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BOR - Papers in Press, published online ahead of print November 7, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.064584
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BIOLOGY OF REPRODUCTION 78, 290–298 (2008)
DOI: 10.1095/biolreprod.107.064584
© 2008 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract; 

The MUC1 HMFG1 Glycoform Is a Precursor to the 214D4 Glycoform in the Human Uterine Epithelial Cell Line, HES1

Peng Wang , JoAnne A Julian , and Daniel D Carson  2

Department of Biological Sciences, University of Delaware, Newark, Delaware 19716

ABSTRACT

MUC1, a type I transmembrane glycoprotein expressed on most epithelia and many cancer cells, is involved in embryo implantation and tumor progression. A series of antibodies directed against the MUC1 ectodomain have been used to study MUC1 expression in the female reproductive tract, sometimes with apparently contradictory results. In the current study, we used two monoclonal MUC1 antibodies, 214D4 and HMFG1, to study the relationship between these MUC1 glycoforms in the human uterine epithelial cell line, HES, and human endometrial extracts. In response to tumor necrosis factor stimulation, accumulation of the HMFG1-reactive forms preceded that of the 214D4-reactive forms. Following inhibition of protein synthesis by cycloheximide, HMFG1-reactive species were lost rapidly (metabolic half-life [T1/2] = 20 min), while there was no change in the level of the 214D4-reactive forms even after 80 min. HMFG1-reactive forms had smaller oligosaccharide chains than the 214D4-reactive forms, and could not be detected on the cell surface of intact cells or in the shed (media) fraction, although they were readily detected in permeabilized cells. Both 214D4- and HMFG1-reactive species were detected in human endometrial extracts throughout the cycle; however, consistent with the HES cell studies, the HMFG1-reactive species were both smaller and less abundant than the 214D4-reactive species. Consistent with this observation, we found that HMFG1-reactive species were difficult to detect in tissue sections unless predigested with neuraminidase, indicating that these structures are rapidly sialylated during synthesis. In contrast, 214D4-reactive species were robustly detected in both proliferative and secretory stages. Collectively, these studies indicate that the HMFG1-reactive glycoform is a precursor of the 214D4-reactive glycoform in HES cells and normal uterine epithelia. Therefore, discrepancies in patterns of MUC1 expression in other studies may be due to failure to account for these glycoform relationships.

214D4, epitope, glycoform, HMFG1, MUC1

INTRODUCTION

The polymorphic mucin, MUC1, is expressed by a variety of carcinomas and normal epithelial cells, including the female reproductive tract [1–4]. MUC1 functions in conjunction with other cell surface and secreted mucins to lubricate and protect the apical cell surface from attack by micro-organisms, toxins, and proteases [5, 6]. In the uterus, the protective function of MUC1 constitutes a potential barrier to embryo adhesion. For implantation to occur, MUC1 is downregulated in most species, including old world, nonhuman primates [5, 7, 8]. The human uterus appears to be an exception, since MUC1 has been detected at the apical surface of luminal epithelia during the receptive period of the secretory phase of the menstrual cycle [9, 10]. To account for this apparent contradiction, it has been suggested that MUC1 is either locally removed in response to embryonic signals or functionally altered from antiadhesive to adhesive [11–13].

The barrier function of MUC1 requires a minimum number of tandem repeats [14]. These tandem repeats are rich in serine, threonine, and proline, providing sites for O-glycosylation [14, 15]. The highly glycosylated ectodomain projects 200–500 nm above the cell surface, extending far above most other cell surface components [6]. Early studies of MUC1 expression in the uterus primarily utilized antibodies that recognize peptide epitopes within the tandem repeats [16–18]. The ability of these antibodies to detect MUC1 is known to be restricted to varying degrees by the carbohydrate chains associated with the tandem repeat sequences, which has complicated interpretation of the expression pattern. MUC1 glycoforms are expressed in a temporally and compartmentally distinct fashion [9, 10]. The nature of the carbohydrate chains associated with the various glycoforms ultimately defines antibody reactivity and perhaps even the function of the subset of MUC1 core proteins that carry them. Characterization of carbohydrate chains associated with various MUC1 glycoforms will allow us to determine if there is a functional change in the MUC1 expression during the receptive phase at the apical surface of luminal epithelia.

The polymorphic nature of MUC1 has presented a particular challenge to investigators attempting to characterize MUC1-associated glycans in other tissues. Allelic polymorphisms generating different size variants complicate a direct comparison of MUC1 glycoforms expressed in either normal or malignant tissues or cell lines from different individuals [19–23]. Both the size of the glycoform and number of attached glycans per molecule vary with the number of tandem repeats. Tissue-specific patterns of glycosylation make it necessary to evaluate MUC1 glycoforms in different tissues independently [24]. Finally, differences in structure of the glycans and extent of substitution that occur between normal epithelia and their malignant counterparts dictate analyses of MUC1 from normal tissue or cell lines to determine structure-function relationships [24, 25].

