MUC1 and Polarity Markers INADL and SCRIB Identify Salivary Ductal Cells.

Current treatments for xerostomia/dry mouth are palliative and largely ineffective. A permanent clinical resolution is being developed to correct hyposalivation using implanted hydrogel-encapsulated salivary human stem/progenitor cells (hS/PCs) to restore functional salivary components and increase salivary flow. Pluripotent epithelial cell populations derived from hS/PCs, representing a basal stem cell population in tissue, can differentiate along either secretory acinar or fluid-transporting ductal lineages. To develop tissue-engineered salivary gland replacement tissues, it is critical to reliably identify cells in tissue and as they enter these alternative lineages. The secreted protein α-amylase, the transcription factor MIST1, and aquaporin-5 are typical markers for acinar cells, and K19 is the classical ductal marker in salivary tissue. We found that early ductal progenitors derived from hS/PCs do not express K19, and thus earlier markers were needed to distinguish these cells from acinar progenitors. Salivary ductal cells express distinct polarity complex proteins that we hypothesized could serve as lineage biomarkers to distinguish ductal cells from acinar cells in differentiating hS/PC populations. Based on our studies of primary salivary tissue, both parotid and submandibular glands, and differentiating hS/PCs, we conclude that the apical marker MUC1 along with the polarity markers INADL/PATJ and SCRIB reliably can identify ductal cells in salivary glands and in ductal progenitor populations of hS/PCs being used for salivary tissue engineering. Other markers of epithelial maturation, including E-cadherin, ZO-1, and partition complex component PAR3, are present in both ductal and acinar cells, where they can serve as general markers of differentiation but not lineage markers.

Expression of the transmembrane mucins, MUC1, MUC4 and MUC16, in normal endometrium and in endometriosis.

STUDY QUESTION: Are the transmembrane mucins, MUC1, MUC4 and MUC16, differentially expressed in endometriosis compared with normal endometrium? SUMMARY ANSWER: This study revealed that transmembrane mucin expression does not vary significantly in normal endometrium during the menstrual cycle and is not altered in endometriosis relative to the epithelial marker, cytokeratin-18 (KRT18). WHAT IS KNOWN ALREADY: Increased serum levels of the transmembrane mucin fragments MUC1, MUC4 and MUC16 that normally dominate the apical surface of simple epithelia are found in several pathological conditions, including endometriosis. Altered mucin expression in gynecologic diseases may promote infertility or endometrial pathologies. STUDY DESIGN, SIZE, DURATION: This was a laboratory-based study of samples from 12 endometriosis patients as well as non-endometriosis control samples obtained from 31 patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was isolated from endometrial biopsies of ectopic and eutopic endometrium from women with endometriosis and control patients from different stages of the menstrual cycle. Quantitative (q)-RT-PCR analyses were performed for the mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, cytokeratin-18 (KRT18), or ß-actin (ACTB). Frozen sections from endometrial biopsies of proliferative and mid-secretory stage women with endometriosis were immunostained for MUC1, MUC4 and MUC16. MAIN RESULTS AND THE ROLE OF CHANCE: qRT-PCR analyses of MUC1 and MUC16 mRNA revealed that these mucins do not vary significantly during the menstrual cycle nor are they altered in women with endometriosis relative to the epithelial marker, KRT18. MUC4 mRNA is expressed at very low levels relative to MUC1 and MUC16 under all conditions. There was little difference in MUC1 and MUC16 expression between eutopic endometrial and ectopic endometriotic tissues. MUC4 expression also was not significantly higher in the ectopic endometriotic tissues. Immunostaining for all three mucins reveals robust expression of MUC1 and MUC16 at the apical surfaces of endometrial epithelia, but little to no staining for MUC4. LIMITATIONS, REASONS FOR CAUTION: qRT-PCR analysis was the main method used for mucin detection. Additional studies with stage III-IV endometriotic tissue would be useful to determine if changes in MUC1 and MUC16 expression occur, or if MUC4 expression increases, at later stages of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: We report a comprehensive comparative profile of the major transmembrane mucins, MUC1, MUC4 and MUC16, relative to the epithelial marker, KRT18, in normal cycling endometrium and in endometriosis, and indicate constitutive expression. Previous studies have profiled the expression of individual mucins relative to ß-actin and indicate accumulation in the luteal phase. Thus, these differences in interpretation appear to reflect the increased epithelial content of endometrium during the luteal phase. STUDY FUNDING: This study was supported by: NIH R01HD29963 to D.D.C.; NIH U54HD007495 to S.M.H.; and NIH R01HD067721 to S.L.Y. and B.A.L. The authors have no competing interests to declare.

