ARTICLE |
Correspondence to: Gwynneth D. Offner, Boston U. Medical Center, Section of Gastroenterology, 650 Albany St., X-510, Boston, MA 02118. E-mail: goffner@bu.edu
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Summary |
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Mucins are high molecular weight glycoproteins secreted by salivary glands and epithelial cells lining the digestive, respiratory, and reproductive tracts. These glycoproteins, encoded in at least 13 distinct human genes, can be subdivided into gel-forming and membrane-associated forms. The gel-forming mucin MUC5B is secreted by mucous acinar cells in major and minor salivary glands, but little is known about the expression pattern of membrane-associated mucins. In this study, RT-PCR and Northern blotting demonstrated the presence of transcripts for MUC1 and MUC4 in both parotid and submandibular glands, and in situ hybridization localized these transcripts to epithelial cells lining striated and excretory ducts and in some serous acinar cells. The same cellular distribution was observed by immunohistochemistry. Soluble forms of both mucins were detected in parotid secretion after immunoprecipitation with mucin-specific antibodies. These studies have shown that membrane-associated mucins are produced in both parotid and submandibular glands and that they are expressed in different cell types than gel-forming mucins. Although the function of these mucins in the oral cavity remains to be elucidated, it is possible that they both contribute to the epithelial protective mucin layer and act as receptors initiating one or more intracellular signal transduction pathways.
(J Histochem Cytochem 50:811820, 2002)
Key Words: human mucin genes, salivary mucins, parotid and submandibular, glands, epithelial protection
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Introduction |
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MUCINS ARE HIGH molecular weight glycoproteins secreted by epithelial surfaces lining the gastrointestinal, respiratory, and reproductive tracts. These glycoproteins protect mucosal surfaces from desiccation, mechanical injury, and microbial assault. In recent years, genes for 13 distinct human mucins have been identified and have been designated MUC1-4, MUC5AC, MUC5B, MUC6-8, MUC11-13, and MUC16 (
By contrast, membrane-associated mucins are monomeric. The best characterized of the membrane-associated mucins are MUC1 and sialomucin complex (SMC), the rat homologue of MUC4. These proteins are synthesized as a single polypeptide chain which is cleaved in the ER to generate two subunits that assemble non-covalently as a heterodimer at the cell surface (
Human submandibular, sublingual, and minor salivary gland secretions contain high and low molecular weight mucins named MG1 (mucous glycoprotein 1) and MG2 (mucous glycoprotein 2), respectively (
Because the parotid gland is considered a purely serous gland on the basis of histological criteria, it has been widely assumed that this gland does not synthesize mucins. The parotid gland does not express MUC5B, the major component of MG1 (
The present investigation was initiated to determine the expression patterns of membrane-associated mucins in the human parotid gland and to compare the expression pattern with that in human submandibular gland. MUC1 and MUC4 transcripts were identified in parotid gland by RT-PCR and by Northern blotting and were localized to epithelial cells lining striated and excretory ducts by in situ hybridization (ISH). MUC1 and MUC4 gene products displayed a similar cellular distribution by immunohistochemistry (IHC). Finally, immunoprecipitation (IP) experiments demonstrated that these mucins are components of parotid secretion.
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Materials and Methods |
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Tissue Samples
Human parotid, submandibular, and sublingual gland tissue was obtained from the National Disease Research Interchange (Philadelphia, PA). All samples were snap-frozen in liquid nitrogen less than 12 hr post mortem, shipped on dry ice, and stored at -80C until used. Tissue samples used for ISH and IHC were thawed on ice, fixed in 4% paraformaldehyde at 4C overnight, embedded in paraffin, sectioned, and mounted on microscope slides. Buccal epithelial cells were obtained from whole saliva sediment as described (
PCR and Northern Blotting Analyses
RNA was isolated from tissue samples using TriPure Reagent (Roche; Indianapolis, IN). RT-PCR reactions were carried out under standard conditions (30 cycles of 1 min at 95C, 1 min at 62C, 1.5 min at 72C) using primers corresponding to non-tandem repeat regions of the membrane-associated mucins MUC1, MUC3, MUC4, MUC12, and the gel-forming mucin MUC5B (Table 1). Northern blots of salivary gland RNAs (15 µg) were hybridized with 32P-labeled MUC1 and MUC4 probes containing mucin tandem repeat sequences. The 0.4-kb MUC1 probe was derived from a clone isolated from a human gallbladder cDNA library (
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In Situ Hybridization
MUC1 and MUC4 riboprobes were synthesized using the Riboprobe In Vitro Transcription System (Promega; Madison, WI) according to the manufacturer's instructions. pBluescript plasmids containing the MUC1 and MUC4 tandem repeat probes used for Northern analysis (see above) were linearized with BamH1 and antisense riboprobes were synthesized using T7 RNA polymerase. The corresponding sense riboprobes were synthesized from plasmids linearized with XhoI using T3 RNA polymerase. Riboprobes (labeled with [-35S]-UTP) were resuspended in 20 µl of 1 M DTT and 180 µl of hybridization buffer (HB) containing 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM Na2HPO4, 50% deionized formamide, 300 mM NaCl, 1 x Denhardt's solution, 10% dextran sulfate, and 0.5 mg/ml of total yeast RNA.