Many monoclonal antibodies have been developed against MUC1, and several of these have been used in previous studies profiling uterine MUC1 expression during the human menstrual cycle. Nonetheless, the different reactivities of these antibodies may contribute to some confusion, since the expression patterns they produce are distinct [16, 17]. In general, HMFG1 reactivity, although higher than HMFG2 and SM3, is low relative to that of other MUC1-directed antibodies, especially during the luteal phase; however, glycosidase digestion reveals cryptic HMFG1 epitopes, indicating that this epitope is masked by additional sugar modifications. The International Society for Oncodevelopmental Biology and Medicine TD-4 Workshop investigated the reactivity and specificity of 56 monoclonal MUC1 antibodies, and showed the majority of antibodies (34/56) defined epitopes located within the 20 amino acid tandem repeat sequence of the MUC1 protein core. Moreover, it was found that the epitopes of 16 antibodies involve carbohydrate residues that regulate accessibility to mask, or stabilize epitope conformations [26]. The monoclonal antibody, HMFG1, was generated against delipidated preparations of human milk fat globule. The tandem repeat peptide (proline-glutamate-threonine-arginine [PDTR]) epitope recognized by HMFG1 is masked to varying degrees by glycosylation in either normal or malignant cells [27]. While the 214D4 antibody is also directed against the MUC1 ectodomain, its epitope has not been determined [28, 29]. MUC1 structural differences must underlie the different antibody reactivities; however, the relationships among these reactivities have not been defined in most cases, generating confusing interpretations.

In previous studies, we have shown that MUC1 core protein expression is regulated at a transcriptional level by steroid hormones and cytokines, including progesterone [30], interferon-gamma (IFNG) and tumor necrosis factor (TNF) [31, 32]. In the course of continuing our studies of the regulation of MUC1 expression and metabolism in uterine epithelia, we found that HMFG1 and 214D4 antibodies recognize distinct subsets of MUC1. This observation led us to speculate that this distinction might account for differences in MUC1 expression and distribution observed in uterine tissues when using HMFG1 versus other antibodies. The present study indicates that the HMFG1-reactive species are precursors to the 214D4-reactive species in HES cells and, we propose, normal human endometrium.

MATERIALS AND METHODS

Cell Culture, TNF Treatment, and Shedding Assay

The human uterine epithelial cell line, HES, was kindly provided by Dr. Doug Kniss (Ohio State University, Columbus, OH). HES cells were maintained in Dulbecco modified Eagle medium (DMEM [GIBCO 11965–092]; Invitrogen, Carlsbad, CA), supplemented with 10% (v/v) charcoal-stripped fetal bovine serum (FBS; HyClone, Logan, UT), and 100 µM sodium pyruvate (Sigma-Aldrich, St. Louis, MO). Cells were seeded on 24-well tissue culture plates and maintained until cells reached 80% confluence. To stimulate MUC1 expression, HES cells were serum starved for 24 h and then treated with 25 ng/ml TNF (Roche Biochemicals, Indianapolis, IN) in fresh serum-free medium for 48 h. Medium was removed and centrifuged 20 min at 3000 x g at 4°C. Shed and secreted proteins in cell culture medium, with 2 µl FBS added per milliliter as carrier, were precipitated with 10% (w/v) trichloroacetic acid, and the precipitate rinsed with acetone as previously described [33]. The resulting pellet was solubilized in sample extraction buffer (SEB; 0.05 M Tris, pH 7, 8 M urea, 1% [w/v] SDS, 0.01% [v/v] PMSF, 1% [v/v] β-mercaptoethanol) for Western blot analysis.

Cycloheximide Inhibition Experiments

HES cells were cultured and treated with TNF, as described above. Subsequently, the cells were treated with cycloheximide (0.1–5.0 µg/ml, as indicated, dissolved in serum-free medium) for 2.5 h prior to harvest. To examine the efficiency of cycloheximide inhibition of protein synthesis, 3H-amino acid mixture (20 µCi/ml) was added 2 h prior to harvest. Parallel control cultures were incubated for the same period in medium lacking cycloheximide. For time-course studies of the effects on MUC1 expression, 5 µg/ml of cycloheximide was used for 10–80 min on TNF-treated HES cells.