Elafin expression in mucosa of fallopian tubes is altered by hydrosalpinx.

Elafin is a natural antimicrobial molecule and a member of the antileukoproteinase (Trappin) family. It is normally expressed in the mucosae of fallopian tubes. Hydrosalpinx is a chronic inflammatory process of the fallopian tubes. The objective of this study is to compare the localization of elafin protein and levels of elafin messenger RNA (mRNA) in the mucosa of oviducts with and without hydrosalpinx. Immunohistochemical analysis was performed on tissue sections of hydrosalpinx (n = 10) and normal tubes (n = 22) from paraffin-embedded blocks, obtained from patients who underwent salpingectomy for benign conditions. The main outcome measure was the intensity of staining with 3,3'-diaminobenzidine calculated by ImageJ software and mRNA expression by real-time polymerase chain reaction. The mean intensity of elafin (mean ± standard deviation) in mucosae of the fallopian tubes was 69.68 ± 24.55 in controls and 32.03±18.16 in patients with hydrosalpinx (P < .0001). Elafin mRNA levels were reduced in hydrosalpinx, although not significantly (P = .05, n = 9 from each group). Therefore, tubal epithelium of women with hydrosalpinx seems to have a lower expression of elafin, an elastase inhibitor and a natural antimicrobial molecule, compared to normal tubes.

Heparanase expression and activity influences chondrogenic and osteogenic processes during endochondral bone formation.

Endochondral bone formation is a highly orchestrated process involving coordination among cell-cell, cell-matrix and growth factor signaling that eventually results in the production of mineralized bone from a cartilage template. Chondrogenic and osteogenic differentiation occur in sequence during this process, and the temporospatial patterning clearly requires the activities of heparin binding growth factors and their receptors. Heparanase (HPSE) plays a role in osteogenesis, but the mechanism by which it does so is incompletely understood. We used a combination of ex vivo and in vitro approaches and a well described HPSE inhibitor, PI-88 to study HPSE in endochondral bone formation. In situ hybridization and immunolocalization with HPSE antibodies revealed that HPSE is expressed in the peri-chondrium, peri-osteum, and at the chondro-osseous junction, all sites of key signaling events and tissue morphogenesis. Transcripts encoding Hpse also were observed in the pre-hypertrophic zone. Addition of PI-88 to metatarsals in organ culture reduced growth and suggested that HPSE activity aids the transition from chondrogenic to osteogenic processes in growth of long bones. To study this, we used high density cultures of ATDC5 pre-chondrogenic cells grown under conditions favoring chondrogenesis or osteogenesis. Under chondrogenic conditions, HPSE/Hpse was expressed at high levels during the mid-culture period, at the onset of terminal chondrogenesis. PI-88 addition reduced chondrogenesis and accelerated osteogenesis, including a dramatic up-regulation of osteocalcin levels. In normal growth medium, addition of PI-88 reduced migration of ATDC-5 cells, suggesting that HPSE facilitates cartilage replacement by bone at the chondro-osseous junction by removing the HS component of proteoglycans, such as perlecan/HSPG2, that otherwise prevent osteogenic cells from remodeling hypertrophic cartilage.

Perlecan domain I promotes fibroblast growth factor 2 delivery in collagen I fibril scaffolds.

Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17 , 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.

MUC1 expression in human prostate cancer cell lines and primary tumors.

MUC1 expression was evaluated in normal prostate epithelial cells (PrEC), and prostate cancer cell lines in response to dihydrotestosterone (DHT), interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) treatment. Expression of MUC1 core protein was stimulated in PrEC and PC-3 cells after cytokine treatment, but was highly and constitutively expressed by DU-145 cells. MUC1 was not expressed by LNCaP, C4-2 or C4-2B cells under any condition. DHT alone or in combination with cytokines had no effect on MUC1 expression in any cell line tested. Using antibodies capable of detecting all isoforms of MUC1 core protein independent of their glycosylation state, immunohistochemical staining of tissue microarrays containing both nontumor and tumor tissue revealed that only 17% of tumor tissues and 41% of nontumor tissues stained positively for MUC1. Staining patterns in tumor tissue varied from focal apical staining to diffuse cytoplasmic staining. Neither the presence of MUC1 core protein nor its subcellular distribution correlated with Gleason grade. These data indicate that MUC1 is a poor marker of prostate cancer progression. Furthermore, IFN-gamma and TNF-alpha strongly induce MUC1 expression in both normal prostate epithelia and certain prostate tumor cell lines and may exacerbate pathologies associated with MUC1-positive prostate cancers.