Tissue sections on glass microscope slides were deparaffinized in xylene, rehydrated using a graded ethanol series (100% to 30%), rinsed in 150 mM NaCl and PBS, each for 5 min, and postfixed in 4% paraformaldehyde in PBS for 30 min. Tissue sections were treated with proteinase K (Roche; 20 µg/ml) in 50 mM Tris-HCl, pH 7.2, containing 5 mM EDTA at room temperature (RT) for 7.5 min and fixed in 4% paraformaldehyde in PBS. Acetylation was carried out for 10 min with 0.25% acetic anhydride in 100 mM triethanolamine, pH 8.0, and slides were rinsed sequentially in PBS and 150 mM NaCl, dehydrated through a graded ethanol series, and allowed to dry for at least 2 hr before hybridization.
Hybridization was carried out as previously described (
Immunohistochemistry
Sections were deparaffinized and rehydrated as described above and endogenous peroxidase activity was quenched by treatment with 0.3% H2O2 in 100% methanol for 30 min at RT. Antigen retrieval was carried out by autoclaving (liquid cycle) the slides in 10 mM citrate buffer, pH 6.0, for 15 min and subsequently allowing slides to stand at RT for 20 min.
IHC was carried out with a series of specific antibodies directed against MUC1 and MUC4 (Table 2). MUC1 was detected using a monoclonal anti-MUC1 antibody (clone E29) purchased from NeoMarkers (Fremont, CA) and MUC4 was detected using rabbit polyclonal antibodies SG9 and MUC4-CT. SG9 (kindly provided by Dr. Sandra Gendler, Mayo Clinic Scottsdale) is directed against a synthetic peptide corresponding to a sequence in the tandem repeat domain and MUC4-CT is directed against a synthetic peptide, SGARFSYFLNSAEAL, corresponding to a sequence in the cytoplasmic domain. A similar antibody directed against an analogous portion of the cytoplasmic domain of the rat homologue (i.e., ASGP-2 subunit of SMC) of human MUC4 has been used previously to detect the membrane-associated form of SMC in a variety of tissues (
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Tissue sections mounted on glass slides were incubated with blocking buffer for 30 min at 37C to mask nonspecific binding sites. For all antibodies, the blocking buffer was PBS containing 3% BSA and 10% normal goat serum. Slides were rinsed three times with PBS containing 0.1% Tween-20 for 5 min and incubated with primary antibodies for 30 min at 37C. E29 (1:100), SG9 (1:100), and MUC4-CT (1:500) were diluted in PBS, 1% BSA, and 3% normal goat serum. Sections were rinsed and incubated with either biotinylated goat anti-mouse IgG (1:1000) or biotinylated goat anti-rabbit IgG (1:200) at 37C for 30 min. Slides were rinsed three times and incubated at 37C for 30 min with ABC reagent (avidin/biotin complex Elite Vectastain; Vector Laboratories, Burlingame, CA) in PBS containing 0.1% Tween-20 to enhance the color reaction. Color development was achieved using diaminobenzidine (Vector Laboratories) for 210 min at RT and sections were stained with 0.68% hematoxylin before examination under the light microscope. Control experiments were carried out in which incubation with primary antibodies was omitted. Sections from four different parotid gland and four different submandibular gland specimens were examined.
Immunoprecipitation and Western Blotting
Stimulated parotid secretion was collected using Curby cups as described (
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Protein A/G beads (Santa Cruz Biotechnology; Santa Cruz, CA) were added and the mixture was incubated for 1 hr at 4C on a rotary mixer. Beads were collected by centrifugation, washed twice in IP buffer and twice in wash buffer containing 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 900 mM NaCl, and 0.5% NP-40. Bound proteins eluted by boiling in sample loading buffer were electrophoresed on 6% polyacrylamide gels or on tricine gels, transferred to nitrocellulose, and the blots incubated as described below. To detect MUC1, blots containing HMFG-2 or CT-2 IPs were probed with the same antibodies. To detect MUC4, blots containing anti-MG1 IPs were incubated with either the anti-MG1 antibody (1:2500) or with the MUC4-specific antibody SG9 (1:200). Blots were incubated with peroxidase-conjugated species-specific secondary antibodies with detection using the Renaissance chemiluminescence reagent (NEN; Boston, MA).