Western Blotting

Cells were cultured and treated with TNF as described above. After treatment, cells were solubilized with SEB, then diluted 1:1 in Laemmli sample buffer [34]. Proteins (15% of SEB extract from each well) were separated by SDS-PAGE using a 3.5% (w/v) polyacrylamide stacking gel and a 10% (w/v) polyacrylamide resolving gel [35]. Proteins were then transferred to a nitrocellulose membrane at 4°C, as previously described [33]. Blots were blocked at 4°C in Dulbecco phosphate-buffered saline (PBS) plus 0.1% (v/v) Tween-20 and 3% (w/v) BSA. The MUC1 primary antibody, 214D4 (kindly provided by Dr. John Hilkens, The Netherlands Cancer Institute, Amsterdam, The Netherlands) [14], was added to a final dilution of 1:2000. MUC1 primary antibody, HMFG1 (kindly provided by Dr. Sandra Gendler, Mayo Clinic, Scottsdale, AZ) [27], was added to a final dilution of 1:1000. MUC1 primary antibody, CT1 [9], was added to a final dilution of 1:2000. ACTB primary antibody (Abcam, Cambridge, MA) was added to a final dilution of 1:10 000. Blots were incubated with the primary antibody overnight at 4°C with constant rotary agitation. Subsequently, blots were incubated for 1 h at room temperature with a secondary antibody, peroxidase-conjugated donkey anti-mouse immunoglobulin (Ig) G (Jackson Immunoresearch, West Grove, PA), at a final dilution of 1:200 000 in blocking solution. SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL) was used for detection per the manufacturer's instructions. Blots were exposed to x-ray film, and signal intensities were quantified using the Alpha Imager 1D-Multi Function (Alpha Innotech, San Leandro, CA).

Immunoprecipitation

HES cells were cultured and treated with TNF, as described above, and extracted in 300 µl boiling lysis buffer (0.5% [v/v] NP40, 1 mM EDTA, plus 1% [v/v] protease inhibitors [P8340; Sigma-Aldrich] in PBS). After preclearing by centrifugation, the extracts were incubated by constant rotary agitation overnight at 4°C with primary antibody (214D4 [10–40 µl], HMFG1 [30–60 µl]). The antigen-antibody complex was incubated for 6 h at 4°C with constant rotary agitation after the addition of 100 µl of a 50% (v/v) slurry of protein G-agarose (Kirkegaard & Perry Laboratories, Gaithersburg, MD) that had been preblocked with heat-inactivated serum. The resin was separated from the solution by centrifugation at 8000 x g for 3 min and the pellet rinsed twice with 1 ml lysis buffer and twice with 1 ml PBS. The immunoprecipitated MUC1 was solubilized from the resin by the addition of 50 µl of SEB followed by boiling for 5 min. After cooling and centrifugation, 50 µl of Laemmli sample buffer was added prior to SDS-PAGE.

3H-Glucosamine Labeling, β-Elimination, and Molecular Exclusion Chromatography

HES cells were cultured to confluence on a Matrigel-coated tissue culture plate, and then incubated for 2 days in low glucosamine DMEM (GIBCO 11885–084; Invitrogen), in which 25 µCi/ml 3H-glucosamine (American Radiolabeled Chemicals, Inc., St. Louis, MO), 25 ng/ml TNF, and 200 U/ml IFNG (Roche Biochemicals, Indianapolis, IN) were added. To maximize the amount of 3H-glucosamine-labeled MUC1, IFNG was included, as previous studies indicated that this further enhanced MUC1 expression in HES cells, as has been found in other systems [32, 36]. The cells were lysed and immunoprecipitated with HMFG1 or 214D4 antibody, as described above. The immunoprecipitates (HMFG1 or 214D4) on protein G-agarose beads were incubated in β-elimination buffer (0.1 M NaOH, 0.25 M NaBH4) at 37°C for 2 days and neutralized with 1 M HCl. After removal of the resin by centrifugation, supernates were dried under vacuum, redissolved in 0.5 ml PBS, and then applied to a Sephadex G-50 column (0.8 cm x 60 cm) equilibrated in PBS. Fractions (15 drops per tube) were collected and radioactivity in each fraction determined by liquid scintillation (TRI-CARB 2900TR; PerkinElmer Life Analytical Sciences Inc., Wellesley, MA). Void volume (VO) and total column volume (VT) were determined by the elution positions of blue dextran and potassium dichromate, respectively.