Heparan sulfate interacting protein (HIP/L29) negatively regulates growth responses to basic fibroblast growth factor in gingival fibroblasts.

Basic fibroblast growth factor (bFGF) modulates gingival growth, and its release from heparan sulfate (HS) in the extracellular matrix (ECM) governs local tissue bioavailability. We identified a heparin/HS interacting protein (HIP/L29) that recognizes specific HS sequences. We hypothesize that HIP/L29, by modulating the interactions of bFGF with HS chains on proteoglycans, could regulate bFGF bioavailability. To investigate interactions between bFGF and HIP/L29, we isolated and cultured fibroblasts from normal gingiva and overgrown gingiva from patients on cyclosporine (CSA). bFGF significantly stimulated gingival fibroblast proliferation with or without heparin. Recombinant human HIP/L29 dramatically decreased bFGF-induced proliferation, but did not alter responses to insulin-like growth factor-1 (IGF-1). Analysis of mitogen-activated protein kinase (MAPK) phosphorylation patterns showed that bFGF stimulation of p44 (Erk-1), but not p42 (Erk-2), also was inhibited by HIP/L29 in a dose-dependent manner. Together, these results support our hypothesis that HIP/L29 modulates the bioavailability and action of bFGF.

Perlecan: an important component of the cartilage pericellular matrix.

Perlecan (Pln) is a large proteoglycan that can bear HS (heparan sulfate) and chondroitin sulfate glycosaminoglycans. Previous studies have demonstrated that Pln can interact with growth factors and cell surfaces either via its constituent glycosaminoglycan chains or core protein. Herein, we summarize studies demonstrating spatially and temporally regulated expression of Pln mRNA and protein in developing and mature cartilage. Mutations either in the Pln gene or in genes involved in glycosaminoglycan assembly result in severe cartilage phenotypes seen in both human syndromes and mouse model systems. In vitro studies demonstrate that Pln can trigger chondrogenic differentiation of multipotential mouse CH310T1/2 stem cells as well as maintain the phenotype of adult human chondrocytes. Structural mapping indicates that these activities lie entirely within domain I, a region unique to Pln, and that they require glycosaminoglycans. We also discuss data indicating that Pln cooperates with the key chondrogenic growth factor, BMP-2, to promote expression of hypertrophic chondrocyte markers. Collectively, these studies indicate that Pln is an important component of human cartilage and may have useful applications in tissue engineering and cartilage-directed therapeutics.

Expression of heparin/heparan sulfate interacting protein/ribosomal protein l29 during the estrous cycle and early pregnancy in the mouse.

Using a variety of approaches, we have examined the expression of the heparin/heparan sulfate (Hp/HS) interacting protein/ribosomal protein L29 (HIP/RPL29) in mouse uteri during the estrous cycle and early pregnancy. HIP/RPL29 selectively binds heparin and HS and may promote HS-dependent embryo adhesion. HIP/RPL29 was prominently expressed in both luminal and glandular epithelia under almost all conditions, including the phase of embryo attachment. In contrast, differences were noted in HIP/RPL29 expression in the stromal compartment both during the estrous cycle and during early pregnancy. Most notably, HIP/RPL29 accumulated in decidua, where it displayed a pattern complementary to that of pericellular deposition of the HS proteoglycan, perlecan. HIP/RPL29 protein was detected in implanted embryos at both initial and later stages of implantation; however, embryonic HIP/RPL29 mRNA accumulation was more pronounced at later stages (Day 7.5 postcoitum). In situ hybridization revealed similar spatial changes for HIP/RPL29 mRNA during these different physiological states. Whereas differences in the spatial pattern of HIP/RPL29 protein and mRNA expression were demonstrable, little change was detected in the level of HIP/RPL29 mRNA or protein in total endometrial extracts. Mouse blastocysts attached, but did not outgrow, on surfaces coated with recombinant murine HIP/RPL29. Surprisingly, soluble glycosaminoglycans including heparin, low molecular weight heparin, or chondroitin sulfate were not able to inhibit embryo attachment to HIP/RPL29-coated surfaces. These latter observations indicate that embryonic cell surface components other than HS proteoglycans can promote binding to HIP/RPL29 expressed by uterine cells.

