1, 2004; 66(2): 337 - 346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Moniaux, G. C. Varshney, S. C. Chauhan, M. C. Copin, M. Jain, U. A. Wittel, M. Andrianifahanana, J.-P. Aubert, and S. K. Batra Generation and Characterization of Anti-MUC4 Monoclonal Antibodies Reactive with Normal and Cancer Cells in Humans J. Histochem. Cytochem., February 1, 2004; 52(2): 253 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. M. Fischer, J. G. Cuellar, M. L. Diehl, A. M. deFreytas, J. Zhang, K. L. Carraway, and J. A. Voynow Neutrophil elastase increases MUC4 expression in normal human bronchial epithelial cells Am J Physiol Lung Cell Mol Physiol, April 1, 2003; 284(4): L671 - L679. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Perrais, P. Pigny, M.-C. Copin, J.-P. Aubert, and I. Van Seuningen Induction of MUC2 and MUC5AC Mucins by Factors of the Epidermal Growth Factor (EGF) Family Is Mediated by EGF Receptor/Ras/Raf/Extracellular Signal-regulated Kinase Cascade and Sp1* J. Biol. Chem., August 23, 2002; 277(35): 32258 - 32267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. D. Leikauf, M. T. Borchers, D. R. Prows, and L. G. Simpson Mucin Apoprotein Expression in COPD* Chest, May 1, 2002; 121(5_suppl): 166S - 182S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M-P Buisine, P Desreumaux, E Leteurtre, M-C Copin, J-F Colombel, N Porchet, and J-P Aubert Mucin gene expression in intestinal epithelial cells in Crohn's disease Gut, October 1, 2001; 49(4): 544 - 551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Roger, J-P. Gascard, J. Bara, E. Dulmet, and C. Brink EGTA treatment of human airways in vitro unmasks M1/MUC5AC mucin in submucosal glands Eur. Respir. J., July 1, 2001; 18(1): 176 - 183. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-P. Buisine, L. Devisme, V. Maunoury, E. Deschodt, B. Gosselin, M.-C. Copin, J.-P. Aubert, and N. Porchet Developmental Mucin Gene Expression in the Gastroduodenal Tract and Accessory Digestive Glands. I. Stomach: A Relationship to Gastric Carcinoma J. Histochem. Cytochem., December 1, 2000; 48(12): 1657 - 1666. [Abstract] [Full Text]
|
|
|
|
|
|
M.-P. Buisine, L. Devisme, P. Degand, M.-C. Dieu, B. Gosselin, M.-C. Copin, J.-P. Aubert, and N. Porchet Developmental Mucin Gene Expression in the Gastroduodenal Tract and Accessory Digestive Glands. II. Duodenum and Liver, Gallbladder, and Pancreas J. Histochem. Cytochem., December 1, 2000; 48(12): 1667 - 1676. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. E. Vinall, J. C. Fowler, A. L. Jones, H. J. Kirkbride, C. de Bolós, A. Laine, N. Porchet, J. R. Gum, Y. S. Kim, F. M. Moss, et al. Polymorphism of Human Mucin Genes in Chest Disease . Possible Significance of MUC2 Am. J. Respir. Cell Mol. Biol., November 1, 2000; 23(5): 678 - 686. [Abstract] [Full Text]
|
|
|
|
 |
|
 J.-L. Desseyn, J.-P. Aubert, N. Porchet, and A. Laine Evolution of the Large Secreted Gel-Forming Mucins Mol. Biol. Evol., August 1, 2000; 17(8): 1175 - 1184. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Louahed, M. Toda, J. Jen, Q. Hamid, J.-C. Renauld, R. C. Levitt, and N. C. Nicolaides Interleukin-9 Upregulates Mucus Expression in the Airways Am. J. Respir. Cell Mol. Biol., June 1, 2000; 22(6): 649 - 656. [Abstract] [Full Text]
|
|
|
|
|
|
D. J. Thornton, T. Gray, P. Nettesheim, M. Howard, J. S. Koo, and J. K. Sheehan Characterization of mucins from cultured normal human tracheobronchial epithelial cells Am J Physiol Lung Cell Mol Physiol, June 1, 2000; 278(6): L1118 - L1128. [Abstract] [Full Text] [PDF]
|
|