Immunostaining

HES cells were plated on glass coverslips in 24-well plates and maintained as described above until cells reached 80% confluence. After treatment with TNF, as described above, cells were rinsed with PBS and fixed with 2.5% (w/v) paraformaldehyde (EMS, Hatfield, PA) diluted in PBS for 10 min at room temperature. The samples then were rinsed with PBS and excess formaldehyde quenched with 50 mM NH4Cl diluted in PBS for 15 min at room temperature. Some samples were subsequently permeabilized with 0.25% (v/v) Triton X-100 diluted in PBS for 2 min at room temperature. After a further rinse in PBS, samples were incubated with the MUC1 primary antibody, 214D4 (used at 2.7 µg/ml mouse IgG in hybridoma medium diluted 1:1 with PBS), HMFG1 (undiluted hybridoma medium, approximately 3 µg/ml mouse IgG), or nonimmune mouse IgG (2.7 µg/ml) for 1 h at 37°C. After rinsing three times with PBS, samples were incubated with fluorescein-conjugated sheep anti-mouse IgG (Amersham Biosciences, Pittsburgh, PA). Unbound secondary antibody was removed by rinsing three times with PBS. Slides were then imaged on a Zeiss Axioskop 2 equipped with a Spot cooled color digital camera and Spot software v2.1 (Diagnostic Instruments Inc., Sterling Heights, MI).

Immunohistochemistry

Frozen 6-µm sections of staged, normal cycling human uterine endometrium were fixed for 10 min at room temperature in 100% methanol and rehydrated for 30 min in Ca++- and Mg++-free PBS. Sections were treated for 1 h at 37°C with 3 U/ml neuraminidase (Sigma N7885, from Vibrio cholerae) and rinsed twice for 5 min with PBS. Sections were incubated for 3 h at room temperature with undiluted primary antibodies, 214D4 or HMFG1, as described above (both in hybridoma medium containing 10% [v/v] FBS) and rinsed three times for 5 min with PBS. Sections were incubated with fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (Amersham) diluted 1:10 (v/v) in PBS containing 0.1 µg/ml 4',6'-diamidino-2-phenylindole for 1.5 h at room temperature, and rinsed three times for 5 min in PBS. Samples were mounted and imaged as described above.

Human Endometrial Tissues

Human endometrial tissues were collected with informed consent from a pool of fertile volunteers; the collection was staged as previously described [37]. The use of human subjects and the procedures were approved by the institutional review boards of the University of Delaware and the University of North Carolina at Chapel Hill. Samples were prepared for analysis by solubilization in SEB.

RESULTS

HMFG1 and 214D4 Detect Distinct MUC1 Isoforms in TNF-Stimulated HES Cells

Previous studies from our lab showed TNF stimulates MUC1 expression through the NF-{kappa}B site from –589 to –580 of the human MUC1 promoter [31]. We also found that this treatment greatly enhanced expression of a form of MUC1 detected with the HMFG1 antibody. The increase in HMFG1 immunoactivity could reflect either the appearance of HMFG1-reactive epitopes on MUC1 molecules that were also 214D4-reactive, or the accumulation of a new, distinct MUC1 population. Therefore, we performed experiments to distinguish between these possibilities. First, lysates of TNF-stimulated HES cells were electrophoresed on SDS-PAGE and blotted with HMFG1, 214D4, or both (Fig. 1A). Both antibodies recognized multiple, diffuse bands due to allelic polymorphism and the inherent heterogeneity of glycosylation; however, the bands recognized by HMFG1 were both more diffuse and generally migrated faster than those recognized by 214D4. Next, we used either HMFG1 or 214D4 antibodies to immunoprecipitate extracts of TNF-stimulated HES cells. Although we were unable to immunoprecipitate the 214D4-reactve forms completely, the immunoprecipitable forms were not reactive with HMFG1 (Fig. 1B). Conversely, immunoprecipitation with HMFG1 failed to immunoprecipitate 214D4-reactive forms (Fig. 1C). Predigestion of these samples with neuraminidase did not convert either form into species that were recognized by the other antibody (data not shown). Therefore, it appeared that HMFG1- and 214D4-reactive species represented distinct forms of MUC1 and that these forms differed by more than the degree of sialylation.


Figure 1
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  		FIG. 1 HMFG1- and 214D4-reactive species are distinct isoforms of MUC1 in TNF-stimulated HES cells. HES cells were treated with 25 ng/ml TNF for 48 h in serum-free medium prior to extraction, immunoprecipitation, and Western blot analyses, as described in Materials and Methods. A) Triplicate samples of HES cell lysates were probed with 214D4 antibody alone and 214D4 and HMFG1 antibodies together or HMFG1 antibody alone by Western blotting. B) Duplicate extracts were immunoprecipitated with the indicated amounts of 214D4 antibody and the material in the immunoprecipitates (IP by 214D4), nonimmunoprecipitated material (supernatant), or total lysate (lysate) were analyzed by Western blotting with 214D4 or HMFG1 antibody, as indicated in the figure. C) Duplicate extracts were immunoprecipitated with the indicated amounts of HMFG1 antibody and the material in the immunoprecipitates (IP by HMFG1), nonimmunoprecipitated material (supernatant), or total lysate (lysate) were analyzed by Western blotting with 214D4 or HMFG1 antibody, as indicated in the figure. The migration position of a 250-kDa molecular weight marker is indicated to the left of each blot.