Multiple domains contribute to heparin/heparan sulfate binding by human HIP/L29.

Human heparin/heparan sulfate interacting protein/L29 (HIP/L29) is thought to be involved in the promotion of cell adhesion, the promotion of cell growth in the cancerous state, and the modulation of blood coagulation. These activities are consistent with the proposed function of HIP/L29 as a heparin/heparan sulfate (Hp/HS) binding growth factor that has a preference for anticoagulantly active Hp/HS. Previous studies showed that a peptide derived from the C terminus of human HIP/L29 (HIP peptide-1) can selectively bind anticoagulant Hp and support cell adhesion. However, a murine ortholog does not have an identical HIP peptide-1 sequence, yet still retains the ability to bind Hp, suggesting that there may be additional Hp/HS binding sites outside of the HIP peptide-1 domain. To test this hypothesis, a systematic study of the domains within human and murine HIP/L29 responsible for Hp/HS binding activity was undertaken. Using deletion mutants, proteolytic fragments, and protease protection of HIP/L29 by Hp, we demonstrate that multiple binding domains contribute to the overall Hp/HS binding activity of HIP/L29 proteins. Furthermore, a conformational change is induced in human HIP/L29 upon Hp binding as detected by circular dichroism spectroscopy. These studies demonstrate the multiplicity of Hp/HS binding sequences within human and murine HIP/L29.

Cloning, expression, and chromosome mapping of the murine Hip/Rpl29 gene.

We previously have identified murine heparin/heparan sulfate-interacting protein (HIP) identical to mouse ribosomal protein L29 that is, like its human orthologue, distinctively expressed both on the cell surface and intracellularly in different adult tissues and cell types. In the present study, we show that mouse HIP/RPL29 is encoded by a single mRNA and that it is expressed to different extents in most of the tissues of the developing embryo without restriction to a specific cell type. We isolated the single-copy gene coding for murine Hip/Rpl29 among a large number of pseudogenes, established its structure, and assigned its location to distal chromosome 9. Similar to other ribosomal protein promoters, the promoter of Hip/Rpl29 is rich in polypyrimidine tracts, contains binding motifs for ubiquitously expressed transcription factors, and lacks a TATA box. Progressive 5' deletion analyses identified a strong enhancer region that includes CT-rich sequences and a potential consensus binding site for NF-kappaB. These data will provide valuable tools to progress the understanding of HIP/RPL29 function as a ribosomal protein and/or as a regulator of growth and cell adhesion through interaction with heparan sulfate proteoglycans.

Cytoskeletal abnormalities in chondrocytes with EXT1 and EXT2 mutations.

The EXT genes are a group of putative tumor suppressor genes that previously have been shown to participate in the development of hereditary multiple exostoses (HME), HME-associated and isolated chondrosarcomas. Two HME disease genes, EXT1 and EXT2, have been identified and are expressed ubiquitously. However, the only known effect of mutations in the EXT genes is on chondrocyte function as evidenced by aberrant proliferation of chondrocytes leading to formation of bony, cartilage-capped projections (exostoses). In this study, we have characterized exostosis chondrocytes from three patients with HME (one with EXT1 and two with EXT2 germline mutations) and from one individual with a non-HME, isolated exostosis. At the light microscopic level, exostosis chondrocytes have a stellate appearance with elongated inclusions in the cytoplasm. Confocal and immunofluorescence of in vitro and in vivo chondrocytes showed that these massive accumulations are composed of actin bundled by 1.5-microm repeat cross-bridges of alpha-actinin. Western blot analysis shows that exostosis chondrocytes from two out of three patients aberrantly produce high levels of muscle-specific alpha-actin, whereas beta-actin levels are similar to normal chondrocytes. These findings suggest that mutations in the EXT genes cause abnormal processing of cytoskeleton proteins in chondrocytes.

Mammalian reproductive tract mucins.