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Results |
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On the basis of previous reports suggesting that mucins might be synthesized in human parotid gland, the present work was undertaken to determine whether genes for membrane-associated mucins were expressed in this gland and to compare the pattern of mucin gene expression in parotid and submandibular glands.
RT-PCR and Northern Analyses
Expression of mucin genes in major salivary glands was investigated by RT-PCR. Parotid gland contained transcripts for the membrane-associated mucins MUC1 and MUC4 (Fig 1, Lane P), whereas transcripts for MUC3 and MUC12 were not detectable (data not shown). Transcripts for the gel-forming mucin MUC5B were also not detectable in parotid gland using this technique (Fig 1, Lane P). In contrast, submandibular and sublingual gland contained transcripts for all three mucins (Fig 1, Lanes SM, SL). Interestingly, buccal epithelial cells also contained MUC1 and MUC4 transcripts with lower but detectable levels of MUC5B transcripts (Fig 1, Lane BEC).
To determine the relative expression levels of MUC1 and MUC4 transcripts in parotid, submandibular, and sublingual glands, Northern blots of RNA from these glands were hybridized with DNA probes containing MUC1 and MUC4 tandem repeats. Two discrete MUC1 transcripts of approximately 6.4 and 4.7 kb were observed in all three major salivary glands (Fig 2A). The 6.4-kb transcript most likely represents full-length MUC1 RNA, while the 4.7-kb transcript may correspond to an allelic variation containing a lesser number of tandem repeats (
In Situ Hybridization
Having identified MUC1 and MUC4 transcripts in major salivary glands (Fig 2A and Fig 2B), ISH was carried out to localize these mRNAs within glandular tissue. The data obtained with antisense riboprobes are shown in Fig 3A, Fig 3C, Fig 3E, Fig 3G, Fig 3I, Fig 3K, Fig 3M, and Fig 3O, and those with sense probes (negative controls) are shown in Fig 3B, Fig 3D, Fig 3F, Fig 3H, Fig 3J, Fig 3L, Fig 3N, and Fig 3P. In parotid gland, weak hybridization of the MUC1 antisense probe was detected in cells lining striated ducts (Fig 3A), whereas in submandibular gland a much stronger signal was observed in striated duct cells as well as in some serous acinar cells (Fig 3C). A similar distribution of mucin transcripts was observed in both glands with the MUC4 antisense probe (Fig 3E and Fig 3G), while the intensity of hybridization in both glands was nearly the same. The MUC4 antisense probe also hybridized strongly to cells lining major excretory ducts in both parotid (Fig 3I) and submandibular gland (Fig 3K). As expected, ISH carried out with the MUC5B antisense probe showed no significant hybridization to transcripts in parotid gland acinar cells (Fig 3M), although transcripts were clearly present in mucous acinar cells of submandibular gland (panel O). No MUC5B transcripts were detected in duct cells in either salivary gland (Fig 3M and Fig 3O). In addition, no significant hybridization signal was detected with the sense riboprobes for any of the mucin genes examined.
Immunohistochemistry
To determine whether MUC1 and MUC4 proteins were expressed in salivary glands, IHC experiments were performed using a monoclonal antibody directed against an epitope in the tandem repeat domain of MUC1 (E29) and polyclonal antibodies directed against peptide sequences in both the tandem repeat (SG9) and cytoplasmic (MUC4-CT) domains of MUC4.
Using the anti-MUC1 antibody, strong immunoreactivity was detected in epithelial cells lining striated ducts in parotid and submandibular gland (Fig 4A and Fig 4B). Immunoreactive anti-MUC1 material was also observed in the lumen of striated ducts and diffuse staining was detected in some clusters of serous acinar cells in both glands. In other sections, immunoreactive MUC1 material was also detected in major excretory ducts in both parotid and submandibular glands (data not shown). Both anti-MUC4 antibodies reacted strongly with material in striated ductal epithelial cells of both parotid (Fig 4C and Fig 4E) and submandibular (Fig 4D and Fig 4F) gland. Some immunoreactive material was also observed in serous acinar cells in both glands. The similarity in the pattern of immunochemical staining observed with SG9 (extracellular domain; Fig 4C and Fig 4D) and MUC4-CT (cytoplasmic domain; Fig 4E and Fig 4F) indicated that the MUC4 present on duct epithelial cells and serous acinar cells represents intact mucin molecules containing the tandem repeat, transmembrane, and cytoplasmic regions. Control experiments carried out in which each of the primary antibodies was omitted did not demonstrate immunoreactive protein in either parotid or submandibular gland sections (data not shown).