HMFG1 Is a Precursor of the 214D4 Isoform

One possible cause for the occurrence of these two immunologically defined subpopulations was that a precursor-product relationship existed between them. Consistent with this idea, we found that the HMFG1-reactive species accumulated more rapidly in response to TNF treatment than the 214D4-reactive species (Fig. 2). If the HMFG1-reactive species were metabolic precursors to the 214D4-reactive species, then the HMFG1-reactive forms should be lost first when further MUC1 synthesis is inhibited. Therefore, we inhibited de novo protein synthesis and monitored expression of both MUC1 isoforms. Cycloheximide inhibited more than 95% of general protein synthesis in HES cells at a concentration of 5 µg/ml (Fig. 3A). When HES cells were treated with this dose of cycloheximide for 0–80 min, no significant difference in the cellular levels of 214D4-reactive isoforms was detected; however, after a 10-min lag period, the HMFG1-reactive isoforms were lost rapidly with a half-life of approximately 20 min (Fig. 3, B and C). Almost all HMFG1-reactive material was lost by 80 min. In contrast, there was no apparent decrease in the levels of 214D4-reactive material over this time period. The cytoplasmic tail-reactive MUC1 antibody, CT-1, was used to detect all cell-associated forms of MUC1, and showed a similar pattern of stability as the 214D4-reactive forms. These observations indicate that HMFG1-reactive forms are likely to be metabolic intermediates and precursors to the 214D4-reactive forms.


Figure 2
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  		FIG. 2 Time course of TNF stimulation of MUC1 expression. HES cells were treated with 25 ng/ml TNF for the indicated times in serum-free medium extracts analyzed by Western blotting with either 214D4 (A) or HMFG1 (B) antibody, and ACTB antibody as loading control, as described in Materials and Methods. The bar graphs below the images represent the densitometric analyses of these data provided as means ± SEM in each case.


Figure 3
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  		FIG. 3 HMFG1 is a precursor of the 214D4 isoform. A) HES cells were treated with the indicated concentrations of cycloheximide for 30 min, then incubated with a 3H-amino acid mixture for 2 h, and incorporation into protein assessed as described in Materials and Methods. B) Duplicate series of HES cells were incubated without (vehicle) or with 25 ng/ml TNF for 2 days. One series of TNF-treated cells was then incubated with 5 µg/ml cycloheximide for 10–80 min prior to extraction. Equivalent amounts of extracts in each series were then analyzed by Western blotting with HMFG1, 214D4, CT-1, or ACTB antibody, as described in Materials and Methods. C) Densitometry of Western blots, normalized to ACTB. The values determined for samples treated with TNF, but without cycloheximde, were defined as 1. CPMA, counts per minute, channel A.

HMFG1-Reactive Species Contain Smaller Oligosaccharides than Those of 214D4-Reactive Species

If the HMFG1-reactive MUC1 forms were precursors to the 214D4-reactive forms, then we considered that the oligosaccharides chains of the HMFG1-reactive forms would be smaller than those of the 214D4-reactive forms. HES cells were labeled with 3H-glucosamine and immunoprecipitated with HMFG1 or 214D4 antibodies. The immunoprecipitates were then subjected to β elimination and the released O-linked oligosaccharides analyzed by molecular exclusion chromatography (Fig. 4). The oligosaccharides released from the 214D4-reactive material exhibited a larger size relative to those released from the HMFG1-reactive forms. This observation was consistent with the notion that the HMFG1-reactive forms were precursors to the 214D4-reactive forms and, therefore, did not contain oligosaccharides as fully extended as the 214D4-reactive forms.


Figure 4
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  		FIG. 4 Oligosaccharides of HMFG1-reactive species are smaller than those of 214D4-reactive species. HES cells were incubated with 25 ng/ml TNF and 200 U/ml IFNG for 2 days. Equivalent amounts of cell lysates were immunoprecipitated with HMFG1 or 214D4 antibody, then the HMFG1 or 214D4 immunoprecipitates were released by β elimination and analyzed by Sephadex G-50 molecular exclusion chromatography, as described in Materials and Methods. DPM, disintegrations per minute.