Mucin glycoproteins are major constituents of the glycocalyx that covers mucosal epithelium. Two broad classes of mucins exist: membrane-associated and secretory. Of the secreted mucins, those with cysteine-rich regions are thought to polymerize through disulphide bonds. Among these gel-forming mucins are MUC2, MUC5AC, MUC5B and possibly MUC6. MUC7 lacks cysteine-rich domains and is thought to be secreted as a soluble monomer. Incomplete sequence information prevents classification of other mucins. Tandem repeats of amino acids rich in serine, threonine and proline are a common element in mucin core proteins, giving rise to relatively rigid, linear molecules with great potential for glycosylation. Ten distinct mucin genes have been identified in humans so far. Patterns of expression vary greatly. While MUC9, or oviductin, appears to be restricted to oviduct, the transmembrane mucin MUC1 is widely expressed. Proven functions for the different mucins are largely unknown, although potential functions are addressed in this review. Genetic and protein sequence information and expression profiles are also summarized, followed by a description of mucin assembly. Special attention is given to mucin expression in male and female reproductive tracts.

Bacterial conjunctivitis in Muc1 null mice.

PURPOSE: In contrast to wild-type mice, genetically engineered Mucin1 (Muc1) null animals display a marked propensity for development of blepharitis and conjunctivitis. Molecular approaches confirmed the presence of Muc1 mRNA and protein in the conjunctival tissue of wild-type mice and identified the bacterial species in Muc1 null symptomatic mice. METHODS: Muc1 null animals housed in a conventional facility were examined for visually apparent inflammation of the eye and surrounding tissue. Blood taken from overtly affected animals was assayed for antibodies to common murine viral agents. Swabs of infected eyes and whole eye preparations were used to detect and speciate bacterial pathogens. Frozen sections of whole eye, lid margin, and Harderian gland were immunostained with antibodies to Muc1 and cytokeratin 14, both epithelial cell markers. Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were performed on RNA isolated from conjunctiva and Harderian gland of wild-type mice to compare relative levels of transcript. RESULTS: Student's unpaired t-test performed on the eye inflammation frequency of Muc1 null mice confirmed a statistical significance (P < 0.01) when compared to wild-type background animals housed in the same room. Analysis of blood samples from affected Muc1 null animals detected no common murine viral pathogens. Bacterial analysis of conjunctival swabs and whole eye preparations demonstrated the presence of coagulase-negative Staphylococcus, Streptococcus type alpha, and Corynebacterium group G2. Muc1 antibody staining of wild-type sections revealed the presence of Muc1 on conjunctival goblet and non-goblet cells and on the epithelium of the Harderian gland. Serial sections stained with cytokeratin 14 antibody confirmed the epithelial nature of cells expressing the Muc1 protein. RNA from conjunctiva and Harderian gland subjected to RT-PCR and northern blot analysis showed an abundance of Muc1 transcript in these tissues. CONCLUSIONS: Muc1 mRNA and protein are present in murine conjunctival and Harderian gland epithelia. Animals lacking Muc1 mRNA and protein are predisposed to developing eye inflammation when compared to wild-type animals with an intact Muc1 gene. Muc1 appears to play a critical protective role at the ocular surface, presumably by acting as a barrier to infection by certain bacterial strains.

Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.

MUC1/episialin: a critical barrier in the female reproductive tract.

The female reproductive tract must resist microbial infections as well as support embryonic development, implantation and placentation. Reproductive tract mucins, in general, and Muc1/episialin, in particular, play key roles in implantation related events and in protection from microbial infection. High levels of mucin expression in the lower reproductive tract presumably affords protection against infection while down-regulation of uterine mucins has been suggested to provide access to the uterine surface. The present studies demonstrate that mucins, particularly Muc1, are effective barriers to embryo attachment. Furthermore, a strain of female Muc1 null mice in normal housing displays chronic infection and inflammation of the lower reproductive tract and markedly reduced fertility rates. This phenotype is not observed when Muc1 nulls are housed in a pathogen-free environment indicating that this phenotype results from chronic microbial exposure. Only normal endogenous flora were isolated from the reproductive tracts of affected Muc1 null mice, suggesting that these bacterial species become opportunistic with loss of the mucin barrier. Staphylococcal adherence to lower reproductive tract epithelia was found to be mediated by cell surface mucin carbohydrates. Collectively, these studies demonstrate a critical barrier role for Muc1 in various aspects of female reproductive tract physiology.

Dynamics of uterine glycoconjugate expression and function.