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Immunoprecipitation and Western Blotting
To determine whether MUC1 and MUC4 proteins are normal constituents of parotid secretion, a series of IP experiments were carried out. Western blots of material precipitated with HMFG-2, an antibody directed against the MUC1 tandem repeat domain, and probed with the same antibody revealed a faint smear of high molecular weight protein in the upper portion of the separating gel. This material had a similar electrophoretic mobility as authentic MUC1 in IPs from a lysate of MCF-7 breast cancer cells probed with the same antibody (Fig 5A, Lanes 1, 2). Control IPs were prepared using an irrelevant monoclonal antibody (anti-glutathione-S-transferase; GST), and when blots were probed with HMFG-2 (against MUC1 tandem repeats) no immunoreactive material was detected (Fig 5A, Lane 3). This demonstrates that the extracellular portion of MUC1 is present in parotid secretion. Western blots of anti-MG1 IPs, probed with the same antibody, revealed several discrete protein bands all greater than 200 kD (Fig 5A, Lane 4). A similar pattern of immunoreactive proteins was observed when blots of anti-MG1 IPs were probed with SG9, which recognizes an epitope in the tandem repeat domain of MUC4 (Fig 5A, Lane 5). This demonstrates that the extracellular portion of MUC4 was also detectable in parotid secretion, and we concluded from these studies that MUC1 and MUC4 proteins are normal constituents of this fluid.
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Because MUC1 is a transmembrane protein containing both a mucin-like extracellular domain and a short cytoplasmic domain, the immunoprecipitation experiment described above does not distinguish between intact MUC1 (containing the cytoplasmic domain) released into parotid secretion by turnover of duct epithelial cells and soluble MUC1 (lacking the cytoplasmic domain) released into parotid secretion by proteolytic cleavage of the membrane-associated mucin. Therefore, additional IP experiments were carried out using CT-2, an antibody directed against a synthetic peptide corresponding to a sequence in the cytoplasmic domain of MUC1. IPs of parotid secretion prepared with CT-2 revealed no detectable immunoreactive protein when blots of IPs were probed with the same antibody (Fig 5B, Lane 1). Control IPs of MCF-7 breast cancer cell lysates probed with CT-2 contained three major proteins in the 1824-kD range (Fig 5B, Lane 2). It has been suggested that these immunoreactive bands represent different glycoforms of the membrane-associated MUC1 subunit which contains the cytoplasmic domain (
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Discussion |
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The structures and properties of salivary mucins have been studied extensively during the past two decades. These proteins were first identified as biochemical entities MG1 and MG2 and later were found to represent the products of the MUC5B and MUC7 genes, respectively (
Early histochemical studies identified a mucin-like substance in serous acini and duct epithelial cells in both parotid and submandibular glands (
In view of earlier evidence summarized above that epithelial membrane antigen occurred in both parotid and submandibular glands, it seemed likely that the membrane-associated mucins MUC1 and MUC4 would also be expressed in parotid gland. In a preliminary communication, we reported that transcripts for MUC1 and MUC4, as well as the corresponding proteins, were synthesized in parotid gland (
After having identified both MUC1 and MUC4 transcripts and proteins in parotid gland tissue, we were interested to determine whether these mucins were present in parotid secretion and, if so, to ascertain whether they were soluble forms arising by proteolytic cleavage of membrane-associated forms or were full-length species arising from turnover of duct epithelial cells. It was found that both MUC1 and MUC4 could be immunoprecipitated from parotid secretion with antibodies directed against extracellular portions of the respective molecules (see Fig 5). However, MUC1 could not be detected in immunoprecipitates using CT-2, an antibody directed against the cytoplasmic domain of this mucin. This provides strong evidence that MUC1 in parotid secretion is probably derived proteolytically from the membrane-associated form. The origin of MUC4 in parotid secretion remains to be determined.
The physiological function of MUC1 and MUC4 in salivary gland duct cells is not known. By analogy to breast tissue, it has been suggested that in minor salivary glands MUC1 may facilitate flow of secretion through the duct system (
In addition, it is possible that MUC1 and MUC4, synthesized in duct epithelial cells, are released proteolytically and enter the oral cavity, where they interact with one or more receptor-like molecules on oral epithelial surfaces. The resulting membrane-associated mucin complexes could interact with the gel-forming mucin MUC5B and form a "scaffold" (
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Acknowledgments |
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Supported in part by NIH grants DK 44619, DE 11691, and DE 07652.
We wish to thank Dr Sandra Gendler (Mayo Clinic; Scottsdale, AZ) for providing antibodies to MUC1 and MUC4.
Received for publication July 17, 2001; accepted December 27, 2001.
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