214D4-Reactive, but Not the HMFG1-Reactive, MUC1 Forms Are Accessible at the Cell Surface

If the HMFG1-reactive forms represented intermediates, these forms should be in the intracellular secretory pathway, and not found on the cell surface. To test this idea, intact TNF-stimulated HES cells were immunostained with HMFG1 or 214D4 antibodies (Fig. 5). Only 214D4 demonstrated reactivity under these conditions (Fig. 5, a and c). In contrast, both antibodies displayed reactivity in permeabilized cells (Fig. 5, b and d). These observations were consistent with the notion that HMFG1-reactive forms represent intracellular forms, presumably in the secretory pathway, while the 214D4-reactive forms were mature forms that reached the cell surface.


Figure 5
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  		FIG. 5 The 214D4 isoform, but not the HMFG1 isoform, is accessible at the cell surface. HES cells were incubated with 25 ng/ml TNF for 48 h in serum-containing medium and then formalin fixed prior to immunostaining with 214D4 (a, b), HMFG1 (c, d, f) or a nonimmune mouse IgG control (e) as described in Materials and Methods. One set of monolayers (b, d, e, and f) was permeabilized with Triton X-100 prior to immunostaining to reveal intracellular signal. a–e) Bar = 40 µm; f) Bar = 10 µm.

214D4-Reactive Forms, but Not HMFG1-Reactive Forms, Are Shed

Previous studies have demonstrated that cell surface sheddases release MUC1 ectodomain fragments from HES cells [33, 38]. If both HMFG1- and 214D4-reactive forms reached the cell surface, then both forms of ectodomains would be expected to be found in the conditioned media. Nonetheless, Western blotting revealed that only 214D4-reactive forms accumulated in the media in a time-dependent fashion. Even after 72 h of incubation, we observed no HMFG1-reactive material in the media (i.e., shed) fraction (Fig. 6). These data were consistent with the hypothesis that HMFG1-reactive forms never reach the cell surface and, therefore, are not shed.


Figure 6
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  		FIG. 6 The 214D4 isoform, but not the HMFG1 isoform, is shed. Triplicate samples of HES cells were incubated with 25 ng/ml TNF for the indicated times in serum-free medium. Media then were collected, trichloroacetic acid (TCA) precipitated, and analyzed by Western blotting with either HMFG1 or 214D4 antibody, as described in Materials and Methods. The migration position of a 250-kDa molecular weight marker is indicated to the left of each panel.

Expression of 214D4- and HMFG1-Reactive Species During the Menstrual Cycle

Human endometrial extracts were obtained and used in Western blotting experiments. As shown in Figure 7, reactivity with both antibodies was low in early- and mid-proliferative stage samples; however, throughout the secretory phase, reactivity with 214D4 was robust. In contrast, the species recognized by HMFG1 were both smaller, and reactivity generally was weaker, particularly at the late-secretory phase. Differences in size of species recognized by each antibody among the different samples are attributed to the well-described allelic polymorphism of MUC1 in humans [22]. This pattern of expression is consistent with the notion that the HMFG1-reactive species represent low-abundance precursors to 214D4-reactive species in normal human endometrium. As shown in Figure 8, 214D4-reactive species were readily detectable in epithelia of sections from mid-proliferative- or mid-secretory-stage endometria. In contrast, very little reactivity with HMFG1 was detected at these stages unless sections were first digested with neuraminidase. Reactivity in the proliferative phase was particularly weak and only slightly above the nonspecific background signal evident in the underlying stroma (Fig. 8B). These observations indicate that the HMFG1-reactive epitopes were likely to be transient species that are rapidly sialylated in normal uterine epithelium, and are consistent with the notion that they represent precursors to more mature (i.e., 214D4-reactive) forms.


Figure 7
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  		FIG. 7 Western blotting of 214D4- and HMFG1-reactive species in human endometrial extracts. Staged human endometrial extracts were obtained and analyzed by Western blotting as described in Materials and Methods. Western blots with (A) 214D4 and (B) HMFG1 antibodies. Identical amounts of extracts were used in each case. The migration position of a 203 000 molecular weight marker is indicated to the left of each panel (203). The identities of each lane are as follows: 1, early proliferative; 2, mid-proliferative; 3, early secretory; 4, mid-secretory; 5 and 6, late secretory. Differences in sizes of bands recognized by either antibody in a set of extracts is attributed to the well-described allelic polymorphism of the MUC1 tandem repeat region [22].


Figure 8
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  		FIG. 8 Immunostaining for 214D4- and HMFG1-reactive epitopes in human endometrial sections. MUC1 expression in d9 (A and B) and d24 (C and D) human endometrium was detected by antibodies, 214D4 (A and C) and HMFG1 (B and D), as described in Materials and Methods. Sections stained with HMFG1 required pretreatment with neuraminidase to reveal these epitopes. Staining for MUC1 epitopes is in green, and DAP1 staining for nuclei is in blue. ge, Glandular epithelium; le, luminal epithelium; s, stroma. The magnification bar in C represents 100 µm, and magnification is the same in all panels. The same gland is marked in A and B to facilitate comparison. The uterine lumen is at the top of A, B, C, and to the left in D.