Embryo attachment to the apical surface of the uterine epithelium is an event found in all mammalian species. Consequently, aspects of this process may be shared and considered as general principles in implantation strategies across species. This review focuses on studies implicating mucin glycoproteins disposed at the apical surface of uterine epithelia as antiadhesive molecules that block embryo attachment. As such, mucins must be removed, at least locally, to permit intimate contact between trophectoderm and uterine epithelia. Subsequently, we consider the role that heparan sulfate proteoglycans (HSPGs) and HSPG-binding proteins play in tethering embryos to the apical surface of uterine epithelia during the attachment process.

Regional specialization of the cell membrane-associated, polymorphic mucin (MUC1) in human uterine epithelia.

The cell membrane-associated, polymorphic mucin, MUC1, has been proposed to hinder implantation by virtue of its anti-adhesive properties. Consistent with this proposal is the observation of a dramatic decrease in MUC1 protein and mRNA expression in the uterine epithelium of several species at the time of implantation. In contrast, little change in glandular epithelial expression of MUC1 protein or its mRNA during the peri-implantation period has been detected in humans. However, expression in the luminal epithelium, i.e. the epithelium involved in embryo attachment, has not been reported. Using tissue samples with a clearly defined luminal epithelium and antibodies directed against the cytoplasmic domain found in all cell-associated MUC1 species (CT-1) and against two MUC1 ectodomain epitopes, HMFG-1 and HMFG-2, we demonstrate that MUC1 expression in the luminal epithelium is maintained throughout the menstrual cycle. The staining observed with CT-1 correlates with that seen with HMFG-2, but not HMFG-1. HMFG-1 reactivity was high in all regions except basal glands in the mid proliferative endometrium and fell to very low levels throughout the tissue in the mid secretory phase. In all cases, HMFG-1 reactivity could be restored by predigestion with keratanase or neuraminidase which removes keratan sulphates and sialic acids, respectively. These observations suggest that regionally restricted glycosylation generates an altered external structure of MUC1. These alterations appear to decrease accessibility to the MUC1 protein core region and are maximal in luminal epithelium at the receptive phase. Due to their large highly extended structures, MUC1 ectodomains are very likely to be among the first cell surface components encountered during human blastocyst attachment to the luminal epithelium. Thus, MUC1 either must be locally removed during the attachment process or functions actually to promote the initial steps in embryo adhesion to the apical surface of the human uterine epithelium.

Estrogen receptor does not directly regulate the murine Muc-1 promoter.

Muc-1 is a heavily O-glycosylated, type 1 membrane glycoprotein present on the surface of polarized secretory uterine epithelial cells. Previous studies have shown that treatment of ovariectomized mice with 17-beta-estradiol (E2) strongly induces Muc-1 mRNA expression in an estrogen receptor (ER)-mediated fashion in the uterus. In this study, the 5.4 kb Muc-1 gene promoter has been isolated from a mouse genomic library and the proximal 1.85 kb region has been sequenced. Sequence analysis revealed the presence of one potential full estrogen response element (ERE) (GCTCGCGGTGACC) located at -748 to -735 bp in the Muc-1 promoter and several potential ERE half sites. Electrophoretic mobility shift assays (EMSA) showed that neither ERalpha nor ERbeta bind efficiently to this sequence. Transient cotransfection assays using constructs containing various deletion mutations of the 5' Muc-1 flanking sequences showed that E2 had no direct stimulation on promoter-driven reporter in NMuMG cells or primary mouse uterine epithelial cells, but did stimulate a consensus ERE CAT-reporter gene activity. In addition, E2-treatment of Weg-ER cells, a mouse uterine epithelial cell line stably expressing human ERalpha, did not restore endogenous Muc-1 expression or activate Muc-1 promoter-driven CAT activity. These results indicate that regions of the Muc-1 gene promoter within -1838 to +43 bp do not respond to E2 and ER stimulation and that ER alone is not sufficient to restore Muc1 gene expression. Deletion analyses also revealed that the sequence between -73 and +43 bp of the Muc-1 promoter is the minimal promoter region required for maximal Muc-1 promoter activity. Collectively, these results demonstrate that ER does not directly regulate the 1.85 kb murine Muc-1 gene promoter. Therefore, E2 control of uterine Muc-1 gene expression is likely to be indirect, i.e. mediated by stromal cell-derived factors.