DISCUSSION

Regardless of whether it is expressed in normal or malignant epithelia, full length MUC1 undergoes a complex but ordered series of intracellular processing events. MUC1 is self-cleaved to two polypeptides within the SEA (sea urchin sperm protein, enterokinase and agrin) domain in the endoplasmic reticulum [39]. This heterodimer remains tightly, but noncovalently, associated in both malignant and normal cells as MUC1 transits the metabolic pathway [40, 41]. MUC1 is highly O-glycosylated, with oligosaccharides constituting >50% of the molecular weight of the mature glycoprotein [42]. O-glycosylation begins in the Golgi apparatus with the addition of N-acetylgalactosamine to serine or threonine residues in the tandem repeat residues of MUC1, followed by elongation with N-acetylglucosamine, N-acetylgalactosamine, or galactose residues, to form linear or branched structures, and termination by sialylation, fucosylation, and sulfation. This stepwise process involves many glycosyltransferases and sulfotransferases [43]. Subsequently, MUC1 is transported to the cell surface, where it may be shed [33, 38] or recycled through the trans-Golgi network [44]. Recently, recycling of MUC1 was found to be dependent on palmitoylation at the boundary of the transmembrane and cytoplasmic domains [45], but it is not yet clear whether recycling occurs in normal cells. A variety of monoclonal antibodies, including HMFG1, recognize aberrant MUC1 forms expressed by tumor cells. In this regard, many malignant cells overexpress MUC1 in an underglycosylated form, accumulate MUC1 in intracellular vesicles, and lose apical restriction of cell surface MUC1 [46, 47]. Inhibition of glycosylation in cultured human mucosal cells also leads to accumulation of MUC1 in intracellular vesicles [48]. In previous studies of human endometrial tissue, we found that 214D4 reacts almost exclusively with material at the apical aspect of epithelia, while HMFG1 reacts with intracellular material, presumably in the secretory pathway, with only modest HMFG1 reactivity at the apical aspect [9, 33]. Both findings are consistent with our current result demonstrating that the HMFG1 glycoform expressed by TNF-stimulated HES has shorter O-glycans, is primarily found intracellularly, and is not transported to the cell surface or shed. We also observed that HMFG1-reactive forms generally were smaller and expressed at much lower levels than the 214D4-reactive forms in extracts of normal human endometrium throughout the cycle. Taken together with previous immunostaining studies of human endometrium, these observations suggest that, during the mid-proliferative phase, MUC1 is less glycosylated and, therefore, may not be as effective a barrier as during the secretory phase. The finding of a substantial amount of intracellular HMFG1 reactivity during both the mid-proliferative and mid-luteal phases is consistent with these being precursors to the cell surface forms. During the luteal phase, very little HMFG1 reactivity is observed either by immunostaining or by Western blotting, although glycosidase digestion unmasks these epitopes [9, 17]. We saw reduced staining of lumenal epithelia with 214D4 antibody at the mid-luteal phase as well. In other studies, we were able to detect some 214D4 reactivity in lumenal epithelia at the mid-luteal phase [33, 34]. Overall, these studies suggest that local loss of MUC1 occurs in certain regions of human lumenal epithelia that may provide zones for embryo attachment.

Collectively, our current data indicate that: 1) HMFG1 antibodies do not recognize MUC1 containing fully processed (i.e., 214D4 reactive) O-glycans; 2) 214D4 antibodies do not recognize MUC1 species lacking fully processed O-glycans; and 3) HMFG1-reactive species are metabolic precursors to the 214D4-reactive species. As expected for precursors, the HMFG1 glycoforms accumulated faster than the 214D4-reactive forms in response to TNF, and were barely detectable in nonstimulated cells. Conversely, HMFG1-reactive forms were lost rapidly (metabolic half-life [T1/2] = 20 min) when protein synthesis was inhibited, while no change in cellular levels of 214D4-reactive forms were observed. These data are consistent with the HMFG1-reactive species being metabolic intermediates, while the 214D4-reactive species represent mature MUC1. Previous studies of Muc1 in mouse uterine epithelial cells demonstrated a transit time of 21 ± 15 min from the rough endoplasmic reticulum to Golgi apparatus, a transit time of 111 ± 28 min from the Golgi apparatus to the cell surface, and an overall metabolic half-life of 16.5 ± 0.8 h [49]. This is consistent with our current results demonstrating that the HMFG1-reactive species started to decrease within 10 min (the minimum transit time required for MUC1 from the rough endoplasmic reticulum to the Golgi apparatus), and with a subsequent half-life of 20 min, representing the time required for conversion of the HMFG1 glycoforms to 214D4 glycoforms in the Golgi apparatus.

Initially, the HMFG1 epitope was thought to include both carbohydrate residues and PDTR sequence in the tandem repeat, an interpretation that was revised after the observation that HMFG1 was capable of recognizing PDTRs devoid of glycans [27, 50]. The 214D4 epitope has not been determined [14, 28]. Neuraminidase digestion increases MUC1 detection by HMFG1 on cancer cell lines [51] as well as secretory-phase human uterine epithelia [9, 17, 18]. Nonetheless, we found that neuraminidase digestion of 214D4 immunoprecipitates did not increase recognition by HMFG1 (data not shown). Thus, it appears that the 214D4-reactive species in HES cells contain modifications other than sialylation that prevent recognition by HMFG1. In HES cells, the HMFG1 glycoform was not detected on the cell surface or in shed material; however, HMFG1-reactive forms have been detected on cell surfaces and secreted fractions from certain normal tissues, including the uterus and many malignant cells [9, 51]. In a scanning immunoelectron microscopy study, HMFG1-reactive species were detected in association with microvilli of ciliated luminal epithelial cells of receptive-stage uteri, while microvilli of nonciliated cells were not recognized [52]. Neuraminidase digestion increased intensity of detection on ciliated cells, but uncovered no recognition sites on nonciliated cells. Furthermore, HMFG1 was unable to immunoprecipitate MUC1 released in vitro from secretory-phase endometrial tissue [10], in agreement with our results with HES cells. Collectively, these studies indicate that HMFG1-reactive species fail to reach the cell surface under most conditions. Open questions are whether there are functional differences between these MUC1 species, and whether the ratio of HMFG1- and 214D4-reactive species in secretions might serve as a useful marker of certain physiological or pathological states (e.g., cancer or endometriosis).

Differences in the levels and location of mucin glycosyltransferases among different cell types may lead to more efficient completion or extension of O-linked oligosaccharides. It is possible that the glycosylation apparatus is overwhelmed with substrate under conditions in which MUC1 is highly overexpressed (e.g., in various tumors) or when expression is maximally stimulated by steroid hormones or cytokines. It also is possible that controls exist in cells to prevent cell surface transport of underglycosylated MUC1 in some contexts. For example, MUC1 recycling could represent an aspect of such "proof reading" [44]. We have recently shown that human uterine MUC1 can carry selectin ligands associated with 214D4-reactive species [13]. Selectin ligands are complex structures requiring several terminal steps in O-glycosylation [53]. Although efficient completion of MUC1 oligosaccharides may be characteristic of uterine epithelia, normal in vivo compartmental distinctions have been observed. Keratan sulfate chains containing terminal sialic acid are increased in the secretory phase [54, 55] and are associated with MUC1 [10]. During the receptive period, keratan sulfate disappears from some regions of luminal epithelial apical surface, although MUC1 core protein remains detectable [10]. Sialyl Tn was not detected in luminal epithelia at any stage of the cycle, although it is abundant in glandular epithelia [10, 56]. Interestingly, malignant uterine epithelial cell lines displayed both cell-to-cell and cell line-to-cell line heterogeneity when MUC1 and the selectin ligands, sialyl-Lewis x and sialyl-Lewis a, were examined [57]. In unstimulated HES, selectin ligand epitopes were not recognized in association with MUC1 immunoprecipitated by 214D4, although MUC1 core protein was detected [13]. Thus, independent components of the glycosylation machinery and MUC1 core protein may be subject to regulatory mechanisms dependent on cell context and microenvironment.

ACKNOWLEDGMENTS

We thank Dr. Sandra Gendler for HMFG1 antibody and Dr. John Hilkens for 214D4 antibody. We also greatly appreciate the generous provision of the human endometrial tissue samples by Dr. Bruce Lessey (Greenville Hospital System, Greenville, SC), and the secretarial and graphics assistance of Ms. Sharron Kingston. We also thank Neeraja Dharmaraj and Drs. Melissa Brayman and Mary C. Farach-Carson for many helpful discussions and suggestions.

FOOTNOTES

1Supported by National Institutes of Health grant UO1 HD 29963 to D.D.C. Back

Correspondence: 2Daniel D. Carson, Department of Biological Sciences, University of Delaware, Newark, DE 19716. FAX: 302 831 2281; e-mail: dcarson{at}udel.edu

Received: 27 July 2007.

First decision: 25 August 2007.

Accepted: 25 October 2007.

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